WO1999053101A1 - Identification of genetic targets for modulation by oligonucleotides and generation of oligonucleotides for gene modulation - Google Patents

Identification of genetic targets for modulation by oligonucleotides and generation of oligonucleotides for gene modulation Download PDF

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Publication number
WO1999053101A1
WO1999053101A1 PCT/US1999/008268 US9908268W WO9953101A1 WO 1999053101 A1 WO1999053101 A1 WO 1999053101A1 US 9908268 W US9908268 W US 9908268W WO 9953101 A1 WO9953101 A1 WO 9953101A1
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Prior art keywords
oligonucleotides
nucleic acid
compounds
sequences
silico
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PCT/US1999/008268
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French (fr)
Inventor
Lex M. Cowsert
Brenda F. Baker
John Mcneil
Susan M. Freier
Henri M. Sasmor
Douglas G. Brooks
Cara Ohasi
Jacqueline R. Wyatt
Alexander H. Borchers
Timothy A. Vickers
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Isis Pharmaceuticals, Inc.
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Priority to CA002325013A priority Critical patent/CA2325013A1/en
Priority to DE69933205T priority patent/DE69933205T2/en
Priority to JP2000543647A priority patent/JP2002511276A/en
Priority to AU39650/99A priority patent/AU762397B2/en
Priority to EP99922713A priority patent/EP1071826B1/en
Publication of WO1999053101A1 publication Critical patent/WO1999053101A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811Selection methods for production or design of target specific oligonucleotides or binding molecules
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034Isolating an individual clone by screening libraries
    • C12N15/1048SELEX
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G16B30/10Sequence alignment; Homology search
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B35/00ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B35/00ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
    • G16B35/20Screening of libraries
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16CCOMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
    • G16C20/60In silico combinatorial chemistry
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068Means for controlling the apparatus of the process
    • B01J2219/007Simulation or vitual synthesis
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00ICT specially adapted for sequence analysis involving nucleotides or amino acids

Definitions

  • the present invention relates generally to the generation and identification of synthetic compounds having defined physical, chemical or bioactive properties. More particularly, the present invention relates to the automated generation of oligonucleotide compounds targeted to a given nucleic acid sequence via computer-based, iterative robotic synthesis of synthetic oligonucleotide compounds and robotic or robot-assisted analysis of the activities of such compounds. Information gathered from assays of such compounds is used to identify nucleic acid sequences that are tractable to a variety of nucleotide sequence-based technologies, for example, antisense drug discovery and target validation. 2
  • Synthetic oligonucleotides of complementarity to targets are known to hybridize with particular, target nucleic acids in a sequence-specific manner.
  • compounds complementary to the "sense" strand of nucleic acids that encode polypeptides are referred to as "antisense oligonucleotides.”
  • a subset of such compounds may be capable of modulating the expression of a target nucleic acid; such synthetic compounds are described herein as “active oligonucleotide compounds.”
  • Oligonucleotide compounds are commonly used in vitro as research reagents and diagnostic aids, and in vivo as therapeutic and bioactive agents. Oligonucleotide compounds can exert their effect by a variety of means. One such means takes advantage of an endogenous nuclease, such as RNase H in eukaryotes or RNase P in prokaryotes, to degrade the DNA/RNA hybrid formed between the oligonucleotide sequence and mRNA (Chiang et al., J. Biol. Chem., 1991, 266, 18162; Forster et al, Science, 1990, 249, 783).
  • RNase H endogenous nuclease
  • RNase P in prokaryotes
  • Another means involves covalently linking of a synthetic moiety having nuclease activity to an oligonucleotide having an antisense sequence. This does not rely upon recruitment of an endogenous nuclease to modulate target activity.
  • Synthetic moieties having nuclease activity include, but are not limited to, enzymatic RNAs, lanthanide ion complexes, and other reactive species.
  • RNA structure can inhibit duplex formation with antisense compounds, so much so that "moving" the target nucleotide sequence even a few bases can drastically decrease the activity of such compounds (Lima et al., Biochemistry, 1992, 31, 12055).
  • the preferred method of searching for lead antisense compounds has been the manual synthesis and analysis of such compounds. Consequently, a fundamental limitation of the conventional approach is its dependence upon the availability, number and cost of antisense compounds produced by manual, or at best semi-automated, means.
  • the assaying of such compounds has traditionally been performed by tedious manual techniques.
  • the traditional approach to generating active antisense compounds is limited by the relatively high cost and long time required to synthesize and screen a relatively small number of candidate antisense compounds.
  • antisense sequences hybridizable to the sequence can be generated using techniques known in the art.
  • a large number of candidate antisense oligonucleotides are synthesized having sequences that are more-or-less randomly spaced across the length of the target nucleic acid sequence (e.g., a "gene walk") and their ability to modulate the expression of the target nucleic acid is assayed.
  • Cells or animals can then be treated with one or more active antisense oligonucleotides, and the resulting effects determined in order to determine the function(s) of the target gene.
  • the present disclosure answers this need by providing systems and methods for automatically generating active antisense compounds to a target nucleotide sequence via robotic means.
  • Such active antisense compounds are contacted with cells, cell-free extracts, tissues or animals capable of expressing the gene of interest and subsequent biochemical or biological parameters are measured. The results are compared to those obtained from a control cell culture, cell-free extract, tissue or animal which has not been contacted with an active antisense compound in order to determine the function of the gene of interest. 4.
  • Target Validation Determining the nucleotide sequence of a gene is no longer an end unto itself; rather, it is "merely a means to an end.
  • the critical next step is to validate the gene and its [gene] product as a potential drug target" (Glasser, Genetic Engineering News, 1997, 17, 1). This process, i.e., confirming that modulation of a gene that is suspected of being involved in a disease or disorder actually results in an effect that is consistent with a causal relationship between the gene and the disease or disorder, is known as target validation.
  • Efforts such as the Human Genome Project are yielding a vast number of complete or partial nucleotide sequences, many of which might correspond to or encode targets useful for new drug discovery efforts.
  • the present disclosure answers this need by providing systems and methods for automatically generating active oligonucleotide and other compounds, especially antisense compounds, to a target nucleotide sequence via robotic or other automated means.
  • Such active compounds are contacted with a cell culture, cell-free extract, tissue or animal capable of expressing the gene of interest, and subsequent biochemical or biological parameters indicative of the potential gene product function are measured. These results are compared to those obtained with a control cell system, cell-free extract, tissue or animal which has not been contacted with an active antisense compound in order to determine whether or not modulation of the gene of interest affects a specific cellular function.
  • the resulting active antisense compounds may be used as positive controls when other, non antisense-based agents directed to the same target nucleic acid, or to its gene product, are screened.
  • embodiments of the invention drawn to gene function analysis and target validation have parameters that are shared with other embodiments of the invention, but also have unique parameters.
  • antisense drug discovery naturally requires that the toxicity of the antisense compounds be manageable, whereas, for gene function analysis or target validation, overt toxicity resulting from the antisense compounds is acceptable unless it interferes with the assay being used to evaluate the effects of treatment with such compounds.
  • U.S. Patent 5,563,036 to Peterson et al. describes systems and methods of screening for compounds that inhibit the binding of a transcription factor to a nucleic acid.
  • an assay portion of the process is stated to be performed by a computer controlled robot.
  • U.S. Patent 5,708,158 to Hoey describes systems and methods for identifying pharmacological agents stated to be useful for diagnosing or treating a disease associated with a gene the expression of which is modulated by a human nuclear factor of activated T cells. The methods are stated to be particularly suited to high-thoughput screening wherein one or more steps of the process are performed by a computer controlled robot.
  • U.S. Patents 5,693,463 and 5,716,780 to Edwards et al. describe systems and methods for identifying non-oligonucleotide molecules that specifically bind to a DNA molecule based on their ability to compete with a DNA-binding protein that recognizes the
  • the present invention is directed to automated systems and methods for defining sets of compounds that modulate the expression of target nucleic acid sequences, and generating sets of oligonucleotides that modulate the expression of target nucleic acid sequences.
  • the present invention is also directed to identifying nucleic acid sequences amenable to antisense binding of oligonucleotides to those nucleic acid sequences by the systems and methods of the invention.
  • the present invention is described herein with respect to the production and identification of active antisense oligonucleotides; however, the present invention is not limited to this embodiment.
  • the present invention is directed to iterative processes for defining chemical compounds with prescribed sets of physical, chemical and/or biological properties, and to systems for implementing these processes.
  • a target nucleic acid sequence is provided or selected, and a library of (candidate) virtual compounds is generated in silico (that is in a computer manipulative and reliable form) according to defined criteria.
  • a library of virtual compounds is generated. These virtual compounds are reviewed and compounds predicted to have particular desired properties are selected.
  • the selected compounds are synthesized, preferably in a robotic, batchwise manner; and then they are robotically assayed for a desired physical, chemical or biological activity in order to identify compounds with the desired properties.
  • Active compounds are, thus, generated and, at the same time, preferred sequences and regions of the target nucleic acid that are amenable to modulation are identified.
  • the preferred compounds of the invention are oligonucleotides that bind to a target nucleic acid sequence.
  • second libraries of candidate compounds are generated and/or selected to give rise to a second virtual compound library.
  • a library of target nucleic acid sequences that are tractable to modulation via binding of these compounds to the nucleic acid sequence are identified.
  • modulation includes, but is not limited to, antisense technology, gene function analysis and target validation.
  • the present invention is also directed to processes for validating the function of a gene or the product of the gene comprising generating in silico a library of nucleobase sequences targeted to the gene and robotically assaying a plurality of synthetic compounds having at least some of the nucleobase sequences for effects on biological function.
  • Figures 1 and 2 are a flow diagram of one method according to the present invention depicting the overall flow of data and materials among various elements of the invention.
  • Figure 3 is a flow diagram depicting the flow of data and materials among elements of step 200 of Figure 1.
  • Figures 4 and 5 are a flow diagram depicting the flow of data and materials among elements of step 300 of Figure 1.
  • Figure 6 is a flow diagram depicting the flow of data and materials among elements of step 306 of Figure 4.
  • FIG. 7 is another flow diagram depicting the flow of data and materials among elements of step 306 of Figure 4.
  • Figure 8 is a another flow diagram depicting the flow of data and materials among elements of step 306 of Figure 4.
  • Figure 9 is a flow diagram depicting the flow of data and materials among elements of step 350 of Figure 5.
  • Figures 10 and 11 are flow diagrams depicting a logical analysis of data and materials among elements of step 400 of Figure 1.
  • Figure 12 is a flow diagram depicting the flow of data and materials among the elements of step 400 of Figure 1.
  • Figures 13 and 14 are flow diagrams depicting the flow of data and materials among elements of step 500 of Figure 1.
  • Figure 15 is a flow diagram depicting the flow of data and materials among elements of step 600 of Figure 1.
  • Figure 16 is a flow diagram depicting the flow of data and materials among elements of step 700 of Figure 1.
  • Figure 17 is a flow diagram depicting the flow of data and materials among the elements of step 1100 of Figure 2.
  • Figure 18 is a block diagram showing the interconnecting of certain devices utilized in conjunction with a preferred method of the invention.
  • Figure 19 is a flow diagram showing a representation of data storage in a relational database utilized in conjunction with one method of the invention.
  • Figure 20 is a flow diagram depicting the flow of data and materials in effecting a preferred embodiment of the invention as set forth in Example 14;
  • Figure 21 is a flow diagram depicting the flow of data and materials in effecting a preferred embodiment of the invention as set forth in Example 15;
  • Figure 22 is a flow diagram depicting the flow of data and materials in effecting a preferred embodiment of the invention as set forth in Example 2;
  • Figure 23 is a pictorial elevation view of a preferred apparatus used to robotically synthesize oligonucleotides.
  • Figure 24 is a pictorial plan view of an apparatus used to robotically synthesize oligonucleotides.
  • the target selection process provides a target nucleotide sequence that is used to help guide subsequent steps of the process. It is generally desired to modulate the expression of the target nucleic acid for any of a variety of purposes, such as, e.g., drug discovery, target validation and/or gene function analysis.
  • One of the primary objectives of the target selection process, step 100 is to identify molecular targets that represent significant therapeutic opportunities, to provide new and efficacious means of drug discovery and to determine the function of genes that are uncharacterized except for nucleotide sequence. To meet these objectives, genes are classified based upon specific sets of selection criteria.
  • target nucleotide sequence There must be sufficient target nucleic acid sequence information 10 available for oligonucleotide design. Moreover, such information must be of sufficient quality to give rise to an acceptable level of confidence in the data to perform the methods described herein. Thus, the data must not contain too many missing or incorrect base entries.
  • target sequence that encodes a polypeptide such errors can often be detected by virtually translating all three reading frames of the sense strand of the target sequence and confirming the presence of a continuous polypeptide sequence having predictable attributes, e.g., encoding a polypeptide of known size, or encoding a polypeptide that is about the same length as a homologous protein. In any event, only a very high frequency of sequence errors will frustrate the methods of the invention; most oligonucleotides to the target sequence will avoid such errors unless such errors occur frequently throughout the entire target sequence.
  • a cell line can be transfected with an expression vector comprising the target gene in order to generate an appropriate cell line for assay purposes.
  • a target nucleic acid for gene function analysis might be absolutely uncharacterized, or might be thought to have a function based on minimal data or homology to another gene.
  • active compounds that modulate the expression of the gene can be developed and applied to cells. The resulting cellular, biochemical or molecular biological responses are observed, and this information is used by those skilled in the art to elucidate the function of the target gene.
  • Level 1 Targets are target nucleic acids for which there is a strong correlation with disease. This correlation can come from multiple scientific disciplines including, but not limited to, epidemiology, wherein frequencies of gene abnormalities are associated with disease incidence; molecular biology, wherein gene expression and function are associated with cellular events correlated with a disease; and biochemistry, wherein the in vitro activities of a gene product are associated with disease parameters. Because there is a strong therapeutic rationale for focusing on Level 1 Targets, these targets are most preferred for drug discovery and/or target validation.
  • Level 2 Targets are nucleic acid targets for which the combined epidemiological, molecular biological, and/or biochemical correlation with disease is not so clear as for Level 1.
  • Level 3 Targets are targets for which there is little or no data to directly link the target with a disease process, but there is indirect evidence for such a link, i.e., homology with a Level 1 or Level 2 target nucleic acid sequence or with the gene product thereof.
  • nucleic acids can also classified by biological processes. For example, programmed cell death ("apoptosis") has recently emerged as an important biological process that is perturbed in a wide variety of diseases. Accordingly, nucleic acids that encode factors that play a role in the apoptotic process are identified as candidate targets. Similarly, potential target nucleic acids can be classified as being involved in inflammation, autoimmune disorders, cancer, or other pathological or dysfunctional processes. 12
  • genes can often be grouped into families based on sequence homology and biological function. Individual family members can act redundantly, or can provide specificity through diversity of interactions with downstream effectors, or through expression being restricted to specific cell types. When one member of a gene family is associated with a disease process then the rationale for targeting other members of the same family is reasonably strong. Therefore, members of such gene families are preferred target nucleic acids to which the methods and systems of the invention may be applied. Indeed, the potent specificity of antisense compounds for different gene family members makes the invention particularly suited for such targets (Albert et al., Trends Pharm. Sci., 1994, 15, 250).
  • PCR polymerase chain reaction
  • primers designed to be common to all members of a given gene family.
  • nucleotide sequences from cloned DNAs, or from complementary DNAs (cDNAs) derived from mRNAs may be used in the process of the invention, there is no requirement that the target nucleotide sequence be isolated from a cloned nucleic acid. Any nucleotide sequence, no matter how determined, of any nucleic acid, isolated or prepared in any fashion, may be used as a target nucleic acid in the process of the invention.
  • nucleotide sequences of structural or enzymatic RNAs may be utilized for drug discovery and/or target validation when such RNAs are associated with a disease state, or for gene function analysis when their biological role is not known.
  • Figure 3 is a block diagram detailing the steps of the target nucleotide sequence assembly process, process step 200 in acccordance with one embodiment of the invention.
  • the oligonucleotide design process, process step 300 is facilitated by the availability of accurate target sequence information.
  • target nucleic acid assembly process take advantage of computerized homology search algorithms and sequence fragment assembly algorithms to search available databases for related sequence information and incorporate available sequence information into the best possible representation of the target nucleic acid molecule, for example a RNA transcript. This representation is then used to design oligonucleotides, process step 300, which can be tested for biological activity in process step 700.
  • each distinct transcript is a unique target nucleic acid for purposes of step 300.
  • the target nucleotide sequence is limited to those sequences that are unique to that transcript isoform. In another embodiment of the invention, if it is desired to modulate two or more transcript isoforms in concert, the target nucleotide sequence is limited to sequences that are shared between the two or more transcripts.
  • full-length cDNA be used in the oligonucleotide design process step 300 (with full-length cDNA being defined as reading from the 5' cap to the poly A tail).
  • full-length cDNA is preferred, it is possible to design oligonucleotides using partial sequence information. Therefore it is not necessary for the assembly process to generate a complete cDNA sequence. Further in some cases it may be desirable to design oligonucleotides targeting introns. In this case the process can be used to identify individual introns at process step 220. The process can be initiated by entering initial sequence information on a selected molecular target at process step 205.
  • the full-length cDNA sequence is generally preferred for use in oligonucleotide design strategies at process step 300.
  • the first step is to determine if the initial sequence information represents the full-length cDNA, decision step 210. In the case where the full- length cDNA sequence is available the process advances directly to the oligonucleotide design step 300. When the full-length cDNA sequence is not available, databases are 14 searched at process step 212 for additional sequence information.
  • BLAST Altschul, et al., J. Mol. Biol, 1990, 215, 403
  • Gapped BLAST Altschul et al, Nucl Acids Res., 1997, 25, 3389.
  • GenBank GenBank, EMBL, DDBJ and Information at the National Institutes
  • GenBank GenBank, EMBL, DDBJ and Information at the National Institutes
  • Phrap at process step 250 If multiple complete exons are not identified at decision step 225, then sequences can be analyzed for partial sequence information in decision step 228. ESTs identified in the database dbEST are examples of such partial sequence information. If additional partial information is not found, then the process is advanced to process step 230 at decision step 228. If partial sequence information is found in process 212 then that information is advanced to process step 230 via decision step 228.
  • Process step 230, decision step 240, decision step 260 and process step 250 define a loop designed to extend iteratively the amount of sequence information available for targeting. At the end of each iteration of this loop, the results are analyzed in decision steps 240 and 260. If no new information is found then the process advances at decision step 240 to process step 300. If there is an unexpectedly large amount of sequence information identified, suggesting that the process moved outside the boundary of the gene into repetitive genomic sequence, then the process is preferably cycled back one iteration and that sequence is advanced at decision step 240 to process step 300. If a small amount of new sequence information is identified, then the loop is iterated such as by taking the
  • each oligonucleotide chemistry is first assigned to each oligonucleotide sequence. Then, each combination of oligonucleotide chemistry and sequence is evaluated according to the parameters of step
  • This embodiment has the desirable feature of taking into account the effect of 16 alternative oligonucleotide chemistries on such parameters. For example, substitution of 5-methyl cytosine (5MeC or m5c) for cytosine in an antisense compound may enhance the stability of a duplex formed between that compound and its target nucleic acid.
  • Other oligonucleotide chemistries that enhance oligonucleotide: [target nucleic acid] duplexes are known in the art (see for example, Freier et al, Nucleic Acids Research, 1997, 25, 4429).
  • oligonucleotide chemistries may be preferred for different target nucleic acids. That is, the optimal oligonucleotide chemistry for binding to a target DNA might be suboptimal for binding to a target RNA having the same nucleotide sequence.
  • a list of oligonucleotide sequences is generated as represented in the flowchart shown in Figures 4 and 5.
  • the desired oligonucleotide length is chosen.
  • oligonucleotide length is between from about 8 to about 30, more preferably from about 12 to about 25, nucleotides.
  • step 304 all possible oligonucleotide sequences of the desired length capable of hybridizing to the target sequence obtained in step 200 are generated.
  • a series of oligonucleotide sequences are generated, simply by determining the most 5' oligonucleotide possible and "walking" the target sequence in increments of one base until the 3' most oligonucleotide possible is reached.
  • a virtual oligonucleotide chemistry is applied to the nucleobase sequences of step 304 in order to yield a set of virtual oligonucleotides that can be evaluated in silico.
  • Default virtual oligonucleotide chemistries include those that are well- characterized in terms of their physical and chemical properties, e.g., 2'-deoxyribonucleic acid having naturally occurring bases (A, T, C and G), unmodified sugar residues and a phosphodiester backbone.
  • thermodynamic properties are calculated as represented in Figure 6.
  • the desired thermodynamic properties are selected. As many or as few as desired can be 17 selected; optionally, none will be selected.
  • the desired properties will typically include step 309, calculation of the free energy of the target structure. If the oligonucleotide is a DNA molecule, then steps 310, 312, and 314 are performed. If the oligonucleotide is an RNA molecule, then steps 311, 313 and 315 are performed.
  • a free energy of oligonucleoti de-target binding is preferably calculated at step 316.
  • thermodynamic and kinetic properties may be calculated for oligonucleotides as represented at step 317.
  • Such other thermodynamic and kinetic properties may include melting temperatures, association rates, dissociation rates, or any other physical property that may be predictive of oligonucleotide activity.
  • the free energy of the target structure is defined as the free energy needed to disrupt any secondary structure in the target binding site of the targeted nucleic acid.
  • This region includes any intra-target nucleotide base pairs that need to be disrupted in order for an oligonucleotide to bind to its complementary sequence.
  • the effect of this localized disruption of secondary structure is to provide accessibility by the oligonucleotide.
  • Such structures will include double helices, terminal unpaired and mismatched nucleotides, loops, including hairpin loops, bulge loops, internal loops and multibranch loops (Serra et al, Methods in Enzymology, 1995, 259, 242).
  • the intermolecular free energies refer to inherent energy due to the most stable structure formed by two oligonucleotides; such structures include dimer formation. Intermolecular free energies should also be taken into account when, for example, two or more oligonucleotides, of different sequence are to be administered to the same cell in an assay.
  • the intramolecular free energies refer to the energy needed to disrupt the most stable secondary structure within a single oligonucleotide. Such structures include, for example, hairpin loops, bulges and internal loops. The degree of intramolecular base pairing is indicative of the energy needed to disrupt such base pairing.
  • the free energy of duplex formation is the free energy of denatured oligonucleotide binding to its denatured target sequence.
  • the oligonucleotide-target binding is the total binding involved, and includes the energies involved in opening up 18 intra- and inter- molecular oligonucleotide structures, opening up target structure, and duplex formation.
  • RNA structure is predicted based on nearest neighbor analysis (Xia, T., et al, Biochemistry, 1998, 37, 14719-14735; Serra et al, Methods in Enzymology, 1995, 259, 242). This analysis is based on the assumption that stability of a given base pair is determined by the adjacent base pairs. For each possible nearest neighbor combination, thermodynamic properties have been determined and are provided. For double helical regions, two additional factors need to be considered, an entropy change required to initiate a helix and a entropy change associated with self-complementary strands only. Thus, the free energy of a duplex can be calculated using the equation:
  • AG is the free energy of duplex formation
  • AH is the enthalpy change for each nearest neighbor
  • AS is the entropy change for each nearest neighbor
  • T is temperature.
  • the AH and AS for each possible nearest neighbor combination have been experimentally determined. These letter values are often available in published tables. For terminal unpaired and mismatched nucleotides, enthalpy and entropy measurements for each possible nucleotide combination are also available in published tables. Such results are added directly to values determined for duplex formation.
  • one known model determines the free energy of loop formation as the sum of free energy based on loop size, the closing base pair, the interactions between the first mismatch of the loop with the closing base pair, and additional factors including being closed by AU or UA or a first mismatch of GA or UU.
  • Such equations may also be used for oligoribonucleotide-target RNA interactions.
  • DNA duplex stability is used in the case of intra- or intermolecular oligodeoxyribonucleotide interactions.
  • DNA duplex stability is calculated using similar equations as RNA stability, except experimentally determined values differ between nearest neighbors in DNA and RNA and helix initiation tends to be more favorable in 19
  • thermodynamic parameters are used in the case of RNA/DNA hybrid duplexes. This would be the case for an RNA target and oligodeoxynucleotide. Such parameters were determined by Sugimoto et al. ⁇ Biochemistry, 1995, 34, 11211). In addition to values for nearest neighbors, differences were seen for values for enthalpy of helix initiation.
  • Target accessibility is believed to be an important consideration in selecting oligonucleotides. Such a target site will possess minimal secondary structure and thus, will require minimal energy to disrupt such structure. In addition, secondary structure in oligonucleotides, whether inter- or intra-molecular, is undesirable due to the energy required to disrupt such structures. Oligonucleotide-target binding is dependent on both these factors. It is desirable to minimize the contributions of secondary structure based on these factors. The other contribution to oligonucleotide-target binding is binding affinity. Favorable binding affinities based on tighter base pairing at the target site is desirable.
  • thermodynamic properties Following the calculation of thermodynamic properties ending at step 317, the desired sequence properties to be scored are selected at step 324. As many or as few as desired can be selected; optionally, none will be selected. These properties include the number of strings of four guanosine residues in a row at step 325 or three guanosine in a row at step 326, the length of the longest string of adenosines at step 327, cytidines at step
  • uri dines or thymidines at step 329, the length of the longest string of purines at step 330 or pyrimidine at step 331, the percent composition of adenosine at step 332, cytidine at step 333, guanosine at step 334 or uridines or thymidines at step 335, the percent composition of purines at step 336 or pyrimidines at step 337, the number of CG dinucleotide repeats at step 338, CA dinucleotide repeats at step 339 or UA or TA dinucleotide repeats at step 340.
  • other sequence properties may be used as found to be relevant and predictive of antisense efficacy, as represented at step 341.
  • sequence properties may be important in predicting oligonucleotide activity, or lack thereof.
  • U.S. Patent 5,523,389 discloses oligonucleotides containing stretches of three or four guanosine residues in a row. Oligonucleotides having such sequences may act in a sequence-independent manner. For an antisense approach, such a 20 mechanism is not usually desired. In addition, high numbers of dinucleotide repeats may be indicative of low complexity regions which may be present in large numbers of unrelated genes. Unequal base composition, for example, 90% adenosine, can also give non-specific effects.
  • the homology scores to be calculated are selected in step 342.
  • Homology to nucleic acids encoding protein isoforms of the target may be desired.
  • oligonucleotides specific for an isoform of protein kinase C can be selected.
  • oligonucleotides can be selected to target multiple isoforms of such genes.
  • Homology to analogous target sequences may also be desired.
  • an oligonucleotide can be selected to a region common to both humans and mice to facilitate testing of the oligonucleotide in both species.
  • Homology to splice variants of the target nucleic acid as represented at step 345, may be desired.
  • a desired range is selected to select the most promising oligonucleotides, as represented at step 347.
  • step 349 A decision is made whether to target such regions, as represented in decision step 349. If it is desired to target functional regions then process step 350 occurs as seen in greater detail in Figure 9. If it is not desired then the process proceeds to step 375. In step 350, as seen in Figure 9, the desired functional regions are selected.
  • Such regions include the transcription start site or 5' cap at step 353, the 5' untranslated region 21 at step 354, the start codon at step 355, the coding region at step 356, the stop codon at step 357, the 3' untranslated region at step 358, 5' splice sites at step 359 or 3' splice sites at step 360, specific exons at step 361 or specific introns at step 362, mRNA stabilization signal at step 363, mRNA destabihzation signal at step 364, poly-adenylation signal at step 365, poly-A addition site at step 366, poly-A tail at step 367, or the gene sequence 5' of known pre-mRNA at step 368.
  • additional functional sites may be selected, as represented at step 369.
  • AUG start codon is commonly targeted because it is necessary to initiate translation.
  • splice sites are thought to be attractive targets because these regions are important for processing of the mRNA.
  • Other known sites may be more accessible because of interactions with protein factors or other regulatory molecules.
  • oligonucleotides are selected based on hybridization to only those desired functional regions, as represented by step 370. 7. Uniform Distribution of Oligonucleotides. Whether or not targeting functional sites is desired, a large number of oligonucleotide sequences may result from the process thus far. In order to reduce the number of oligonucleotide sequences to a manageable number, a decision is made whether to uniformly distribute selected oligonucleotides along the target, as represented in step 375. A uniform distribution of oligonucleotide sequences will aim to provide complete coverage throughout the complete target nucleic acid or the selected functional regions.
  • a computer-based program is used to automate the distribution of sequences, as represented in step 380.
  • Such a program factors in parameters such as length of the target nucleic acid, total number of oligonucleotide sequences desired, oligonucleotide sequences per unit length, number of oligonucleotide sequences per functional region.
  • Manual selection of oligonucleotide sequences is also provided for by step 385. In some cases, it may be desirable to manually select oligonucleotide sequences. For example, it may be useful to determine the effect of small base shifts on activity.
  • oligonucleotide sequences are passed onto step 400 of the process, where oligonucleotide chemistries are assigned.
  • actual oligonucleotide chemistry is assigned to the sequences.
  • An "actual oligonucleotide chemistry” or simply “chemistry” is a chemical motif that is common to a particular set of robotically synthesized oligonucleotide compounds.
  • Preferred chemistries include, but are not limited to, oligonucleotides in which every linkage is a phosphorothioate linkage, and chimeric oligonucleotides in which a defined number of 5' and/or 3' terminal residues have a 2'-methoxyethoxy modification.
  • Chemistries can be assigned to the nucleobase sequences during general procedure step 400 ( Figure 1).
  • the logical basis for chemistry assignment is illustrated in Figures 10 and 11 and an iterative routine for stepping through an oligonucleotide nucleoside by nucleoside is illustrated in Figure 12.
  • Chemistry assignment can be effected by assignment directly into a word processing program, via an interactive word processing program or via automated programs and devices. In each of these instances, the output file is selected to be in a format that can serve as an input file to automated synthesis devices.
  • oligonucleotide in reference to oligonucleotides, is used to refer to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally- occurring portions which function similarly.
  • backbone covalent internucleoside
  • Such modified or substituted oligonucleotides are often preferred over native forms, i.e., phosphodiester linked A, C, G,
  • T and U nucleosides because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.
  • oligonucleotide compounds in accordance with this invention can be of various lengths depending on various parameters, including but not limited to those discussed above in reference to the selection criteria of general procedure 300.
  • antisense oligonucleotides compounds of the invention preferably are from about 8 to about 30 nucleobases in length (i.e. from about 8 to about 30 linked nucleosides).
  • antisense oligonucleotides comprising from about 12 to about 25 nucleobases.
  • oligonucleotides might be selected for non-antisense targeting strategies, for instance using the oligonucleotides as ribozymes. Such ribozymes normally require oligonucleotides of longer length as is known in the art.
  • a nucleoside is a base-sugar combination.
  • the base portion of the nucleoside is normally a heterocyclic base.
  • the two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar.
  • the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred.
  • the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
  • the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • oligonucleotides useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages.
  • oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. 10. Selection of Oligonucleotide Chemistries.
  • a base is selected.
  • base chemistry 1 can be selected at process step 412 or one or more alternative bases are selected at process steps 414, 416 and 418.
  • the sugar portion of the nucleoside is selected.
  • a sugar is selected that together with the select base will complete the nucleoside.
  • sugar chemistry 1 can be selected at process 422 or one or more alternative sugars are selected at process steps 424, 426 and 428.
  • the internucleoside linker is selected.
  • the linker chemistry for the internucleoside linker can be linker chemistry 1 selected at process step 432 or one or more alternative internucleoside linker chemistries are selected at process steps 434, 436 and 438.
  • one or more conjugate groups can be attached to the oligonucleotide via attachment to the nucleoside or attachment to the internucleoside linkage.
  • the addition of a conjugate group is integrated at process step 440 and the assignment of the conjugate group is effected at process step 450.
  • chemistries 1 though n are illustrated.
  • the number of alternate chemistries between chemistry 1 and alternative chemistry n, for each of the bases, the sugars, the internucleoside linkages and the conjugates is variable and includes, but is not limited to, each of the specific alternative bases, sugar, internucleoside linkers and conjugates identified in this specification as well as equivalents known in the art.
  • chemistry is assigned, as is shown in Figure 12, to the list of oligonucleotides from general procedure 300.
  • a pointer can be set at process step 452 to the first oligonucleotide in the list and at step 453 to the first nucleotide of that first oligonucleotide.
  • the base chemistry is selected at step 410, as described above, the sugar chemistry is selected at step 420, also as described above, followed by selection of the internucleoside linkage at step 430, also as described above. 25
  • the process branches depending on whether a conjugate will be added at the current nucleotide position. If a conjugate is desired, the conjugate is selected at step 450, also as described above.
  • an inquiry is made at decision step 454. This inquiry asks if the pointer resides at the last nucleotide in the current oligonucleotide. If the result at decision step 454 is "No,” the pointer is moved to the next nucleotide in the current oligonucleotide and the loop including steps 410, 420, 430, 440 and 454 is repeated. This loop is reiterated until the result at decision step 454 is "Yes.” When the result at decision step 454 is "Yes,” a query is made at decision step 460 concerning the location of the pointer in the list of oligonucleotides.
  • the "No" path of the decision step 460 is followed and the pointer is moved to the first nucleotide of the next oligonucleotide in the list at process step 458. With the pointer set to the next oligonucleotide in the list, the loop that starts at process steps 453 is reiterated. When the result at decision step 460 is "Yes,” chemistry has been assigned to all of the nucleotides in the list of oligonucleotides. 11. Description of Oligonucleotide Chemistries.
  • chemistry selection includes selection of the base forming the nucleoside from a large palette of different base units available.
  • These may be "modified” or “natural” bases (also reference herein as nucleobases) including the natural purine bases adenine (A) and guanine (G), and the natural pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • modified nucleobases including other synthetic and natural nucleobases such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo uracils and cytosines particularly 5-bromo, 5- trifluoromethyl and other 5-substituted ura
  • nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Rroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • These include 5 -substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyl- adenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T.
  • adenine might be selected however other alternative bases that can effect hybridization in a manner mimicking an "A" base such as 2-aminoadenine might be selected should other consideration, e.g., stronger hybridization (relative to hybridization achieved with adenine), be desired.
  • chemistry selection includes selection of the sugar forming the nucleoside from a large palette of different sugar or sugar surrogate units available. These may be modified sugar groups, for instance sugars containing one or more substituent groups.
  • Preferred substituent groups comprise the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N- alkenyl; or O, S- or N-alkynyl; wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • substituent groups comprise one of the following at the 2' position: Cj to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3, OCF 3 ⁇ SOCH 3 ⁇ SO 2 CH 3, ONO 2, NO 2, N 3, NH 2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • a preferred modification includes 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , also known as 2'- O-(2-methoxyethyl), 2'-O-methoxyethyl, or 2'-MOE) (Martin et al, Helv. Chim. Ada, 1995, 78, 486) i.e., an alkoxyalkoxy group.
  • a further preferred modification includes 2'- dimethylamino oxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMAOE, as described in co-owned United States patent application Serial Number 09/016,520, filed on January 30, 1998, which is inco ⁇ orated herein by reference in its entirety.
  • modifications include 2'-methoxy (2'-O-CH 3 ), 2'-aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2 ) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the sugar group, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
  • the nucleosides of the oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • chemistry selection includes selection of the internucleoside linkage.
  • These internucleoside linkages are also referred to as linkers, backbones or oligonucleotide backbones.
  • linkers backbones or oligonucleotide backbones.
  • a palette of different internucleoside linkages or backbones is available.
  • modified oligonucleotide backbones for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalklyphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 '-5' to 5'-3' or 2'-5' to 5'-2'.
  • modified oligonucleotide backbones for example, phosphorothioates, chiral phosphorothi
  • Preferred internucleoside linkages for oligonucleotides that do not include a phosphorus atom therein, i.e., for oligonucleosides, have backbones that are formed by short chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatomic or heterocyclic intersugar linkages.
  • mo ⁇ holino linkages formed in part from 29 the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • both the sugar and the intersugar linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • a peptide nucleic acid (PNA) is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar-phosphate backbone of an oligonucleotide is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone.
  • nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S.: 5,539,082; 5,714,331; and 5,719,262, each of which is inco ⁇ orated herein by reference in its entirety. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497.
  • the most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular -CH 2 -NH-O-CH 2 -, -CH 2 -N(CH 3 )-O-CH 2 - [known as a methylene (methylimino) or MMI backbone], -CH 2 -O-N(CH 3 )-CH 2 -,- CH 2 -N(CH 3 )- N(CH 3 )-CH 2 - and -O-N(CH 3 )-CH 2 -CH 2 - (wherein the native phosphodiester backbone is represented as -O-P-O-CH 2 -) of the above referenced U.S.
  • oligonucleotide In attaching a conjugate group to one or more nucleosides or internucleoside linkages of an oligonucleotide, various properties of the oligonucleotide are modified.
  • oligonucleotides of the invention to chemically link one or more moieties or conjugates to the oligonucleotide are intended to enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al, Bioorg. Med. Chem. Let., 1994,
  • a thioether e.g., hexyl-S-tritylthiol (Manoharan et al, Ann. N Y. Acad. Sci., 1992, 660, 306; Manoharan et al, Bioorg. Med. Chem. Let., 1993, 3, 2765), a thiocholesterol (Oberhauser et al, Nucl.
  • the present invention also includes compounds which are chimeric compounds.
  • "Chimeric” compounds or “chimeras,” in the context of this invention, are compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound.
  • oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
  • An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
  • Chimeric antisense compounds of the invention may be formed as composite structures representing the union of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as “hybrids” or "gapmers". Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Patents 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and
  • oligonucleotides are synthesized on an automated synthesizer.
  • the synthesizer is preferably a variation of the synthesizer described in United States patents 5,472,672 and 5,529,756, each of which is inco ⁇ orated herein by reference in its entirety.
  • the synthesizer described in those patents is modified to include movement in along the Y axis in addition to movement along the X axis. As so modified, a 96-well array of compounds can be synthesized by the synthesizer.
  • the synthesizer further includes temperature control and the ability to maintain an inert atmosphere during all phases of synthesis.
  • the reagent array delivery format employs orthogonal X-axis motion of a matrix of reaction vessels and Y-axis motion of an array of reagents.
  • Each reagent has its own dedicated plumbing system to eliminate the possibility of cross- contamination of reagents and line flushing and/or pipette washing. This in combined with a high delivery speed obtained with a reagent mapping system allows for the extremely rapid delivery of reagents. This further allows long and complex reaction sequences to be performed in an efficient and facile manner.
  • the software that operates the synthesizer allows the straightforward programming of the parallel synthesis of a large number of compounds.
  • the software utilizes a general synthetic procedure in the form of a command (.cmd) file, which calls upon certain reagents to be added to certain wells via lookup in a sequence (.seq) file.
  • the bottle position, flow rate, and concentration of each reagent is stored in a lookup table (.tab) file.
  • a plate of compounds is made by permutating a set of reagents, and writing the resulting output to a text file.
  • the text file is input directly into the synthesizer and used for the synthesis of the plate of compounds.
  • the synthesizer is interfaced with a relational database allowing data output related to the synthesized compounds to be registered in a highly efficient manner.
  • a synthesis file i.e., a .cmd file
  • This file can be built fresh to reflect a completely new set of machine commands reflecting a set of chemical synthesis steps or it can modify an existing file stored at process step 504 by editing that stored file in process step 508.
  • the .cmd files are built using a word processor and a command set of instructions as outlined below.
  • control software and data files are within the routine skill of persons skilled in annotated nucleotide synthesis. The same will depend upon the hardware employed, the chemistries adopted and the design paradigm selected by the operator.
  • .tab files are built to reflect the necessary reagents used in the automatic synthesizer for the particular chemistries that have been selected for the linkages, bases, sugars and conjugate chemistries.
  • a .tab file is built at process step 512 and stored at process step 514.
  • an existing .tab file can be edited at process step 516.
  • Both the .cmd files and the .tab files are linked together at process step 518 and stored for later retrieval in an appropriate sample database 520.
  • Linking can be as simple as using like file names to associate a .cmd file to its appropriate .tab file, e.g., synthesis 1.cmd is linked to synthesis_l .tab by use of the same preamble in their names.
  • the automated, multi-well parallel array synthesizer employs a reagent array delivery format, in which each reagent utilized has a dedicated plumbing system. As seen in Figures 23 and 24, an inert atmosphere 522 is maintained during all phases of a synthesis. Temperature is controlled via a thermal transfer plate 524, which holds an injection molded reaction block 526. The reaction plate assembly slides in the X-axis direction, while for example eight nozzle blocks (528, 530, 532, 534, 536, 538, 540 and
  • the target reaction vessels In synthesizing oligonucleotides for screening, the target reaction vessels, a 96 well plate 556 (a 2-dimensional array), moves in one direction along the X axis, while the 34 series of independently controlled reagent delivery nozzles (528, 530, 532, 534, 536, 538, 540 and 542) move along the Y-axis relative to the reaction vessel 558.
  • the reaction plate 556 and reagent nozzles (528, 530, 532, 534, 536, 538, 540 and 542) can be moved independently at the same time, this arrangement facilitates the extremely rapid delivery of up to 72 reagents independently to each of the 96 reaction vessel wells.
  • the system software allows the straightforward programming of the synthesis of a large number of compounds by supplying the general synthetic procedure in the form of the command file to call upon certain reagents to be added to specific wells via lookup in the sequence file with the bottle position, flow rate, and concentration of each reagent being stored in the separate reagent table file.
  • Compounds can be synthesized on various scales. For oligonucleotides, a 200 nmole scale is typically selected while for other compounds larger scales, as for example a 10 ⁇ mole scale (3-5 mg), might be utilized.
  • the resulting crude compounds are generally >80% pure, and are utilized directly for high throughput screening assays. Alternatively, prior to use the plates can be subjected to quality control (see general procedure 600 and Example 9) to ascertain their exact purity.
  • the software inputs accept tab delimited text files (as discussed above for file 504 and 512) from any text editor.
  • a typical command file, a .cmd file, is shown in Example 3 at Table 2.
  • Typical sequence files, .seq files, are shown in Example 3 at Tables 3 and 4
  • Example 3 (.SEQ file), and a typical reagent file, a .tab file, is shown in Example 3 at Table 5.
  • Table 3 illustrates the sequence file for an oligonucleotide having 2'-deoxy nucleotides at each position with a phosphorothioate backbone throughout.
  • Table 4 illustrates the sequence file for an oligonucleotide, again having a phosphorothioate backbone throughout, however, certain modified nucleoside are utilized in portions of the oligonucleotide.
  • 2'-O-(2-methoxyethyl) modified nucleosides are utilized in a first region (a wing) of the oligonucleotide, followed by a second region (a gap) of 2'-deoxy nucleotides and finally a third region (a further wing) that has the same chemistry as the first region.
  • a wing a first region of the oligonucleotide
  • a gap of 2'-deoxy nucleotides
  • a third region a further wing
  • some of the wells of the 96 well plate 556 may be left empty (depending on the number of oligonucleotides to be made during an individual synthesis) or some of the wells may have oligonucleotides that will serve as standards for comparison 35 or analytical pu ⁇ oses.
  • moisture sensitive reagent lines Prior to loading reagents, moisture sensitive reagent lines are purged with argon at 522 for 20 minutes. Reagents are dissolved to appropriate concentrations and installed on the synthesizer.
  • Large bottles, collectively identified as 558 in Figure 23 (containing 8 delivery lines) are used for wash solvents and the delivery of general activators, trityl group cleaving reagents and other reagents that may be used in multiple wells during any particular synthesis.
  • Small septa bottles collectively identified as 560 in Figure 23, are utilized to contain individual nucleotide amidite precursor compounds. This allows for anhydrous preparation and efficient installation of multiple reagents by using needles to pressurize the bottle, and as a delivery path.
  • the lines are primed with reagent, flow rates measured, then entered into the reagent table (.tab file). A dry resin loaded plate is removed from vacuum and installed in the machine for the synthesis.
  • the modified 96 well polypropylene plate 556 is utilized as the reaction vessel.
  • the working volume in each well is approximately 700 ⁇ l.
  • the bottom of each well is provided with a pressed- fit 20 ⁇ m polypropylene frit and a long capillary exit into a lower collection chamber as is illustrated in Figure 5 of the above referenced United States Patent 5,372,672.
  • the solid support for use in holding the growing oligonucleotide during synthesis is loaded into the wells of the synthesis plate 556 by pipetting the desired volume of a balanced density slurry of the support suspended in an appropriate solvent, typically an acetonitrile-methylene chloride mixture. Reactions can be run on various scales as for instance the above noted 200 nmole and 10 ⁇ mol scales.
  • a CPG support is preferred, however other medium loading polystyrene-PEG supports such as TENTAGELTM or ARGOGELTM can also be used.
  • the synthesis plate is transported back and forth in the X- direction under an array of 8 moveable banks (530, 532, 534, 536, 538, 540, 542 and 544) of 8 nozzles (64 total) in the Y-direction, and 6 banks (544, 546, 548, 550, 552 and 554) of 48 fixed nozzles, so that each well can receive the appropriate amounts of reagents and/or solvents from any reservoir (large bottle or smaller septa bottle).
  • a sliding balloon-type seal 562 surrounds this nozzle array and joins it to the reaction plate headspace 564.
  • a slow sweep of nitrogen or argon 522 at ambient pressure across the plate headspace is used 36 to preserve an anhydrous environment.
  • the liquid contents in each well do not drip out until the headspace pressure exceeds the capillary forces on the liquid in the exit nozzle.
  • a slight positive pressure in the lower collection chamber can be added to eliminate residual slow leakage from filled wells, or to effect agitation by bubbling inert gas through the suspension.
  • the headspace gas outlet valve is closed and the internal pressure raised to about 2 psi. Normally, liquid contents are blown directly to waste 566.
  • a 96 well microtiter plate can be inserted into the lower chamber beneath the synthesis plate in order to collect the individual well eluents for spectrophotometric monitoring (trityl, etc.) of reaction progress and yield.
  • the basic plumbing scheme for the machine is the gas-pressurized delivery of reagents.
  • Each reagent is delivered to the synthesis plate through a dedicated supply line, collectively identified at 568, solenoid valve collectively identified at 570 and nozzle, collectively identified at 572.
  • Reagents never cross paths until they reach the reaction well. Thus, no line needs to be washed or flushed prior to its next use and there is no possibility of cross-contamination of reagents.
  • the liquid delivery velocity is sufficiently energetic to thoroughly mix the contents within a well to form a homogeneous solution, even when employing solutions having drastically different densities. With this mixing, once reactants are in homogeneous solution, diffusion carries the individual components into and out of the solid support matrix where the desired reaction takes place.
  • Each reagent reservoir can be plumbed to either a single nozzle or any combination of up to 8 nozzles.
  • Each nozzle is also provided with a concentric nozzle washer to wash the outside of the delivery nozzles in order to eliminate problems of crystallized reactant buildup due to slow evaporation of solvent at the tips of the nozzles.
  • the nozzles and supply lines can be primed into a set of dummy wells directly to waste at any time.
  • the entire plumbing system is fabricated with teflon tubing, and reagent reservoirs are accessed via syringe needle/septa or direct connection into the higher capacity bottles.
  • the septum vials 560 are held in removable 8-bottle racks to facilitate easy setup and cleaning.
  • the priming volume for each line is about 350 ⁇ l.
  • the minimum delivery volume is about 2 ⁇ l, and flow rate accuracy is ⁇ 5%.
  • the actual amount of material delivered depends on a timed flow of liquid.
  • the flow rate for a particular solvent will 37 depend on its viscosity and wetting characteristics of the teflon tubing.
  • the flow rate (typically 200-350 ⁇ l per sec) is experimentally determined, and this information is contained in the reagent table setup file.
  • Heating and cooling of the reaction block 526 is effected utilizing a recirculating heat exchanger plate 524, similar to that found in PCR thermocyclers, that nests with the polypropylene synthesis plate 556 to provide good thermal contact.
  • the liquid contents in a well can be heated or cooled at about 10°C per minute over a range of +5 to +80°C, as polypropylene begins to soften and deform at about 80°C.
  • a non-disposable synthesis plate machined from stainless steel or monel with replaceable frits can be utilized.
  • the hardware controller can be any of a wide variety, but conveniently can be designed around a set of three 1 MHz 86332 chips. This controller is used to drive the single X-axis and 8 Y-axis stepper motors as well as provide the timing functions for a total of 154 solenoid valves. Each chip has 16 bidirectional timer I/O and 8 interrupt channels in its timer processing unit (TPU). These are used to provide the step and direction signals, and to read 3 encoder inputs and 2 limit switches for controlling up to three motors per chip. Each 86332 chip also drives a serial chain of 8 UNC5891A darlington array chips to provide power to 64 valves with msec resolution. The controller communicates with the Windows software interface program running on a PC via a 19200 Hz serial channel, and uses an elementary instruction set to communicate valve number, time open, motor_number and position_data.
  • TPU timer processing unit
  • the three components of the software program that run the array synthesizer are the generalized procedure or command (.cmd) file which specifies the synthesis instructions to be performed, the sequence (.seq) file which specifies the scale of the reaction and the order in which variable groups will be added to the core synthon, and the reagent table (.tab) file which specifies the name of a chemical, its location (bottle number), flow rate, and concentration are utilized in conjunction with a basic set of command instructions.
  • .cmd generalized procedure or command
  • sequence .seq
  • reagent table reagent table
  • One basic set of command instructions can be: ADD
  • the ADD instruction has two forms, and is intended to have the look and feel of a standard chemical equation.
  • Reagents are specified to be added by a molar amount if the number proceeds the name identifier, or by an absolute volume in micro liters if the number follows the identifier.
  • the number of reagents to be added is a parsed list, separated by the "+" sign.
  • the key word, ⁇ seq> means look in the sequence table for the identity of the reagent to be added, while the key word, ⁇ act>, means add the reagent which is associated with that particular ⁇ seq>.
  • Reagents are delivered in the order specified in the list.
  • ADD ACN 300 means: Add 300 ⁇ l of the named reagent acetonitrile; ACN to each well of active synthesis ADD ⁇ seq> 300 means: If the sequence pointer in the .seq file is to a reagent in the list of reagents, independent of scale, add 300 ⁇ l of that particular reagent specified for that well.
  • ADD 1.1 PYR + 1.0 ⁇ seq> + 1.1 ⁇ actl> means: If the sequence pointer in the .seq file is to a reagent in the list of acids in the Class ACIDS 1 , and PYR is the name of pyridine, and ethyl chloroformate is defined in the .tab file to activate the class, ACIDS 1, then this instruction means: Add 1.1 equiv. pyridine
  • the IF command allows one to test what type of reagent is specified in the ⁇ seq> variable 39 and process the succeeding block of commands accordingly.
  • IF CLASS ACIDS_2 ADD 1.0 ⁇ seq> + 1.2 ⁇ actl> + 1.2 TEA
  • WAIT 60 DRAIN 10 END means: Operate on those wells for which reagents contained in the Acid_l class are specified, WAIT 60 sec, then operate on those wells for which reagents contained in the Acid_2 class are specified, then WAIT 60 sec longer, then DRAIN the whole plate. Note that the Acid 1 group has reacted for a total of 120 sec, while the Acid_2 group has reacted for only 60 sec.
  • the REPEAT command is a simple way to execute the same block of commands multiple times.
  • ADD ACN 300 DRAIN 15 END_REPEAT END means: repeats the add acetonitrile and drain sequence for each well three times.
  • the PRIME command will operate either on specific named reagents or on nozzles 40 which will be used in the next associated ⁇ seq> operation.
  • the ⁇ l amount dispensed into a prime port is a constant that can be specified in a config.dat file.
  • the NOZZLE_WASH command for washing the outside of reaction nozzles free from residue due to evaporation of reagent solvent will operate either on specific named reagents or on nozzles which have been used in the preceding associated ⁇ seq> operation.
  • the machine is plumbed such that if any nozzle in a block has been used, all the nozzles in that block will be washed into the prime port.
  • the WAIT and DRAIN commands are by seconds, with the drain command applying a gas pressure over the top surface of the plate in order to drain the wells.
  • the LOAD and REMOVE commands are instructions for the machine to pause for operator action.
  • the NEXT_SEQUENCE command increments the sequence pointer to the next group of substituents to be added in the sequence file.
  • the general form of a .seq file entry is the definition:
  • the sequence information is conveyed by a series of columns, each of which represents a variable reagent to be added at a particular position.
  • the scale ( ⁇ mole) variable is included so that reactions of different scale can be run at the same time if desired.
  • the reagents are defined in a lookup table (the .tab file), which specifies the name of the reagent as referred to in the sequence and command files, its location (bottle number), flow rate, and concentration. This information is then used by the controller software and hardware to determine both the appropriate slider motion to position the plate and slider arms for delivery of a specific reagent, as well as the specific valve and time required to deliver the appropriate reagents.
  • the adept classification of reagents allows the use of conditional IF loops from within a command file to perform addition of different reagents differently during a "single step" performed across 96 wells simultaneously.
  • the special class ACTIVATORS defines certain reagents that always get added with a particular class of reagents (for example tetrazole during a phosphitylation reaction in adding the next nucleotide to a growing oligonucleotide).
  • the LOOP BEGLN and LOOP_END commands define the block of commands which will continue to operate until a NEXT_SEQUENCE command points past the end of the longest list of reactants in any well.
  • a MOVE command Not included in the command set is a MOVE command.
  • the controller software and hardware determines the correct nozzle(s) and well(s) required for a particular reagent addition, then synchronizes the position of the requisite nozzle and well prior to adding the reagent.
  • a MANUAL mode can also be utilized in which the synthesis plate and nozzle blocks can be "homed” or moved to any position by the operator, the nozzles primed or washed, the various reagent bottles depressurized or washed with solvent, the chamber pressurized, etc.
  • the automatic COMMAND mode can be interrupted at any point, MANUAL commands executed, and then operation resumed at the appropriate location.
  • the sequence pointer can be incremented to restart a synthesis anywhere within a command file.
  • the list of oligonucleotides for synthesis can be rearranged or grouped for optimization of synthesis.
  • the oligonucleotides are grouped according to a factor on which to base the optimization of synthesis. As illustrated in the Examples below, one such factor is the 3' most nucleoside of the oligonucleotide.
  • a nucleotide bearing a 3' phosphoramite is added to the 5' hydroxyl group of a growing nucleotide chain.
  • the first nucleotide (at the 3' terminus of the oligonucleotide - the 3' most nucleoside) is first connected to a solid support. This is normally done batchwise on a large scale as is standard practice during oligonucleotide synthesis. Such solid supports pre-loaded with a nucleoside are commercially available. In utilizing the multi well format for oligonucleotide synthesis, for each oligonucleotide to be 42 synthesized, an aliquot of a solid support bearing the proper nucleoside thereon is added to the well for synthesis.
  • oligonucleotides to be synthesized in the .seq file Prior to loading the sequence of oligonucleotides to be synthesized in the .seq file, they are sorted by the 3' terminal nucleotide. Based on that sorting, all of the oligonucleotide sequences having an "A" nucleoside at their 3' end are grouped together, those with a "C” nucleoside are grouped together as are those with "G” or “T” nucleosides. Thus in loading the nucleoside-bearing solid support into the synthesis wells, machine movements are conserved.
  • the oligonucleotides can be grouped by the above described parameter or other parameters that facilitate the synthesis of the oligonucleotides.
  • sorting is noted as being effected by some parameter of type 1, as for instance the above described
  • the .seq file is associated with the respective .cmd and .tab files needed for synthesis of the particular chemistries specified for the oligonucleotides at process step 584 by retrieval of the .cmd and .tab files at process step 586 from the sample database 520. These files are then input into the multi well synthesizer at process step 588 for oligonucleotide synthesis.
  • each well contains the oligonucleotides located therein as a dry compound. 14. Quality Control.
  • quality control is performed on the oligonucleotides at process step 600 after a decision is made (decision step 550) to perform quality control.
  • quality control may be desired when there is some reason to think that some aspect of the synthetic process step 500 has been compromised.
  • samples of the oligonucleotides may be taken and stored in the event that the results of assays conducted using the oligonucleotides (process step 700) yield confusing results or 43 suboptimal data. In the latter event, for example, quality control might be performed after decision step 800 if no oligonucleotides with sufficient activity are identified.
  • decision step 650 follows quality control step process 600. If one or more of the oligonucleotides do not pass quality control, process step 500 can be repeated, i.e., the oligonucleotides are synthesized for a second time.
  • step 610 sterile, double-distilled water is transferred by an automated liquid handler (2040 of Figure 18) to each well of a multi -well plate containing a set of lyophilized antisense oligonucleotides.
  • the automated liquid handler (2040 of Figure 18) reads the barcode sticker on the multi-well plate to obtain the plate's identification number.
  • Automated liquid handler 2040 queries Sample Database 520 (which resides in Database Server 2002 of Figure 18) for the quality control assay instruction set for that plate and executes the appropriate steps.
  • Sample Database 520 which resides in Database Server 2002 of Figure 18
  • the first illustrative quality control process quantitates the concentration of oligonucleotide in each well. If this quality control step is performed, an automated liquid handler (2040 of Figure 18) is instructed to remove an aliquot from each well of the master plate and generate a replicate daughter plate for transfer to the UV spectrophotometer (2016 of Figure 18).
  • the UV spectrophotometer (2016 of Figure 18) then measures the optical density of each well at a wavelength of 260 nanometers.
  • a microprocessor within UV spectrophotometer (2016 of Figure 18) then calculates a concentration value from the measured absorbance value for each well and output the results to Sample Database 520.
  • the second illustrative quality control process steps 632 to 636) quantitates the percent of total oligonucleotide in each well that is full length. If this quality control step is performed, an automated liquid handler (2040 of Figure 18) is instructed to remove an aliquot from each well of the master plate and generate a replicate daughter plate for transfer to the multichannel capillary gel electrophoresis apparatus (2022 of Figure 18). 44
  • the apparatus electrophoretically resolves in capillary tube gels the oligonucleotide product in each well.
  • a detection window dynamically measures the optical density of the product that passes by it.
  • the value of percent product that passed by the detection window with respect to time is utilized by a built in microprocessor to calculate the relative size distribution of oligonucleotide product in each well.
  • the third illustrative quality control process steps 632 to 636) quantitates the mass of the oligonucleotide in each well that is full length. If this quality control step is performed, an automated liquid handler (2040 of Figure 18) is instructed to remove an aliquot from each well of the master plate and generate a replicate daughter plate for transfer to the multichannel liquid electrospray mass spectrometer (2018 of Figure 18). The apparatus then uses electrospray technology to inject the oligonucleotide product into the mass spectrometer. A built in microprocessor calculates the mass-to-charge ratio to arrive at the mass of oligonucleotide product in each well. The results are then output to
  • the output data is manually examined or is examined using an appropriate algorithm and a decision is made as to whether or not the plate receives "Pass" or "Fail” status.
  • the current criteria for acceptance for 18 mer oligonucleotides, is that at least 85% of the oligonucleotides in a multi-well plate must be 85% or greater full length product as measured by both capillary gel electrophoresis and mass spectrometry.
  • An input (manual or automated) is then made into Sample Database 520 as to the pass/fail status of the plate. If a plate fails, the process cycles back to step 500, and a new plate of the same oligonucleotides is automatically placed in the plate synthesis request queue (process 554 of Figure 15). If a plate receives
  • T-24 cells The transitional cell bladder carcinoma cell line T-24 is obtained from the American Type Culture Collection (ATCC) (Manassas, VA). T-24 cells were routinely cultured in complete McCoy's 5 A basal media (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal calf serum, penicillin 100 units per milliliter, and streptomycin 100 micrograms per milliliter (all from Life Technologies). Cells are routinely passaged by trypsinization and dilution when they reach 90% confluence.
  • ATCC American Type Culture Collection
  • VA Manassas, VA
  • McCoy's 5 A basal media (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal calf serum, penicillin 100 units per milliliter, and streptomycin 100 micrograms per milliliter (all from Life Technologies). Cells are routinely passaged by trypsinization and dilution when they reach 90% confluence.
  • A549 cells The human lung carcinoma cell line A549 is obtained from the
  • ATCC Manassas, VA.
  • A549 cells were routinely cultured in DMEM basal media (Life Technologies) supplemented with 10% fetal calf serum, penicillin 100 units per milliliter, and streptomycin 100 micrograms per milliliter (all from Life Technologies). Cells are routinely passaged by trypsinization and dilution when they reach 90% confluence.
  • NHDF cells Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Co ⁇ oration (WalkersviUe, MD). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Co ⁇ .) as provided by the supplier. Cells are maintained for up to 10 passages as recommended by the supplier.
  • HEK cells Human embryonic keratinocytes (HEK) were obtained from the Clonetics Co ⁇ . HEKs were routinely maintained in Keratinocyte Growth Medium
  • oligonucleotide When cells reach about 80% confluency, they are treated with oligonucleotide. For cells grown in 96-well plates, wells are washed once with 200 ⁇ l OPTI-MEM-1TM reduced-serum medium (Life Technologies) and then treated with 130 ⁇ l of OPTI-MEM- 46
  • oligonucleotide 1TM containing 3.75 ⁇ g/ml LIPOFECTINTM (Life Technologies) and the desired oligonucleotide at a final concentration of 150 nM. After 4 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16 hours after oligonucleotide treatment.
  • oligonucleotides can be introduced by electroporation. Electroporation conditions must be optimized for every cell type. In general, oligonucleotide is added directly to complete growth media to a final concentration between 1 and 20 micromolar.
  • Oligonucleotide-mediated modulation of expression of a target nucleic acid can be assayed in a variety of ways known in the art.
  • target RNA levels can be quantitated by, e.g., Northern blot analysis, competitive PCR, or reverse transcriptase polymerase chain reaction (RT-PCR).
  • RNA analysis can be performed on total cellular RNA or, preferably in the case of polypeptide-encoding nucleic acids, poly(A)+ mRNA.
  • poly(A)+ mRNA is preferred.
  • Methods of RNA isolation are taught in, for example, Ausubel et al. ⁇ Short Protocols in Molecular Biology, 2nd Ed., pp. 4-1 to 4-13, Greene Publishing Associates and John Wiley & Sons, New York, 1992).
  • Northern blot analysis is routine in the art ⁇ Id., pp. 4-14 to 4-29).
  • total RNA can be prepared from cultured cells or tissue using the QIAGEN RNeasy®-96 kit for the high throughput preparation of RNA (QIAGEN, Inc., Valencia, CA). Essentially, protocols are carried out according to the manufacturer's directions. Optionally, a DNase step is included to remove residual DNA prior to RT-
  • the repetitive pipeting steps and elution step have been automated using a QIAGEN Bio-Robot 9604. Essentially after lysing of the oligonucleotide treated cell cultures in situ, the plate is transferred to the robot deck where the pipeting, DNase treatment, and elution steps are carried out.
  • RT-PCR Reverse transcriptase polymerase chain reaction
  • PE- Applied Biosystems Foster City, CA
  • Other methods of PCR are also known in the art.
  • Target protein levels can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), Enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS).
  • Antibodies directed to a protein encoded by a target nucleic acid can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies, (Aerie Co ⁇ oration, Birmingham, MI or via the internet at http://www.ANTIBODIES-PROBES.com/), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal, monospecific (“antipeptide") and monoclonal antisera are taught by, for example, Ausubel et al. ⁇ Short Protocols in Molecular Biology, 2nd Ed., pp. 11-3 to 11- 54, Greene Publishing Associates and John Wiley & Sons, New York, 1992).
  • Immunoprecipitation methods are standard in the art and are described by, for example, Ausubel et al ⁇ Id., pp. 10-57 to 10-63).
  • Western blot (immunoblot) analysis is standard in the art ⁇ Id., pp. 10-32 to 10-10-35).
  • Enzyme-linked immunosorbent assays (ELISA) are standard in the art ⁇ Id., pp. 11-5 to 11-17).
  • preferred means of assaying are suitable for use in 96-well plates and with robotic means.
  • automated RT-PCR is preferred for assaying target nucleic acid levels
  • automated ELISA is preferred for assaying target protein levels.
  • the assaying step is described in detail in Figure 16.
  • a decision is made at decision step 714 as to whether RT-PCR will be the only method by which the activity of the compounds is evaluated.
  • alternative assay methods for example, when it is desired to assess target polypeptide levels as well as target RNA levels, an immunoassay such as an ELISA is run in parallel with the RT-PCR assays.
  • such assays are tractable to semi-automated or robotic means.
  • RT-PCR When RT-PCR is used to evaluate the activities of the compounds, cells are plated into multi-well plates (typically, 96-well plates) in process step 720 and treated with test or 48 control oligonucleotides in process step 730. Then, the cells are harvested and lysed in process step 740 and the lysates are introduced into an apparatus where RT-PCR is carried out in process step 750. A raw data file is generated, and the data is downloaded and compiled at step 760. Spreadsheet files with data charts are generated at process step 770, and the experimental data is analyzed at process step 780. Based on the results, a decision is made at process step 785 as to whether it is necessary to repeat the assays and, if so, the process begins again with step 720. In any event, data from all the assays on each oligonucleotide are compiled and statistical parameters are automatically determined at process step 790. 18. Classification of Compounds Based on Their Activity:
  • oligonucleotide compounds are classified according to one or more desired properties. Typically, three classes of compounds are used: active compounds, marginally active (or "marginal”) compounds and inactive compounds. To some degree, the selection criteria for these classes vary from target to target, and members of one or more classes may not be present for a given set of oligonucleotides.
  • inactive compounds will typically comprise those compounds having 5% or less inhibition of target expression (relative to basal levels). Active compounds will typically cause at least 30% inhibition of target expression, although lower levels of inhibition are acceptable in some instances.
  • Marginal compounds will have activities intermediate between active and inactive compounds, with preferred marginal compounds having activities more like those of active compounds.
  • oligonucleotide compounds are optimized for activity is by varying their nucleobase sequences so that different regions of the target nucleic acid are targeted. Some such regions will be more accessible to oligonucleotide compounds than others, and "sliding" a nucleobase sequence along a target nucleic acid only a few bases can have significant effects on activity. Accordingly, varying or adjusting the nucleobase sequences of the compounds of the invention is one means by which suboptimal compounds can be made optimal, or by which new active compounds can be generated. 49
  • the term "gene walk” is defined as the process by which a specified oligonucleotide sequence x that binds to a specified nucleic acid target y is used as a frame of reference around which a series of new oligonucleotides sequences capable of hybridizing to nucleic acid target y are generated that are sequence shifted increments of oligonucleotide sequence x. Gene walking can be done “downstream”, “upstream” or in both directions from a specified oligonucleotide.
  • step 1104 the user manually enters the identification number of the oligonucleotide sequence around which it is desired to execute gene walk process 1100 and the name of the corresponding target nucleic acid.
  • the user then enters the scope of the gene walk at step 1104, by which is meant the number of oligonucleotide sequences that it is desired to generate.
  • the user then enters in step 1108 a positive integer value for the sequence shift increment.
  • the gene walk is effected. This causes a subroutine to be executed that automatically generates the desired list of sequences by walking along the target sequence. At that point, the user proceeds to process 400 to assign chemistries to the selected oligonucleotides.
  • Example 16 below details a gene walk.
  • this new set of nucleobase sequences generated by the gene walk is used to direct the automated synthesis at general procedure step 500 of a second set of candidate oligonucleotides. These compounds are then taken through subsequent process steps to yield active compounds or reiterated as necessary to optimize activity of the compounds.
  • an oligonucleotide chemistry different from that of the first set of oligonucleotides is assigned at general procedure step 400.
  • the nucleobase sequences of marginal compounds are used to direct the synthesis at general procedure step 500 of a second set of oligonucleotides having the second assigned chemistry.
  • the resulting second set of oligonucleotide compounds is assayed in the same manner as the first set at 50 procedure process step 700 and the results are examined to determine if compounds having sufficient activity have been generated at decision step 800.
  • a second oligonucleotide chemistry is assigned at procedure step 400 to the nucleobase sequences of all of the oligonucleotides (or, at least, all of the active and marginal compounds) and a second set of oligonucleotides is synthesized at procedure step 500 having the same nucleobase sequences as the first set of compounds.
  • the resulting second set of oligonucleotide compounds is assayed in the same manner as the first set at procedure step 700 and active and marginal compounds are identified at procedure steps 800 and 1000.
  • FIG. 18 An embodiment of computer, network and instrument resources for effecting the methods of the invention is shown in Figure 18.
  • four computer servers are provided.
  • a large database server 2002 stores all chemical structure, sample tracking and genomic, assay, quality control, and program status data. Further, this database server serves as the platform for a document management system.
  • a compute engine 2004 runs computational programs including RNA folding, oligonucleotide walking, and genomic searching.
  • a file server 2006 allows raw instrument output storage and sharing of robot instructions.
  • a groupware server 2008 enhances staff communication and process scheduling.
  • a redundant high-speed network system is provided between the main servers and the bridges 2026, 2028 and 2030. These bridges provide reliable network access to the 51 many workstations and instruments deployed for this process.
  • the instruments selected to support this embodiment are all designed to sample directly from standard 96 well microtiter plates, and include an optical density reader 2016, a combined liquid chromatography and mass spectroscopy instrument 2018, a gel fluorescence and scintillation imaging system 2032 and 2042, a capillary gel electrophoreses system 2022 and a real-time PCR system 2034.
  • FIG. 19 illustrates the data structure of a sample relational database. Various elements of data are segregated among linked storage elements of the database.
  • Cell-cell interactions are a feature of a variety of biological processes.
  • one of the earliest detectable events in a normal inflammatory response is adhesion of leukocytes to the vascular endothelium, followed by migration of leukocytes out of the vasculature to the site of infection or injury.
  • the adhesion of leukocytes to vascular endothelium is an obligate step in their migration out of the vasculature (for a review, see Albelda et al, FASEB J, 1994, 8, 504).
  • CD40 was first characterized as a receptor expressed on B-lymphocytes. It was later found that engagement of B-cell CD40 with CD40L expressed on activated T-cells is essential for T-cell dependent B-cell activation (i.e. proliferation, immunoglobulin secretion, and class switching) (for a review, see Gruss et al. Leuk. Lymphoma, 1997, 24, 393).
  • a full cDNA sequence for CD40 is available (GenBank accession number X60592, inco ⁇ orated herein by reference as SEQ ID NO:85).
  • CD40 As interest in CD40 mounted, it was subsequently revealed that functional CD40 is expressed on a variety of cell types other than B-cells, including macrophages, dendritic cells, thymic epithelial cells, Langerhans cells, and endothelial cells ⁇ Ibid.). These studies have led to the current belief that CD40 plays a much broader role in immune regulation by mediating interactions of T-cells with cell types other than B-cells. In support of this notion, it has been shown that stimulation of CD40 in macrophages and dendritic results is required for T-cell activation during antigen presentation ⁇ Id.). Recent evidence points to a role for CD40 in tissue inflammation as well.
  • CD40 Due to the pivotal role that CD40 plays in humoral immunity, the potential exists that therapeutic strategies aimed at downregulating CD40 may provide a novel class of agents useful in treating a number of immune associated disorders, including but not limited to graft versus host disease, graft rejection, and autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus, and certain forms of arthritis.
  • Inhibitors 53 of CD40 may also prove useful as an anti-inflammatory compound, and could therefore be useful as treatment for a variety of diseases with an inflammatory component such as asthma, rheumatoid arthritis, allograft rejections, inflammatory bowel disease, and various dermatological conditions, including psoriasis.
  • inhibitors of CD40 may prove useful as anti-tumor agents as well.
  • CD40 a variety of agents that disrupt CD40/CD40L binding.
  • agents that disrupt CD40/CD40L binding include monoclonal antibodies directed against either CD40 or CD40L, soluble forms of CD40, and synthetic peptides derived from a second CD40 binding protein, A20.
  • the use of neutralizing antibodies against CD40 and/or CD40L in animal models has provided evidence that inhibition of CD40 stimulation would have therapeutic benefit for GVHD, allograft rejection, rheumatoid arthritis, SLE, MS, and B-cell lymphoma (Buhlmann et al, J. Clin. Immunol, 1996, 16, 83).
  • Oligonucleotides compounds avoid many of the pitfalls of current agents used to block CD40/CD40L interactions and may therefore prove to be uniquely useful in a number of therapeutic applications.
  • the process of the invention was used to select oligonucleotides targeted to CD40, generating the list of oligonucleotide sequences with desired properties as shown in Figure 22. From the assembled CD40 sequence, the process began with determining the desired oligonucleotide length to be eighteen nucleotides, as represented in step 2500. All possible oligonucleotides of this length were generated by Oligo 5.0TM, as represented in step 2504. Desired thermodynamic properties were selected in step 2508. The single parameter used was oligonucleotides of melting temperature less than or equal to 40°C were discarded. In step 2512, oligonucleotide melting temperatures were calculated by Oligo 5.0 TM .
  • Oligonucleotide sequences possessing an undesirable score were discarded. It 54 is believed that oligonucleotides with melting temperatures near or below physiological and cell culture temperatures will bind poorly to target sequences. All oligonucleotide sequences remaining were exported into a spreadsheet.
  • desired sequence properties are selected. These include discarding oligonucleotides with at least one stretch of four guanosines in a row and stretches of six of any other nucleotide in a row.
  • a spreadsheet macro removed all oligonucleotides containing the text string "GGGG.”
  • another spreadsheet macro removed all oligonucleotides containing the text strings "AAAAAA” or “CCCCCC” or « TTTTTT .”
  • 84 sequences were selected manually with the criteria of having an uniform distribution of oligonucleotide sequences throughout the target sequence, as represented in step 2528. These oligonucleotide sequences were then passed to the next step in the process, assigning actual oligonucleotide chemistries to the sequences.
  • Table 2 is a command file for synthesis of an oligonucleotide having regions of 2'- O-(2-methoxyethyl) nucleosides and a central region of 2'-deoxy nucleosides each linked by phosphorothioate internucleotide linkages.
  • Table 3 is a .seq file for oligonucleotides having 2'-deoxy nucleosides linked by phosphorothioate internucleotide linkages.
  • Table 4 is a .seq file for oligonucleotides having regions of 2'-O-(2-methoxyethyl)- nucleosides and a central region of 2'-deoxy nucleosides each linked by phosphorothioate internucleotide linkages.
  • Table 5 is a .tab file for reagents necessary for synthesizing an oligonucleotides having both 2'-O-(2-methoxyethyl)nucleosides and 2'-deoxy nucleosides located therein.
  • Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry using a multi well automated synthesizer utilizing input files as described in EXAMPLE 3 above.
  • the oligonucleotides were synthesized by assembling 96 sequences simultaneously in a standard 96 well format.
  • Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine.
  • Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile.
  • Standard base-protected beta-cyanoethyl- diisopropyl phosphoramidites were purchased from commercial vendors (e.g. PE/ABI, Pharmacia). Non-standard nucleosides are synthesized as per known literature or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.
  • oligonucleotides were cleaved from support and deprotected with concentrated NH 4 OH at elevated temperature (55-60 °C) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.
  • Unsubstituted and substituted phosphodiester oligonucleotides are alternately synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine.
  • Phosphorothioates are synthesized as per the phosphodiester oligonucleotides except the standard oxidation bottle was replaced by 0.2 M solution of 3H-1,2- benzodithiole-3-one 1,1 -dioxide in acetonitrile for the stepwise thiation of the phosphite linkages.
  • the thiation wait step was increased to 68 sec and was followed by the capping 73 step.
  • the oligonucleotides were purified by precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution.
  • Phosphinate oligonucleotides are prepared as described in U.S. Patent 5,508,270, inco ⁇ orated herein by reference in its entirety.
  • Alkyl phosphonate oligonucleotides are prepared as described in U.S. Patent 4,469,863, inco ⁇ orated herein by reference in its entirety.
  • 3 '-Deoxy-3 '-methylene phosphonate oligonucleotides are prepared as described in U.S. Patents 5,610,289 or 5,625,050, each of which is inco ⁇ orated herein by reference in its entirety.
  • Phosphoramidite oligonucleotides are prepared as described in U.S. Patent, 5,256,775 or U.S. Patent 5,366,878, inco ⁇ orated herein by reference in its entirety.
  • Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), each of which is inco ⁇ orated herein by reference in its entirety.
  • 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared as described in U.S. Patent 5,476,925, inco ⁇ orated herein by reference in its entirety.
  • Phosphotriester oligonucleotides are prepared as described in U.S. Patent 5,023,243, inco ⁇ orated herein by reference in its entirety.
  • Boranophosphate oligonucleotides are prepared as described in U.S. Patents 5,130,302 and 5,177,198, each of which is inco ⁇ orated herein by reference in its entirety.
  • Methylenemethylimino linked oligonucleosides also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and PO or PS linkages are prepared as described in U.S. Patents 5,378,825; 5,386,023; 5,489,677; 5,602,240 and 5,610,289, each of which is inco ⁇ orated herein by reference in its entirety.
  • Formacetal and thioformacetal linked oligonucleosides are prepared as described in 74
  • Ethylene oxide linked oligonucleosides are prepared as described in U.S. Patent 5,223,618, inco ⁇ orated herein by reference in its entirety.
  • PNAs Peptide nucleic acids
  • PNA Peptide nucleic acids
  • Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the "gap" segment of linked nucleosides is positioned between 5' and 3' "wing" segments of linked nucleosides and a second "open end" type wherein the
  • “gap” segment is located at either the 3' or the 5' terminus of the oligomeric compound.
  • Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides.
  • Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers.”
  • Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate and 2'-deoxy phosphorothioate oligonucleotide segments are synthesized using 2'-deoxy-5'- dimethoxytrityl-3'-O-phosphoramidites for the DNA portion and 5'-dimethoxytrityl-2'-O- methyl-3'-O-phosphoramidites for 5' and 3' wings.
  • the standard synthesis cycle is modified by increasing the wait step after the delivery of tetrazole and base to 600 s repeated four times for DNA and twice for 2'-0-methyl.
  • the fully protected oligonucleotide was cleaved from the support and the phosphate group is deprotected in 3:1 Ammonia Ethanol at room temperature overnight then lyophilized to dryness. 75
  • [2'-O-(2-methoxyethyl phosphodiester] ⁇ [2'-deoxy phosphorothioate]— [2'-O-(2- methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2'-O-methyl chimeric oligonucleotide with the substitution of 2'-O-(2- methoxy ethyl) amidites for the 2'-O-methyl amidites in the wing portions.
  • Sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) is used to generate the phosphorothioate internucleotide linkages within the wing portions of the chimeric structures.
  • Oxidization with iodine is used to generate the phosphodiester internucleotide linkages for the center gap.
  • oligonucleotides were prepared as per the protocol of the 96 well format of Example 4.
  • the oligonucleotides were prepared utilizing phosphorothioate chemistry to give in one instance a first library of phosphorothioate oligodeoxynucleotides.
  • the oligonucleotides were prepared in a second instance as a second library of hybrid oligonucleotides having phosphorothioate backbones with a first and third "wing" region of 2'-O-(2- methoxyethyl)nucleotides on either side of a center gap region of 2'-deoxy nucleotides.
  • the two libraries contained the same set of oligonucleotide sequences. Thus the two 76 libraries are redundant with respect to sequence but are unique with respect to the combination of sequence and chemistry. Because the sequences of the second library of compounds is the same as the first (however the chemistry is different), for brevity sake, the second library is not shown.
  • Tables 6-a and 6-b show the sequences of an initial first library, i.e., a library of phosphorothioate oligonucleotides targeted to a CD40 target.
  • the compounds of Table 6-a shows the members of this library listed in compliance with the established rule for listing SEQ ID NO:, i.e., in numerical SEQ ID NO: order.
  • GTTTGTGCCTGCCTGTTG 59 GTCTTGTTTGTGCCTGCC 60
  • CTCTTTGCCATCCTCCTG 79 CTGTCTCTCCTGCACTGA 80
  • AACTGCCTGTTTGCCCAC 82 CTTCTGCCTGCACCCCTG 83 ACTGACTGGGCATAGCTC 84
  • a to G The location of the well holding each particular oligonucleotides is indicated by row and column. There are eight rows designated A to G and twelve columns designated
  • Table 6-b the oligonucleotides of Table 6-a are shown reordered according to the Well No. on their synthesis plate.
  • the order shown in Table 6-b is the actually order as synthesized on an automated synthesizer taking advantage of the preferred placement of the first nucleoside according to the above alignment criteria.
  • the concentration of oligonucleotide in each well was assessed by dilution of samples and UV abso ⁇ tion spectroscopy.
  • the full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96 well format (Beckman MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors.
  • oligonucleotides or oligonucleosides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides are analyzed by polyacrylamide gel electrophoresis on denaturing gels. Oligonucleotide purity is checked by 31 P nuclear 82 magnetic resonance spectroscopy, and/or by HPLC, as described by Chiang et al, J. Biol. Chem. 1991, 266, 18162.
  • EXAMPLE 10 Automated Assay of CD40 Oligonucleotide Activity A. Poly(A)+ mRNA isolation.
  • Poly(A)+ mRNA was isolated according to Miura et al. ⁇ Clin. Chem., 1996, 42, 1758). Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 ⁇ l cold PBS. 60 ⁇ l lysis buffer (10 mM Tris- HC1, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 ⁇ l of lysate was transferred to Oligo d(T) coated 96 well plates (AGCT Inc., Irvine, CA).
  • Total mRNA was isolated using an RNEASY 960 kit and buffers purchased from Qiagen Inc. (Valencia CA) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 mL cold PBS. 100 mL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 mL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 960 well plate attached to a QIAVACO manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds.
  • Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes.
  • the plate was then removed from the QIAVACO manifold and blotted dry on paper towels.
  • the plate was then re-attached to the QIAVACO manifold fitted with a collection tube rack containing 1.2 mL collection tubes.
  • RNA was then eluted by pipetting 60 mL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 mL water.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • PE- Applied Biosystems Foster City, CA
  • This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in realtime.
  • PCR polymerase chain reaction
  • amplification products are quantitated after the PCR is completed
  • products in RT-PCR are quantitated as they accumulate.
  • a reporter dye e.g., JOE or FAM, PE-Applied Biosystems, Foster City, CA
  • a quencher dye e.g., TAMRA, PE-Applied
  • Biosystems, Foster City, CA is attached to the 3' end of the probe.
  • reporter dye emission is quenched by the proximity of the 3' quencher dye.
  • annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5'-exonuclease activity of Taq polymerase.
  • cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated.
  • RT-PCR reagents were obtained from PE-Applied Biosystems, Foster City, CA. RT-PCR reactions were carried out by adding 25 ml PCR cocktail (lx TAQMANTM buffer A, 5.5 mM MgCl 2 , 300 mM each of dATP, dCTP and dGTP, 600 mM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 U RNAse inhibitor, 1.25 units AMPLITAQ GOLDTM, and 12.5 U MuLV reverse transcriptase) to 96 well plates containing 25 ml poly(A) mRNA solution. The RT reaction was carried out by incubation for 30 minutes at 48°C. following a 10 minute incubation at 95°C to activate the AMPLITAQ GOLDTM, 40 cycles of a two-step PCR protocol were carried out: 95 °C for
  • the PCR primers were: forward: 5' CAGAGTTCACTGAAACGGAATGC 3'
  • PCR probe 5' E4 -TTCCTTGCGGTGAAAGCGAATTCCT-7 i 4 3' (SEQ ID NO:88) where FAM (PE-Applied Biosystems, Foster City, CA) is the fluorescent reporter dye and TAMRA (PE-Applied Biosystems, Foster City, CA) is the quencher dye.
  • FAM PE-Applied Biosystems, Foster City, CA
  • TAMRA PE-Applied Biosystems, Foster City, CA
  • the PCR primers were: forward: 5' GAAGGTGAAGGTCGGAGTC 3' (SEQ ID NO:89) reverse: 5' GAAGATGGTGATGGGATTTC 3' (SEQ ID NO:90), and
  • PCR probe 5* JOE-CAAGCTTCCCGTTCTCAGCC-7 ⁇ 4 3' (S ⁇ Q ID NO. 91) where JOE (P ⁇ - Applied Biosystems, Foster City, CA) is the fluorescent reporter dye and TAMRA (P ⁇ - Applied Biosystems, Foster City, CA) is the quencher dye.
  • oligonucleotides complementary to mRNA were designed to target different regions of the human CD40 mRNA, using published sequences (GenBank accession number X60592, inco ⁇ orated herein by reference as SEQ ID NO: 85). The oligonucleotides are shown in Table 7. 85
  • Target sites are indicated by the beginning nucleotide numbers, as given in the sequence source reference (X60592), to which the oligonucleotide binds. All compounds in Table 7 are oligodeoxynucleotides with phosphorothioate backbones (internucleoside linkages) throughout. Data are averages from three experiments.
  • SEQ ID NOS: 1, 2, 7, 47 and 82 demonstrated at least 25% inhibition of CD40 expression and are therefore preferred compounds of the invention.
  • EXAMPLE 12 Inhibition of CD40 Expression by Phosphorothioate 2'-MOE Gapmer Oligonucleotides
  • a second series of oligonucleotides complementary to mRNA were designed to target different regions of the human CD40 mRNA, using published sequence X60592.
  • the oligonucleotides are shown in Table 8. Target sites are indicated by the beginning or initial nucleotide numbers, as given in the sequence source reference (X60592), to which the oligonucleotide binds.
  • All compounds in Table 8 are chimeric oligonucleotides ("gapmers”) 18 nucleotides in length, composed of a central "gap" region consisting often 2'- deoxynucleo tides, which is flanked on both sides (5' and 3' directions) by four-nucleotide "wings.”
  • the wings are composed of 2'-O-(2-methoxyethyl) (2'-MOE) nucleotides.
  • SEQ ID NOS: 1 and 2 overlap each other and both map to the 5- untranslated region (5'-UTR) of CD40. Accordingly, this region of CD40 is particularly preferred for modulation via sequence-based technologies.
  • SEQ ID NOS: 7 and 47 map to the open reading frame of CD40, whereas SEQ ID NO: 82 maps to the 3'- untranslated region (3'-UTR).
  • the ORF and 3'-UTR of CD40 may be targeted by sequence-based technologies as well.
  • the reverse complements of the active CD40 compounds are easily determined by those skilled in the art and may be assembled to yield nucleotide sequences corresponding 90 to accessible sites on the target nucleic acid.
  • the assembled reverse complement of SEQ ID NOS: 1 and 2 is represented below as SEQ ID NO:92: 5'- AGTGGTCCTGCCGCCTGGTC -3' SEQ ID NO:92 TCACCAGGACGGCGGACC -5' SEQ ID NO: 1 ACCAGGACGGCGGACCAG -5' SEQ ID NO:2
  • footprints are generated. A library of this information is compiled and may be used by those skilled in the art in a variety of sequence-based technologies to study the molecular and biological functions of CD40 and to investigate or confirm its role in various diseases and disorders.
  • an application is deployed which facilitates the selection process for determining the target positions of the oligos to be synthesized, or "sites.”
  • This program is written using a three-tiered object-oriented approach. All aspects of the software described, therefore, are tightly integrated with the relational database. For this reason, explicit database read and write steps are not shown. It should be assumed that each step described includes database access. The description below illustrates one way the program can be used. The actual interface allows users to skip from process to process at will, in any order.
  • the target Before running the site picking program, the target must have all relevant properties computed as described previously and indicated in process step 2204.
  • the site picking program When the site picking program is launched at process step 2206 the user is presented with a panel showing targets which have previously been selected and had their properties calculated. The user selects one target to work with at process step 2208 and proceeds to decide if any derived properties will be needed at process step 2210. Derived properties are calculated by performing mathematical operations on combinations of pre-calculated properties as defined by the user at process step 2212.
  • the derived properties are made available as peers with all the pre-calculated properties.
  • the user selects one of the properties to view plotted versus target position at process step 2214. This graph is shown above a linear representation of the target. The 91 horizontal or position axis of both the graph and target are linked and scalable by the user. The zoom range goes from showing the full target length to showing individual target bases as letters and individual property points.
  • the user next selects a threshold value below or above which all sites will be eliminated from future consideration at process step 2216.
  • the user decides whether to eliminate more sites based on any other properties at process step 2218. If they choose to eliminate more, they return to pick another property to display at process step 2214 and threshold at process step 2216.
  • the user selects from the remaining list by choosing any property at process step 2220 and then choosing a manual or automatic selection technique at process step 2222.
  • the automatic technique the user decides whether they want to pick from maxima or minima and the number of maxima or minima to be selected as sites at process step 2224.
  • the software automatically finds and picks the points.
  • When picking manually the user must decide if they wish to use automatic peak finding at process step 2226. If the user selects automatic peak finding, then user must click on the graphed property with the mouse at process step 2236. The nearest maxima or minima, depending on the modifier key held down, to the selected point will be picked as the site. Without the peak finding option, the user must pick a site at process step 2238 by clicking on its position on the linear representation of target.
  • a dynamic property is calculated for all possible sites (not yet eliminated) at process step 2230. This property indicates the nearness of the site to a picked site allowing the user to pick sites in subsequent iterations based on target coverage.
  • the user determines if the desired number of sites has been picked. If too few sites have been picked the user returns to pick more 2220. If too many sites have been picked, the user may eliminate them by selecting and deleting them on the target display at process step 2234. If the correct number of sites is picked, and the user is satisfied with the set of picked sites, the user registers these sites to the database along with their name, notebook number, and page number at process step 2238. The database time stamps this registration event. 92

Abstract

Iterative, preferably computer based iterative processes for generating synthetic compounds with desired physical, chemical and/or bioactive properties, i.e., active compounds, are provided. During iterations of the processes, a target nucleic acid sequence is provided or selected, and a library of candidate nucleobase sequences is generated in silico according to defined criteria. A 'virtual' oligonucleotide chemistry is chosen and a library of virtual oligonucleotide compounds having the selected nucleobase sequences is generated. These virtual compounds are reviewed and compounds predicted to have particular properties are selected. The selected compounds are robotically synthesized and are preferably robotically assayed for a desired physical, chemical or biological activity. Active compounds are thus generated and, at the same time, preferred sequences and regions of the target nucleic acid that are amenable to oligonucleotide or sequence-based modulation are identified.

Description

IDENTIFICATION OF GENETIC TARGETS FOR MODULATION BY OLIGONUCLEOTIDES AND GENERATION OF OLIGONUCLEOTIDES FOR GENE MODULATION
CROSS REFERENCE TO RELATED APPLICATIONS The present application is a continuation-in-part of U.S. Serial No. 09/067,638 filed April 28, 1998, which claims priority to provisional application Serial No. 60/081,483 filed April 13, 1998, each of which is incoφorated herein by reference in its entirety.
FIELD OF THE INVENTION The present invention relates generally to the generation and identification of synthetic compounds having defined physical, chemical or bioactive properties. More particularly, the present invention relates to the automated generation of oligonucleotide compounds targeted to a given nucleic acid sequence via computer-based, iterative robotic synthesis of synthetic oligonucleotide compounds and robotic or robot-assisted analysis of the activities of such compounds. Information gathered from assays of such compounds is used to identify nucleic acid sequences that are tractable to a variety of nucleotide sequence-based technologies, for example, antisense drug discovery and target validation. 2
BACKGROUND OF THE INVENTION
1. Oligonucleotide Technology
Synthetic oligonucleotides of complementarity to targets are known to hybridize with particular, target nucleic acids in a sequence-specific manner. In one example, compounds complementary to the "sense" strand of nucleic acids that encode polypeptides, are referred to as "antisense oligonucleotides." A subset of such compounds may be capable of modulating the expression of a target nucleic acid; such synthetic compounds are described herein as "active oligonucleotide compounds."
Oligonucleotide compounds are commonly used in vitro as research reagents and diagnostic aids, and in vivo as therapeutic and bioactive agents. Oligonucleotide compounds can exert their effect by a variety of means. One such means takes advantage of an endogenous nuclease, such as RNase H in eukaryotes or RNase P in prokaryotes, to degrade the DNA/RNA hybrid formed between the oligonucleotide sequence and mRNA (Chiang et al., J. Biol. Chem., 1991, 266, 18162; Forster et al, Science, 1990, 249, 783). Another means involves covalently linking of a synthetic moiety having nuclease activity to an oligonucleotide having an antisense sequence. This does not rely upon recruitment of an endogenous nuclease to modulate target activity. Synthetic moieties having nuclease activity include, but are not limited to, enzymatic RNAs, lanthanide ion complexes, and other reactive species. (Haseloff et al., Nature, 1988, 334, 585; Baker et al., J. Am. Chem. Soc, 1997, 119, 8749).
Despite the advances made in utilizing antisense technology to date, it is still common to identify target sequences amenable to antisense technologies through an empirical approach (Szoka, Nature Biotechnology, 1997, 15, 509). Accordingly, the need exists for systems and methods for efficiently and effectively identifying target nucleotide sequences that are suitable for antisense modulation. The present disclosure answers this need by providing systems and methods for automatically identifying such sequences via in silico, robotic or other automated means.
2. Identification of Active Oligonucleotide Compounds
Traditionally, new chemical entities with useful properties are generated by (1) identifying a chemical compound (called a "lead compound") with some desirable property or activity, (2) creating variants of the lead compound, and (3) evaluating the property and activity of such variant compounds. The process has been called "SAR," i.e., structure activity relationship. Although "SAR" and its handmaiden, rational drug design, has been utilized with some degree of success, there are a number of limitations to these approaches to lead compound generation, particularly as it pertains to the discovery of bioactive oligonucleotide compounds. In attempting to use SAR with oligonucleotides, it has been recognized that RNA structure can inhibit duplex formation with antisense compounds, so much so that "moving" the target nucleotide sequence even a few bases can drastically decrease the activity of such compounds (Lima et al., Biochemistry, 1992, 31, 12055). Heretofore, the preferred method of searching for lead antisense compounds has been the manual synthesis and analysis of such compounds. Consequently, a fundamental limitation of the conventional approach is its dependence upon the availability, number and cost of antisense compounds produced by manual, or at best semi-automated, means. Moreover, the assaying of such compounds has traditionally been performed by tedious manual techniques. Thus, the traditional approach to generating active antisense compounds is limited by the relatively high cost and long time required to synthesize and screen a relatively small number of candidate antisense compounds.
Accordingly, the need exists for systems and methods for efficiently and effectively generating new active antisense and other oligonucleotide compounds targeted to specific nucleic acid sequences. The present disclosure answers this need by providing systems and methods for automatically generating and screening active antisense compounds via robotic and other automated means. 3. Gene Function Analysis Efforts such as the Human Genome Project are making an enormous amount of nucleotide sequence information available in a variety of forms, e.g., genomic sequences, cDNAs, expressed sequence tags (ESTs) and the like. This explosion of information has led one commentator to state that "genome scientists are producing more genes than they can put a function to" (Kahn, Science, 1995, 270, 369). Although some approaches to this problem have been suggested, no solution has yet emerged. For example, methods of looking at gene expression in different disease states or stages of development only provide, at best, an association between a gene and a disease or stage of development (Nowak, Science, 1995, 270, 368). Another approach, looking at the proteins encoded by genes, is developing but "this approach is more complex and big obstacles remain" (Kahn, Science, 1995, 270, 369). Furthermore, neither of these approaches allows one to directly utilize nucleotide sequence information to perform gene function analysis. In contrast, antisense technology does allow for the direct utilization of nucleotide sequence information for gene function analysis. Once a target nucleic acid sequence has been selected, antisense sequences hybridizable to the sequence can be generated using techniques known in the art. Typically, a large number of candidate antisense oligonucleotides (ASOs) are synthesized having sequences that are more-or-less randomly spaced across the length of the target nucleic acid sequence (e.g., a "gene walk") and their ability to modulate the expression of the target nucleic acid is assayed. Cells or animals can then be treated with one or more active antisense oligonucleotides, and the resulting effects determined in order to determine the function(s) of the target gene. Although the practicality and value of this empirical approach to determining gene function has been acknowledged in the art, it has also been stated that this approach "is beyond the means of most laboratories and is not feasible when a new gene sequence is identified, but whose function and therapeutic potential are unknown" (Szoka, Nature Biotechnology, 1997, 15, 509).
Accordingly, the need exists for systems and methods for efficiently and effectively determining the function of a gene that is uncharacterized except that its nucleotide sequence, or a portion thereof, is known. The present disclosure answers this need by providing systems and methods for automatically generating active antisense compounds to a target nucleotide sequence via robotic means. Such active antisense compounds are contacted with cells, cell-free extracts, tissues or animals capable of expressing the gene of interest and subsequent biochemical or biological parameters are measured. The results are compared to those obtained from a control cell culture, cell-free extract, tissue or animal which has not been contacted with an active antisense compound in order to determine the function of the gene of interest. 4. Target Validation Determining the nucleotide sequence of a gene is no longer an end unto itself; rather, it is "merely a means to an end. The critical next step is to validate the gene and its [gene] product as a potential drug target" (Glasser, Genetic Engineering News, 1997, 17, 1). This process, i.e., confirming that modulation of a gene that is suspected of being involved in a disease or disorder actually results in an effect that is consistent with a causal relationship between the gene and the disease or disorder, is known as target validation. Efforts such as the Human Genome Project are yielding a vast number of complete or partial nucleotide sequences, many of which might correspond to or encode targets useful for new drug discovery efforts. The challenge represented by this plethora of information is how to use such nucleotide sequences to identify and rank valid targets for drug discovery. Antisense technology provides one means by which this might be accomplished; however, the many manual, labor-intensive and costly steps involved in traditional methods of developing active antisense compounds has limited their use in target validation (Szoka, Nature Biotechnology, 1997, 15, 509). Nevertheless, the great target specificity that is characteristic of antisense compounds makes them ideal choices for target validation, especially when the functional roles of proteins that are highly related are being investigated (Albert et al., Trends in Pharm. Sci., 1994, 15, 250).
Accordingly, the need exists for systems and methods for developing compounds efficiently and effectively that modulate a gene, wherein such compounds can be directly developed from nucleotide sequence information. Such compounds are needed to confirm that modulation of a gene that is thought to be involved in a disease or disorder will in fact cause an in vitro or in vivo effect indicative of the origin, development, spread or growth of the disease or disorder.
The present disclosure answers this need by providing systems and methods for automatically generating active oligonucleotide and other compounds, especially antisense compounds, to a target nucleotide sequence via robotic or other automated means. Such active compounds are contacted with a cell culture, cell-free extract, tissue or animal capable of expressing the gene of interest, and subsequent biochemical or biological parameters indicative of the potential gene product function are measured. These results are compared to those obtained with a control cell system, cell-free extract, tissue or animal which has not been contacted with an active antisense compound in order to determine whether or not modulation of the gene of interest affects a specific cellular function. The resulting active antisense compounds may be used as positive controls when other, non antisense-based agents directed to the same target nucleic acid, or to its gene product, are screened.
It should be noted that embodiments of the invention drawn to gene function analysis and target validation have parameters that are shared with other embodiments of the invention, but also have unique parameters. For example, antisense drug discovery naturally requires that the toxicity of the antisense compounds be manageable, whereas, for gene function analysis or target validation, overt toxicity resulting from the antisense compounds is acceptable unless it interferes with the assay being used to evaluate the effects of treatment with such compounds. U.S. Patent 5,563,036 to Peterson et al. describes systems and methods of screening for compounds that inhibit the binding of a transcription factor to a nucleic acid.
In a preferred embodiment, an assay portion of the process is stated to be performed by a computer controlled robot.
U.S. Patent 5,708,158 to Hoey describes systems and methods for identifying pharmacological agents stated to be useful for diagnosing or treating a disease associated with a gene the expression of which is modulated by a human nuclear factor of activated T cells. The methods are stated to be particularly suited to high-thoughput screening wherein one or more steps of the process are performed by a computer controlled robot. U.S. Patents 5,693,463 and 5,716,780 to Edwards et al. describe systems and methods for identifying non-oligonucleotide molecules that specifically bind to a DNA molecule based on their ability to compete with a DNA-binding protein that recognizes the
DNA molecule.
U.S. Patents 5,463,564 and 5,684,711 to Agrafiotis et al. describe computer based iterative processes for generating chemical entities with defined physical, chemical and/or bioactive properties.
SUMMARY OF THE INVENTION
The present invention is directed to automated systems and methods for defining sets of compounds that modulate the expression of target nucleic acid sequences, and generating sets of oligonucleotides that modulate the expression of target nucleic acid sequences. The present invention is also directed to identifying nucleic acid sequences amenable to antisense binding of oligonucleotides to those nucleic acid sequences by the systems and methods of the invention. For purposes of illustration, the present invention is described herein with respect to the production and identification of active antisense oligonucleotides; however, the present invention is not limited to this embodiment. The present invention is directed to iterative processes for defining chemical compounds with prescribed sets of physical, chemical and/or biological properties, and to systems for implementing these processes. During each iteration of a process as contemplated herein, a target nucleic acid sequence is provided or selected, and a library of (candidate) virtual compounds is generated in silico (that is in a computer manipulative and reliable form) according to defined criteria. A library of virtual compounds is generated. These virtual compounds are reviewed and compounds predicted to have particular desired properties are selected. The selected compounds are synthesized, preferably in a robotic, batchwise manner; and then they are robotically assayed for a desired physical, chemical or biological activity in order to identify compounds with the desired properties. Active compounds are, thus, generated and, at the same time, preferred sequences and regions of the target nucleic acid that are amenable to modulation are identified. The preferred compounds of the invention are oligonucleotides that bind to a target nucleic acid sequence.
In subsequent iterations of the process, second libraries of candidate compounds are generated and/or selected to give rise to a second virtual compound library. Through multiple iterations of the process, a library of target nucleic acid sequences that are tractable to modulation via binding of these compounds to the nucleic acid sequence are identified. Such modulation includes, but is not limited to, antisense technology, gene function analysis and target validation. The present invention is also directed to processes for validating the function of a gene or the product of the gene comprising generating in silico a library of nucleobase sequences targeted to the gene and robotically assaying a plurality of synthetic compounds having at least some of the nucleobase sequences for effects on biological function.
Further features and advantages of the present invention, as well as the structure and operation of various embodiments of the present invention, are described in detail below with reference to the accompanying drawings. In the drawings, like reference 8 numbers indicate identical or functionally similar elements.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be described with reference to the accompanying drawings, wherein: Figures 1 and 2 are a flow diagram of one method according to the present invention depicting the overall flow of data and materials among various elements of the invention.
Figure 3 is a flow diagram depicting the flow of data and materials among elements of step 200 of Figure 1. Figures 4 and 5 are a flow diagram depicting the flow of data and materials among elements of step 300 of Figure 1.
Figure 6 is a flow diagram depicting the flow of data and materials among elements of step 306 of Figure 4.
Figure 7 is another flow diagram depicting the flow of data and materials among elements of step 306 of Figure 4.
Figure 8 is a another flow diagram depicting the flow of data and materials among elements of step 306 of Figure 4.
Figure 9 is a flow diagram depicting the flow of data and materials among elements of step 350 of Figure 5. Figures 10 and 11 are flow diagrams depicting a logical analysis of data and materials among elements of step 400 of Figure 1.
Figure 12 is a flow diagram depicting the flow of data and materials among the elements of step 400 of Figure 1.
Figures 13 and 14 are flow diagrams depicting the flow of data and materials among elements of step 500 of Figure 1.
Figure 15 is a flow diagram depicting the flow of data and materials among elements of step 600 of Figure 1.
Figure 16 is a flow diagram depicting the flow of data and materials among elements of step 700 of Figure 1. Figure 17 is a flow diagram depicting the flow of data and materials among the elements of step 1100 of Figure 2.
Figure 18 is a block diagram showing the interconnecting of certain devices utilized in conjunction with a preferred method of the invention;
Figure 19 is a flow diagram showing a representation of data storage in a relational database utilized in conjunction with one method of the invention;
Figure 20 is a flow diagram depicting the flow of data and materials in effecting a preferred embodiment of the invention as set forth in Example 14;
Figure 21 is a flow diagram depicting the flow of data and materials in effecting a preferred embodiment of the invention as set forth in Example 15; Figure 22 is a flow diagram depicting the flow of data and materials in effecting a preferred embodiment of the invention as set forth in Example 2;
Figure 23 is a pictorial elevation view of a preferred apparatus used to robotically synthesize oligonucleotides; and
Figure 24 is a pictorial plan view of an apparatus used to robotically synthesize oligonucleotides.
DETAILED DESCRIPTION OF THE INVENTION
Certain preferred methods of this invention are now described with reference to the flow diagram of Figures 1 and 2.
1. Target Nucleic Acid Selection. The target selection process, process step 100, provides a target nucleotide sequence that is used to help guide subsequent steps of the process. It is generally desired to modulate the expression of the target nucleic acid for any of a variety of purposes, such as, e.g., drug discovery, target validation and/or gene function analysis.
One of the primary objectives of the target selection process, step 100, is to identify molecular targets that represent significant therapeutic opportunities, to provide new and efficacious means of drug discovery and to determine the function of genes that are uncharacterized except for nucleotide sequence. To meet these objectives, genes are classified based upon specific sets of selection criteria.
One such set of selection criteria concerns the quantity and quality of target nucleotide sequence. There must be sufficient target nucleic acid sequence information 10 available for oligonucleotide design. Moreover, such information must be of sufficient quality to give rise to an acceptable level of confidence in the data to perform the methods described herein. Thus, the data must not contain too many missing or incorrect base entries. In the case of a target sequence that encodes a polypeptide, such errors can often be detected by virtually translating all three reading frames of the sense strand of the target sequence and confirming the presence of a continuous polypeptide sequence having predictable attributes, e.g., encoding a polypeptide of known size, or encoding a polypeptide that is about the same length as a homologous protein. In any event, only a very high frequency of sequence errors will frustrate the methods of the invention; most oligonucleotides to the target sequence will avoid such errors unless such errors occur frequently throughout the entire target sequence.
Another preferred criterion is that appropriate culturable cell lines or other source of reproducible genetic expression should be available. Such cell lines express, or can be induced to express, the gene comprising the target nucleic acid sequence. The oligonucleotide compounds generated by the process of the invention are assayed using such cell lines and, if such assaying is performed robotically, the cell line is preferably tractable to robotic manipulation such as by growth in 96 well plates. Those skilled in the art will recognize that if an appropriate cell line does not exist, it will nevertheless be possible to construct an appropriate cell line. For example, a cell line can be transfected with an expression vector comprising the target gene in order to generate an appropriate cell line for assay purposes.
For gene function analysis, it is possible to operate upon a genetic system having a lack of information regarding, or incomplete characterization of, the biological function(s) of the target nucleic acid or its gene product(s). This is a powerful agent of the invention. A target nucleic acid for gene function analysis might be absolutely uncharacterized, or might be thought to have a function based on minimal data or homology to another gene. By application of the process of the invention to such a target, active compounds that modulate the expression of the gene can be developed and applied to cells. The resulting cellular, biochemical or molecular biological responses are observed, and this information is used by those skilled in the art to elucidate the function of the target gene.
For target validation and drug discovery, another selection criterion is disease 11 association. Candidate target genes are placed into one of several broad categories of known or deduced disease association. Level 1 Targets are target nucleic acids for which there is a strong correlation with disease. This correlation can come from multiple scientific disciplines including, but not limited to, epidemiology, wherein frequencies of gene abnormalities are associated with disease incidence; molecular biology, wherein gene expression and function are associated with cellular events correlated with a disease; and biochemistry, wherein the in vitro activities of a gene product are associated with disease parameters. Because there is a strong therapeutic rationale for focusing on Level 1 Targets, these targets are most preferred for drug discovery and/or target validation. Level 2 Targets are nucleic acid targets for which the combined epidemiological, molecular biological, and/or biochemical correlation with disease is not so clear as for Level 1. Level 3 Targets are targets for which there is little or no data to directly link the target with a disease process, but there is indirect evidence for such a link, i.e., homology with a Level 1 or Level 2 target nucleic acid sequence or with the gene product thereof. In order not to prejudice the target selection process, and to ensure that the maximum number of nucleic acids actually involved in the causation, potentiation, aggravation, spread, continuance or after-effects of disease states are investigated, it is preferred to examine a balanced mix of Level 1, 2 and 3 target nucleic acids.
In order to carry out drug discovery, experimental systems and reagents shall be available in order for one to evaluate the therapeutic potential of active compounds generated by the process of the invention. Such systems may be operable in vitro (e.g., in vitro models of cellxell association) or in vivo (e.g., animal models of disease states). It is also desirable, but not obligatory, to have available animal model systems which can be used to evaluate drug pharmacology. Candidate targets nucleic acids can also classified by biological processes. For example, programmed cell death ("apoptosis") has recently emerged as an important biological process that is perturbed in a wide variety of diseases. Accordingly, nucleic acids that encode factors that play a role in the apoptotic process are identified as candidate targets. Similarly, potential target nucleic acids can be classified as being involved in inflammation, autoimmune disorders, cancer, or other pathological or dysfunctional processes. 12
Moreover, genes can often be grouped into families based on sequence homology and biological function. Individual family members can act redundantly, or can provide specificity through diversity of interactions with downstream effectors, or through expression being restricted to specific cell types. When one member of a gene family is associated with a disease process then the rationale for targeting other members of the same family is reasonably strong. Therefore, members of such gene families are preferred target nucleic acids to which the methods and systems of the invention may be applied. Indeed, the potent specificity of antisense compounds for different gene family members makes the invention particularly suited for such targets (Albert et al., Trends Pharm. Sci., 1994, 15, 250). Those skilled in the art will recognize that a partial or complete nucleotide sequence of such family members can be obtained using the polymerase chain reaction (PCR) and "universal" primers, i.e., primers designed to be common to all members of a given gene family.
PCR products generated from universal primers can be cloned and sequenced or directly sequenced using techniques known in the art. Thus, although nucleotide sequences from cloned DNAs, or from complementary DNAs (cDNAs) derived from mRNAs, may be used in the process of the invention, there is no requirement that the target nucleotide sequence be isolated from a cloned nucleic acid. Any nucleotide sequence, no matter how determined, of any nucleic acid, isolated or prepared in any fashion, may be used as a target nucleic acid in the process of the invention.
Furthermore, although polypeptide-encoding nucleic acids provide the target nucleotide sequences in one embodiment of the invention, other nucleic acids may be targeted as well. Thus, for example, the nucleotide sequences of structural or enzymatic RNAs may be utilized for drug discovery and/or target validation when such RNAs are associated with a disease state, or for gene function analysis when their biological role is not known.
2. Assembly of Target Nucleotide Sequence. Figure 3 is a block diagram detailing the steps of the target nucleotide sequence assembly process, process step 200 in acccordance with one embodiment of the invention. The oligonucleotide design process, process step 300, is facilitated by the availability of accurate target sequence information.
Because of limitations of automated genome sequencing technology, gene sequences are 13 often accumulated in fragments. Further, because individual genes are often being sequenced by independent laboratories using different sequencing strategies, sequence information corresponding to different fragments is often deposited in different databases. The target nucleic acid assembly process take advantage of computerized homology search algorithms and sequence fragment assembly algorithms to search available databases for related sequence information and incorporate available sequence information into the best possible representation of the target nucleic acid molecule, for example a RNA transcript. This representation is then used to design oligonucleotides, process step 300, which can be tested for biological activity in process step 700. In the case of genes directing the synthesis of multiple transcripts, i.e. by alternative splicing, each distinct transcript is a unique target nucleic acid for purposes of step 300. In one embodiment of the invention, if active compounds specific for a given transcript isoform are desired, the target nucleotide sequence is limited to those sequences that are unique to that transcript isoform. In another embodiment of the invention, if it is desired to modulate two or more transcript isoforms in concert, the target nucleotide sequence is limited to sequences that are shared between the two or more transcripts.
In the case of a polypeptide-encoding nucleic acid, it is generally preferred that full-length cDNA be used in the oligonucleotide design process step 300 (with full-length cDNA being defined as reading from the 5' cap to the poly A tail). Although full-length cDNA is preferred, it is possible to design oligonucleotides using partial sequence information. Therefore it is not necessary for the assembly process to generate a complete cDNA sequence. Further in some cases it may be desirable to design oligonucleotides targeting introns. In this case the process can be used to identify individual introns at process step 220. The process can be initiated by entering initial sequence information on a selected molecular target at process step 205. In the case of a polypeptide-encoding nucleic acid, the full-length cDNA sequence is generally preferred for use in oligonucleotide design strategies at process step 300. The first step is to determine if the initial sequence information represents the full-length cDNA, decision step 210. In the case where the full- length cDNA sequence is available the process advances directly to the oligonucleotide design step 300. When the full-length cDNA sequence is not available, databases are 14 searched at process step 212 for additional sequence information.
The algorithm preferably used in process steps 212 and 230 is BLAST (Altschul, et al., J. Mol. Biol, 1990, 215, 403), or "Gapped BLAST" (Altschul et al, Nucl Acids Res., 1997, 25, 3389). These are database search tools based on sequence homology used to identify related sequences in a sequence database. The BLAST search parameters are set to only identify closely related sequences. Some preferred databases searched by BLAST are a combination of public domain and proprietary databases. The databases, their contents, and sources are listed in Table 1.
Table 1: Database Sources of Target Sequences
Database Contents Source
NR All non-redundant GenBank, National Center for Biotechnology
EMBL, DDBJ and PDB Information at the National Institutes sequences of Health
Month All new or revised GenBank, National Center for Biotechnology
EMBL, DDBJ and PDB Information at the National Institutes sequences released in the last of Health
30 days
Dbest Non-redundant database of National Center for Biotechnology
GenBank, EMBL, DDBJ and Information at the National Institutes
EST divisions of Health
Dbsts Non-redundant database of National Center for Biotechnology
GenBank, EMBL, DDBJ and Information at the National Institutes
STS divisions of Health
Htgs High throughput genomic National Center for Biotechnology sequences Information at the National Institutes of Health
When genomic sequence information is available at decision step 215, introns are removed and exons are assembled into continuous sequence representing the cDNA sequence in process step 220. Exon assembly occurs using the Phragment Assembly Program "Phrap" (Copyright University of Washington Genome Center, Seattle, WA). The Phrap algorithm analyzes sets of overlapping sequences and assembles them into one 15 continuous sequence referred to as a "contig." The resulting contig is preferably used to search databases for additional sequence information at process step 230. When genomic information is not available, the results of process step 212 are analyzed for individual exons at decision step 225. Exons are frequently recorded individually in databases. If multiple complete exons are identified, they are prferably assembled into a contig using
Phrap at process step 250. If multiple complete exons are not identified at decision step 225, then sequences can be analyzed for partial sequence information in decision step 228. ESTs identified in the database dbEST are examples of such partial sequence information. If additional partial information is not found, then the process is advanced to process step 230 at decision step 228. If partial sequence information is found in process 212 then that information is advanced to process step 230 via decision step 228.
Process step 230, decision step 240, decision step 260 and process step 250 define a loop designed to extend iteratively the amount of sequence information available for targeting. At the end of each iteration of this loop, the results are analyzed in decision steps 240 and 260. If no new information is found then the process advances at decision step 240 to process step 300. If there is an unexpectedly large amount of sequence information identified, suggesting that the process moved outside the boundary of the gene into repetitive genomic sequence, then the process is preferably cycled back one iteration and that sequence is advanced at decision step 240 to process step 300. If a small amount of new sequence information is identified, then the loop is iterated such as by taking the
100 most 5-prime (5') and 100 most 3-prime (3') bases and interating them through the BLAST homology search at process step 230. New sequence information is added to the existing contig at process step 250.
3. In Silico Generation of a Set of Nucleobase Sequences and Virtual Oligonucleotides.
For the following steps 300 and 400, they may be performed in the order described below, i.e., step 300 before step 400, or, in an alternative embodiment of the invention, step 400 before step 300. In this alternate embodiment, each oligonucleotide chemistry is first assigned to each oligonucleotide sequence. Then, each combination of oligonucleotide chemistry and sequence is evaluated according to the parameters of step
300. This embodiment has the desirable feature of taking into account the effect of 16 alternative oligonucleotide chemistries on such parameters. For example, substitution of 5-methyl cytosine (5MeC or m5c) for cytosine in an antisense compound may enhance the stability of a duplex formed between that compound and its target nucleic acid. Other oligonucleotide chemistries that enhance oligonucleotide: [target nucleic acid] duplexes are known in the art (see for example, Freier et al, Nucleic Acids Research, 1997, 25, 4429).
As will be appreciated by those skilled in the art, different oligonucleotide chemistries may be preferred for different target nucleic acids. That is, the optimal oligonucleotide chemistry for binding to a target DNA might be suboptimal for binding to a target RNA having the same nucleotide sequence. In effecting the process of the invention in the order step 300 before step 400 as seen in Figure 1, from a target nucleic acid sequence assembled at step 200, a list of oligonucleotide sequences is generated as represented in the flowchart shown in Figures 4 and 5. In step 302, the desired oligonucleotide length is chosen. In a preferred embodiment, oligonucleotide length is between from about 8 to about 30, more preferably from about 12 to about 25, nucleotides. In step 304, all possible oligonucleotide sequences of the desired length capable of hybridizing to the target sequence obtained in step 200 are generated. In this step, a series of oligonucleotide sequences are generated, simply by determining the most 5' oligonucleotide possible and "walking" the target sequence in increments of one base until the 3' most oligonucleotide possible is reached. In step 305, a virtual oligonucleotide chemistry is applied to the nucleobase sequences of step 304 in order to yield a set of virtual oligonucleotides that can be evaluated in silico. Default virtual oligonucleotide chemistries include those that are well- characterized in terms of their physical and chemical properties, e.g., 2'-deoxyribonucleic acid having naturally occurring bases (A, T, C and G), unmodified sugar residues and a phosphodiester backbone.
4. In Silico Evaluation of Thermodynamic Properties of Virtual Oligonucleotides.
In step 306, a series of thermodynamic, sequence, and homology scores are preferably calculated for each virtual oligonucleotide obtained from step 305. Thermodynamic properties are calculated as represented in Figure 6. In step 308, the desired thermodynamic properties are selected. As many or as few as desired can be 17 selected; optionally, none will be selected. The desired properties will typically include step 309, calculation of the free energy of the target structure. If the oligonucleotide is a DNA molecule, then steps 310, 312, and 314 are performed. If the oligonucleotide is an RNA molecule, then steps 311, 313 and 315 are performed. In both cases, these steps correspond to calculation of the free energy of intramolecular oligonucleotide interactions, intermolecular interactions and duplex formation. In addition, a free energy of oligonucleoti de-target binding is preferably calculated at step 316.
Other thermodynamic and kinetic properties may be calculated for oligonucleotides as represented at step 317. Such other thermodynamic and kinetic properties may include melting temperatures, association rates, dissociation rates, or any other physical property that may be predictive of oligonucleotide activity.
The free energy of the target structure is defined as the free energy needed to disrupt any secondary structure in the target binding site of the targeted nucleic acid. This region includes any intra-target nucleotide base pairs that need to be disrupted in order for an oligonucleotide to bind to its complementary sequence. The effect of this localized disruption of secondary structure is to provide accessibility by the oligonucleotide. Such structures will include double helices, terminal unpaired and mismatched nucleotides, loops, including hairpin loops, bulge loops, internal loops and multibranch loops (Serra et al, Methods in Enzymology, 1995, 259, 242). The intermolecular free energies refer to inherent energy due to the most stable structure formed by two oligonucleotides; such structures include dimer formation. Intermolecular free energies should also be taken into account when, for example, two or more oligonucleotides, of different sequence are to be administered to the same cell in an assay. The intramolecular free energies refer to the energy needed to disrupt the most stable secondary structure within a single oligonucleotide. Such structures include, for example, hairpin loops, bulges and internal loops. The degree of intramolecular base pairing is indicative of the energy needed to disrupt such base pairing.
The free energy of duplex formation is the free energy of denatured oligonucleotide binding to its denatured target sequence. The oligonucleotide-target binding is the total binding involved, and includes the energies involved in opening up 18 intra- and inter- molecular oligonucleotide structures, opening up target structure, and duplex formation.
The most stable RNA structure is predicted based on nearest neighbor analysis (Xia, T., et al, Biochemistry, 1998, 37, 14719-14735; Serra et al, Methods in Enzymology, 1995, 259, 242). This analysis is based on the assumption that stability of a given base pair is determined by the adjacent base pairs. For each possible nearest neighbor combination, thermodynamic properties have been determined and are provided. For double helical regions, two additional factors need to be considered, an entropy change required to initiate a helix and a entropy change associated with self-complementary strands only. Thus, the free energy of a duplex can be calculated using the equation:
AG°τ = ΔH° - TAS° where:
AG is the free energy of duplex formation, AH is the enthalpy change for each nearest neighbor, AS is the entropy change for each nearest neighbor, and T is temperature. The AH and AS for each possible nearest neighbor combination have been experimentally determined. These letter values are often available in published tables. For terminal unpaired and mismatched nucleotides, enthalpy and entropy measurements for each possible nucleotide combination are also available in published tables. Such results are added directly to values determined for duplex formation. For loops, while the available data is not as complete or accurate as for base pairing, one known model determines the free energy of loop formation as the sum of free energy based on loop size, the closing base pair, the interactions between the first mismatch of the loop with the closing base pair, and additional factors including being closed by AU or UA or a first mismatch of GA or UU. Such equations may also be used for oligoribonucleotide-target RNA interactions.
The stability of DNA duplexes is used in the case of intra- or intermolecular oligodeoxyribonucleotide interactions. DNA duplex stability is calculated using similar equations as RNA stability, except experimentally determined values differ between nearest neighbors in DNA and RNA and helix initiation tends to be more favorable in 19
DNA than in RNA (SantaLucia et al, Biochemistry, 1996, 35, 3555).
Additional thermodynamic parameters are used in the case of RNA/DNA hybrid duplexes. This would be the case for an RNA target and oligodeoxynucleotide. Such parameters were determined by Sugimoto et al. {Biochemistry, 1995, 34, 11211). In addition to values for nearest neighbors, differences were seen for values for enthalpy of helix initiation.
5. In Silico Evaluation of Target Accessibility
Target accessibility is believed to be an important consideration in selecting oligonucleotides. Such a target site will possess minimal secondary structure and thus, will require minimal energy to disrupt such structure. In addition, secondary structure in oligonucleotides, whether inter- or intra-molecular, is undesirable due to the energy required to disrupt such structures. Oligonucleotide-target binding is dependent on both these factors. It is desirable to minimize the contributions of secondary structure based on these factors. The other contribution to oligonucleotide-target binding is binding affinity. Favorable binding affinities based on tighter base pairing at the target site is desirable.
Following the calculation of thermodynamic properties ending at step 317, the desired sequence properties to be scored are selected at step 324. As many or as few as desired can be selected; optionally, none will be selected. These properties include the number of strings of four guanosine residues in a row at step 325 or three guanosine in a row at step 326, the length of the longest string of adenosines at step 327, cytidines at step
328 or uri dines or thymidines at step 329, the length of the longest string of purines at step 330 or pyrimidine at step 331, the percent composition of adenosine at step 332, cytidine at step 333, guanosine at step 334 or uridines or thymidines at step 335, the percent composition of purines at step 336 or pyrimidines at step 337, the number of CG dinucleotide repeats at step 338, CA dinucleotide repeats at step 339 or UA or TA dinucleotide repeats at step 340. In addition, other sequence properties may be used as found to be relevant and predictive of antisense efficacy, as represented at step 341.
These sequence properties may be important in predicting oligonucleotide activity, or lack thereof. For example, U.S. Patent 5,523,389 discloses oligonucleotides containing stretches of three or four guanosine residues in a row. Oligonucleotides having such sequences may act in a sequence-independent manner. For an antisense approach, such a 20 mechanism is not usually desired. In addition, high numbers of dinucleotide repeats may be indicative of low complexity regions which may be present in large numbers of unrelated genes. Unequal base composition, for example, 90% adenosine, can also give non-specific effects. From a practical standpoint, it may be desirable to remove oligonucleotides that possess long stretches of other nucleotides due to synthesis considerations. Other sequences properties, either listed above or later found to be of predictive value may be used to select oligonucleotide sequences.
Following step 341, the homology scores to be calculated are selected in step 342. Homology to nucleic acids encoding protein isoforms of the target, as represented at step 343, may be desired. For example, oligonucleotides specific for an isoform of protein kinase C can be selected. Also, oligonucleotides can be selected to target multiple isoforms of such genes. Homology to analogous target sequences, as represented at step 344, may also be desired. For example, an oligonucleotide can be selected to a region common to both humans and mice to facilitate testing of the oligonucleotide in both species. Homology to splice variants of the target nucleic acid, as represented at step 345, may be desired. In addition, it may be desirable to determine homology to other sequence variants as necessary, as represented in step 346.
Following step 346, from which scores were obtained in each selected parameter, a desired range is selected to select the most promising oligonucleotides, as represented at step 347. Typically, only several parameters will be used to select oligonucleotide sequences. As structure prediction improves, additional parameters may be used. Once the desired score ranges are chosen, a list of all oligonucleotides having parameters falling within those ranges will be generated, as represented at step 348.
6. Targeting Oligonucleotides to Functional Regions of a Nucleic Acid. It may be desirable to target oligonucleotide sequences to specific functional regions of the target nucleic acid. A decision is made whether to target such regions, as represented in decision step 349. If it is desired to target functional regions then process step 350 occurs as seen in greater detail in Figure 9. If it is not desired then the process proceeds to step 375. In step 350, as seen in Figure 9, the desired functional regions are selected. Such regions include the transcription start site or 5' cap at step 353, the 5' untranslated region 21 at step 354, the start codon at step 355, the coding region at step 356, the stop codon at step 357, the 3' untranslated region at step 358, 5' splice sites at step 359 or 3' splice sites at step 360, specific exons at step 361 or specific introns at step 362, mRNA stabilization signal at step 363, mRNA destabihzation signal at step 364, poly-adenylation signal at step 365, poly-A addition site at step 366, poly-A tail at step 367, or the gene sequence 5' of known pre-mRNA at step 368. In addition, additional functional sites may be selected, as represented at step 369.
Many functional regions are important to the proper processing of the gene and are attractive targets for antisense approaches. For example, the AUG start codon is commonly targeted because it is necessary to initiate translation. In addition, splice sites are thought to be attractive targets because these regions are important for processing of the mRNA. Other known sites may be more accessible because of interactions with protein factors or other regulatory molecules.
After the desired functional regions are selected and determined, then a subset of all previously selected oligonucleotides are selected based on hybridization to only those desired functional regions, as represented by step 370. 7. Uniform Distribution of Oligonucleotides. Whether or not targeting functional sites is desired, a large number of oligonucleotide sequences may result from the process thus far. In order to reduce the number of oligonucleotide sequences to a manageable number, a decision is made whether to uniformly distribute selected oligonucleotides along the target, as represented in step 375. A uniform distribution of oligonucleotide sequences will aim to provide complete coverage throughout the complete target nucleic acid or the selected functional regions. A computer-based program is used to automate the distribution of sequences, as represented in step 380. Such a program factors in parameters such as length of the target nucleic acid, total number of oligonucleotide sequences desired, oligonucleotide sequences per unit length, number of oligonucleotide sequences per functional region. Manual selection of oligonucleotide sequences is also provided for by step 385. In some cases, it may be desirable to manually select oligonucleotide sequences. For example, it may be useful to determine the effect of small base shifts on activity. Once the desired number of oligonucleotide sequences is obtained either from step 380 or step 385, then these 22 oligonucleotide sequences are passed onto step 400 of the process, where oligonucleotide chemistries are assigned.
8. Assignment of Actual Oligonucleotide Chemistry.
Once a set of select nucleobase sequences has been generated according to the preceding process and decision steps, actual oligonucleotide chemistry is assigned to the sequences. An "actual oligonucleotide chemistry" or simply "chemistry" is a chemical motif that is common to a particular set of robotically synthesized oligonucleotide compounds. Preferred chemistries include, but are not limited to, oligonucleotides in which every linkage is a phosphorothioate linkage, and chimeric oligonucleotides in which a defined number of 5' and/or 3' terminal residues have a 2'-methoxyethoxy modification.
Chemistries can be assigned to the nucleobase sequences during general procedure step 400 (Figure 1). The logical basis for chemistry assignment is illustrated in Figures 10 and 11 and an iterative routine for stepping through an oligonucleotide nucleoside by nucleoside is illustrated in Figure 12. Chemistry assignment can be effected by assignment directly into a word processing program, via an interactive word processing program or via automated programs and devices. In each of these instances, the output file is selected to be in a format that can serve as an input file to automated synthesis devices.
9. Oligonucleotide Compounds.
In the context of this invention, in reference to oligonucleotides, the term "oligonucleotide" is used to refer to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. Thus this term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally- occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms, i.e., phosphodiester linked A, C, G,
T and U nucleosides, because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.
The oligonucleotide compounds in accordance with this invention can be of various lengths depending on various parameters, including but not limited to those discussed above in reference to the selection criteria of general procedure 300. For use as 23 antisense oligonucleotides compounds of the invention preferably are from about 8 to about 30 nucleobases in length (i.e. from about 8 to about 30 linked nucleosides). Particularly preferred are antisense oligonucleotides comprising from about 12 to about 25 nucleobases. A discussion of antisense oligonucleotides and some desirable modifications can be found in De Mesmaeker et al, Ace. Chem. Res., 1995, 28, 366. Other lengths of oligonucleotides might be selected for non-antisense targeting strategies, for instance using the oligonucleotides as ribozymes. Such ribozymes normally require oligonucleotides of longer length as is known in the art.
A nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a normal (where normal is defined as being found in RNA and DNA) pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
Specific examples of preferred oligonucleotides useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. 10. Selection of Oligonucleotide Chemistries. In a general logic scheme as illustrated in Figures 10 and 11 , for each nucleoside position, the user or automated device is interrogated first for a base assignment, followed 24 by a sugar assignment, a linker assignment and finally a conjugate assignment. Thus for each nucleoside, at process step 410 a base is selected. In selecting the base, base chemistry 1 can be selected at process step 412 or one or more alternative bases are selected at process steps 414, 416 and 418. After base selection is effected, the sugar portion of the nucleoside is selected. Thus for each nucleoside, at process step 420 a sugar is selected that together with the select base will complete the nucleoside. In selecting the sugar, sugar chemistry 1 can be selected at process 422 or one or more alternative sugars are selected at process steps 424, 426 and 428. For each two adjacent nucleoside units, at process step 430, the internucleoside linker is selected. The linker chemistry for the internucleoside linker can be linker chemistry 1 selected at process step 432 or one or more alternative internucleoside linker chemistries are selected at process steps 434, 436 and 438.
In addition to the base, sugar and internucleoside linkage, at each nucleoside position, one or more conjugate groups can be attached to the oligonucleotide via attachment to the nucleoside or attachment to the internucleoside linkage. The addition of a conjugate group is integrated at process step 440 and the assignment of the conjugate group is effected at process step 450.
For illustrative purposes in Figures 10 and 11, for each of the bases, the sugars, the internucleoside linkers, or the conjugates, chemistries 1 though n are illustrated. As described in this specification, it is understood that the number of alternate chemistries between chemistry 1 and alternative chemistry n, for each of the bases, the sugars, the internucleoside linkages and the conjugates, is variable and includes, but is not limited to, each of the specific alternative bases, sugar, internucleoside linkers and conjugates identified in this specification as well as equivalents known in the art. Utilizing the logic as described in conjunction with Figures 10 and 11, chemistry is assigned, as is shown in Figure 12, to the list of oligonucleotides from general procedure 300. In assigning chemistries to the oligonucleotides in this list, a pointer can be set at process step 452 to the first oligonucleotide in the list and at step 453 to the first nucleotide of that first oligonucleotide. The base chemistry is selected at step 410, as described above, the sugar chemistry is selected at step 420, also as described above, followed by selection of the internucleoside linkage at step 430, also as described above. 25
At decision 440, the process branches depending on whether a conjugate will be added at the current nucleotide position. If a conjugate is desired, the conjugate is selected at step 450, also as described above.
Whether or not a conjugate was added at decision step 440, an inquiry is made at decision step 454. This inquiry asks if the pointer resides at the last nucleotide in the current oligonucleotide. If the result at decision step 454 is "No," the pointer is moved to the next nucleotide in the current oligonucleotide and the loop including steps 410, 420, 430, 440 and 454 is repeated. This loop is reiterated until the result at decision step 454 is "Yes." When the result at decision step 454 is "Yes," a query is made at decision step 460 concerning the location of the pointer in the list of oligonucleotides. If the pointer is not at the last oligonucleotide of the list, the "No" path of the decision step 460 is followed and the pointer is moved to the first nucleotide of the next oligonucleotide in the list at process step 458. With the pointer set to the next oligonucleotide in the list, the loop that starts at process steps 453 is reiterated. When the result at decision step 460 is "Yes," chemistry has been assigned to all of the nucleotides in the list of oligonucleotides. 11. Description of Oligonucleotide Chemistries.
As is illustrated in Figure 10, for each nucleoside of an oligonucleotide, chemistry selection includes selection of the base forming the nucleoside from a large palette of different base units available. These may be "modified" or "natural" bases (also reference herein as nucleobases) including the natural purine bases adenine (A) and guanine (G), and the natural pyrimidine bases thymine (T), cytosine (C) and uracil (U). They further can include modified nucleobases including other synthetic and natural nucleobases such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo uracils and cytosines particularly 5-bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7- methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 26
3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Rroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and
Applications, pages 289-302, Crooke, S.T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5 -substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyl- adenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred for selection as the base. These are particularly useful when combined with a 2'-O-methoxyethyl sugar modifications, described below.
Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Patent 3,687,808, as well as U.S. Patents 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and
5,681,941, each of which is incorporated herein by reference in its entirety. Reference is also made to allowed United States patent application 08/762,488, filed on December 10, 1996, commonly owned with the present application and which is incorporated herein by reference in its entirety. In selecting the base for any particular nucleoside of an oligonucleotide, consideration is first given to the need of a base for a particular specificity for hybridization to an opposing strand of a particular target. Thus if an "A" base is required, adenine might be selected however other alternative bases that can effect hybridization in a manner mimicking an "A" base such as 2-aminoadenine might be selected should other consideration, e.g., stronger hybridization (relative to hybridization achieved with adenine), be desired. 27
As is illustrated in Figure 10, for each nucleoside of an oligonucleotide, chemistry selection includes selection of the sugar forming the nucleoside from a large palette of different sugar or sugar surrogate units available. These may be modified sugar groups, for instance sugars containing one or more substituent groups. Preferred substituent groups comprise the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N- alkenyl; or O, S- or N-alkynyl; wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, 0(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2; where n and m are from 1 to about 10. Other preferred substituent groups comprise one of the following at the 2' position: Cj to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF SOCH SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'- O-(2-methoxyethyl), 2'-O-methoxyethyl, or 2'-MOE) (Martin et al, Helv. Chim. Ada, 1995, 78, 486) i.e., an alkoxyalkoxy group. A further preferred modification includes 2'- dimethylamino oxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in co-owned United States patent application Serial Number 09/016,520, filed on January 30, 1998, which is incoφorated herein by reference in its entirety.
Other preferred modifications include 2'-methoxy (2'-O-CH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the sugar group, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. The nucleosides of the oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
Representative United States patents that teach the preparation of such modified sugars structures include, but are not limited to, U.S. Patents 4,981,957; 5,118,800;
5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 28
5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the present application, each of which is incoφorated herein by reference in its entirety, together with allowed United States patent application 08/468,037, filed on June 5, 1995, which is commonly owned with the present application and which is incoφorated herein by reference in its entirety.
As is illustrated in Figure 10, for each adjacent pair of nucleosides of an oligonucleotide, chemistry selection includes selection of the internucleoside linkage. These internucleoside linkages are also referred to as linkers, backbones or oligonucleotide backbones. For forming these nucleoside linkages, a palette of different internucleoside linkages or backbones is available. These include modified oligonucleotide backbones, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalklyphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 '-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. Representative United States patents that teach the preparation of the above phosphorus containing linkages include, but are not limited to, U.S. Patents 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; and 5,697,248, certain of which are commonly owned with this application, each of which is incoφorated herein by reference in its entirety.
Preferred internucleoside linkages for oligonucleotides that do not include a phosphorus atom therein, i.e., for oligonucleosides, have backbones that are formed by short chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatomic or heterocyclic intersugar linkages. These include those having moφholino linkages (formed in part from 29 the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patents 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, certain of which are commonly owned with this application, each of which is incoφorated herein by reference in its entirety.
In other preferred oligonucleotides, i.e., oligonucleotide mimetics, both the sugar and the intersugar linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-phosphate backbone of an oligonucleotide is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S.: 5,539,082; 5,714,331; and 5,719,262, each of which is incoφorated herein by reference in its entirety. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497.
For the internucleoside linkages, the most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- [known as a methylene (methylimino) or MMI backbone], -CH2-O-N(CH3)-CH2-,- CH2-N(CH3)- N(CH3)-CH2- and -O-N(CH3)-CH2-CH2- (wherein the native phosphodiester backbone is represented as -O-P-O-CH2-) of the above referenced U.S. patent 5,489,677, and the amide 30 backbones of the above referenced U.S. patent 5,602,240. Also preferred are oligonucleotides having moφholino backbone structures of the above-referenced U.S. Patent 5,034,506.
In attaching a conjugate group to one or more nucleosides or internucleoside linkages of an oligonucleotide, various properties of the oligonucleotide are modified.
Thus modification of the oligonucleotides of the invention to chemically link one or more moieties or conjugates to the oligonucleotide are intended to enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al, Bioorg. Med. Chem. Let., 1994,
4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al, Ann. N Y. Acad. Sci., 1992, 660, 306; Manoharan et al, Bioorg. Med. Chem. Let., 1993, 3, 2765), a thiocholesterol (Oberhauser et al, Nucl. Acids Res., 1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al, EMBO J, 1991, 10, 111; Kabanov et al, FEBS Lett., 1990, 259, 327; Svinarchuk et al, Biochimie, 1993, 75, 49), a phospholipid, e.g., di-hexadecyl-rαe-glycerol or triethylammonium 1 ,2-di-O-hexadecyl- rac-glycero-3-H-phosphonate (Manoharan et al, Tetrahedron Lett., 1995, 36, 3651; Shea et al, Nucl. Acids Res., 1990, 18, 3111), a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety (Mishra et al,
Biochim. Biophys. Ada, 1995, 1264, 229), or an octadecylamine or hexylamino-carbonyl- oxycholesterol moiety (Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277, 923).
Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Patents 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731:
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785
5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 31
5,599,928 and 5,688,941, certain of which are commonly owned with the present application, and each of which is herein incoφorated by reference in its entirety. 12. Chimeric Compounds.
It is not necessary for all positions in a given compound to be uniformly modified. In fact, more than one of the aforementioned modifications may be incoφorated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes compounds which are chimeric compounds. "Chimeric" compounds or "chimeras," in the context of this invention, are compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
Chimeric antisense compounds of the invention may be formed as composite structures representing the union of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as "hybrids" or "gapmers". Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Patents 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and
5,700,922, certain of which are commonly owned with the present application and each of 32 which is incoφorated herein by reference in its entirety, together with commonly owned and allowed United States patent application serial number 08/465,880, filed on June 6, 1995, which is incoφorated herein by reference in its entirety.
13. Description of Automated Oligonucleotide Synthesis. In the next step of the overall process (illustrated in Figures 1 and 2), oligonucleotides are synthesized on an automated synthesizer. Although many devices may be employed, the synthesizer is preferably a variation of the synthesizer described in United States patents 5,472,672 and 5,529,756, each of which is incoφorated herein by reference in its entirety. The synthesizer described in those patents is modified to include movement in along the Y axis in addition to movement along the X axis. As so modified, a 96-well array of compounds can be synthesized by the synthesizer. The synthesizer further includes temperature control and the ability to maintain an inert atmosphere during all phases of synthesis. The reagent array delivery format employs orthogonal X-axis motion of a matrix of reaction vessels and Y-axis motion of an array of reagents. Each reagent has its own dedicated plumbing system to eliminate the possibility of cross- contamination of reagents and line flushing and/or pipette washing. This in combined with a high delivery speed obtained with a reagent mapping system allows for the extremely rapid delivery of reagents. This further allows long and complex reaction sequences to be performed in an efficient and facile manner. The software that operates the synthesizer allows the straightforward programming of the parallel synthesis of a large number of compounds. The software utilizes a general synthetic procedure in the form of a command (.cmd) file, which calls upon certain reagents to be added to certain wells via lookup in a sequence (.seq) file. The bottle position, flow rate, and concentration of each reagent is stored in a lookup table (.tab) file. Thus, once any synthetic method has been outlined, a plate of compounds is made by permutating a set of reagents, and writing the resulting output to a text file. The text file is input directly into the synthesizer and used for the synthesis of the plate of compounds. The synthesizer is interfaced with a relational database allowing data output related to the synthesized compounds to be registered in a highly efficient manner. Building of the .seq, .cmd and .tab files is illustrated in Figure 13. Thus as a part of the general oligonucleotide synthesis procedure 500, for each linker chemistry at 33 process step 502, a synthesis file, i.e., a .cmd file, is built at process step 504. This file can be built fresh to reflect a completely new set of machine commands reflecting a set of chemical synthesis steps or it can modify an existing file stored at process step 504 by editing that stored file in process step 508. The .cmd files are built using a word processor and a command set of instructions as outlined below.
It will be appreciated that the preparation of control software and data files is within the routine skill of persons skilled in annotated nucleotide synthesis. The same will depend upon the hardware employed, the chemistries adopted and the design paradigm selected by the operator. In a like manner to the building the .cmd files, .tab files are built to reflect the necessary reagents used in the automatic synthesizer for the particular chemistries that have been selected for the linkages, bases, sugars and conjugate chemistries. Thus for each of a set of these chemistries at process step 510, a .tab file is built at process step 512 and stored at process step 514. As with the .cmd files, an existing .tab file can be edited at process step 516.
Both the .cmd files and the .tab files are linked together at process step 518 and stored for later retrieval in an appropriate sample database 520. Linking can be as simple as using like file names to associate a .cmd file to its appropriate .tab file, e.g., synthesis 1.cmd is linked to synthesis_l .tab by use of the same preamble in their names. The automated, multi-well parallel array synthesizer employs a reagent array delivery format, in which each reagent utilized has a dedicated plumbing system. As seen in Figures 23 and 24, an inert atmosphere 522 is maintained during all phases of a synthesis. Temperature is controlled via a thermal transfer plate 524, which holds an injection molded reaction block 526. The reaction plate assembly slides in the X-axis direction, while for example eight nozzle blocks (528, 530, 532, 534, 536, 538, 540 and
542) holding the reagent lines slide in the Y-axis direction, allowing for the extremely rapid delivery of any of 64 reagents to 96 wells. In addition, there are for example, six banks of fixed nozzle blocks (544, 546, 548, 550, 552 and 554) which deliver the same reagent or solvent to eight wells at once, for a total of 72 possible reagents. In synthesizing oligonucleotides for screening, the target reaction vessels, a 96 well plate 556 (a 2-dimensional array), moves in one direction along the X axis, while the 34 series of independently controlled reagent delivery nozzles (528, 530, 532, 534, 536, 538, 540 and 542) move along the Y-axis relative to the reaction vessel 558. As the reaction plate 556 and reagent nozzles (528, 530, 532, 534, 536, 538, 540 and 542) can be moved independently at the same time, this arrangement facilitates the extremely rapid delivery of up to 72 reagents independently to each of the 96 reaction vessel wells.
The system software allows the straightforward programming of the synthesis of a large number of compounds by supplying the general synthetic procedure in the form of the command file to call upon certain reagents to be added to specific wells via lookup in the sequence file with the bottle position, flow rate, and concentration of each reagent being stored in the separate reagent table file. Compounds can be synthesized on various scales. For oligonucleotides, a 200 nmole scale is typically selected while for other compounds larger scales, as for example a 10 μmole scale (3-5 mg), might be utilized. The resulting crude compounds are generally >80% pure, and are utilized directly for high throughput screening assays. Alternatively, prior to use the plates can be subjected to quality control (see general procedure 600 and Example 9) to ascertain their exact purity.
Use of the synthesizer results in a very efficient means for the parallel synthesis of compounds for screening.
The software inputs accept tab delimited text files (as discussed above for file 504 and 512) from any text editor. A typical command file, a .cmd file, is shown in Example 3 at Table 2. Typical sequence files, .seq files, are shown in Example 3 at Tables 3 and 4
(.SEQ file), and a typical reagent file, a .tab file, is shown in Example 3 at Table 5. Table 3 illustrates the sequence file for an oligonucleotide having 2'-deoxy nucleotides at each position with a phosphorothioate backbone throughout. Table 4 illustrates the sequence file for an oligonucleotide, again having a phosphorothioate backbone throughout, however, certain modified nucleoside are utilized in portions of the oligonucleotide. As shown in this table, 2'-O-(2-methoxyethyl) modified nucleosides are utilized in a first region (a wing) of the oligonucleotide, followed by a second region (a gap) of 2'-deoxy nucleotides and finally a third region (a further wing) that has the same chemistry as the first region. Typically some of the wells of the 96 well plate 556 may be left empty (depending on the number of oligonucleotides to be made during an individual synthesis) or some of the wells may have oligonucleotides that will serve as standards for comparison 35 or analytical puφoses.
Prior to loading reagents, moisture sensitive reagent lines are purged with argon at 522 for 20 minutes. Reagents are dissolved to appropriate concentrations and installed on the synthesizer. Large bottles, collectively identified as 558 in Figure 23 (containing 8 delivery lines) are used for wash solvents and the delivery of general activators, trityl group cleaving reagents and other reagents that may be used in multiple wells during any particular synthesis. Small septa bottles, collectively identified as 560 in Figure 23, are utilized to contain individual nucleotide amidite precursor compounds. This allows for anhydrous preparation and efficient installation of multiple reagents by using needles to pressurize the bottle, and as a delivery path. After all reagents are installed, the lines are primed with reagent, flow rates measured, then entered into the reagent table (.tab file). A dry resin loaded plate is removed from vacuum and installed in the machine for the synthesis.
The modified 96 well polypropylene plate 556 is utilized as the reaction vessel. The working volume in each well is approximately 700 μl. The bottom of each well is provided with a pressed- fit 20 μm polypropylene frit and a long capillary exit into a lower collection chamber as is illustrated in Figure 5 of the above referenced United States Patent 5,372,672. The solid support for use in holding the growing oligonucleotide during synthesis is loaded into the wells of the synthesis plate 556 by pipetting the desired volume of a balanced density slurry of the support suspended in an appropriate solvent, typically an acetonitrile-methylene chloride mixture. Reactions can be run on various scales as for instance the above noted 200 nmole and 10 μmol scales. For oligonucleotide synthesis a CPG support is preferred, however other medium loading polystyrene-PEG supports such as TENTAGEL™ or ARGOGEL™ can also be used. As seen in Figure 24, the synthesis plate is transported back and forth in the X- direction under an array of 8 moveable banks (530, 532, 534, 536, 538, 540, 542 and 544) of 8 nozzles (64 total) in the Y-direction, and 6 banks (544, 546, 548, 550, 552 and 554) of 48 fixed nozzles, so that each well can receive the appropriate amounts of reagents and/or solvents from any reservoir (large bottle or smaller septa bottle). A sliding balloon-type seal 562 surrounds this nozzle array and joins it to the reaction plate headspace 564. A slow sweep of nitrogen or argon 522 at ambient pressure across the plate headspace is used 36 to preserve an anhydrous environment.
The liquid contents in each well do not drip out until the headspace pressure exceeds the capillary forces on the liquid in the exit nozzle. A slight positive pressure in the lower collection chamber can be added to eliminate residual slow leakage from filled wells, or to effect agitation by bubbling inert gas through the suspension. In order to empty the wells, the headspace gas outlet valve is closed and the internal pressure raised to about 2 psi. Normally, liquid contents are blown directly to waste 566. However, a 96 well microtiter plate can be inserted into the lower chamber beneath the synthesis plate in order to collect the individual well eluents for spectrophotometric monitoring (trityl, etc.) of reaction progress and yield.
The basic plumbing scheme for the machine is the gas-pressurized delivery of reagents. Each reagent is delivered to the synthesis plate through a dedicated supply line, collectively identified at 568, solenoid valve collectively identified at 570 and nozzle, collectively identified at 572. Reagents never cross paths until they reach the reaction well. Thus, no line needs to be washed or flushed prior to its next use and there is no possibility of cross-contamination of reagents. The liquid delivery velocity is sufficiently energetic to thoroughly mix the contents within a well to form a homogeneous solution, even when employing solutions having drastically different densities. With this mixing, once reactants are in homogeneous solution, diffusion carries the individual components into and out of the solid support matrix where the desired reaction takes place. Each reagent reservoir can be plumbed to either a single nozzle or any combination of up to 8 nozzles. Each nozzle is also provided with a concentric nozzle washer to wash the outside of the delivery nozzles in order to eliminate problems of crystallized reactant buildup due to slow evaporation of solvent at the tips of the nozzles. The nozzles and supply lines can be primed into a set of dummy wells directly to waste at any time.
The entire plumbing system is fabricated with teflon tubing, and reagent reservoirs are accessed via syringe needle/septa or direct connection into the higher capacity bottles. The septum vials 560 are held in removable 8-bottle racks to facilitate easy setup and cleaning. The priming volume for each line is about 350 μl. The minimum delivery volume is about 2 μl, and flow rate accuracy is ±5%. The actual amount of material delivered depends on a timed flow of liquid. The flow rate for a particular solvent will 37 depend on its viscosity and wetting characteristics of the teflon tubing. The flow rate (typically 200-350 μl per sec) is experimentally determined, and this information is contained in the reagent table setup file.
Heating and cooling of the reaction block 526 is effected utilizing a recirculating heat exchanger plate 524, similar to that found in PCR thermocyclers, that nests with the polypropylene synthesis plate 556 to provide good thermal contact. The liquid contents in a well can be heated or cooled at about 10°C per minute over a range of +5 to +80°C, as polypropylene begins to soften and deform at about 80°C. For temperatures greater than this, a non-disposable synthesis plate machined from stainless steel or monel with replaceable frits can be utilized.
The hardware controller can be any of a wide variety, but conveniently can be designed around a set of three 1 MHz 86332 chips. This controller is used to drive the single X-axis and 8 Y-axis stepper motors as well as provide the timing functions for a total of 154 solenoid valves. Each chip has 16 bidirectional timer I/O and 8 interrupt channels in its timer processing unit (TPU). These are used to provide the step and direction signals, and to read 3 encoder inputs and 2 limit switches for controlling up to three motors per chip. Each 86332 chip also drives a serial chain of 8 UNC5891A darlington array chips to provide power to 64 valves with msec resolution. The controller communicates with the Windows software interface program running on a PC via a 19200 Hz serial channel, and uses an elementary instruction set to communicate valve number, time open, motor_number and position_data.
The three components of the software program that run the array synthesizer are the generalized procedure or command (.cmd) file which specifies the synthesis instructions to be performed, the sequence (.seq) file which specifies the scale of the reaction and the order in which variable groups will be added to the core synthon, and the reagent table (.tab) file which specifies the name of a chemical, its location (bottle number), flow rate, and concentration are utilized in conjunction with a basic set of command instructions.
One basic set of command instructions can be: ADD
IF {block of instructions} END_IF 38
REPEAT {block of instructions} END_REPEAT
PRIME, NOZZLE WASH WAIT, DRAIN LOAD, REMOVE NEXT SEQUENCE
LOOP_BEGΓN, LOOP_END
The ADD instruction has two forms, and is intended to have the look and feel of a standard chemical equation. Reagents are specified to be added by a molar amount if the number proceeds the name identifier, or by an absolute volume in micro liters if the number follows the identifier. The number of reagents to be added is a parsed list, separated by the "+" sign. For variable reagent identifiers, the key word, <seq>, means look in the sequence table for the identity of the reagent to be added, while the key word, <act>, means add the reagent which is associated with that particular <seq>. Reagents are delivered in the order specified in the list. Thus:
ADD ACN 300 means: Add 300 μl of the named reagent acetonitrile; ACN to each well of active synthesis ADD <seq> 300 means: If the sequence pointer in the .seq file is to a reagent in the list of reagents, independent of scale, add 300 μl of that particular reagent specified for that well. ADD 1.1 PYR + 1.0 <seq> + 1.1 <actl> means: If the sequence pointer in the .seq file is to a reagent in the list of acids in the Class ACIDS 1 , and PYR is the name of pyridine, and ethyl chloroformate is defined in the .tab file to activate the class, ACIDS 1, then this instruction means: Add 1.1 equiv. pyridine
1.0 equiv. of the acid specified for that well and 1.1 equiv. of the activator, ethyl chloroformate
The IF command allows one to test what type of reagent is specified in the <seq> variable 39 and process the succeeding block of commands accordingly. Thus:
ACYLATION {the procedure name}
BEGIN IF CLASS = ACIDS J
ADD 1.0 <seq> + 1.1 <actl> + 1.1 PYR WAIT 60 ENDIF
IF CLASS = ACIDS_2 ADD 1.0 <seq> + 1.2 <actl> + 1.2 TEA
ENDIF WAIT 60 DRAIN 10 END means: Operate on those wells for which reagents contained in the Acid_l class are specified, WAIT 60 sec, then operate on those wells for which reagents contained in the Acid_2 class are specified, then WAIT 60 sec longer, then DRAIN the whole plate. Note that the Acid 1 group has reacted for a total of 120 sec, while the Acid_2 group has reacted for only 60 sec. The REPEAT command is a simple way to execute the same block of commands multiple times. Thus:
WASH_1 {the procedure name}
BEGIN REPEAT 3
ADD ACN 300 DRAIN 15 END_REPEAT END means: repeats the add acetonitrile and drain sequence for each well three times.
The PRIME command will operate either on specific named reagents or on nozzles 40 which will be used in the next associated <seq> operation. The μl amount dispensed into a prime port is a constant that can be specified in a config.dat file.
The NOZZLE_WASH command for washing the outside of reaction nozzles free from residue due to evaporation of reagent solvent will operate either on specific named reagents or on nozzles which have been used in the preceding associated <seq> operation.
The machine is plumbed such that if any nozzle in a block has been used, all the nozzles in that block will be washed into the prime port.
The WAIT and DRAIN commands are by seconds, with the drain command applying a gas pressure over the top surface of the plate in order to drain the wells. The LOAD and REMOVE commands are instructions for the machine to pause for operator action.
The NEXT_SEQUENCE command increments the sequence pointer to the next group of substituents to be added in the sequence file. The general form of a .seq file entry is the definition:
Well No Well D Scale Sequence
The sequence information is conveyed by a series of columns, each of which represents a variable reagent to be added at a particular position. The scale (μmole) variable is included so that reactions of different scale can be run at the same time if desired. The reagents are defined in a lookup table (the .tab file), which specifies the name of the reagent as referred to in the sequence and command files, its location (bottle number), flow rate, and concentration. This information is then used by the controller software and hardware to determine both the appropriate slider motion to position the plate and slider arms for delivery of a specific reagent, as well as the specific valve and time required to deliver the appropriate reagents. The adept classification of reagents allows the use of conditional IF loops from within a command file to perform addition of different reagents differently during a "single step" performed across 96 wells simultaneously. The special class ACTIVATORS defines certain reagents that always get added with a particular class of reagents (for example tetrazole during a phosphitylation reaction in adding the next nucleotide to a growing oligonucleotide). 41
The general form of the .tab file is the definition:
Class Bottle Reagent Name Flo rate Cone.
The LOOP BEGLN and LOOP_END commands define the block of commands which will continue to operate until a NEXT_SEQUENCE command points past the end of the longest list of reactants in any well.
Not included in the command set is a MOVE command. For all of the above commands, if any plate or nozzle movement is required, this is automatically executed in order to perform the desired solvent or reagent delivery operation. This is accomplished by the controller software and hardware, which determines the correct nozzle(s) and well(s) required for a particular reagent addition, then synchronizes the position of the requisite nozzle and well prior to adding the reagent.
A MANUAL mode can also be utilized in which the synthesis plate and nozzle blocks can be "homed" or moved to any position by the operator, the nozzles primed or washed, the various reagent bottles depressurized or washed with solvent, the chamber pressurized, etc. The automatic COMMAND mode can be interrupted at any point, MANUAL commands executed, and then operation resumed at the appropriate location. The sequence pointer can be incremented to restart a synthesis anywhere within a command file.
In reference to Figure 14, the list of oligonucleotides for synthesis can be rearranged or grouped for optimization of synthesis. Thus at process step 574, the oligonucleotides are grouped according to a factor on which to base the optimization of synthesis. As illustrated in the Examples below, one such factor is the 3' most nucleoside of the oligonucleotide. Using the amidite approach for oligonucleotide synthesis, a nucleotide bearing a 3' phosphoramite is added to the 5' hydroxyl group of a growing nucleotide chain. The first nucleotide (at the 3' terminus of the oligonucleotide - the 3' most nucleoside) is first connected to a solid support. This is normally done batchwise on a large scale as is standard practice during oligonucleotide synthesis. Such solid supports pre-loaded with a nucleoside are commercially available. In utilizing the multi well format for oligonucleotide synthesis, for each oligonucleotide to be 42 synthesized, an aliquot of a solid support bearing the proper nucleoside thereon is added to the well for synthesis. Prior to loading the sequence of oligonucleotides to be synthesized in the .seq file, they are sorted by the 3' terminal nucleotide. Based on that sorting, all of the oligonucleotide sequences having an "A" nucleoside at their 3' end are grouped together, those with a "C" nucleoside are grouped together as are those with "G" or "T" nucleosides. Thus in loading the nucleoside-bearing solid support into the synthesis wells, machine movements are conserved.
The oligonucleotides can be grouped by the above described parameter or other parameters that facilitate the synthesis of the oligonucleotides. Thus in Figure 14, sorting is noted as being effected by some parameter of type 1, as for instance the above described
3' most nucleoside, or other types of parameters from type 2 to type n at process steps 576, 578 and 580. Since synthesis will be from the 3' end of the oligonucleotides to the 5' end, the oligonucleotide sequences are reverse sorted to read 3' to 5'. The oligonucleotides are entered in the .seq file in this form, i.e., reading 3' to 5'. Once sorted into types, the position of the oligonucleotides on the synthesis plates is specified at process step 582 by the creation of a .seq file as described above. The .seq file is associated with the respective .cmd and .tab files needed for synthesis of the particular chemistries specified for the oligonucleotides at process step 584 by retrieval of the .cmd and .tab files at process step 586 from the sample database 520. These files are then input into the multi well synthesizer at process step 588 for oligonucleotide synthesis.
Once physically synthesized, the list of oligonucleotides again enters the general procedure flow as indicated in Figure 1. For shipping, storage or other handling puφoses, the plates can be lyophilized at this point if desired. Upon lyophilization, each well contains the oligonucleotides located therein as a dry compound. 14. Quality Control.
In an optional step, quality control is performed on the oligonucleotides at process step 600 after a decision is made (decision step 550) to perform quality control. Although optional, quality control may be desired when there is some reason to think that some aspect of the synthetic process step 500 has been compromised. Alternatively, samples of the oligonucleotides may be taken and stored in the event that the results of assays conducted using the oligonucleotides (process step 700) yield confusing results or 43 suboptimal data. In the latter event, for example, quality control might be performed after decision step 800 if no oligonucleotides with sufficient activity are identified. In either event, decision step 650 follows quality control step process 600. If one or more of the oligonucleotides do not pass quality control, process step 500 can be repeated, i.e., the oligonucleotides are synthesized for a second time.
The operation of the quality control system general procedure 600 is detailed in steps 610-660 of Figure 15. Also referenced in the following discussion are the robotics and associated analytical instrumentation as shown in Figure 18.
During step 610 (Figure 15), sterile, double-distilled water is transferred by an automated liquid handler (2040 of Figure 18) to each well of a multi -well plate containing a set of lyophilized antisense oligonucleotides. The automated liquid handler (2040 of Figure 18) reads the barcode sticker on the multi-well plate to obtain the plate's identification number. Automated liquid handler 2040 then queries Sample Database 520 (which resides in Database Server 2002 of Figure 18) for the quality control assay instruction set for that plate and executes the appropriate steps. Three quality control processes are illustrated, however, it is understood that other quality control processes or steps maybe practiced in addition to or in place of the processes illustrated.
The first illustrative quality control process (steps 622 to 626) quantitates the concentration of oligonucleotide in each well. If this quality control step is performed, an automated liquid handler (2040 of Figure 18) is instructed to remove an aliquot from each well of the master plate and generate a replicate daughter plate for transfer to the UV spectrophotometer (2016 of Figure 18). The UV spectrophotometer (2016 of Figure 18) then measures the optical density of each well at a wavelength of 260 nanometers. Using standardized conversion factors, a microprocessor within UV spectrophotometer (2016 of Figure 18) then calculates a concentration value from the measured absorbance value for each well and output the results to Sample Database 520.
The second illustrative quality control process steps 632 to 636) quantitates the percent of total oligonucleotide in each well that is full length. If this quality control step is performed, an automated liquid handler (2040 of Figure 18) is instructed to remove an aliquot from each well of the master plate and generate a replicate daughter plate for transfer to the multichannel capillary gel electrophoresis apparatus (2022 of Figure 18). 44
The apparatus electrophoretically resolves in capillary tube gels the oligonucleotide product in each well. As the product reaches the distal end of the tube gel during electrophoresis, a detection window dynamically measures the optical density of the product that passes by it. Following electrophoresis, the value of percent product that passed by the detection window with respect to time is utilized by a built in microprocessor to calculate the relative size distribution of oligonucleotide product in each well. These results are then output to the Sample Database (520.
The third illustrative quality control process steps 632 to 636) quantitates the mass of the oligonucleotide in each well that is full length. If this quality control step is performed, an automated liquid handler (2040 of Figure 18) is instructed to remove an aliquot from each well of the master plate and generate a replicate daughter plate for transfer to the multichannel liquid electrospray mass spectrometer (2018 of Figure 18). The apparatus then uses electrospray technology to inject the oligonucleotide product into the mass spectrometer. A built in microprocessor calculates the mass-to-charge ratio to arrive at the mass of oligonucleotide product in each well. The results are then output to
Sample Database 520.
Following completion of the selected quality control processes, the output data is manually examined or is examined using an appropriate algorithm and a decision is made as to whether or not the plate receives "Pass" or "Fail" status. The current criteria for acceptance, for 18 mer oligonucleotides, is that at least 85% of the oligonucleotides in a multi-well plate must be 85% or greater full length product as measured by both capillary gel electrophoresis and mass spectrometry. An input (manual or automated) is then made into Sample Database 520 as to the pass/fail status of the plate. If a plate fails, the process cycles back to step 500, and a new plate of the same oligonucleotides is automatically placed in the plate synthesis request queue (process 554 of Figure 15). If a plate receives
"Pass" status, an automated liquid handler (2040 of Figure 18) is instructed to remove appropriate aliquots from each well of the master plate and generate two replicate daughter plates in which the oligonucleotide in each well is at a concentration of 30 micromolar. The plate then moves on to process 700 for oligonucleotide activity evaluation. 15. Cell Lines for Assaying Oligonucleotide Activity. The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types 45 provided that the target nucleic acid, or its gene product, is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following four cell types are provided for illustrative puφoses, but other cell types can be routinely used. T-24 cells: The transitional cell bladder carcinoma cell line T-24 is obtained from the American Type Culture Collection (ATCC) (Manassas, VA). T-24 cells were routinely cultured in complete McCoy's 5 A basal media (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal calf serum, penicillin 100 units per milliliter, and streptomycin 100 micrograms per milliliter (all from Life Technologies). Cells are routinely passaged by trypsinization and dilution when they reach 90% confluence. Cells are routinely seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis. For Northern blotting or other analysis, cells are seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide. A549 cells: The human lung carcinoma cell line A549 is obtained from the
ATCC (Manassas, VA). A549 cells were routinely cultured in DMEM basal media (Life Technologies) supplemented with 10% fetal calf serum, penicillin 100 units per milliliter, and streptomycin 100 micrograms per milliliter (all from Life Technologies). Cells are routinely passaged by trypsinization and dilution when they reach 90% confluence. NHDF cells: Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Coφoration (WalkersviUe, MD). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Coφ.) as provided by the supplier. Cells are maintained for up to 10 passages as recommended by the supplier.
HEK cells: Human embryonic keratinocytes (HEK) were obtained from the Clonetics Coφ. HEKs were routinely maintained in Keratinocyte Growth Medium
(Clonetics Coφ.) as provided by the supplier. Cell are routinely maintained for up to 10 passages as recommended by the supplier.
16. Treatment of Cells with Candidate Compounds:
When cells reach about 80% confluency, they are treated with oligonucleotide. For cells grown in 96-well plates, wells are washed once with 200 μl OPTI-MEM-1™ reduced-serum medium (Life Technologies) and then treated with 130 μl of OPTI-MEM- 46
1™ containing 3.75 μg/ml LIPOFECTIN™ (Life Technologies) and the desired oligonucleotide at a final concentration of 150 nM. After 4 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16 hours after oligonucleotide treatment. Alternatively, for cells resistant to cationic mediated transfection, oligonucleotides can be introduced by electroporation. Electroporation conditions must be optimized for every cell type. In general, oligonucleotide is added directly to complete growth media to a final concentration between 1 and 20 micromolar. An electronic pulse is delivered to the cells using a BTX T820 ELECTRO SQUARE PORATOR™ using a Multi-coaxial 96- well electrode (BT840) (BTX Coφoration, San Diego, California). Following electroporation, the cells are returned to the incubator for 16 hours. 17. Assaying Oligonucleotide Activity:
Oligonucleotide-mediated modulation of expression of a target nucleic acid can be assayed in a variety of ways known in the art. For example, target RNA levels can be quantitated by, e.g., Northern blot analysis, competitive PCR, or reverse transcriptase polymerase chain reaction (RT-PCR). RNA analysis can be performed on total cellular RNA or, preferably in the case of polypeptide-encoding nucleic acids, poly(A)+ mRNA. For RT-PCR, poly(A)+ mRNA is preferred. Methods of RNA isolation are taught in, for example, Ausubel et al. {Short Protocols in Molecular Biology, 2nd Ed., pp. 4-1 to 4-13, Greene Publishing Associates and John Wiley & Sons, New York, 1992). Northern blot analysis is routine in the art {Id., pp. 4-14 to 4-29).
Alternatively, total RNA can be prepared from cultured cells or tissue using the QIAGEN RNeasy®-96 kit for the high throughput preparation of RNA (QIAGEN, Inc., Valencia, CA). Essentially, protocols are carried out according to the manufacturer's directions. Optionally, a DNase step is included to remove residual DNA prior to RT-
PCR.
To improve efficiency and accuracy the repetitive pipeting steps and elution step have been automated using a QIAGEN Bio-Robot 9604. Essentially after lysing of the oligonucleotide treated cell cultures in situ, the plate is transferred to the robot deck where the pipeting, DNase treatment, and elution steps are carried out.
Reverse transcriptase polymerase chain reaction (RT-PCR) can be conveniently 47 accomplished using the commercially available ABI PRISM® 7700 Sequence Detection System (PE- Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Other methods of PCR are also known in the art.
Target protein levels can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), Enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodies directed to a protein encoded by a target nucleic acid can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies, (Aerie Coφoration, Birmingham, MI or via the internet at http://www.ANTIBODIES-PROBES.com/), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal, monospecific ("antipeptide") and monoclonal antisera are taught by, for example, Ausubel et al. {Short Protocols in Molecular Biology, 2nd Ed., pp. 11-3 to 11- 54, Greene Publishing Associates and John Wiley & Sons, New York, 1992).
Immunoprecipitation methods are standard in the art and are described by, for example, Ausubel et al {Id., pp. 10-57 to 10-63). Western blot (immunoblot) analysis is standard in the art {Id., pp. 10-32 to 10-10-35). Enzyme-linked immunosorbent assays (ELISA) are standard in the art {Id., pp. 11-5 to 11-17).
Because it is preferred to assay the compounds of the invention in a batchwise fashion, i.e., in parallel to the automated synthesis process described above, preferred means of assaying are suitable for use in 96-well plates and with robotic means.
Accordingly, automated RT-PCR is preferred for assaying target nucleic acid levels, and automated ELISA is preferred for assaying target protein levels.
The assaying step, general procedure step 700, is described in detail in Figure 16. After an appropriate cell line is selected at process step 710, a decision is made at decision step 714 as to whether RT-PCR will be the only method by which the activity of the compounds is evaluated. In some instances, it is desirable to run alternative assay methods at process step 718; for example, when it is desired to assess target polypeptide levels as well as target RNA levels, an immunoassay such as an ELISA is run in parallel with the RT-PCR assays. Preferably, such assays are tractable to semi-automated or robotic means. When RT-PCR is used to evaluate the activities of the compounds, cells are plated into multi-well plates (typically, 96-well plates) in process step 720 and treated with test or 48 control oligonucleotides in process step 730. Then, the cells are harvested and lysed in process step 740 and the lysates are introduced into an apparatus where RT-PCR is carried out in process step 750. A raw data file is generated, and the data is downloaded and compiled at step 760. Spreadsheet files with data charts are generated at process step 770, and the experimental data is analyzed at process step 780. Based on the results, a decision is made at process step 785 as to whether it is necessary to repeat the assays and, if so, the process begins again with step 720. In any event, data from all the assays on each oligonucleotide are compiled and statistical parameters are automatically determined at process step 790. 18. Classification of Compounds Based on Their Activity:
Following assaying, general procedure step 700, oligonucleotide compounds are classified according to one or more desired properties. Typically, three classes of compounds are used: active compounds, marginally active (or "marginal") compounds and inactive compounds. To some degree, the selection criteria for these classes vary from target to target, and members of one or more classes may not be present for a given set of oligonucleotides.
However, some criteria are constant. For example, inactive compounds will typically comprise those compounds having 5% or less inhibition of target expression (relative to basal levels). Active compounds will typically cause at least 30% inhibition of target expression, although lower levels of inhibition are acceptable in some instances.
Marginal compounds will have activities intermediate between active and inactive compounds, with preferred marginal compounds having activities more like those of active compounds.
19. Optimization of Lead Compounds by Sequence. One means by which oligonucleotide compounds are optimized for activity is by varying their nucleobase sequences so that different regions of the target nucleic acid are targeted. Some such regions will be more accessible to oligonucleotide compounds than others, and "sliding" a nucleobase sequence along a target nucleic acid only a few bases can have significant effects on activity. Accordingly, varying or adjusting the nucleobase sequences of the compounds of the invention is one means by which suboptimal compounds can be made optimal, or by which new active compounds can be generated. 49
The operation of the gene walk process 1100 detailed in steps 1104-1112 of Figure 17 is detailed as follows. As used herein, the term "gene walk" is defined as the process by which a specified oligonucleotide sequence x that binds to a specified nucleic acid target y is used as a frame of reference around which a series of new oligonucleotides sequences capable of hybridizing to nucleic acid target y are generated that are sequence shifted increments of oligonucleotide sequence x. Gene walking can be done "downstream", "upstream" or in both directions from a specified oligonucleotide. During step 1104 the user manually enters the identification number of the oligonucleotide sequence around which it is desired to execute gene walk process 1100 and the name of the corresponding target nucleic acid. The user then enters the scope of the gene walk at step 1104, by which is meant the number of oligonucleotide sequences that it is desired to generate. The user then enters in step 1108 a positive integer value for the sequence shift increment. Once this data is generated, the gene walk is effected. This causes a subroutine to be executed that automatically generates the desired list of sequences by walking along the target sequence. At that point, the user proceeds to process 400 to assign chemistries to the selected oligonucleotides.
Example 16 below, details a gene walk. In subsequent steps, this new set of nucleobase sequences generated by the gene walk is used to direct the automated synthesis at general procedure step 500 of a second set of candidate oligonucleotides. These compounds are then taken through subsequent process steps to yield active compounds or reiterated as necessary to optimize activity of the compounds. 20. Optimization of Lead Compounds by Chemistry. Another means by which oligonucleotide compounds of the invention are optimized is by reiterating portions of the process of the invention using marginal or active compounds from the first iteration and selecting additional chemistries to the nucleobase sequences thereof.
Thus, for example, an oligonucleotide chemistry different from that of the first set of oligonucleotides is assigned at general procedure step 400. The nucleobase sequences of marginal compounds are used to direct the synthesis at general procedure step 500 of a second set of oligonucleotides having the second assigned chemistry. The resulting second set of oligonucleotide compounds is assayed in the same manner as the first set at 50 procedure process step 700 and the results are examined to determine if compounds having sufficient activity have been generated at decision step 800.
21. Identification of Sites Amenable to Antisense Technologies.
In a related process, a second oligonucleotide chemistry is assigned at procedure step 400 to the nucleobase sequences of all of the oligonucleotides (or, at least, all of the active and marginal compounds) and a second set of oligonucleotides is synthesized at procedure step 500 having the same nucleobase sequences as the first set of compounds. The resulting second set of oligonucleotide compounds is assayed in the same manner as the first set at procedure step 700 and active and marginal compounds are identified at procedure steps 800 and 1000.
In order to identify sites on the target nucleic acid that are amenable to a variety of antisense technologies, the following mathematically simple steps are taken. The sequences of active and marginal compounds from two or more such automated syntheses/assays are compared and a set of nucleobase sequences that are active, or marginally so, in both sets of compounds is identified. The reverse complements of these nucleobase sequences corresponds to sequences of the target nucleic acid that are tractable to a variety of antisense and other sequence-based technologies. These antisense-sensitive sites are assembled into contiguous sequences (contigs) using the procedures described for assembling target nucleotide sequences (at procedure step 200). 22. Systems for Executing Preferred Methods of the Invention.
An embodiment of computer, network and instrument resources for effecting the methods of the invention is shown in Figure 18. In this embodiment, four computer servers are provided. First, a large database server 2002 stores all chemical structure, sample tracking and genomic, assay, quality control, and program status data. Further, this database server serves as the platform for a document management system. Second, a compute engine 2004 runs computational programs including RNA folding, oligonucleotide walking, and genomic searching. Third, a file server 2006 allows raw instrument output storage and sharing of robot instructions. Fourth, a groupware server 2008 enhances staff communication and process scheduling. A redundant high-speed network system is provided between the main servers and the bridges 2026, 2028 and 2030. These bridges provide reliable network access to the 51 many workstations and instruments deployed for this process. The instruments selected to support this embodiment are all designed to sample directly from standard 96 well microtiter plates, and include an optical density reader 2016, a combined liquid chromatography and mass spectroscopy instrument 2018, a gel fluorescence and scintillation imaging system 2032 and 2042, a capillary gel electrophoreses system 2022 and a real-time PCR system 2034.
Most liquid handling is accomplished automatically using robots with individually controllable robotic pipetters 2038 and 2020 as well as a 96-well pipette system 2040 for duplicating plates. Windows NT or Macintosh workstations 2044, 2024, and 2036 are deployed for instrument control, analysis and productivity support.
23. Relational Database.
Data is stored in an appropriate database. For use with the methods of the invention, a relational database is preferred. Figure 19 illustrates the data structure of a sample relational database. Various elements of data are segregated among linked storage elements of the database.
EXAMPLES
The following examples illustrate the invention and are not intended to limit the same. Those skilled in the art will recognize, or be able to ascertain through routine experimentation, numerous equivalents to the specific procedures, materials and devices described herein. Such equivalents are considered to be within the scope of the present invention.
EXAMPLE 1: Selection of CD40 as a Target
Cell-cell interactions are a feature of a variety of biological processes. In the activation of the immune response, for example, one of the earliest detectable events in a normal inflammatory response is adhesion of leukocytes to the vascular endothelium, followed by migration of leukocytes out of the vasculature to the site of infection or injury. The adhesion of leukocytes to vascular endothelium is an obligate step in their migration out of the vasculature (for a review, see Albelda et al, FASEB J, 1994, 8, 504). As is well known in the art, cell-cell interactions are also critical for propagation of both B- 52 lymphocytes and T-lymphocytes resulting in enhanced humoral and cellular immune responses, respectively (for a reviews, see Makgoba et al, Immunol. Today, 1989, 10, 417; Janeway, Sci. Amer., 1993, 269, 72).
CD40 was first characterized as a receptor expressed on B-lymphocytes. It was later found that engagement of B-cell CD40 with CD40L expressed on activated T-cells is essential for T-cell dependent B-cell activation (i.e. proliferation, immunoglobulin secretion, and class switching) (for a review, see Gruss et al. Leuk. Lymphoma, 1997, 24, 393). A full cDNA sequence for CD40 is available (GenBank accession number X60592, incoφorated herein by reference as SEQ ID NO:85). As interest in CD40 mounted, it was subsequently revealed that functional CD40 is expressed on a variety of cell types other than B-cells, including macrophages, dendritic cells, thymic epithelial cells, Langerhans cells, and endothelial cells {Ibid.). These studies have led to the current belief that CD40 plays a much broader role in immune regulation by mediating interactions of T-cells with cell types other than B-cells. In support of this notion, it has been shown that stimulation of CD40 in macrophages and dendritic results is required for T-cell activation during antigen presentation {Id.). Recent evidence points to a role for CD40 in tissue inflammation as well. Production of the inflammatory mediators IL-12 and nitric oxide by macrophages has been shown to be CD40 dependent (Buhlmann et al, J. Clin. Immunol, 1996, 16, 83). In endothelial cells, stimulation of CD40 by CD40L has been found to induce surface expression of E-selectin, ICAM-1, and VCAM-1, promoting adhesion of leukocytes to sites of inflammation (Buhlmann et al, J. Clin. Immunol, 1996, 16, 83; Gruss et al, Leuk Lymphoma, 1997, 24, 393). Finally, a number of reports have documented overexpression of CD40 in epithelial and hematopoietic tumors as well as tumor infiltrating endothelial cells, indicating that CD40 may play a role in tumor growth and or angiogenesis as well (Gruss et al, Leuk Lymphoma, 1997, 24, 393-
422; Kluth et al Cancer Res, 1997, 57, 891).
Due to the pivotal role that CD40 plays in humoral immunity, the potential exists that therapeutic strategies aimed at downregulating CD40 may provide a novel class of agents useful in treating a number of immune associated disorders, including but not limited to graft versus host disease, graft rejection, and autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus, and certain forms of arthritis. Inhibitors 53 of CD40 may also prove useful as an anti-inflammatory compound, and could therefore be useful as treatment for a variety of diseases with an inflammatory component such as asthma, rheumatoid arthritis, allograft rejections, inflammatory bowel disease, and various dermatological conditions, including psoriasis. Finally, as more is learned about the association between CD40 overexpression and tumor growth, inhibitors of CD40 may prove useful as anti-tumor agents as well.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of CD40. To date, strategies aimed at inhibiting CD40 function have involved the use of a variety of agents that disrupt CD40/CD40L binding. These include monoclonal antibodies directed against either CD40 or CD40L, soluble forms of CD40, and synthetic peptides derived from a second CD40 binding protein, A20. The use of neutralizing antibodies against CD40 and/or CD40L in animal models has provided evidence that inhibition of CD40 stimulation would have therapeutic benefit for GVHD, allograft rejection, rheumatoid arthritis, SLE, MS, and B-cell lymphoma (Buhlmann et al, J. Clin. Immunol, 1996, 16, 83). However, due to the expense, short half-life, and bioavailability problems associated with the use of large proteins as therapeutic agents, there is a long felt need for additional agents capable of effectively inhibiting CD40 function. Oligonucleotides compounds avoid many of the pitfalls of current agents used to block CD40/CD40L interactions and may therefore prove to be uniquely useful in a number of therapeutic applications.
EXAMPLE 2: Generation of Virtual Oligonucleotides Targeted to CD40
The process of the invention was used to select oligonucleotides targeted to CD40, generating the list of oligonucleotide sequences with desired properties as shown in Figure 22. From the assembled CD40 sequence, the process began with determining the desired oligonucleotide length to be eighteen nucleotides, as represented in step 2500. All possible oligonucleotides of this length were generated by Oligo 5.0™, as represented in step 2504. Desired thermodynamic properties were selected in step 2508. The single parameter used was oligonucleotides of melting temperature less than or equal to 40°C were discarded. In step 2512, oligonucleotide melting temperatures were calculated by Oligo 5.0. Oligonucleotide sequences possessing an undesirable score were discarded. It 54 is believed that oligonucleotides with melting temperatures near or below physiological and cell culture temperatures will bind poorly to target sequences. All oligonucleotide sequences remaining were exported into a spreadsheet. In step 2516, desired sequence properties are selected. These include discarding oligonucleotides with at least one stretch of four guanosines in a row and stretches of six of any other nucleotide in a row. In step
2520, a spreadsheet macro removed all oligonucleotides containing the text string "GGGG." In step 2524, another spreadsheet macro removed all oligonucleotides containing the text strings "AAAAAA" or "CCCCCC" or « TTTTTT." From the remaining oligonucleotide sequences, 84 sequences were selected manually with the criteria of having an uniform distribution of oligonucleotide sequences throughout the target sequence, as represented in step 2528. These oligonucleotide sequences were then passed to the next step in the process, assigning actual oligonucleotide chemistries to the sequences.
EXAMPLE 3: Input Files For Automated Oligonucleotide Synthesis Command File (.cmd File)
Table 2 is a command file for synthesis of an oligonucleotide having regions of 2'- O-(2-methoxyethyl) nucleosides and a central region of 2'-deoxy nucleosides each linked by phosphorothioate internucleotide linkages.
Table 2 SOLID_SUPPORT_SKIP
BEGIN
Next_Sequence END
INITIAL-WASH
BEGIN
Add ACN 300 Drain 10 END 55
LOOP-BEGIN DEBLOCK
BEGIN
Prime TCA Load Tray Repeat 2
Add TCA 150 Wait 10 Drain 8 End Repeat
Remove Tray
Add TCA 125 Wait 10 Drain 8
END
WASH_AFTER_DEBLOCK BEGIN
Repeat 3
Add ACN 250 To_All Drain 10
End Repeat END
COUPLING
BEGIN if class = DEOXY_THIOATE
Nozzle wash <actl> prime <actl> prime <seq> Add <actl> 70 + <seq> 70 56
Wait 40
Drain 5 end-if if class = MOE THIOATE Nozzle wash <act 1 >
Prime <actl> prime <seq>
Add <actl> 120 + <seq> 120 Wait 230 Drain 5
End_if END
WASH_AFTER_COUPLING BEGIN Add ACN 200 To_All
Drain 10 END
OXIDIZE BEGIN if class = DEOXY THIOATE Add BEAU 180 Wait 40
Drain 7 end_if if class = MOE_THIOATE Add BEAU 200 Wait 120 Drain 7 end if 57 END
CAP
BEGIN Add CAP_B 80 + CAP_A 80
Wait 20 Drain 7 END
WASH_AFTER_CAP
BEGIN
AddACN150To_All Drain 5
Add ACN 250 To_All Drain 11
END
BASE_COUNTER
BEGIN Next_Sequence
END
LOOP_END
DEBLOCKFΓNAL BEGIN
Prime TCA
Load Tray
Repeat 2 AddTCA150To_All Wait 10 58
Drain 8
End_Repeat Remove Tray Add TCA 125 To All Wait 10
Drain 10 END
FLNAL_WASH
BEGIN Repeat 4
Add ACN 300 to All Drain_12 End_Repeat END END ALL
BEGIN
Wait 3 END
Sequence files (.seq Files) Table 3 is a .seq file for oligonucleotides having 2'-deoxy nucleosides linked by phosphorothioate internucleotide linkages.
Table 3
Identity of columns: Syn #, Well, Scale, Nucleotide at particular position (identified using base identifier followed by backbone identifier where "s" is phosphorothioate). Note the columns wrap around to next line when longer than one line.
1 A01 200 As Cs Cs As Gs Gs As Cs Gs
Figure imgf000060_0001
Gs Cs Gs Gs As Cs Cs As G 59
2 A02 200 As Cs Gs Gs Cs Gs Gs As Cs
Cs As Gs As Gs Ts Gs Gs A
3 A03 200 As Cs Cs As As Gs Cs As Gs
As Cs Gs Gs As Gs As Cs G
4 A04 200 As Gs Gs As Gs As Cs Cs Cs
Cs Gs As Cs Gs As As Cs G
5 A05 200 As Cs Cs Cs Cs Gs As Cs Gs
As As Cs Gs As Cs Ts Gs G
6 A06 200 As Cs Gs As As Cs Gs As Cs
Ts Gs Gs Cs Gs As Cs As G
7 A07 200 As Cs Gs As Cs Ts Gs Gs Cs
Gs As Cs As Gs Gs Ts As G
8 A08 200 As Cs As Gs Gs Ts As Gs Gs
Ts Cs Ts Ts Gs Gs Ts Gs G
9 A A0099 220000 AAss GGss GGss T Tss C Css T Tss Ts Gs Gs
Ts Gs Gs Gs Ts Gs As Cs G
10 A10 200 As Gs Ts Cs As Cs Gs As Cs
As As Gs As As As Cs As C
11 All 200 As Cs Gs As Cs As As Gs As
As As Cs As Cs Gs Gs Ts C
12 A12 200 As Gs As As As Cs As Cs Gs
Gs Ts Cs Gs Gs Ts Cs Cs T
13 B01 200 As As Cs As Cs Gs Gs Ts Cs
Gs Gs Ts Cs Cs Ts Gs Ts C
1144 BB0022 220000 AAss CCss TTss CCss AAss CCss Ts Gs As
Cs Gs Ts Gs Ts Cs Ts Cs A
15 B03 200 As Cs Gs Gs As As Gs Gs As
As Cs Gs Cs Cs As Cs Ts T
16 B04 200 As Ts Cs Ts Gs Ts Gs Gs As
Cs Cs Ts Ts Gs Ts Cs Ts C
Figure imgf000061_0001
17 B05 200 As Cs As Cs Ts Ts Cs Ts Ts 60
Cs Cs Gs As Cs Cs Gs Ts G
18 B06 200 As Cs Ts Cs Ts Cs Gs As Cs
As Cs As Gs Gs As Cs Gs T
19 B07 200 As As As Cs Cs Cs Cs As Gs
Ts Ts Cs Gs Ts Cs Ts As A
20 B08 200 As Ts Gs Ts Cs Cs Cs Cs As
As As Gs As Cs Ts As Ts G
21 B09 200 As Cs Gs Cs Ts Cs Gs Gs Gs
As Cs Gs Gs Gs Ts Cs As G
22 BIO 200 As Gs Cs Cs Gs As As Gs As
As Gs As Gs Gs Ts Ts As C
23 Bll 200 As Cs As Cs As Gs Ts As Gs
As Cs Gs As As As Gs Cs T
24 B12 200 As Cs As Cs Ts Cs Ts Gs Gs
Ts Ts Ts Cs Ts Gs Gs As C
25 C01 200 As Cs Gs As Cs Cs As Gs As
As As Ts As Gs Ts Ts Ts T
26 C02 200 As Gs Ts Ts As As As As Gs
Gs Gs Cs Ts Gs Cs Ts As G
27 C03 200 As Gs Gs Ts Ts Gs Ts Gs As
Cs Gs As Cs Gs As Gs Gs T
28 C04 200 As As Ts Gs Ts As Cs Cs Ts
As Cs Gs Gs Ts Ts Gs Gs C
29 C05 200 As Gs Ts Cs As Cs Gs Ts Cs
Cs Ts Cs Ts Cs Ts Gs Ts C
30 C06 200 Cs Ts Gs Gs Cs Gs As Cs As
Gs Gs Ts As Gs Gs Ts Cs T
31 C07 200 Cs Ts Cs Ts Gs Ts Gs Ts Gs
As Cs Gs Gs Ts Gs Gs Ts C
32 C08 200 Cs As Gs Gs Ts Cs Gs Ts Cs
Figure imgf000062_0001
Ts Ts Cs Cs Cs Gs Ts Gs G 61
33 C09 200 Cs Ts Gs Ts Gs Gs Ts As Gs
As Cs Gs Ts Gs Gs As Cs A
34 CIO 200 Cs Ts As As Cs Gs As Ts Gs
Ts Cs Cs Cs Cs As As As G
35 Cll 200 Cs Ts Gs Ts Ts Cs Gs As Cs
As Cs Ts Cs Ts Gs Gs Ts T
36 C12 200 Cs Ts Gs Gs As Cs Cs As As
Cs As Cs Gs Ts Ts Gs Ts C
37 D01 200 Cs Cs Gs Ts Cs Cs Gs Ts Gs
Ts Ts Ts Gs Ts Ts Cs Ts G
38 D02 200 Cs Ts Gs As Cs Ts As Cs As
As Cs As Gs As Cs As Cs C
39 D03 200 Cs As As Cs As Gs As Cs As
Cs Cs As Gs Gs Gs Gs Ts C
4 400 D D0044 220000 CCss AAss GGss GGss GGss GGss Ts Cs Cs
Ts As Gs Cs Cs Gs As Cs T
41 D05 200 Cs Ts Cs Ts As Gs Ts Ts As
As As As Gs Gs Gs Cs Ts G
42 D06 200 Cs Ts Gs Cs Ts As Gs As As
Gs Gs As Cs Cs Gs As Gs G
43 D07 200 Cs Ts Gs As As As Ts Gs Ts
As Cs Cs Ts As Cs Gs Gs T
44 D08 200 Cs As Cs Cs Cs Gs Ts Ts Ts
Gs Ts Cs Cs Gs Ts Cs As A
4455 DD0099 220000 CCss TTss CCss GGss AAss TTss As Cs Gs
Gs Gs Ts Cs As Gs Ts Cs A
46 D10 200 Gs Gs Ts As Gs Gs Ts Cs Ts
Ts Gs Gs Ts Gs Gs Gs Ts G
47 Dll 200 Gs As Cs Ts Ts Ts Gs Cs Cs
Ts Ts As Cs Gs Gs As As G
Figure imgf000063_0001
48 D12 200 Gs Ts Gs Gs As Gs Ts Cs Ts 62
Ts Ts Gs Ts Cs Ts Gs Ts G
49 E01 200 Gs Gs As Gs Ts Cs Ts Ts Ts
Gs Ts Cs Ts Gs Ts Gs Gs T
50 E02 200 Gs Gs As Cs As Cs Ts Cs Ts
Cs Gs As Cs As Cs As Gs G
51 E03 200 Gs As Cs As Cs As Gs Gs As
Cs Gs Ts Gs Gs Cs Gs As G
52 E04 200 Gs As Gs Ts As Cs Gs As Gs
Cs Gs Gs Gs Cs Cs Gs As A
5 533 E E0055 2 20000 G Gss A Ass C Css T Tss A Ass T Tss Gs Gs Ts
As Gs As Cs Gs Cs Ts Cs G
54 E06 200 Gs As As Gs As Gs Gs Ts Ts
As Cs As Cs As Gs Ts As G
55 E07 200 Gs As Gs Gs Ts Ts As Cs As
Cs As Gs Ts As Gs As Cs G
56 E08 200 Gs Ts Ts Gs Ts Cs Cs Gs Ts
Cs Cs Gs Ts Gs Ts Ts Ts G
57 E09 200 Gs As Cs Ts Cs Ts Cs Gs Gs
Gs As Cs Cs As Cs Cs As C
5588 EE1100 220000 GGss TTss AAss GGss GGss AAss Gs As As
Cs Cs As Cs Gs As Cs Cs A
59 Ell 200 Gs Gs Ts Ts Cs Ts Ts Cs Gs
Gs Ts Ts Gs Gs Ts Ts As T
60 E12 200 Gs Ts Gs Gs Gs Gs Ts Ts Cs
Gs Ts Cs Cs Ts Ts Gs Gs G
61 F01 200 Gs Ts Cs As Cs Gs Ts Cs Cs
Ts Cs Ts Gs As As As Ts G
62 F02 200 Gs Ts Cs Cs Ts Cs Cs Ts As
Cs Cs Gs Ts Ts Ts Cs Ts C
6633 FF0033 220000 GGss TTss CCss CCss CCss CCss As Cs Gs
Figure imgf000064_0001
Ts Cs Cs Gs Ts Cs Ts Ts C 63
64 F04 200 Ts Cs As Cs Cs As Gs Gs As
Cs Gs Gs Cs Gs Gs As Cs C
65 F05 200 Ts As Cs Cs As As Gs Cs As
Gs As Cs Gs Gs As Gs As C
66 F06 200 Ts Cs Cs Ts Gs Ts Cs Ts Ts
Ts Gs As Cs Cs As Cs Ts C
67 F07 200 Ts Gs Ts Cs Ts Ts Ts Gs As
Cs Cs As Cs Ts Cs As Cs T
68 F08 200 Ts Gs As Cs Cs As Cs Ts Cs
As Cs Ts Gs As Cs Gs Ts G
69 F09 200 Ts Gs As Cs Gs Ts Gs Ts Cs
Ts Cs As As Gs Ts Gs As C
70 F10 200 Ts Cs As As Gs Ts Gs As Cs
Ts Ts Ts Gs Cs Cs Ts Ts A
7 711 F Fllll 220000 TTss G Gss TTss T Tss T Tss A Ass Ts Gs As
Cs Gs Cs Ts Gs Gs Gs Gs T
72 F12 200 Ts Ts As Ts Gs As Cs Gs Cs
Ts Gs Gs Gs Gs Ts Ts Gs G
73 G01 200 Ts Gs As Cs Gs Cs Ts Gs Gs
Gs Gs Ts Ts Gs Gs As Ts C
74 G02 200 Ts Cs Gs Ts Cs Ts Ts Cs Cs
Cs Gs Ts Gs Gs As Gs Ts C
75 G03 200 Ts Gs Gs Ts As Gs As Cs Gs
Ts Gs Gs As Cs As Cs Ts T
7766 GG0044 220000 TTss TTss CCss TTss TTss CCss Cs Gs As
Cs Cs Gs Ts Gs As Cs As T
77 G05 200 Ts Gs Gs Ts As Gs As Cs Gs
Cs Ts Cs Gs Gs Gs As Cs G
78 G06 200 Ts As Gs As Cs Gs Cs Ts Cs
Gs Gs Gs As Cs Gs Gs Gs T
Figure imgf000065_0001
79 G07 200 Ts Ts Ts Ts As Cs As Gs Ts 64
Gs Gs Gs As As Cs Cs Ts G
80 G08 200 Ts Gs Gs Gs As As Cs Cs Ts
Gs Ts Ts Cs Gs As Cs As C
81 G09 200 Ts Cs Gs Gs Gs As Cs Cs As
Cs Cs As Cs Ts As Gs Gs G
82 G10 200 Ts As Gs Gs As Cs As As As
Cs Gs Gs Ts As Gs Gs As G
83 Gil 200 Ts Gs Cs Ts As Gs As As Gs
Gs As Cs Cs Gs As Gs Gs T
8844 GG1122 220000 TTss CCss TTss GGss TTss CCss As Cs Ts
Figure imgf000066_0001
Cs Cs Gs As Cs Gs Ts Gs G
Table 4 is a .seq file for oligonucleotides having regions of 2'-O-(2-methoxyethyl)- nucleosides and a central region of 2'-deoxy nucleosides each linked by phosphorothioate internucleotide linkages.
Table 4
Identity of columns: Syn #, Well, Scale, Nucleotide at particular position (identified using base identifier followed by backbone identifier where "s" is phosphorothioate and "moe" indicated a 2'-O-(2-methoxyethyl) substituted nucleoside). The columns wrap around to next line when longer than one line.
1 A01 200 moeAs moeCs moeCs moeAs Gs Gs As Cs Gs Gs Cs Gs Gs As moeCs moeCs moeAs moeG
2 A02200 moeAs moeCs moeGs moeGs Cs Gs Gs As Cs Cs As Gs As Gs moeTs moeGs moeGs moeA 3 A03200 moeAs moeCs moeCs moeAs As Gs Cs As Gs As Cs Gs Gs As moeGs moeAs moeCs moeG
4 A04200 moeAs moeGs moeGs moeAs Gs As Cs Cs Cs Cs Gs As Cs Gs moeAs moeAs moeCs moeG
5 A05200 moeAs moeCs moeCs moeCs Cs Gs As Cs Gs As As Cs Gs As 65 moeCs moeTs moeGs moeG
6 A06 200 moeAs moeCs moeGs moeAs As Cs Gs As Cs Ts Gs Gs Cs Gs moeAs moeCs moeAs moeG
7 A07 200 moeAs moeCs moeGs moeAs Cs Ts Gs Gs Cs Gs As Cs As Gs moeGs moeTs moeAs moeG
8 A08 200 moeAs moeCs moeAs moeGs Gs Ts As Gs Gs Ts Cs Ts Ts Gs moeGs moeTs moeGs moeG
9 A09 200 moeAs moeGs moeGs moeTs Cs Ts Ts Gs Gs Ts Gs Gs Gs Ts moeGs moeAs moeCs moeG 10 AlO 200 moeAs moeGs moeTs moeCs As Cs Gs As Cs As As Gs As As moeAs moeCs moeAs moeC
11 Al l 200 moeAs moeCs moeGs moeAs Cs As As Gs As As As Cs As Cs moeGs moeGs moeTs moeC
12 A12 200 moeAs moeGs moeAs moeAs As Cs As Cs Gs Gs Ts Cs Gs Gs moeTs moeCs moeCs moeT
13 B01 200 moeAs moeAs moeCs moeAs Cs Gs Gs Ts Cs Gs Gs Ts Cs Cs moeTs moeGs moeTs moeC
14 B02 200 moeAs moeCs moeTs moeCs As Cs Ts Gs As Cs Gs Ts Gs Ts moeCs moeTs moeCs moeA 15 B03 200 moeAs moeCs moeGs moeGs As As Gs Gs As As Cs Gs Cs Cs moeAs moeCs moeTs moeT
16 B04 200 moeAs moeTs moeCs moeTs Gs Ts Gs Gs As Cs Cs Ts Ts Gs moeTs moeCs moeTs moeC
17 B05 200 moeAs moeCs moeAs moeCs Ts Ts Cs Ts Ts Cs Cs Gs As Cs moeCs moeGs moeTs moeG
18 B06 200 moeAs moeCs moeTs moeCs Ts Cs Gs As Cs As Cs As Gs Gs moeAs moeCs moeGs moeT
19 B07 200 moeAs moeAs moeAs moeCs Cs Cs Cs As Gs Ts Ts Cs Gs Ts moeCs moeTs moeAs moeA 20 B08 200 moeAs moeTs moeGs moeTs Cs Cs Cs Cs As As As Gs As Cs moeTs moeAs moeTs moeG 66
21 B09 200 moeAs moeCs moeGs moeCs Ts Cs Gs Gs Gs As Cs Gs Gs Gs moeTs moeCs moeAs moeG
22 BIO 200 moeAs moeGs moeCs moeCs Gs As As Gs As As Gs As Gs Gs moeTs moeTs moeAs moeC 23 Bl l 200 moeAs moeCs moeAs moeCs As Gs Ts As Gs As Cs Gs As As moeAs moeGs moeCs moeT
24 B12 200 moeAs moeCs moeAs moeCs Ts Cs Ts Gs Gs Ts Ts Ts Cs Ts moeGs moeGs moeAs moeC
25 COl 200 moeAs moeCs moeGs moeAs Cs Cs As Gs As As As Ts As Gs moeTs moeTs moeTs moeT
26 C02 200 moeAs moeGs moeTs moeTs As As As As Gs Gs Gs Cs Ts Gs moeCs moeTs moeAs moeG
27 C03 200 moeAs moeGs moeGs moeTs Ts Gs Ts Gs As Cs Gs As Cs Gs moeAs moeGs moeGs moeT 28 C04 200 moeAs moeAs moeTs moeGs Ts As Cs Cs Ts As Cs Gs Gs Ts moeTs moeGs moeGs moeC
29 C05 200 moeAs moeGs moeTs moeCs As Cs Gs Ts Cs Cs Ts Cs Ts Cs moeTs moeGs moeTs moeC
30 C06 200 moeCs moeTs moeGs moeGs Cs Gs As Cs As Gs Gs Ts As Gs moeGs moeTs moeCs moeT
31 C07 200 moeCs moeTs moeCs moeTs Gs Ts Gs Ts Gs As Cs Gs Gs Ts moeGs moeGs moeTs moeC
32 C08 200 moeCs moeAs moeGs moeGs Ts Cs Gs Ts Cs Ts Ts Cs Cs Cs moeGs moeTs moeGs moeG 33 C09 200 moeCs moeTs moeGs moeTs Gs Gs Ts As Gs As Cs Gs Ts Gs moeGs moeAs moeCs moeA
34 CIO 200 moeCs moeTs moeAs moeAs Cs Gs As Ts Gs Ts Cs Cs Cs Cs moeAs moeAs moeAs moeG
35 Cl l 200 moeCs moeTs moeGs moeTs Ts Cs Gs As Cs As Cs Ts Cs Ts moeGs moeGs moeTs moeT
36 C12 200 moeCs moeTs moeGs moeGs As Cs Cs As As Cs As Cs Gs Ts 67 moeTs moeGs moeTs moeC
37 D01 200 moeCs moeCs moeGs moeTs Cs Cs Gs Ts Gs Ts Ts Ts Gs Ts moeTs moeCs moeTs moeG
38 D02 200 moeCs moeTs moeGs moeAs Cs Ts As Cs As As Cs As Gs As moeCs moeAs moeCs moeC
39 D03 200 moeCs moeAs moeAs moeCs As Gs As Cs As Cs Cs As Gs Gs moeGs moeGs moeTs moeC
40 D04 200 moeCs moeAs moeGs moeGs Gs Gs Ts Cs Cs Ts As Gs Cs Cs moeGs moeAs moeCs moeT 41 D05 200 moeCs moeTs moeCs moeTs As Gs Ts Ts As As As As Gs Gs moeGs moeCs moeTs moeG
42 D06 200 moeCs moeTs moeGs moeCs Ts As Gs As As Gs Gs As Cs Cs moeGs moeAs moeGs moeG
43 D07 200 moeCs moeTs moeGs moeAs As As Ts Gs Ts As Cs Cs Ts As moeCs moeGs moeGs moeT
44 D08 200 moeCs moeAs moeCs moeCs Cs Gs Ts Ts Ts Gs Ts Cs Cs Gs moeTs moeCs moeAs moeA
45 D09 200 moeCs moeTs moeCs moeGs As Ts As Cs Gs Gs Gs Ts Cs As moeGs moeTs moeCs moeA 46 D10 200 moeGs moeGs moeTs moeAs Gs Gs Ts Cs Ts Ts Gs Gs Ts Gs moeGs moeGs moeTs moeG
47 Dl l 200 moeGs moeAs moeCs moeTs Ts Ts Gs Cs Cs Ts Ts As Cs Gs moeGs moeAs moeAs moeG
48 D12 200 moeGs moeTs moeGs moeGs As Gs Ts Cs Ts Ts Ts Gs Ts Cs moeTs moeGs moeTs moeG
49 E01 200 moeGs moeGs moeAs moeGs Ts Cs Ts Ts Ts Gs Ts Cs Ts Gs moeTs moeGs moeGs moeT
50 E02 200 moeGs moeGs moeAs moeCs As Cs Ts Cs Ts Cs Gs As Cs As moeCs moeAs moeGs moeG 51 E03 200 moeGs moeAs moeCs moeAs Cs As Gs Gs As Cs Gs Ts Gs Gs moeCs moeGs moeAs moeG 68
52 E04 200 moeGs moeAs moeGs moeTs As Cs Gs As Gs Cs Gs Gs Gs Cs moeCs moeGs moeAs moeA
53 E05 200 moeGs moeAs moeCs moeTs As Ts Gs Gs Ts As Gs As Cs Gs moeCs moeTs moeCs moeG 54 E06 200 moeGs moeAs moeAs moeGs As Gs Gs Ts Ts As Cs As Cs As moeGs moeTs moeAs moeG
55 E07 200 moeGs moeAs moeGs moeGs Ts Ts As Cs As Cs As Gs Ts As moeGs moeAs moeCs moeG
56 E08 200 moeGs moeTs moeTs moeGs Ts Cs Cs Gs Ts Cs Cs Gs Ts Gs moeTs moeTs moeTs moeG
57 E09 200 moeGs moeAs moeCs moeTs Cs Ts Cs Gs Gs Gs As Cs Cs As moeCs moeCs moeAs moeC
58 ElO 200 moeGs moeTs moeAs moeGs Gs As Gs As As Cs Cs As Cs Gs moeAs moeCs moeCs moeA 59 El l 200 moeGs moeGs moeTs moeTs Cs Ts Ts Cs Gs Gs Ts Ts Gs Gs moeTs moeTs moeAs moeT
60 E12 200 moeGs moeTs moeGs moeGs Gs Gs Ts Ts Cs Gs Ts Cs Cs Ts moeTs moeGs moeGs moeG
61 F01 200 moeGs moeTs moeCs moeAs Cs Gs Ts Cs Cs Ts Cs Ts Gs As moeAs moeAs moeTs moeG
62 F02 200 moeGs moeTs moeCs moeCs Ts Cs Cs Ts As Cs Cs Gs Ts Ts moeTs moeCs moeTs moeC
63 F03 200 moeGs moeTs moeCs moeCs Cs Cs As Cs Gs Ts Cs Cs Gs Ts moeCs moeTs moeTs moeC 64 F04 200 moeTs moeCs moeAs moeCs Cs As Gs Gs As Cs Gs Gs Cs Gs moeGs moeAs moeCs moeC
65 F05 200 moeTs moeAs moeCs moeCs As As Gs Cs As Gs As Cs Gs Gs moeAs moeGs moeAs moeC
66 F06 200 moeTs moeCs moeCs moeTs Gs Ts Cs Ts Ts Ts Gs As Cs Cs moeAs moeCs moeTs moeC
67 F07 200 moeTs moeGs moeTs moeCs Ts Ts Ts Gs As Cs Cs As Cs Ts 69 moeCs moeAs moeCs moeT
68 F08 200 moeTs moeGs moeAs moeCs Cs As Cs Ts Cs As Cs Ts Gs As moeCs moeGs moeTs moeG
69 F09 200 moeTs moeGs moeAs moeCs Gs Ts Gs Ts Cs Ts Cs As As Gs moeTs moeGs moeAs moeC
70 F10 200 moeTs moeCs moeAs moeAs Gs Ts Gs As Cs Ts Ts Ts Gs Cs moeCs moeTs moeTs moeA
71 Fl l 200 moeTs moeGs moeTs moeTs Ts As Ts Gs As Cs Gs Cs Ts Gs moeGs moeGs moeGs moeT 72 F12 200 moeTs moeTs moeAs moeTs Gs As Cs Gs Cs Ts Gs Gs Gs Gs moeTs moeTs moeGs moeG
73 G01 200 moeTs moeGs moeAs moeCs Gs Cs Ts Gs Gs Gs Gs Ts Ts Gs moeGs moeAs moeTs moeC
74 G02 200 moeTs moeCs moeGs moeTs Cs Ts Ts Cs Cs Cs Gs Ts Gs Gs moeAs moeGs moeTs moeC
75 G03 200 moeTs moeGs moeGs moeTs As Gs As Cs Gs Ts Gs Gs As Cs moeAs moeCs moeTs moeT
76 G04 200 moeTs moeTs moeCs moeTs Ts Cs Cs Gs As Cs Cs Gs Ts Gs moeAs moeCs moeAs moeT 77 G05 200 moeTs moeGs moeGs moeTs As Gs As Cs Gs Cs Ts Cs Gs Gs moeGs moeAs moeCs moeG
78 G06 200 moeTs moeAs moeGs moeAs Cs Gs Cs Ts Cs Gs Gs Gs As Cs moeGs moeGs moeGs moeT
79 G07 200 moeTs moeTs moeTs moeTs As Cs As Gs Ts Gs Gs Gs As As moeCs moeCs moeTs moeG
80 G08 200 moeTs moeGs moeGs moeGs As As Cs Cs Ts Gs Ts Ts Cs Gs moeAs moeCs moeAs moeC
81 G09 200 moeTs moeCs moeGs moeGs Gs As Cs Cs As Cs Cs As Cs Ts moeAs moeGs moeGs moeG 82 G10 200 moeTs moeAs moeGs moeGs As Cs As As As Cs Gs Gs Ts As moeGs moeGs moeAs moeG 70
83 Gi l 200 moeTs moeGs moeCs moeTs As Gs As As Gs Gs As Cs Cs Gs moeAs moeGs moeGs moeT
84 G12 200 moeTs moeCs moeTs moeGs Ts Cs As Cs Ts Cs Cs Gs As Cs moeGs moeTs moeGs moeG
Reagent file (.tab File)
Table 5 is a .tab file for reagents necessary for synthesizing an oligonucleotides having both 2'-O-(2-methoxyethyl)nucleosides and 2'-deoxy nucleosides located therein.
Table 5 Identity of columns: GroupName, Bottle ID, ReagentName, FlowRate, Concentration. Wherein reagent name is identified using base identifier, "moe" indicated a 2'-O-(2- methoxyethyl) substituted nucleoside and "cpg" indicates a control pore glass solid support medium. The columns wrap around to next line when longer than one line.
SUPPORT
BEGIN
0 moeG moeG cpg 100
0 moe5meC moe5meC cpg 100
0 moeA moeA cpg 100
0 moeT moeT cpg 100
END
DEBLOCK
BEGIN
70 TCA TCA 100 1 END
WASH
BEGIN 65 ACN ACN 190 1 71
END
OXIDIZERS
BEGIN
68 BEAU BEAUCAGE 320 1
END
CAPPING
BEGIN
66 CAP_B CAP B 220 1
67 CAP_A CAP A 230 1
END
DEOXY THIOATE
BEGIN
31,32 Gs deoxyG 270 1
39,40 5meCs 5methyldeoxyC 270 1
37,38 As deoxyA 270 1
29,30 Ts deoxyT 270 1
Figure imgf000073_0001
END
MOE-THIOATE BEGIN
15,16 moeGs methoxyethoxyG 240 1
23,24 moe5meCs methoxyethoxyC 240 1
21,22 moeAs methoxyethoxyA 240 1
13,14 moeTs methoxyethoxyT
Figure imgf000073_0002
240 1 END
ACTIVATORS
BEGIN
5,6,7,8 SET s-ethyl-tet 280 72
Activates
DEOXY_THIOATE MOE_THIOATE END
EXAMPLE 4: Oligonucleotide Synthesis - 96 Well Plate Format
Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry using a multi well automated synthesizer utilizing input files as described in EXAMPLE 3 above. The oligonucleotides were synthesized by assembling 96 sequences simultaneously in a standard 96 well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl- diisopropyl phosphoramidites were purchased from commercial vendors (e.g. PE/ABI, Pharmacia). Non-standard nucleosides are synthesized as per known literature or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.
Following synthesis, oligonucleotides were cleaved from support and deprotected with concentrated NH4OH at elevated temperature (55-60 °C) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.
EXAMPLE 5: Alternative Oligonucleotide Synthesis
Unsubstituted and substituted phosphodiester oligonucleotides are alternately synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine.
Phosphorothioates are synthesized as per the phosphodiester oligonucleotides except the standard oxidation bottle was replaced by 0.2 M solution of 3H-1,2- benzodithiole-3-one 1,1 -dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation wait step was increased to 68 sec and was followed by the capping 73 step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55°C (18 hr), the oligonucleotides were purified by precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution.
Phosphinate oligonucleotides are prepared as described in U.S. Patent 5,508,270, incoφorated herein by reference in its entirety.
Alkyl phosphonate oligonucleotides are prepared as described in U.S. Patent 4,469,863, incoφorated herein by reference in its entirety.
3 '-Deoxy-3 '-methylene phosphonate oligonucleotides are prepared as described in U.S. Patents 5,610,289 or 5,625,050, each of which is incoφorated herein by reference in its entirety.
Phosphoramidite oligonucleotides are prepared as described in U.S. Patent, 5,256,775 or U.S. Patent 5,366,878, incoφorated herein by reference in its entirety.
Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), each of which is incoφorated herein by reference in its entirety.
3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared as described in U.S. Patent 5,476,925, incoφorated herein by reference in its entirety.
Phosphotriester oligonucleotides are prepared as described in U.S. Patent 5,023,243, incoφorated herein by reference in its entirety.
Boranophosphate oligonucleotides are prepared as described in U.S. Patents 5,130,302 and 5,177,198, each of which is incoφorated herein by reference in its entirety.
Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and PO or PS linkages are prepared as described in U.S. Patents 5,378,825; 5,386,023; 5,489,677; 5,602,240 and 5,610,289, each of which is incoφorated herein by reference in its entirety.
Formacetal and thioformacetal linked oligonucleosides are prepared as described in 74
U.S. Patents 5,264,562 and 5,264,564, each of which is incoφorated herein by reference in its entirety.
Ethylene oxide linked oligonucleosides are prepared as described in U.S. Patent 5,223,618, incoφorated herein by reference in its entirety.
EXAMPLE 6: PNA Synthesis
Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5. They may also be prepared in accordance with U.S. Patents 5,539,082; 5,700,922, and 5,719,262, each of which is incoφorated herein by reference in its entirety.
EXAMPLE 7: Chimeric Oligonucleotide Synthesis
Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the "gap" segment of linked nucleosides is positioned between 5' and 3' "wing" segments of linked nucleosides and a second "open end" type wherein the
"gap" segment is located at either the 3' or the 5' terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as "gapmers" or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as "hemimers" or "wingmers." A. [2'-O-Me]"[2'-deoxy]~[2'-O-Me] Chimeric Phosphorothioate
Oligonucleotides
Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate and 2'-deoxy phosphorothioate oligonucleotide segments are synthesized using 2'-deoxy-5'- dimethoxytrityl-3'-O-phosphoramidites for the DNA portion and 5'-dimethoxytrityl-2'-O- methyl-3'-O-phosphoramidites for 5' and 3' wings. The standard synthesis cycle is modified by increasing the wait step after the delivery of tetrazole and base to 600 s repeated four times for DNA and twice for 2'-0-methyl. The fully protected oligonucleotide was cleaved from the support and the phosphate group is deprotected in 3:1 Ammonia Ethanol at room temperature overnight then lyophilized to dryness. 75
Treatment in methanolic ammonia for 24 hrs at room temperature is done to deprotect all bases and the samples are again lyophilized to dryness.
B. [2'-O-(2-Methoxyethyl)]--[2'-deoxy]--[2'-O-(2-Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides [2'-O-(2-methoxyethyl)]~[2'-deoxy]-[-2'-O-(2-methoxyethyl)] chimeric phosphorothioate oligonucleotides are prepared as per the procedure above for the 2'-O- methyl chimeric oligonucleotide, with the substitution of 2'-O-(2-methoxyethyl) amidites for the 2'-O-methyl amidites.
C. [2'-O-(2-Methoxyethyl)Phosphodiester]—[2'-deoxy Phosphorothioate]— [2'-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotide
[2'-O-(2-methoxyethyl phosphodiester]~[2'-deoxy phosphorothioate]— [2'-O-(2- methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2'-O-methyl chimeric oligonucleotide with the substitution of 2'-O-(2- methoxy ethyl) amidites for the 2'-O-methyl amidites in the wing portions. Sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) is used to generate the phosphorothioate internucleotide linkages within the wing portions of the chimeric structures. Oxidization with iodine is used to generate the phosphodiester internucleotide linkages for the center gap.
Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to United States Patent
5,623,065, which is incoφorated herein by reference in its entirety.
EXAMPLE 8: Output Oligonucleotides From Automated Oligonucleotide Synthesis
Using the .seq files, the .cmd files and .tab file of Example 3, oligonucleotides were prepared as per the protocol of the 96 well format of Example 4. The oligonucleotides were prepared utilizing phosphorothioate chemistry to give in one instance a first library of phosphorothioate oligodeoxynucleotides. The oligonucleotides were prepared in a second instance as a second library of hybrid oligonucleotides having phosphorothioate backbones with a first and third "wing" region of 2'-O-(2- methoxyethyl)nucleotides on either side of a center gap region of 2'-deoxy nucleotides. The two libraries contained the same set of oligonucleotide sequences. Thus the two 76 libraries are redundant with respect to sequence but are unique with respect to the combination of sequence and chemistry. Because the sequences of the second library of compounds is the same as the first (however the chemistry is different), for brevity sake, the second library is not shown. For illustrative puφoses Tables 6-a and 6-b show the sequences of an initial first library, i.e., a library of phosphorothioate oligonucleotides targeted to a CD40 target. The compounds of Table 6-a shows the members of this library listed in compliance with the established rule for listing SEQ ID NO:, i.e., in numerical SEQ ID NO: order.
Table 6-a Sequences of Oligonucleotides Targeted to CD40 by SEQ ID NO.:
NUCLEOBASE SEQUENCE SEQ ID NO.
CCAGGCGGCAGGACCACT 1
GACCAGGCGGCAGGACCA 2
AGGTGAGACCAGGCGGCA 3 CAGAGGCAGACGAACCAT 4
GCAGAGGCAGACGAACCA 5
GCAAGCAGCCCCAGAGGA 6
GGTCAGCAAGCAGCCCCA 7
GACAGCGGTCAGCAAGCA 8 GATGGACAGCGGTCAGCA 9
TCTGGATGGACAGCGGTC 10
GGTGGTTCTGGATGGACA 11
GTGGGTGGTTCTGGATGG 12
GCAGTGGGTGGTTCTGGA 13 CACAAAGAACAGCACTGA 14
CTGGCACAAAGAACAGCA 15
TCCTGGCTGGCACAAAGA 16
CTGTCCTGGCTGGCACAA 17
CTCACCAGTTTCTGTCCT 18 TCACTCACCAGTTTCTGT 19 77
GTGCAGTCACTCACCAGT 20
ACTCTGTGCAGTCACTCA 21
CAGTGAACTCTGTGCAGT 22
ATTCCGTTTCAGTGAACT 23 GAAGGCATTCCGTTTCAG 24
TTCACCGCAAGGAAGGCA 25
CTCTGTTCCAGGTGTCTA 26
CTGGTGGCAGTGTGTCTC 27
TGGGGTCGCAGTATTTGT 28 GGTTGGGGTCGCAGTATT 29
CTAGGTTGGGGTCGCAGT 30
GGTGCCCTTCTGCTGGAC 31
CTGAGGTGCCCTTCTGCT 32
GTGTCTGTTTCTGAGGTG 33 TGGTGTCTGTTTCTGAGG 34
ACAGGTGCAGATGGTGTC 35
TTCACAGGTGCAGATGGT 36
GTGCCAGCCTTCTTCACA 37
TACAGTGCCAGCCTTCTT 38 GGACACAGCTCTCACAGG 39
TGCAGGACACAGCTCTCA 40
GAGCGGTGCAGGACACAG 41
AAGCCGGGCGAGCATGAG 42
AATCTGCTTGACCCCAAA 43 GAAACCCCTGTAGCAATC 44
GTATCAGAAACCCCTGTA 45
GCTCGCAGATGGTATCAG 46
GCAGGGCTCGCAGATGGT 47
TGGGCAGGGCTCGCAGAT 48 GACTGGGCAGGGCTCGCA 49
CATTGGAGAAGAAGCCGA 50 78
GATGACACATTGGAGAAG 51
GCAGATGACACATTGGAG 52
TCGAAAGCAGATGACACA 53
GTCCAAGGGTGACATTTT 54 CACAGCTTGTCCAAGGGT 55
TTGGTCTCACAGCTTGTC 56
CAGGTCTTTGGTCTCACA 57
CTGTTGCACAACCAGGTC 58
GTTTGTGCCTGCCTGTTG 59 GTCTTGTTTGTGCCTGCC 60
CCACAGACAACATCAGTC 61
CTGGGGACCACAGACAAC 62
TCAGCCGATCCTGGGGAC 63
CACCACCAGGGCTCTCAG 64 GGGATCACCACCAGGGCT 65
GAGGATGGCAAACAGGAT 66
ACCAGCACCAAGAGGATG 67
TTTTGATAAAGACCAGCA 68
TATTGGTTGGCTTCTTGG 69 GGGTTCCTGCTTGGGGTG 70
GTCGGGAAAATTGATCTC 71
GATCGTCGGGAAAATTGA 72
GGAGCCAGGAAGATCGTC 73
TGGAGCCAGGAAGATCGT 74 TGGAGCAGCAGTGTTGGA 75
GTAAAGTCTCCTGCACTG 76
TGGCATCCATGTAAAGTC 77
CGGTTGGCATCCATGTAA 78
CTCTTTGCCATCCTCCTG 79 CTGTCTCTCCTGCACTGA 80
GGTGCAGCCTCACTGTCT 81 79
AACTGCCTGTTTGCCCAC 82 CTTCTGCCTGCACCCCTG 83 ACTGACTGGGCATAGCTC 84
The sequences shown in Table 6-a, above, and Table 6-b, below, are in a 5' to 3' direction. This is reversed with respect to 3' to 5' direction shown in the .seq files of Example 3. For synthesis puφoses, the .seq files are generated reading from 3' to 5'. This allows for aligning all of the 3' most "A" nucleosides together, all of the 3' most "G" nucleosides together, all of the 3' most "C" nucleosides together and all of the 3' most "T" nucleosides together. Thus when the first nucleoside of each particular oligonucleotide
(attached to the solid support) is added to the wells on the plates, machine movement is reduced since an automatic pipette can move in a linear manner down one row and up another on the 96 well plate.
The location of the well holding each particular oligonucleotides is indicated by row and column. There are eight rows designated A to G and twelve columns designated
1 to 12 in a typical 96 well format plate. Any particular well location is indicated by its "Well No." which is indicated by the combination of the row and the column, e.g. A08 is the well at row A, column 8.
In Table 6-b below, the oligonucleotides of Table 6-a are shown reordered according to the Well No. on their synthesis plate. The order shown in Table 6-b is the actually order as synthesized on an automated synthesizer taking advantage of the preferred placement of the first nucleoside according to the above alignment criteria.
Table 6-b: Sequences of Oligonucleotides Targeted to CD40 Order by Synthesis Well No.
Well No. SEQ ID NO:
A01 GACCAGGCGGCAGGACCA 2
A02 AGGTGAGACCAGGCGGCA 3
A03 GCAGAGGCAGACGAACCA 5
A04 GCAAGCAGCCCCAGAGGA 6
A05 GGTCAGCAAGCAGCCCCA 7
A06 GACAGCGGTCAGCAAGCA 8
A07 GATGGACAGCGGTCAGCA 9
Figure imgf000081_0001
A08 GGTGGTTCTGGATGGACA 11 80
A09 GCAGTGGGTGGTTCTGGA 13
AlO CACAAAGAACAGCACTGA 14
Al l CTGGCACAAAGAACAGCA 15
A12 TCCTGGCTGGCACAAAGA 16
B01 CTGTCCTGGCTGGCACAA 17
B02 ACTCTGTGCAGTCACTCA 21
B03 TTCACCGCAAGGAAGGCA 25
B04 CTCTGTTCCAGGTGTCTA 26
B05 GTGCCAGCCTTCTTCACA 37
B06 TGCAGGACACAGCTCTCA 40
B07 AATCTGCTTGACCCCAAA 43
B08 GTATCAGAAACCCCTGTA 45
B09 GACTGGGCAGGGCTCGCA 49
BIO CATTGGAGAAGAAGCCGA 50
Bl l TCGAAAGCAGATGACACA 53
B12 CAGGTCTTTGGTCTCACA 57
COl TTTTGATAAAGACCAGCA 68
C02 GATCGTCGGGAAAATTGA 72
C03 TGGAGCAGCAGTGTTGGA 75
C04 CGGTTGGCATCCATGTAA 78
C05 CTGTCTCTCCTGCACTGA 80
C06 TCTGGATGGACAGCGGTC 10
C07 CTGGTGGCAGTGTGTCTC 27
C08 GGTGCCCTTCTGCTGGAC 31
C09 ACAGGTGCAGATGGTGTC 35
CIO GAAACCCCTGTAGCAATC 44
Cl l TTGGTCTCACAGCTTGTC 56
C12 CTGTTGCACAACCAGGTC 58
D01 GTCTTGTTTGTGCCTGCC 60
D02 CCACAGACAACATCAGTC 61
D03 CTGGGGACCACAGACAAC 62
D04 TCAGCCGATCCTGGGGAC 63
D05 GTCGGGAAAATTGATCTC 71
D06 GGAGCCAGGAAGATCGTC 73
D07 TGGCATCCATGTAAAGTC 77
D08 AACTGCCTGTTTGCCCAC 82
D09 ACTGACTGGGCATAGCTC 84
D10 GTGGGTGGTTCTGGATGG 12
Dl l GAAGGCATTCCGTTTCAG 24
D12 GTGTCTGTTTCTGAGGTG 33
E01 TGGTGTCTGTTTCTGAGG 34
E02 GGACACAGCTCTCACAGG 39
E03 GAGCGGTGCAGGACACAG 41
E04 AAGCCGGGCGAGCATGAG 42
E05 GCTCGCAGATGGTATCAG 46
E06 GATGACACATTGGAGAAG 51
E07 GCAGATGACACATTGGAG 52
E08 GTTTGTGCCTGCCTGTTG 59
E09 CACCACCAGGGCTCTCAG 64
ElO ACCAGCACCAAGAGGATG 67
Figure imgf000082_0001
El l TATTGGTTGGCTTCTTGG 69 81
E12 GGGTTCCTGCTTGGGGTG 70
F01 GTAAAGTCTCCTGCACTG 76
F02 CTCTTTGCCATCCTCCTG 79
F03 CTTCTGCCTGCACCCCTG 83
F04 CCAGGCGGCAGGACCACT 1
F05 CAGAGGCAGACGAACCAT 4
F06 CTCACCAGTTTCTGTCCT 18
F07 TCACTCACCAGTTTCTGT 19
F08 GTGCAGTCACTCACCAGT 20
F09 CAGTGAACTCTGTGCAGT 22
F10 ATTCCGTTTCAGTGAACT 23
Fll TGGGGTCGCAGTATTTGT 28
F12 GGTTGGGGTCGCAGTATT 29
G01 CTAGGTTGGGGTCGCAGT 30
G02 CTGAGGTGCCCTTCTGCT 32
G03 TTCACAGGTGCAGATGGT 36
G04 TACAGTGCCAGCCTTCTT 38
G05 GCAGGGCTCGCAGATGGT 47
G06 TGGGCAGGGCTCGCAGAT 48
G07 GTCCAAGGGTGACATTTT 54
G08 CACAGCTTGTCCAAGGGT 55
G09 GGGATCACCACCAGGGCT 65
G10 GAGGATGGCAAACAGGAT 66
Gil TGGAGCCAGGAAGATCGT 74
Figure imgf000083_0001
G12 GGTGCAGCCTCACTGTCT 81
EXAMPLE 9: Oligonucleotide Analysis
A. Oligonucleotide Analysis - 96 Well Plate Format
The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absoφtion spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96 well format (Beckman MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors.
B. Alternative Oligonucleotide Analysis
After cleavage from the controlled pore glass support (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55°C for 18 hours, the oligonucleotides or oligonucleosides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides are analyzed by polyacrylamide gel electrophoresis on denaturing gels. Oligonucleotide purity is checked by 31P nuclear 82 magnetic resonance spectroscopy, and/or by HPLC, as described by Chiang et al, J. Biol. Chem. 1991, 266, 18162.
EXAMPLE 10: Automated Assay of CD40 Oligonucleotide Activity A. Poly(A)+ mRNA isolation.
Poly(A)+ mRNA was isolated according to Miura et al. {Clin. Chem., 1996, 42, 1758). Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μl cold PBS. 60 μl lysis buffer (10 mM Tris- HC1, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μl of lysate was transferred to Oligo d(T) coated 96 well plates (AGCT Inc., Irvine, CA). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 ml of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 ml of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70 °C was added to each well, the plate was incubated on a 90° C plate for 5 minutes, and the eluate then transferred to a fresh 96-well plate. Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions. B. Total RNA isolation
Total mRNA was isolated using an RNEASY 960 kit and buffers purchased from Qiagen Inc. (Valencia CA) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 mL cold PBS. 100 mL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 mL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 960 well plate attached to a QIAVACO manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 960 plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY 960 plate and the vacuum applied for a period of 15 seconds. The 83
Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes. The plate was then removed from the QIAVACO manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVACO manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 60 mL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 mL water.
C. RT-PCR Analysis of CD40 mRNA Levels
Quantitation of CD40 mRNA levels was determined by reverse transcriptase polymerase chain reaction (RT-PCR) using the ABI PRISM™_7700 Sequence Detection
System (PE- Applied Biosystems, Foster City, CA) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in realtime. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in RT-PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE or FAM, PE-Applied Biosystems, Foster City, CA) is attached to the 5' end of the probe and a quencher dye (e.g., TAMRA, PE-Applied
Biosystems, Foster City, CA) is attached to the 3' end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3' quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5'-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated.
With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular (six-second) intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from 84 untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.
RT-PCR reagents were obtained from PE-Applied Biosystems, Foster City, CA. RT-PCR reactions were carried out by adding 25 ml PCR cocktail (lx TAQMAN™ buffer A, 5.5 mM MgCl2, 300 mM each of dATP, dCTP and dGTP, 600 mM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 U RNAse inhibitor, 1.25 units AMPLITAQ GOLD™, and 12.5 U MuLV reverse transcriptase) to 96 well plates containing 25 ml poly(A) mRNA solution. The RT reaction was carried out by incubation for 30 minutes at 48°C. following a 10 minute incubation at 95°C to activate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carried out: 95 °C for
15 seconds (denaturation) followed by 60° C for 1.5 minutes (annealing/extension).
For CD40, the PCR primers were: forward: 5' CAGAGTTCACTGAAACGGAATGC 3'
(SEQ ID NO:86) reverse: 5' GGTGGCAGTGTGTCTCTCTGTTC 3' (SEQ ID NO:87), and
PCR probe: 5' E4 -TTCCTTGCGGTGAAAGCGAATTCCT-7 i 4 3' (SEQ ID NO:88) where FAM (PE-Applied Biosystems, Foster City, CA) is the fluorescent reporter dye and TAMRA (PE-Applied Biosystems, Foster City, CA) is the quencher dye. For GAPDH, the PCR primers were: forward: 5' GAAGGTGAAGGTCGGAGTC 3' (SEQ ID NO:89) reverse: 5' GAAGATGGTGATGGGATTTC 3' (SEQ ID NO:90), and
PCR probe: 5* JOE-CAAGCTTCCCGTTCTCAGCC-7 Λ4 3' (SΕQ ID NO. 91) where JOE (PΕ- Applied Biosystems, Foster City, CA) is the fluorescent reporter dye and TAMRA (PΕ- Applied Biosystems, Foster City, CA) is the quencher dye.
EXAMPLE 11: Inhibition of CD40 Expression by Phosphorothioate Oligodeoxynucleotides
In accordance with the present invention, a series of oligonucleotides complementary to mRNA were designed to target different regions of the human CD40 mRNA, using published sequences (GenBank accession number X60592, incoφorated herein by reference as SEQ ID NO: 85). The oligonucleotides are shown in Table 7. 85
Target sites are indicated by the beginning nucleotide numbers, as given in the sequence source reference (X60592), to which the oligonucleotide binds. All compounds in Table 7 are oligodeoxynucleotides with phosphorothioate backbones (internucleoside linkages) throughout. Data are averages from three experiments.
Table 7: Inhibition of CD40 mRNA Levels by Phosphorothioate Oligodeoxynucleotides TARGET SEQ ID
ISIS# SITE SEQUENCE % INHIB. NO.
18623 18 CCAGGCGGCAGGACCA 30.71 1
18624 20 GACCAGGCGGCAGGAC 28.09 2
18625 26 AGGTGAGACCAGGCGG 21.89 3
18626 48 CAGAGGCAGACGAACC 0.00 4
18627 49 GCAGAGGCAGACGAAC 0.00 5
18628 73 GCAAGCAGCCCCAGAG 0.00 6
18629 78 GGTCAGCAAGCAGCCC 29.96 7
18630 84 GACAGCGGTCAGCAAG 0.00 8
18631 88 GATGGACAGCGGTCAG 0.00 9
18632 92 TCTGGATGGACAGCGG 0.00 10
18633 98 GGTGGTTCTGGATGGA 0.00 11
18634 101 GTGGGTGGTTCTGGAT 0.00 12
18635 104 GCAGTGGGTGGTTCTG 0.00 13
18636 152 CACAAAGAACAGCACT 0.00 14
18637 156 CTGGCACAAAGAACAG 0.00 15
18638 162 TCCTGGCTGGCACAAA 0.00 16
18639 165 CTGTCCTGGCTGGCAC 4.99 17
18640 176 CTCACCAGTTTCTGTCC 0.00 18
18641 179 TCACTCACCAGTTTCTG 0.00 19
18642 185 GTGCAGTCACTCACCA 0.00 20
18643 190 ACTCTGTGCAGTCACTC 0.00 21
18644 196 CAGTGAACTCTGTGCA 5.30 22
18645 205 ATTCCGTTTCAGTGAAC 0.00 23
18646 211 GAAGGCATTCCGTTTC 9.00 24
18647 222 TTCACCGCAAGGAAGG 0.00 25
18648 250 CTCTGTTCCAGGTGTCT 0.00 26
18649 267 CTGGTGGCAGTGTGTC 0.00 27
18650 286 TGGGGTCGCAGTATTT 0.00 28
18651 289 GGTTGGGGTCGCAGTA 0.00 29
18652 292 CTAGGTTGGGGTCGCA 0.00 30
18653 318 GGTGCCCTTCTGCTGG 19.67 31
18654 322 CTGAGGTGCCCTTCTGC 15.63 32
18655 332 GTGTCTGTTTCTGAGGT 0.00 33
18656 334 TGGTGTCTGTTTCTGAG 0.00 34
18657 345 ACAGGTGCAGATGGTG 0.00 35
Figure imgf000087_0001
18658 348 TTCACAGGTGCAGATG 0.00 36 86
18659 360 GTGCCAGCCTTCTTCAC 5.67 37
18660 364 TACAGTGCCAGCCTTCT 7.80 38
18661 391 GGACACAGCTCTCACA 0.00 39
18662 395 TGCAGGACACAGCTCT 0.00 40
18663 401 GAGCGGTGCAGGACAC 0.00 41
18664 416 AAGCCGGGCGAGCATG 0.00 42
18665 432 AATCTGCTTGACCCCA 5.59 43
18666 446 GAAACCCCTGTAGCAA 0.10 44
18667 452 GTATCAGAAACCCCTG 0.00 45 1 188666688 4 46633 GCTCGCAGATGGTATC 0.00 46
18669 468 GCAGGGCTCGCAGATG 34.05 47
18670 471 TGGGCAGGGCTCGCAG 0.00 48
18671 474 GACTGGGCAGGGCTCG 2.71 49
18672 490 CATTGGAGAAGAAGCC 0.00 50 1 188667733 4 49977 GATGACACATTGGAGA 0.00 51
18674 500 GCAGATGACACATTGG 0.00 52
18675 506 TCGAAAGCAGATGACA 0.00 53
18676 524 GTCCAAGGGTGACATT 8.01 54
18677 532 CACAGCTTGTCCAAGG 0.00 55 1 188667788 5 53399 TTGGTCTCACAGCTTGT 0.00 56
18679 546 CAGGTCTTTGGTCTCAC 6.98 57
18680 558 CTGTTGCACAACCAGG 18.76 58
18681 570 GTTTGTGCCTGCCTGTT 2.43 59
18682 575 GTCTTGTTTGTGCCTGC 0.00 60 1 188668833 5 59900 CCACAGACAACATCAG 0.00 61
18684 597 CTGGGGACCACAGACA 0.00 62
18685 607 TCAGCCGATCCTGGGG 0.00 63
18686 621 CACCACCAGGGCTCTC 23.31 64
18687 626 GGGATCACCACCAGGG 0.00 65 1 188668888 6 65577 GAGGATGGCAAACAGG 0.00 66
18689 668 ACCAGCACCAAGAGGA 0.00 67
18690 679 TTTTGATAAAGACCAG 0.00 68
18691 703 TATTGGTTGGCTTCTTG 0.00 69
18692 729 GGGTTCCTGCTTGGGG 0.00 70 1 188669933 7 75500 GTCGGGAAAATTGATC 0.00 71
18694 754 GATCGTCGGGAAAATT 0.00 72
18695 765 GGAGCCAGGAAGATCG 0.00 73
18696 766 TGGAGCCAGGAAGATC 0.00 74
18697 780 TGGAGCAGCAGTGTTG 0.00 75 1 188669988 7 79966 GTAAAGTCTCCTGCAC 0.00 76
18699 806 TGGCATCCATGTAAAG 0.00 77
18700 810 CGGTTGGCATCCATGT 0.00 78
18701 834 CTCTTTGCCATCCTCCT 4.38 79
18702 861 CTGTCTCTCCTGCACTG 0.00 80 1 188770033 8 87733 GGTGCAGCCTCACTGT 0.00 81
18704 910 AACTGCCTGTTTGCCCA 33.89 82
18705 954 CTTCTGCCTGCACCCCT 0.00 83
Figure imgf000088_0001
18706 976 ACTGACTGGGCATAGC
Figure imgf000088_0002
0.00 84 87
As shown in Table 7, SEQ ID NOS: 1, 2, 7, 47 and 82 demonstrated at least 25% inhibition of CD40 expression and are therefore preferred compounds of the invention.
EXAMPLE 12: Inhibition of CD40 Expression by Phosphorothioate 2'-MOE Gapmer Oligonucleotides In accordance with the present invention, a second series of oligonucleotides complementary to mRNA were designed to target different regions of the human CD40 mRNA, using published sequence X60592. The oligonucleotides are shown in Table 8. Target sites are indicated by the beginning or initial nucleotide numbers, as given in the sequence source reference (X60592), to which the oligonucleotide binds. All compounds in Table 8 are chimeric oligonucleotides ("gapmers") 18 nucleotides in length, composed of a central "gap" region consisting often 2'- deoxynucleo tides, which is flanked on both sides (5' and 3' directions) by four-nucleotide "wings." The wings are composed of 2'-O-(2-methoxyethyl) (2'-MOE) nucleotides. The intersugar (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2'-MOE wings are 5-methylcytidines.
Data are averaged from three experiments.
Table 8: Inhibition of CD40 mRNA Levels by Chimeric Phosphorothioate Oligonucleotides ISIS# TARGET SEQUENCE % Inhibition SEQ ID
1 199221111 Ϊ 188 CCAGGCGGCAGGACCA 75.71 1
19212 20 GACCAGGCGGCAGGA 77.23 2
19213 26 AGGTGAGACCAGGCG 80.82 3
19214 48 CAGAGGCAGACGAAC 23.68 4
19215 49 GCAGAGGCAGACGAA 45.97 5 1 199221166 7 733 GCAAGCAGCCCCAGAG 65.80 6
19217 78 GGTCAGCAAGCAGCCC 74.73 7
19218 84 GACAGCGGTCAGCAAG 67.21 8
19219 88 GATGGACAGCGGTCAG 65.14 9
19220 92 TCTGGATGGACAGCGG 78.71 10 1 199222211 9 988 GGTGGTTCTGGATGGA 81.33 11
19222 101 GTGGGTGGTTCTGGAT 57.79 12
19223 104 GCAGTGGGTGGTTCTG 73.70 13
19224 152 CACAAAGAACAGCACT 40.25 14
Figure imgf000089_0002
19225 156 CTGGCACAAAGAACAG
Figure imgf000089_0001
60.11 15 88
19226 162 TCCTGGCTGGCACAAA 10.18 16
19227 165 CTGTCCTGGCTGGCAC 24.37 17
19228 176 CTCACCAGTTTCTGTC 22.30 18
19229 179 TCACTCACCAGTTTCT 40.64 19
19230 185 GTGCAGTCACTCACCA 82.04 20
19231 190 ACTCTGTGCAGTCACT 37.59 21
19232 196 CAGTGAACTCTGTGCA 40.26 22
19233 205 ATTCCGTTTCAGTGAA 56.03 23
19234 211 GAAGGCATTCCGTTTC 32.21 24
19235 222 TTCACCGCAAGGAAGG 61.03 25
19236 250 CTCTGTTCCAGGTGTCT 62.19 26
19237 267 CTGGTGGCAGTGTGTC 70.32 27
19238 286 TGGGGTCGCAGTATTT 0.00 28
19239 289 GGTTGGGGTCGCAGTA 19.40 29
19240 292 CTAGGTTGGGGTCGCA 36.32 30
19241 318 GGTGCCCTTCTGCTGG 78.91 31
19242 322 CTGAGGTGCCCTTCTG 69.84 32
19243 332 GTGTCTGTTTCTGAGG 63.32 33
19244 334 TGGTGTCTGTTTCTGA 42.83 34
19245 345 ACAGGTGCAGATGGTG 73.31 35
19246 348 TTCACAGGTGCAGATG 47.72 36
19247 360 GTGCCAGCCTTCTTCA 61.32 37
19248 364 TACAGTGCCAGCCTTC 46.82 38
19249 391 GGACACAGCTCTCACA 0.00 39
19250 395 TGCAGGACACAGCTCT 52.05 40
19251 401 GAGCGGTGCAGGACAC 50.15 41
19252 416 AAGCCGGGCGAGCATG 32.36 42
19253 432 AATCTGCTTGACCCCA 0.00 43
19254 446 GAAACCCCTGTAGCAA 0.00 44
19255 452 GTATCAGAAACCCCTG 36.13 45
19256 463 GCTCGCAGATGGTATC 64.65 46
19257 468 GCAGGGCTCGCAGATG 74.95 47
19258 471 TGGGCAGGGCTCGCAG 0.00 48
19259 474 GACTGGGCAGGGCTCG 82.00 49
19260 490 CATTGGAGAAGAAGCC 41.31 50
19261 497 GATGACACATTGGAGA 13.81 51
19262 500 GCAGATGACACATTGG 78.48 52
19263 506 TCGAAAGCAGATGACA 59.28 53
19264 524 GTCCAAGGGTGACATT 70.99 54
19265 532 CACAGCTTGTCCAAGG 0.00 55
19266 539 TTGGTCTCACAGCTTG 45.92 56
19267 546 CAGGTCTTTGGTCTCA 63.95 57
19268 558 CTGTTGCACAACCAGG 82.32 58
19269 570 GTTTGTGCCTGCCTGTT 70.10 59
19270 575 GTCTTGTTTGTGCCTGC 68.95 60
19271 590 CCACAGACAACATCAG 11.22 61
19272 597 CTGGGGACCACAGACA 9.04 62
19273 607 TCAGCCGATCCTGGGG 0.00 63
19274 621 CACCACCAGGGCTCTC 23.08 64
19275 626 GGGATCACCACCAGGG 57.94 65
Figure imgf000090_0001
19276 657 GAGGATGGCAAACAG
Figure imgf000090_0002
49.14 66 89
19277 668 ACCAGCACCAAGAGG 3.48 67
19278 679 TTTTGATAAAGACCAG 30.58 68
19279 703 TATTGGTTGGCTTCTTG 49.26 69
19280 729 GGGTTCCTGCTTGGGG 13.95 70
19281 750 GTCGGGAAAATTGATC 54.78 71
19282 754 GATCGTCGGGAAAATT 0.00 72
19283 765 GGAGCCAGGAAGATC 69.47 73
19284 766 TGGAGCCAGGAAGATC 54.48 74
19285 780 TGGAGCAGCAGTGTTG 15.17 75
19286 796 GTAAAGTCTCCTGCAC 30.62 76
19287 806 TGGCATCCATGTAAAG 65.03 77
19288 810 CGGTTGGCATCCATGT 34.49 78
19289 834 CTCTTTGCCATCCTCCT 41.84 79
19290 861 CTGTCTCTCCTGCACT 25.68 80
19291 873 GGTGCAGCCTCACTGT 76.27 81
19292 910 AACTGCCTGTTTGCCC 63.34 82
19293 954 CTTCTGCCTGCACCCC 0.00 83
Figure imgf000091_0001
19294 976 ACTGACTGGGCATAGC 11.55 84
As shown in Table 8, SEQ ID NOS: 1, 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 15, 20, 23,
25, 26, 27, 31, 32, 33, 35, 37, 40, 41, 46, 47, 49, 52, 53, 54, 57, 58, 59, 60, 65, 71, 73, 74, 77, 81 and 82 demonstrated at least 50% inhibition of CD40 expression and are therefore preferred compounds of the invention.
EXAMPLE 13: Oligonucleotide-Sensitive Sites of the CD40 Target Nucleic Acid
As the data presented in the preceding two Examples shows, several sequences were present in preferred compounds of two distinct oligonucleotide chemistries. Specifically, compounds having SEQ ID NOS: 1, 2, 7, 47 and 82 are preferred in both instances. These compounds map to different regions of the CD40 transcript but nevertheless define accessible sites of the target nucleic acid.
For example, SEQ ID NOS: 1 and 2 overlap each other and both map to the 5- untranslated region (5'-UTR) of CD40. Accordingly, this region of CD40 is particularly preferred for modulation via sequence-based technologies. Similarly, SEQ ID NOS: 7 and 47 map to the open reading frame of CD40, whereas SEQ ID NO: 82 maps to the 3'- untranslated region (3'-UTR). Thus, the ORF and 3'-UTR of CD40 may be targeted by sequence-based technologies as well.
The reverse complements of the active CD40 compounds are easily determined by those skilled in the art and may be assembled to yield nucleotide sequences corresponding 90 to accessible sites on the target nucleic acid. For example, the assembled reverse complement of SEQ ID NOS: 1 and 2 is represented below as SEQ ID NO:92: 5'- AGTGGTCCTGCCGCCTGGTC -3' SEQ ID NO:92 TCACCAGGACGGCGGACC -5' SEQ ID NO: 1 ACCAGGACGGCGGACCAG -5' SEQ ID NO:2
Through multiple iterations of the process of the invention, more extensive
"footprints" are generated. A library of this information is compiled and may be used by those skilled in the art in a variety of sequence-based technologies to study the molecular and biological functions of CD40 and to investigate or confirm its role in various diseases and disorders.
EXAMPLE 14: Site Selection Program
In a preferred embodiment of the invention, illustrated in Figure 20, an application is deployed which facilitates the selection process for determining the target positions of the oligos to be synthesized, or "sites." This program is written using a three-tiered object-oriented approach. All aspects of the software described, therefore, are tightly integrated with the relational database. For this reason, explicit database read and write steps are not shown. It should be assumed that each step described includes database access. The description below illustrates one way the program can be used. The actual interface allows users to skip from process to process at will, in any order.
Before running the site picking program, the target must have all relevant properties computed as described previously and indicated in process step 2204. When the site picking program is launched at process step 2206 the user is presented with a panel showing targets which have previously been selected and had their properties calculated. The user selects one target to work with at process step 2208 and proceeds to decide if any derived properties will be needed at process step 2210. Derived properties are calculated by performing mathematical operations on combinations of pre-calculated properties as defined by the user at process step 2212.
The derived properties are made available as peers with all the pre-calculated properties. The user selects one of the properties to view plotted versus target position at process step 2214. This graph is shown above a linear representation of the target. The 91 horizontal or position axis of both the graph and target are linked and scalable by the user. The zoom range goes from showing the full target length to showing individual target bases as letters and individual property points. The user next selects a threshold value below or above which all sites will be eliminated from future consideration at process step 2216. The user decides whether to eliminate more sites based on any other properties at process step 2218. If they choose to eliminate more, they return to pick another property to display at process step 2214 and threshold at process step 2216.
After eliminating sites, the user selects from the remaining list by choosing any property at process step 2220 and then choosing a manual or automatic selection technique at process step 2222. In the automatic technique, the user decides whether they want to pick from maxima or minima and the number of maxima or minima to be selected as sites at process step 2224. The software automatically finds and picks the points. When picking manually the user must decide if they wish to use automatic peak finding at process step 2226. If the user selects automatic peak finding, then user must click on the graphed property with the mouse at process step 2236. The nearest maxima or minima, depending on the modifier key held down, to the selected point will be picked as the site. Without the peak finding option, the user must pick a site at process step 2238 by clicking on its position on the linear representation of target.
Each time a site, or group of sites, is picked, a dynamic property is calculated for all possible sites (not yet eliminated) at process step 2230. This property indicates the nearness of the site to a picked site allowing the user to pick sites in subsequent iterations based on target coverage. After new sites are picked, the user determines if the desired number of sites has been picked. If too few sites have been picked the user returns to pick more 2220. If too many sites have been picked, the user may eliminate them by selecting and deleting them on the target display at process step 2234. If the correct number of sites is picked, and the user is satisfied with the set of picked sites, the user registers these sites to the database along with their name, notebook number, and page number at process step 2238. The database time stamps this registration event. 92
EXAMPLE 15: Site Selection Program
In a preferred embodiment of the invention, illustrated in Figure 21, an application is deployed which facilitates the assignment of specific chemical structure to the complement of the sequence of the sites previously picked and facilitates the registration and ordering of these now fully defined antisense compounds. This program is written using a three- tiered object-oriented approach. All aspects of the software described, therefore, are tightly integrated with the relational database. For this reason, explicit database read and write steps are not shown, it being understood that each step described also includes appropriate database read/write access. To begin using the oligonucleotide chemistry assignment program, the user launches it at process step 2302. The user then selects from the previously selected sets of oligonucleotides at process step 2304, registered to the database in site picker's process step 2238. Next, the user must decide whether to manually assign the chemistry a base at a time, or run the sites through a template at process step 2306. If the user chooses to use a template, they must determine if a desired template is available at process step 2308. If a template is not available with the desired chemistry modifications and the correct length, the user can define one at process step 2314.
To define a template, the user must select the length of the oligonucleotide the template is to define. This oligonucleotide is then represented as a bar with selectable regions. The user sets the number of regions on the oligonucleotide, and the positions and lengths of these regions by dragging them back and forth on the bar. Each region is represented by a different color.
For each region, the user defines the chemistry modifications for the sugars, the linkers, and the heterocycles at each base position in the region. At least four heterocycle chemistries must be given, one for each of the four possible base types (A, G, C or T or U) in the site sequence the template will be applied to. A user interface is provided to select these chemistries which show the molecular structure of each component selected and its modification name. By pushing on a pop-up list next to each of the pictures, the user may choose from a list of structures and names, those possible to put in this place. For example, the heterocycle that represents the base type G is shown as a two dimensional structure diagram. If the user clicks on the pop-up list, a row of other possible structures 93 and names is shown. The user drags the mouse to the desired chemistry and releases the mouse. Now the newly selected molecule is displayed as the choice for G type heterocycle modifications.
Once the user has created a template, or selected an existing one, the software applies the template at process step 2312 to each of the complements of the sites in the list.
When the templates are applied, it is possible that chemistries will be defined which are impossible to make with the chemical precursors presently used on the automatic synthesizer. To check this, a database is maintained of all precursors previously designed, and their availability for automated synthesis. When the templates are applied, the resulting molecules are tested at process step 2316 against this database to see if they are readily synthesized.
If a molecule is not readily synthesized, it is added to a list that the user inspects. At process step 2318, the user decides whether to modify the chemistry to make it compatible with the currently recognized list of available chemistries or to ignore it. To modify a chemistry, the user must use the base at a time interface at process step 2322.
The user can also choose to go directly to this step, bypassing templates all together at process step 2306.
The base at a time interface at process step 2322 is very similar to the template editor at process step 2314 except that instead of specifying chemistries for regions, they are defined one base at a time. This interface also differs in that it dynamically checks to see if the design is readily synthesized as the user makes selections. In other words, each choice made limits the choices the software makes available on the pop-up selection lists. To accommodate this function, an additional choice is made available on each pop-up of "not defined." For example, this allows the user to inhibit linker choice from restricting the sugar choices by first setting the linker to "not defined." The user would then pick the sugar, and then pick from the remaining linker choices available.
Once all of the sites on the list are assigned chemistries or dropped, they are registered at process step 2324 to a commercial chemical structure database. Registering to this database makes sure the structure is unique, assigns it a new identifier if it is unique, and allows future structure and substructure searching by creating various hash- tables. The compound definition is also stored at process step 2326 to various hash tables 94 referred to as chemistry/position tables. These allow antisense compound searching and categorization based on oligonucleotide chemistry modification sequences and equivalent base sequences.
The results of the registration are displayed at process step 2328 with the new IDs if they are new compounds and with the old IDs if they have been previously registered. The user next selects which of the compounds processed they wish to order for synthesis at process step 2330 and registers an order list at process step 2332 by including scientist name, notebook number and page number. The database time-stamps this entry. The user may then choose at process step 2334, to quit the program at process step 2338, go back to the beginning and choose a new site list to work with process step 2304, or start the oligonucleotide ordering interface at process step 2336.
EXAMPLE 16: Gene Walk to Optimize Oligonucleotide Sequence
A gene walk is executed using a CD40 antisense oligonucleotide having SEQ ID NO: 15 (5'-CTGGCACAAAGAACAGCA-3'). In effecting this gene walk, the following parameters are used:
Gene Walk Parameter Entered value
Oligonucleotide Sequence ID: 15 Name of Gene Target: CD40 Scope of Gene Walk: 20 Sequence Shift Increment: 1
Entering these values and effecting the gene walk centered on SEQ ID NO: 15 automatically generates the following new oligonucleotides:
Table 9: Oligonucleotide Generated By Gene Walk
SEQ ID Sequence
93 GAACAGCACTGACTG
94 AGAACAGCACTGACT
95 AAGAACAGCACTGAC
96 AAAGAACAGCACTGA
Figure imgf000096_0001
97 CAAAGAACAGCACTG 95
98 ACAAAGAACAGCACT
14 CACAAAGAACAGCAC
100 GCACAAAGAACAGCA
101 GGCACAAAGAACAGC
102 TGGCACAAAGAACAG
15 CTGGCACAAAGAACA
103 GCTGGCACAAAGAAC
104 GGCTGGCACAAAGAA
105 TGGCTGGCACAAAGA
106 CTGGCTGGCACAAAG
107 CCTGGCTGGCACAAA
16 TCCTGGCTGGCACAA
109 GTCCTGGCTGGCACA
110 TGTCCTGGCTGGCACA
17 CTGTCCTGGCTGGCAC
Figure imgf000097_0001
112 TCTGTCCTGGCTGGCA
The list shown above contains 20 oligonucleotide sequences directed against the CD40 nucleic acid sequence. They are ordered by the position along the CD40 sequence at which the 5' terminus of each oligonucleotide hybridizes. Thus, the first ten oligonucleotides are single-base frame shift sequences directed against the CD40 sequence upstream of compound SEQ ID NO: 15 and the latter ten are single-base frame shift sequences directed against the CD40 sequence downstream of compound SEQ ID NO: 15.
EXAMPLE 17: Automated Assay of RhoC Oligonucleotide Activity
RhoC, a member of the Rho subfamily of small GTPases, is a protein that has been shown to be involved in a diverse set of signaling pathways including the ultimate regulation of the dynamic organization of the cytoskeleton.
Oligonucleotides were designed as described in Example 2, synthesized as described in Examples 3 through 8, analyzed as described in Examples 9 and assayed as described in Example 10 except for target specific primer and probes.
RhoC probes and primers were designed to hybridize to the human RhoC sequence, using published sequence information (GenBank accession number L25081, incoφorated herein by reference as SEQ ID NO:l 13). For RhoC the PCR primers were: forward primer: TGATGTCATCCTCATGTGCTTCT (SEQ ID NO: 114) 96 reverse primer: CCAGGATGATGGGCACGTT (SEQ ID NO: 115) and the PCR probe was: FAM-CGACAGCCCTGACAGCCTGGAAA-TAMRA (SEQ ID NO: 116) where FAM (PE-Applied Biosystems, Foster City, CA) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, CA) is the quencher dye.
EXAMPLE 18: Antisense inhibition of RhoC expression- phosphorothioate oligodeoxynucleotides
In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human RhoC RNA, using published sequences (GenBank accession number L25081 , incoφorated herein by reference as SEQ ID NO:
113). The oligonucleotides are shown in Table 10. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. L25081), to which the oligonucleotide binds. All compounds in Table 10 are oligodeoxynucleotides with phosphorothioate backbones (internucleoside linkages) throughout. The compounds were analyzed for effect on RhoC mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments. If present, "N.D." indicates "no data".
Table 10
Inhibition of RhoC mRNA levels by phosphorothioate oligodeoxynucleotides
ISIS# REGION TARGET SEQUENCE % SEQ ID
SITE Inhibition NO.
25304 5' UTR 4 gagctgagatgaagtcaa 29 117
25305 5' UTR 44 gctgaagttcccaggctg 25 118
25306 5' UTR 47 ccggctgaagttcccagg 42 119
25307 Coding 104 ggcaccatccccaacgat 81 120
25308 Coding 105 aggcaccatccccaacga 81 121
25309 Coding 111 tcccacaggcaccatccc 70 122
25310 Coding 117 aggtcttcccacaggcac 40 123
25311 Coding 127 atgaggaggcaggtcttc 41 124
25312 Coding 139 ttgctgaagacgatgagg 23 125
25313 Coding 178 tcaaagacagtagggacg 0 126
25314 Coding 181 ttctcaaagacagtaggg 2 127
25315 Coding 183 agttctcaaagacagtag 38 128
Figure imgf000098_0001
25316 Coding 342 tgttttccaggctgtcag 59 129 97
25317 Coding 433 tcgtcttgcctcaggtcc 79 130
25318 Coding 439 gtgtgctcgtcttgcctc 67 131
25319 Coding 445 ctcctggtgtgctcgtct 67 132
25320 Coding 483 cagaccgaacgggctcct 65 133
25321 Coding 488 ttcctcagaccgaacggg 57 134
25322 Coding 534 actcaaggtagccaaagg 33 135
25323 Coding 566 ctcccgcactccctcctt 91 136
25324 Coding 575 ctcaaacacctcccgcac 34 137
25325 Coding 581 ggccatctcaaacacctc 64 138
25326 Coding 614 cttgttcttgcggacctg 72 139
25327 Coding 625 cccctccgacgcttgttc 66 140
25328 3' UTR 737 gtatggagccctcaggag 60 141
25329 3' UTR 746 gagccttcagtatggagc 63 142
25330 3' UTR 753 gaaaatggagccttcagt 24 143
25331 3' UTR 759 ggaactgaaaatggagcc 2 144
25332 3' UTR 763 ggagggaactgaaaatgg 13 145
25333 3* UTR 766 gcaggagggaactgaaaa 27 146
25334 3' UTR 851 agggcagggcataggcgt 31 147
25335 3' UTR 854 ggaagggcagggcatagg 21 148
25336 3* UTR 859 catgaggaagggcagggc 0 149
25337 3' UTR 920 taaagtgctggtgtgtga 39 150
25338 3' UTR 939 cctgtgagccagaagtgt 69 151
25339 3' UTR 941 ttcctgtgagccagaagt 69 152
25340 3' UTR 945 cactttcctgtgagccag 82 153
25341 3' UTR 948 agacactttcctgtgagc 69 154
25342 3' UTR 966 actctgggtccctactgc 20 155
Figure imgf000099_0001
25343 3* UTR 992 tgcagaaacaactccagg 0 156
Example 19: Antisense inhibition of RhoC expression- phosphorothioate 2'-MOE gapmer oligonucleotides
In accordance with the present invention, a second series of oligonucleotides targeted to human RhoC were synthesized. The oligonucleotide sequences are shown in Table 11. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession No. L25081), to which the oligonucleotide binds. All compounds in Table 11 are chimeric oligonucleotides ("gapmers") 18 nucleotides in length, composed of a central "gap" region consisting often 2'- deoxynucleotides, which is flanked on both sides (5' and 3' directions) by four-nucleotide "wings". The wings are composed of 2'-methoxyethyl (2'-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the 98 oligonucleotide. Cytidine residues in the 2'-MOE wings are 5-methylcytidines.
Data were obtained by real-time quantitative PCR as described in other examples herein and are averaged from three experiments. If present, "N.D." indicates "no data".
Table 11
Inhibition of RhoC mRNA levels by chimeric phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap
ISIS# REGION TARGET SEQUENCE % SEQ ID SITE Inhibition NO.
25344 5' UTR 4 gagctgagatgaagtcaa 0 117
25345 5' UTR 44 gctgaagttcccaggctg 35 118
25346 5' UTR 47 ccggctgaagttcccagg 53 119
25347 Coding 104 ggcaccatccccaacgat 50 120
25348 Coding 105 aggcaccatccccaacga 56 121
25349 Coding 111 tcccacaggcaccatccc 4 122
25350 Coding 117 aggtcttcccacaggcac 11 123
25351 Coding 127 atgaggaggcaggtcttc 6 124
25352 Coding 139 ttgctgaagacgatgagg 15 125
25353 Coding 178 tcaaagacagtagggacg 32 126
25354 Coding 181 ttctcaaagacagtaggg 7 127
25355 Coding 183 agttctcaaagacagtag 39 128
25356 Coding 342 tgttttccaggctgtcag 59 129
25357 Coding 433 tcgtcttgcctcaggtcc 48 130
25358 Coding 439 gtgtgctcgtcttgcctc 36 131
25359 Coding 445 ctcctggtgtgctcgtct 61 132
25360 Coding 483 cagaccgaacgggctcct 50 133
25361 Coding 488 ttcctcagaccgaacggg 14 134
25362 Coding 534 actcaaggtagccaaagg 32 135
25363 Coding 566 ctcccgcactccctcctt 21 136
25364 Coding 575 ctcaaacacctcccgcac 9 137
25365 Coding 581 ggccatctcaaacacctc 66 138
25366 Coding 614 cttgttcttgcggacctg 61 139
25367 Coding 625 cccctccgacgcttgttc 0 140
25368 3' UTR 737 gtatggagccctcaggag 28 141
25369 3' UTR 746 gagccttcagtatggagc 32 142
25370 3' UTR 753 gaaaatggagccttcagt 0 143
25371 3' UTR 759 ggaactgaaaatggagcc 40 144
25372 3' UTR 763 ggagggaactgaaaatgg 45 145
25373 3' UTR 766 gcaggagggaactgaaaa 35 146
25374 3' UTR 851 agggcagggcataggcgt 5 147
25375 3' UTR 854
Figure imgf000100_0001
ggaagggcagggcatagg 0 148 99
25376 3' UTR 859 catgaggaagggcagggc 0 149
25377 3' UTR 920 taaagtgctggtgtgtga 20 150
25378 3' UTR 939 cctgtgagccagaagtgt 67 151
25379 3' UTR 941 ttcctgtgagccagaagt 61 152
25380 3' UTR 945 cactttcctgtgagccag 80 153
25381 3' UTR 948 agacactttcctgtgagc 0 154
25382 3' UTR 966 actctgggtccctactgc 0 155
Figure imgf000101_0001
25383 3' UTR 992 tgcagaaacaactccagg 0 156
EXAMPLE 20: Automated Assay of Cellular Inhibitor of Apoptosis-2 Expression
Oligonucleotide Activity
Cellular Inhibitor of Apoptosis-2 (also known as c-IAP-2, apoptosis inhibitor 2, API-2, hIAP-1, and MIHC) is a member of the inhibitor of apoptosis (IAP) family of anti- apoptotic proteins which interfere with the transmission of intracellular death signals. Oligonucleotides were designed as described in Example 2, synthesized as described in Examples 3 through 8, analyzed as described in Examples 9 and assayed as described in Example 10 except for target specific primer and probes. Cellular Inhibitor of Apoptosis-2 probes and primers were designed to hybridize to the human Cellular Inhibitor of Apoptosis-2 sequence, using published sequence information (GenBank accession number U37546, incoφorated herein by reference as SEQ ID NO: 157).
For Cellular Inhibitor of Apoptosis-2 the PCR primers were: forward primer: GGACTCAGGTGTTGGGAATCTG (SEQ ID NO: 158) reverse primer: CAAGTACTCACACCTTGGAAACCA (SEQ ID NO: 159) and the PCR probe was: FAM-AGATGATCCATGGGTTCAACATGCCAA-TAMRA (SEQ ID NO: 160) where FAM (PE-Applied Biosystems, Foster City, CA) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, CA) is the quencher dye.
EXAMPLE 21 : Antisense inhibition of Cellular Inhibitor of Apoptosis-2 expression- phosphorothioate oligodeoxynucleotides In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human Cellular Inhibitor of Apoptosis-2 RNA, using published sequences (GenBank accession number U37546, incoφorated herein by reference as SEQ ID NO: 157). The oligonucleotides are shown in Table 12. Target sites 100 are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. U37546), to which the oligonucleotide binds. All compounds in Table 12 are oligodeoxynucleotides with phosphorothioate backbones (internucleoside linkages) throughout. The compounds were analyzed for effect on Cellular Inhibitor of Apoptosis-2 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments. If present, "N.D." indicates "no data".
Table 12 Inhibition of Cellular Inhibitor of Apoptosis-2 mRNA levels by phosphorothioate oligodeoxynucleotides
ISIS# REGION TARGET SEQUENCE % SEQ ID
SITE Inhibition NO.
23412 5' UTR 3 actgaagacattttgaat 62 161
23413 5' UTR 37 cttagaggtacgtaaaat 29 162
23414 5' UTR 49 gcacttttatttcttaga 70 163
23415 5' UTR 62 attttaattagaagcact 0 164
23416 5' UTR 139 accatatttcactgattc 70 165
23417 5' UTR 167 ctaactcaaaggaggaaa 0 166
23418 5' UTR 175 cacaagacctaactcaaa 27 167
23419 5' UTR 268 gctctgctgtcaagtgtt 57 168
23420 5' UTR 303 tgtgtgactcatgaagct 23 169
23421 5' UTR 335 ttcagtggcattcaatca 23 170
23422 5' UTR 357 cttctccaggctactaga 50 171
23423 5' UTR 363 ggtcaacttctccaggct 65 172
23424 5' UTR 437 taaaacccttcacagaag 0 173
23425 5* UTR 525 ttaaggaagaaatacaca 0 174
23426 5' UTR 651 gcatggctttgcttttat 0 175
23427 Coding 768 caaacgtgttggcgcttt 35 176
23428 Coding 830 agcaggaaaagtggaata 0 177
23429 Coding 1015 ttaacggaatttagactc 0 178
23430 Coding 1064 atttgttactgaagaagg 0 179
23431 Coding 1118 agagccacggaaatatcc 9 180
23432 Coding 1168 aaatcttgatttgctctg 7 181
23433 Coding 1231 gtaagtaatctggcattt 0 182
23434 Coding 1323 agcaagccactctgtctc 50 183
23435 Coding 1436 tgaagtgtcttgaagctg 0 184
23436 Coding 1580 tttgacatcatcactgtt 0 185
23437 Coding 1716 tggcttgaacttgacgga 0 186
Figure imgf000102_0001
23438 Coding 1771 tcatctcctgggctgtct 40 187 101
23439 Coding 1861 gcagcattaatcacagga 0 188
23440 Coding 2007 tttctctctcctcttccc 10 189
23441 Coding 2150 aacatcatgttcttgttc 9 190
23442 Coding 2273 atataacacagcttcagc 0 191
23443 Coding 2350 aattgttcttccactggt 0 192
23444 Coding 2460 aagaaggagcacaatctt 70 193
23445 3' UTR 2604 gaaaccaaattaggataa 12 194
23446 3' UTR 2753 tgtagtgctacctctttt 69 195
23447 3* UTR 2779 ctgaaattttgattgaat 14 196
23448 3' UTR 2795 tacaatttcaataatgct 38 197
23449 3* UTR 2920 gggtctcagtatgctgcc 21 198
23450 3' UTR 3005 ccttcgatgtataggaca 0 199
Figure imgf000103_0001
23451 3' UTR 3040 catgtccctaaaatgtca 0 200
EXAMPLE 22: Antisense inhibition of Cellular Inhibitor of Apoptosis-2 expression- phosphorothioate 2'-MOE gapmer oligonucleotides
In accordance with the present invention, a second series of oligonucleotides targeted to human Cellular Inhibitor of Apoptosis-2 were synthesized. The oligonucleotide sequences are shown in Table 13. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. U37546), to which the oligonucleotide binds.
All compounds in Table 13 are chimeric oligonucleotides ("gapmers") 18 nucleotides in length, composed of a central "gap" region consisting often 2'- deoxynucleotides, which is flanked on both sides (5' and 3' directions) by four-nucleotide "wings". The wings are composed of 2'-methoxyethyl (2'-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2'-MOE wings are 5-methylcytidines.
Data were obtained by real-time quantitative PCR as described in other examples herein and are averaged from three experiments. If present, "N.D." indicates "no data". 102
Table 13
Inhibition of Cellular Inhibitor of Apoptosis-2 mRNA levels by chimeric phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap
ISIS# REGION TARGET SEQUENCE % SEQ ID
SITE Inhibition NO.
23452 5' UTR 3 actgaagacattttgaat 35 161
23453 5' UTR 37 cttagaggtacgtaaaat 26 162
23454 5' UTR 49 gcacttttatttcttaga 76 163
23455 5' UTR 62 attttaattagaagcact 0 164
23456 5' UTR 139 accatatttcactgattc 0 165
23457 5' UTR 167 ctaactcaaaggaggaaa 5 166
23458 5' UTR 175 cacaagacctaactcaaa 0 167
23459 5' UTR 268 gctctgctgtcaagtgtt 57 168
23460 5' UTR 303 tgtgtgactcatgaagct 67 169
23461 5' UTR 335 ttcagtggcattcaatca 59 170
23462 5' UTR 357 cttctccaggctactaga 0 171
23463 5' UTR 363 ggtcaacttctccaggct 75 172
23464 5' UTR 437 taaaacccttcacagaag 11 173
23465 5' UTR 525 ttaaggaagaaatacaca 0 174
23466 5' UTR 651 gcatggctttgcttttat 46 175
23467 Coding 768 caaacgtgttggcgcttt 47 176
23468 Coding 830 agcaggaaaagtggaata 39 177
23469 Coding 1015 ttaacggaatttagactc 12 178
23470 Coding 1064 atttgttactgaagaagg 34 179
23471 Coding 1118 agagccacggaaatatcc 54 180
23472 Coding 1168 aaatcttgatttgctctg 34 181
23473 Coding 1231 gtaagtaatctggcattt 0 182
23474 Coding 1323 agcaagccactctgtctc 42 183
23475 Coding 1436 tgaagtgtcttgaagctg 0 184
23476 Coding 1580 tttgacatcatcactgtt 57 185
23477 Coding 1716 tggcttgaacttgacgga 23 186
23478 Coding 1771 tcatctcctgggctgtct 66 187
23479 Coding 1861 gcagcattaatcacagga 65 188
23480 Coding 2007 tttctctctcctcttccc 0 189
23481 Coding 2150 aacatcatgttcttgttc 13 190
23482 Coding 2273 atataacacagcttcagc 0 191
23483 Coding 2350 aattgttcttccactggt 60 192
23484 Coding 2460 aagaaggagcacaatctt 65 193
23485 3' UTR 2604 gaaaccaaattaggataa 0 194
23486 3' UTR 2753 tgtagtgctacctctttt 73 195
Figure imgf000104_0001
23487 3' UTR 2779 ctgaaattttgattgaat 4 196 103
23488 3' UTR 2795 tacaatttcaataatgct 0 197
23489 3' UTR 2920 gggtctcagtatgctgcc 42 198
23490 3' UTR 3005 ccttcgatgtataggaca 71 199
Figure imgf000105_0001
23491 3' UTR 3040 catgtccctaaaatgtca 45 200
EXAMPLE 23: Automated Assay of ELK-1 Oligonucleotide Activity
ELK-1 (also known as p62TCF) is a member of the ternary complex factor (TCF) subfamily of Ets domain proteins and utilizes a bipartite recognition mechanism mediated by both protein-DNA and protein-protein interactions. This results in gene regulation not only by direct DNA binding but also by indirect DNA binding through recruitment by other factors (Rao et al., Science, 1989, 244, 66-70). The formation of ternary complexes with an array of proteins allows the differential regulation of many genes. The mechanism by which ELK-1 controls various signal transduction pathways involves regulating the activity of the Egr-1, pip92, nur77 and c-fos promoters by binding to the serum response element (SRE) in these promoters in response to extracellular stimuli such as growth factors, mitogens and oncogene products (Sharrocks et al., Int. J. Biochem. Cell Biol, 1997, 29, 1371-1387). ELK-1 has also been shown to mediate other functions within the cell including apoptosis.
Oligonucleotides were designed as described in Example 2, synthesized as described in Examples 3 through 8, analyzed as described in Examples 9 and assayed as described in Example 10 except for target specific primer and probes. ELK-1 probes and primers were designed to hybridize to the human ELK-1 sequence, using published sequence information (GenBank accession number M25269, incoφorated herein by reference as SEQ ID NO:201). For ELK-1 the PCR primers were: forward primer: GCAAGGCAATGGCCACAT (SEQ ID NO: 202) reverse primer: CTCCTCTGCATCCACCAGCTT (SEQ ID NO: 203) and the PCR probe was: FAM-TCTCCTGGACTTCACGGGATGGTGGT-TAMRA (SEQ ID NO: 204) where FAM (PE-Applied Biosystems, Foster City, CA) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, CA) is the quencher dye. 104
EXAMPLE 24: Antisense inhibition of ELK-1 expression-phosphorothioate oligodeoxynucleotides
In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human ELK-1 RNA, using published sequences (GenBank accession number M25269, incoφorated herein by reference as SEQ ID NO: 201). The oligonucleotides are shown in Table 14. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. M25269), to which the oligonucleotide binds. All compounds in Table 14 are oligodeoxynucleotides with phosphorothioate backbones (internucleoside linkages) throughout. The compounds were analyzed for effect on ELK-1 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments. If present, "N.D." indicates "no data".
Table 14 Inhibition of ELK-1 mRNA levels by phosphorothioate oligodeoxynucleotides
ISIS# REGION TARGET SEQUENCE % SEQ ID
SITE Inhibition NO.
24752 5' UTR 11 cccctgcgtttccctaca 15 205
24753 5' UTR 50 ggtggtggtggcggtggc 29 206
24754 5' UTR 139 ggcgttggcaatgttggc 82 207
24755 5' UTR 167 aagttgaggctgtgtgta 0 208
24756 5' UTR 189 aggccacggacgggtctc 92 209
24757 5' UTR 229 gattgattcgctacgatg 71 210
24758 5' UTR 255 gggatgcggaggagtgcg 74 211
24759 5' UTR 289 agtgctcacgccatccca 22 212
24760 Coding 328 aaactgccacagcgtcac 64 213
24761 Coding 381 gaagtccaggagatgatg 62 214
24762 Coding 395 caccaccatcccgtgaag 88 215
24763 Coding 455 tcttgttcttgcgtagtc 62 216
24764 Coding 512 tgttcttgtcatagtagt 52 217
24765 Coding 527 tcaccttgcggatgatgt 57 218
24766 Coding 582 gagcaccctgcgacctca 72 219
24767 Coding 600 ggcgggcagtcctcagtg 82 220
24768 Coding 787 ggtgaaggtggaatagag 58 221
24769 Coding 993 tccgatttcaggtttggg 55 222
Figure imgf000106_0001
24770 Coding 1110 ttggtggtttctggcaca 67 223 105
24771 Coding 1132 tggagggacttctggctc 69 224
24772 Coding 1376 gcgtaggaagcagggatg 34 225
24773 Coding 1440 gtgctccagaagtgaatg 64 226
24774 Coding 1498 actggatggaaactggaa 34 227
24775 Coding 1541 ggccatccacgctgatag 74 228
24776 3' UTR 1701 ccaccacaatcagagcat 74 229
24777 3' UTR 1711 gatccccaccccaccaca 16 230
24778 3' UTR 1765 tgttttctgtggaggaga 48 231
24779 3' UTR 1790 aaacagagaagttgtgga 11 232
24780 3' UTR 1802 gggactgacagaaaacag 0 233
24781 3' UTR 1860 ataaataaataaaccgcc 18 234
24782 3' UTR 1894 gttaggtcaggctcatcc 56 235
24783 3' UTR 1974 gttctcaagccagacctc 52 236
24784 3' UTR 1992 aataaagaaagaaaggtc 41 237
24785 3' UTR 2006 agggcaggctgagaaata 29 238
24786 3' UTR 2053 cttctactcacatccaaa 54 239
24787 3' UTR 2068 caaaacaaactaactctt 24 240
24788 3' UTR 2080 ggaataataaaacaaaac 40 241
24789 3' UTR 2107 ttcttcctggacccctga 93 242
24790 3' UTR 2161 ccaagggtgtgattcttc 81 243
Figure imgf000107_0001
24791 3' UTR 2200 tgtctgagagaaaggttg 55 244
EXAMPLE 25: Antisense inhibition of ELK-1 expression- phosphorothioate V- MOE gapmer oligonucleotides In accordance with the present invention, a second series of oligonucleotides targeted to human ELK-1 were synthesized. The oligonucleotide sequences are shown in Table 15. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. M25269), to which the oligonucleotide binds. All compounds in Table 15 are chimeric oligonucleotides ("gapmers") 18 nucleotides in length, composed of a central "gap" region consisting often 2'- deoxynucleotides, which is flanked on both sides (5' and 3' directions) by four-nucleotide "wings". The wings are composed of 2'-methoxyethyl (2'-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2'-MOE wings are 5-methylcytidines. Data were obtained by real-time quantitative PCR as described in other examples herein and are averaged from three experiments. If present, "N.D." indicates "no data". 106
Table 15
Inhibition of ELK-1 mRNA levels by chimeric phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap
ISIS# REGION TARGET SEQUENCE % SEQ ID
SITE Inhibition NO.
24792 5' UTR 11 cccctgcgtttccctaca 23 205
24793 5' UTR 50 ggtggtggtggcggtggc 80 206
24794 5' UTR 139 ggcgttggcaatgttggc 91 207
24795 5' UTR 167 aagttgaggctgtgtgta 27 208
24796 5' UTR 189 aggccacggacgggtctc 79 209
24797 5' UTR 229 gattgattcgctacgatg 69 210
24798 5' UTR 255 gggatgcggaggagtgcg 42 211
24799 5' UTR 289 agtgctcacgccatccca 45 212
24800 Coding 328 aaactgccacagcgtcac 57 213
24801 Coding 381 gaagtccaggagatgatg 55 214
24802 Coding 395 caccaccatcccgtgaag 41 215
24803 Coding 455 tcttgttcttgcgtagtc 80 216
24804 Coding 512 tgttcttgtcatagtagt 65 217
24805 Coding 527 tcaccttgcggatgatgt 70 218
24806 Coding 582 gagcaccctgcgacctca 64 219
24807 Coding 600 ggcgggcagtcctcagtg 67 220
24808 Coding 787 ggtgaaggtggaatagag 45 221
24809 Coding 993 tccgatttcaggtttggg 75 222
24810 Coding 1110 ttggtggtttctggcaca 82 223
24811 Coding 1132 tggagggacttctggctc 60 224
24812 Coding 1376 gcgtaggaagcagggatg 49 225
24813 Coding 1440 gtgctccagaagtgaatg 71 226
24814 Coding 1498 actggatggaaactggaa 62 227
24815 Coding 1541 ggccatccacgctgatag 78 228
24816 3' UTR 1701 ccaccacaatcagagcat 54 229
24817 3' UTR 1711 gatccccaccccaccaca 44 230
24818 3' UTR 1765 tgttttctgtggaggaga 74 231
24819 3' UTR 1790 aaacagagaagttgtgga 64 232
24820 3' UTR 1802 gggactgacagaaaacag 16 233
24821 3' UTR 1860 ataaataaataaaccgcc 38 234
24822 3' UTR 1894 gttaggtcaggctcatcc 59 235
24823 3' UTR 1974 gttctcaagccagacctc 62 236
24824 3' UTR 1992 aataaagaaagaaaggtc 35 237
24825 3' UTR 2006 agggcaggctgagaaata 0 238
24826 3' UTR 2053 cttctactcacatccaaa 46 239
24827 3' UTR 2068 caaaacaaactaactctt 38 240
Figure imgf000108_0001
24828 3* UTR 2080 ggaataataaaacaaaac 37 241 107
24829 3' UTR 2107 ttcttcctggacccctga 71 242
24830 3' UTR 2161 ccaagggtgtgattcttc 88 243
Figure imgf000109_0001
24831 3' UTR 2200 tgtctgagagaaaggttg 65 244
EXAMPLE 26: Automated Assay of Gi alpha proteins Oligonucleotide Activity
G-alpha-11 is a member of the Gq subfamily of G proteins whose primary function is to activate PLC-b isoforms producing second messengers and affecting intracellular calcium stores. Oligonucleotides were designed as described in Example 2, synthesized as described in Examples 3 through 8, analyzed as described in Examples 9 and assayed as described in Example 10 except for target specific primer and probes. G- alpha- 11 probes and primers were designed to hybridize to the human G-alpha-11 sequence, using published sequence information (GenBank accession number AF011497, incoφorated herein by reference as SEQ ID NO:245). For G-alpha-11 the PCR primers were: forward primer: TGACCACCTTCGAGCATCAG (SEQ ID NO: 246) reverse primer: CGGTCGTAGCATTCCTGGAT (SEQ ID NO: 247) and the PCR probe was: FAM-TCAGTGCCATCAAGACCCTGTGGGAG-TAMRA (SEQ ID NO: 248) where FAM (PE-Applied Biosystems, Foster City, CA) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, CA) is the quencher dye.
EXAMPLE 27: Antisense inhibition of G-alpha-11 expression- phosphorothioate oligodeoxynucleotides
In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human G- alpha- 11 RNA, using published sequences (GenBank accession number AFOl 1497, incoφorated herein by reference as SEQ ID NO: 245). The oligonucleotides are shown in Table 16. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. AFOl 1497), to which the oligonucleotide binds. All compounds in Table 16 are oligodeoxynucleotides with phosphorothioate backbones (internucleoside linkages) throughout. The compounds were analyzed for effect on G-alpha-11 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from 108 three experiments. If present, "N.D." indicates "no data".
Table 16 Inhibition of G-alpha-11 mRNA levels by phosphorothioate oligodeoxynucleotides
ISIS# REGION TARGET SEQUENCE % SEQ ID
SITE Inhibition NO.
20576 Coding 1 gatggactccagagtcat 0 249
20577 Coding 6 gccatgatggactccaga 75 250
20578 Coding 9 cacgccatgatggactcc 0 251
20579 Coding 25 ctcatcgctcaggcaaca 61 252
20580 Coding 31 cttcacctcatcgctcag 20 253
20581 Coding 36 gactccttcacctcatcg 15 254
20582 Coding 45 atccgcttggactccttc 17 255
20583 Coding 50 cgttgatccgcttggact 0 256
20584 Coding 61 ctcgatctcggcgttgat 0 257
20585 Coding 77 cccgccgcagctgcttct 58 258
20586 Coding 106 cttgagctcgcgccgggc 31 259
20587 Coding 116 gcagcagcagcttgagct 0 260
20588 Coding 127 gcccgtgccgagcagcag 0 261
20589 Coding 146 acgtgctcttcccgctct 28 262
20590 Coding 159 atctgcttgatgaacgtg 0 263
20591 Coding 162 cgcatctgcttgatgaac 0 264
20592 Coding 184 gtagccggcgccgtggat 1 265
20593 Coding 197 tgtcctcctccgagtagc 0 266
20594 Coding 199 cttgtcctcctccgagta 79 267
20595 Coding 207 aagccgcgcttgtcctcc 56 268
20596 Coding 222 tagacgagcttggtgaag 0 269
20597 Coding 230 tgttctggtagacgagct 0 270
20598 Coding 242 tggcggtgaagatgttct 0 271
20599 Coding 258 cggatcatggcctgcatg 1 272
20600 Coding 271 cgtctccatggcccggat 49 273
20601 Coding 285 tagaggatcttgagcgtc 0 274
20602 Coding 287 tgtagaggatcttgagcg 0 275
20603 Coding 297 tgctcgtacttgtagagg 7 276
20604 Coding 306 gccttgttctgctcgtac 25 277
20605 Coding 309 ttggccttgttctgctcg 0 278
20606 Coding 319 caggagcgcattggcctt 0 279
20607 Coding 340 ctccacgtccacctcccg 69 280
20608 Coding 349 ggtcaccttctccacgtc 27 281
20609 Coding 362 gatgctcgaaggtggtca 33 282
20610 Coding 373 actgacgtactgatgctc 36 283
20611 Coding 382 cttgatggcactgacgta 78 284
Figure imgf000110_0001
20612 Coding 388 cagggtcttgatggcact 0 285 109
20613 Coding 409 ctggatgcccgggtcctc 0 286
20614 Coding 411 tcctggatgcccgggtcc 30 287
20615 Coding 429 cgcctgcggtcgtagcat 0 288
20616 Coding 440 gctggtactcgcgcctgc 41 289
20617 Coding 459 tacttggcagagtcggag 34 290
20618 Coding 468 gtcaggtagtacttggca 76 291
20619 Coding 479 ggtcaacgtcggtcaggt 18 292
20620 Coding 489 gtggcgatgcggtcaacg 1 293
20621 Coding 503 gcaggtagcccaaggtgg 20 294
20622 Coding 518 cgtcctgctgggtgggca 40 295
20623 Coding 544 ggtggtgggcacgcggac 0 296
20624 Coding 555 tcgatgatgccggtggtg 0 297
20625 Coding 572 ccaggtcgaaagggtact 0 298
20626 Coding 578 tgttctccaggtcgaaag 33 299
20627 Coding 584 agatgatgttctccaggt 0 300
20628 Coding 591 atccggaagatgatgttc 0 301
20629 Coding 624 ctccgctccgaccgctgg 56 302
20630 Coding 634 gatccacttcctccgctc 59 303
20631 Coding 655 tgtcacgttctcaaagca 0 304
20632 Coding 663 atgatggatgtcacgttc 0 305
20633 Coding 671 cgagaaacatgatggatg 0 306
20634 Coding 682 gctgagggcgacgagaaa 75 307
20635 Coding 709 cgactccaccaggacttg 40 308
20636 Coding 726 atccggttctcgttgtcc 22 309
20637 Coding 728 ccatccggttctcgttgt 19 310
20638 Coding 744 agggctttgctctcctcc 77 311
20639 Coding 754 ggtccggaacagggcttt 26 312
20640 Coding 766 gtaggtgatgatggtccg 0 313
20641 Coding 787 ggaggagttctggaacca 64 314
20642 Coding 803 tgaggaagaggatgacgg 0 315
20643 Coding 818 gcaggtccttcttgttga 6 316
20644 Coding 831 atcttgtcctccagcagg 4 317
20645 Coding 842 gcgagtacaggatcttgt 17 318
20646 Coding 858 aagtagtccaccaggtgc 0 319
20647 Coding 910 gatgaactcccgcgccgc 52 320
20648 Coding 935 ggttcaggtccacgaaca 71 321
20649 Coding 958 gtagatgatcttgtcgct 0 322
20650 Coding 972 cacgtgaagtgtgagtag 0 323
20651 Coding 993 atgttctccgtgtcggtg 0 324
20652 Coding 1014 acggccgcgaacacgaag 6 325
20653 Coding 1027 gatggtgtccttcacggc 0 326
20654 Coding 1043 tcaggttcagctgcagga 3 327
Figure imgf000111_0001
20655 Coding 1059 accagattgtactccttc 0 328 110
EXAMPLE 28: Antisense inhibition of G-alpha-11 expression- phosphorothioate 2'- MOE gapmer oligonucleotides
In accordance with the present invention, a second series of oligonucleotides targeted to human G- alpha- 11 were synthesized. The oligonucleotide sequences are shown in Table 17. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. AFOl 1497), to which the oligonucleotide binds.
All compounds in Table 17 are chimeric oligonucleotides ("gapmers") 18 nucleotides in length, composed of a central "gap" region consisting often 2'- deoxynucleotides, which is flanked on both sides (5' and 3' directions) by four-nucleotide "wings". The wings are composed of 2'-methoxyethyl (2'-MOE)nucleo tides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2'-MOE wings are 5-methylcytidines.
Data were obtained by real-time quantitative PCR as described in other examples herein and are averaged from three experiments. If present, "N.D." indicates "no data".
Table 17
Inhibition of G-alpha-11 mRNA levels by chimeric phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap
ISIS# REGION TARGET SEQUENCE % SEQ ID
SITE Inhibition NO.
20981 Coding 1 gatggactccagagtcat 0 249
20982 Coding 6 gccatgatggactccaga 0 250
20983 Coding 9 cacgccatgatggactcc 0 251
20984 Coding 25 ctcatcgctcaggcaaca 0 252
20985 Coding 31 cttcacctcatcgctcag 2 253
20986 Coding 36 gactccttcacctcatcg 0 254
20987 Coding 45 atccgcttggactccttc 19 255
20988 Coding 50 cgttgatccgcttggact 15 256
20989 Coding 61 ctcgatctcggcgttgat 0 257
20990 Coding 77 cccgccgcagctgcttct 41 258
20991 Coding 106 cttgagctcgcgccgggc 19 259
20992 Coding 116 gcagcagcagcttgagct 23 260
20993 Coding 127 gcccgtgccgagcagcag 38 261
20994 Coding 146 acgtgctcttcccgctct 34 262
Figure imgf000112_0001
20995 Coding 159 atctgcttgatgaacgtg 56 263 111
20996 Coding 162 cgcatctgcttgatgaac 31 264
20997 Coding 184 gtagccggcgccgtggat 0 265
20998 Coding 197 tgtcctcctccgagtagc 42 266
20999 Coding 199 cttgtcctcctccgagta 0 267
21000 Coding 207 aagccgcgcttgtcctcc 73 268
21001 Coding 222 tagacgagcttggtgaag 0 269
21002 Coding 230 tgttctggtagacgagct 61 270
21003 Coding 242 tggcggtgaagatgttct 14 271
21004 Coding 258 cggatcatggcctgcatg 84 272
21005 Coding 271 cgtctccatggcccggat 70 273
21006 Coding 285 tagaggatcttgagcgtc 39 274
21007 Coding 287 tgtagaggatcttgagcg 28 275
21008 Coding 297 tgctcgtacttgtagagg 70 276
21009 Coding 306 gccttgttctgctcgtac 76 277
21010 Coding 309 ttggccttgttctgctcg 0 278
21011 Coding 319 caggagcgcattggcctt 87 279
21012 Coding 340 ctccacgtccacctcccg 0 280
21013 Coding 349 ggtcaccttctccacgtc 69 281
21014 Coding 362 gatgctcgaaggtggtca 0 282
21015 Coding 373 actgacgtactgatgctc 69 283
21016 Coding 382 cttgatggcactgacgta 32 284
21017 Coding 388 cagggtcttgatggcact 19 285
21018 Coding 409 ctggatgcccgggtcctc 63 286
21019 Coding 411 tcctggatgcccgggtcc 56 287
21020 Coding 429 cgcctgcggtcgtagcat 73 288
21021 Coding 440 gctggtactcgcgcctgc 68 289
21022 Coding 459 tacttggcagagtcggag 50 290
21023 Coding 468 gtcaggtagtacttggca 13 291
21024 Coding 479 ggtcaacgtcggtcaggt 64 292
21025 Coding 489 gtggcgatgcggtcaacg 52 293
21026 Coding 503 gcaggtagcccaaggtgg 52 294
21027 Coding 518 cgtcctgctgggtgggca 0 295
21028 Coding 544 ggtggtgggcacgcggac 81 296
21029 Coding 555 tcgatgatgccggtggtg 48 297
21030 Coding 572 ccaggtcgaaagggtact 61 298
21031 Coding 578 tgttctccaggtcgaaag 0 299
21032 Coding 584 agatgatgttctccaggt 0 300
21033 Coding 591 atccggaagatgatgttc 0 301
21034 Coding 624 ctccgctccgaccgctgg 59 302
21035 Coding 634 gatccacttcctccgctc 17 303
21036 Coding 655 tgtcacgttctcaaagca 9 304
21037 Coding 663 atgatggatgtcacgttc 41 305
Figure imgf000113_0001
21038 Coding 671 cgagaaacatgatggatg 0 306 112
21039 Coding 682 gctgagggcgacgagaaa 11 307
21040 Coding 709 cgactccaccaggacttg 0 308
21041 Coding 726 atccggttctcgttgtcc 67 309
21042 Coding 728 ccatccggttctcgttgt 30 310
21043 Coding 744 agggctttgctctcctcc 61 311
21044 Coding 754 ggtccggaacagggcttt 72 312
21045 Coding 766 gtaggtgatgatggtccg 68 313
21046 Coding 787 ggaggagttctggaacca 54 314
21047 Coding 803 tgaggaagaggatgacgg 23 315
21048 Coding 818 gcaggtccttcttgttga 0 316
21049 Coding 831 atcttgtcctccagcagg 39 317
21050 Coding 842 gcgagtacaggatcttgt 74 318
21051 Coding 858 aagtagtccaccaggtgc 36 319
21052 Coding 910 gatgaactcccgcgccgc 67 320
21053 Coding 935 ggttcaggtccacgaaca 37 321
21054 Coding 958 gtagatgatcttgtcgct 64 322
21055 Coding 972 cacgtgaagtgtgagtag 37 323
21056 Coding 993 atgttctccgtgtcggtg 0 324
21057 Coding 1014 acggccgcgaacacgaag 0 325
21058 Coding 1027 gatggtgtccttcacggc 69 326
21059 Coding 1043 tcaggttcagctgcagga 0 327
Figure imgf000114_0001
21060 Coding 1059 accagattgtactccttc 0 328
EXAMPLE 29: Automated Assay of AKT-1 Oligonucleotide Activity Akt-1 (also known as PKB alpha and RAC-PK alpha) is a member of the
AKT/PKB family of serine/threonine kinases and has been shown to be involved in a diverse set of signaling pathways.
Oligonucleotides were designed as described in Example 2, synthesized as described in Examples 3 through 8, analyzed as described in Examples 9 and assayed as described in Example 10 except for target specific primer and probes. AKT-1 probes and primers were designed to hybridize to the human AKT-1 sequence, using published sequence information (GenBank accession number M63167, incoφorated herein by reference as SEQ ID NO:329). For Akt-1 the PCR primers were: forward primer: CGTGACCATGAACGAGTTTGA (SEQ ID NO: 330) reverse primer: CAGGATCACCTTGCCGAAA (SEQ ID NO: 331) and the PCR probe was: FAM-CTGAAGCTGCTGGGCAAGGGCA-TAMRA (SEQ ID NO: 332) where FAM (PE-Applied Biosystems, Foster City, CA) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, CA) is the quencher dye. 113
EXMAMPLE 30: Antisense inhibition of Akt-1 expression- phosphorothioate oligodeoxynucleotides
In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human Akt-1 RNA, using published sequences (GenBank accession number M63167, incoφorated herein by reference as SEQ ID NO: 329). The oligonucleotides are shown in Table 18. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. M63167), to which the oligonucleotide binds. All compounds in Table 18 are oligodeoxynucleotides with phosphorothioate backbones (internucleoside linkages) throughout. The compounds were analyzed for effect on Akt-1 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments. If present, "N.D." indicates "no data".
Table 18
Inhibition of Akt-1 mRNA levels by phosphorothioate oligodeoxynucleotides
ISIS# REGION TARGET SEQUENCE 0 //o SEQ ID
SITE Inhibition NO.
28880 5' UTR 4 ccctgtgccctgtcccag 55 333
28881 5' UTR 27 cctaagcccctggtgaca 15 334
28882 5' UTR 62 ctttgacttctttgaccc 68 335
28883 5' UTR 70 ggcagcccctttgacttc 53 336
28884 Coding 213 caaccctccttcacaata 24 337
28885 Coding 234 tactcccctcgtttgtgc 0 338
28886 Coding 281 tgccatcattcttgagga 65 339
28887 Coding 293 agccaatgaaggtgccat 67 340
28888 Coding 352 cacagagaagttgttgag 22 341
28889 Coding 496 agtctggatggcggttgt 49 342
28890 Coding 531 tcctcctcctcctgcttc 9 343
28891 Coding 570 cctgagttgtcactgggt 49 344
28892 Coding 666 ccgaaagtgcccttgccc 56 345
28893 Coding 744 gccacgatgacttccttc 60 346
28894 Coding 927 cggtcctcggagaacaca 0 347
28895 Coding 990 acgttcttctccgagtgc 30 348
28896 Coding 1116 gtgccgcaaaaggtcttc 66 349
28897 Coding 1125 tactcaggtgtgccgcaa 66 350
28898 Coding 1461 ggcttgaagggtgggctg 41 351
Figure imgf000115_0001
28899 Coding 1497 tcaaaatacctggtgtca 51 352 114
28900 Coding 1512 gccgtgaactcctcatca 56 353
28901 Coding 1541 ggtcaggtggtgtgatgg 0 354
28902 Coding 1573 ctcgctgtccacacactc 61 355
28903 3' UTR 1671 gcctctccatccctccaa 76 356
28904 3' UTR 1739 acagcgtggcttctctca 12 357
28905 3' UTR 1814 ttttcttccctaccccgc 64 358
28906 3' UTR 1819 gatagttttcttccctac 0 359
28907 3' UTR 1831 taaaacccgcaggatagt 74 360
28908 3' UTR 1856 ggagaacaaactggatga 0 361
28909 3' UTR 1987 ctggctgacagagtgagg 59 362
28910 3' UTR 1991 gcggctggctgacagagt 61 363
28911 3' UTR 2031 cccagagagatgacagat 46 364
28912 3' UTR 2127 gctgctgtgtgcctgcca 38 365
28913 3' UTR 2264 cataatacacaataacaa 39 366
28914 3' UTR 2274 atttgaacaacataatac 11 367
28915 3' UTR 2397 aagtgctaccgtggagag 57 368
28916 3' UTR 2407 cgaaaaggtcaagtgcta 41 369
28917 3' UTR 2453 cagggagtcagggagggc 13 370
28918 3' UTR 2545 aaagttgaatgttgtaaa 10 371
Figure imgf000116_0001
28919 3' UTR 2553 aaaatactaaagttgaat 25 372
EXAMPLE 31: Antisense inhibition of Akt-1 expression- phosphorothioate 2'-MOE gapmer oligonucleotides
In accordance with the present invention, a second series of oligonucleotides targeted to human Akt-1 were synthesized. The oligonucleotide sequences are shown in
Table 19. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. M63167), to which the oligonucleotide binds.
All compounds in Table 19 are chimeric oligonucleotides ("gapmers") 18 nucleotides in length, composed of a central "gap" region consisting often 2'- deoxynucleotides, which is flanked on both sides (5' and 3' directions) by four-nucleotide
"wings". The wings are composed of 2'-methoxyethyl (2'-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2'-MOE wings are 5-methylcytidines.
Data were obtained by real-time quantitative PCR as described in other examples herein and are averaged from three experiments. If present, "N.D." indicates "no data". 115
Table 19
Inhibition of Akt-1 mRNA levels by chimeric phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap
ISIS# REGION TARGET SEQUENCE % SEQ ID
SITE Inhibition NO.
28920 5' UTR 4 ccctgtgccctgtcccag 88 333
28921 5' UTR 27 cctaagcccctggtgaca 44 334
28922 5' UTR 62 ctttgacttctttgaccc 61 335
28923 5' UTR 70 ggcagcccctttgacttc 79 336
28924 Coding 213 caaccctccttcacaata 72 337
28925 Coding 234 tactcccctcgtttgtgc 39 338
28926 Coding 281 tgccatcattcttgagga 73 339
28927 Coding 293 agccaatgaaggtgccat 62 340
28928 Coding 352 cacagagaagttgttgag 48 341
28929 Coding 496 agtctggatggcggttgt 43 342
28930 Coding 531 tcctcctcctcctgcttc 49 343
28931 Coding 570 cctgagttgtcactgggt 71 344
28932 Coding 666 ccgaaagtgcccttgccc 64 345
28933 Coding 744 gccacgatgacttccttc 66 346
28934 Coding 927 cggtcctcggagaacaca 77 347
28935 Coding 990 acgttcttctccgagtgc 89 348
28936 Coding 1116 gtgccgcaaaaggtcttc 61 349
28937 Coding 1125 tactcaggtgtgccgcaa 74 350
28938 Coding 1461 ggcttgaagggtgggctg 54 351
28939 Coding 1497 tcaaaatacctggtgtca 78 352
28940 Coding 1512 gccgtgaactcctcatca 88 353
28941 Coding 1541 ggtcaggtggtgtgatgg 71 354
28942 Coding 1573 ctcgctgtccacacactc 83 355
28943 3' UTR 1671 gcctctccatccctccaa 86 356
28944 3' UTR 1739 acagcgtggcttctctca 73 357
28945 3' UTR 1814 ttttcttccctaccccgc 77 358
28946 3' UTR 1819 gatagttttcttccctac 43 359
28947 3' UTR 1831 taaaacccgcaggatagt 64 360
28948 3' UTR 1856 ggagaacaaactggatga 70 361
28949 3' UTR 1987 ctggctgacagagtgagg 90 362
28950 3' UTR 1991 gcggctggctgacagagt 82 363
28951 3' UTR 2031 cccagagagatgacagat 53 364
28952 3' UTR 2127 gctgctgtgtgcctgcca 80 365
28953 3' UTR 2264 cataatacacaataacaa 48 366
28954 3' UTR 2274 atttgaacaacataatac 39 367
28955 3' UTR 2397 aagtgctaccgtggagag 38 368
Figure imgf000117_0001
28956 3' UTR 2407 cgaaaaggtcaagtgcta 83 369 116
28957 3' UTR 2453 cagggagtcagggagggc 59 370
28958 3* UTR 2545 aaagttgaatgttgtaaa 25 371
28959 3' UTR
Figure imgf000118_0001
2553 aaaatactaaagttgaat 45 372

Claims

117What is claimed is:
1. A method of defining a set of compounds that modulate the expression of a target nucleic acid sequence via binding of said compounds with said target nucleic acid sequence comprising generating a library of virtual compounds in silico according to defined criteria, and evaluating in silico the binding of said virtual compounds with said target nucleic acid according to defined criteria.
2. A method of defining a set of oligonucleotides that modulate the expression of a target nucleic acid sequence via binding of said oligonucleotides with said target nucleic acid sequence comprising generating in silico a plurality of virtual oligonucleotides according to defined criteria,and evaluating in silico the binding of said plurality of virtual oligonucleotides with said target nucleic acid according to defined criteria.
3. A method of defining a set of compounds that modulate the expression of a target nucleic acid sequence via binding of said compounds with said target nucleic acid comprising, generating in silico a library of virtual compounds according to defined criteria wherein said virtual compounds modulate the expression of said target nucleic acid sequence, and robotically synthesizing synthetic compounds corresponding to at least some of said virtual compounds.
4. A method of defining a set of compounds that modulate the expression of a target nucleic acid sequence via binding of said compounds with said target nucleic acid comprising generating in silico virtual compounds according to defined criteria wherein said virtual compounds modulate the expression of said target nucleic acid sequence, synthesizing synthetic compounds corresponding to at least some of said virtual compounds, and robotically assaying said synthetic compounds for one or more desired physical, chemical or biological properties.
5. A method of defining a set of compounds that modulate the expression of a target nucleic acid sequence via binding of said compounds with said target nucleic acid sequence comprising generating in silico a library of nucleobase sequences according to 118 defined criteria and evaluating in silico a plurality of virtual oligonucleotides having said nucleobase sequences according to defined criteria.
6. A method of defining a set of compounds that modulate the expression of a target nucleic acid sequence via binding of said compounds with said target nucleic acid sequence comprising evaluating in silico a plurality of virtual compounds according to defined criteria and robotically synthesizing a plurality of synthetic compounds corresponding to said plurality of virtual compounds.
7. A method of defining a set of compounds that modulate the expression of a target nucleic acid sequence via binding of said compounds with said target nucleic acid sequence comprising evaluating in silico a plurality of virtual compounds according to defined criteria and robotically assaying a plurality of synthetic compounds corresponding to at least some of said virtual compounds for one or more desired physical, chemical or biological properties.
8. A method of defining a set of compounds that modulate the expression of a target nucleic acid sequence via binding of said compounds with said target nucleic acid sequence comprising generating a library of nucleobase sequences in silico according to defined criteria and robotically synthesizing a plurality of synthetic compounds compounds having said nucleobase sequences.
9. A method of defining a set of compounds that modulate the expression of a target nucleic acid sequence via binding of said compounds with said target nucleic acid sequence comprising robotically synthesizing a plurality of synthetic compounds and robotically assaying said plurality of synthetic compounds for one or more desired physical, chemical or biological properties.
10. A method of defining a set of compounds that modulate the expression of a target nucleic acid sequence via binding of said compounds with said target nucleic acid sequence comprising generating a library of nucleobase sequences in silico according to 119 defined criteria and robotically assaying a plurality of synthetic compounds having at least some of said nucleobase sequences for one or more desired physical, chemical or biological properties.
11. A method of generating a set of oligonucleotides that modulate the expression of a target nucleic acid sequence via binding of said oligonucleotides with said target nucleic acid sequence, comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined criteria; (b) evaluating in silico a plurality of virtual oligonucleotides having the nucleobase sequences of (a) according to defined criteria; and
(c) robotically synthesizing a plurality of synthetic oligonucleotides corresponding to at least some of said virtual oligonucleotides.
12. A method of generating a set of oligonucleotides that modulate the expression of a target nucleic acid sequence via binding of said oligonucleotides with said target nucleic acid sequence, comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined criteria; (b) evaluating in silico a plurality of virtual oligonucleotides having the nucleobase sequences of (a) according to defined criteria; and
(c) robotically assaying a plurality of synthetic oligonucleotides corresponding to at least some of said virtual oligonucleotides for one or more desired physical, chemical or biological properties.
13. A method of generating a set of oligonucleotides that modulate the expression of a target nucleic acid sequence via binding of said oligonucletoides with said target nucleic acid sequence, comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined criteria;
(b) robotically synthesizing a plurality of synthetic oligonucleotides having at least 120 some of said nucleobase sequences; and
(c) robotically assaying said plurality of synthetic oligonucleotides for one or more desired physical, chemical or biological properties.
14. A method of generating a set of oligonucleotides that modulate the expression of a target nucleic acid sequence via binding of said oligonucleotides with said target nucleic acid sequence, comprising the steps of:
(a) evaluating in silico a plurality of virtual oligonucleotides according to defined criteria; (b) robotically synthesizing a plurality of synthetic oligonucleotides corresponding to at least some of said virtual oligonucleotides; and
(c) robotically assaying said plurality of synthetic oligonucleotides for one or more desired physical, chemical or biological properties.
15. A method of generating a set of oligonucleotides that modulate the expression of a target nucleic acid sequence via binding of said oligonucleotides with said target nucleic acid sequence, comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined criteria; (b) evaluating in silico a plurality of virtual oligonucleotides having the nucleobase sequences of (a) according to defined criteria;
(c) robotically synthesizing a plurality of synthetic oligonucleotides corresponding to at least some of said virtual oligonucleotides; and
(d) robotically assaying said plurality of synthetic oligonucleotides for one or more desired physical, chemical or biological properties.
16. A method of generating a set of oligonucleotides that modulate the expression of a target nucleic acid sequence via binding of said oligonucleotides with said target nucleic acid sequence, comprising the steps of: (a) generating a library of nucleobase sequences in silico according to defined criteria; 121
(b) choosing an oligonucleotide chemistry;
(c) robotically synthesizing a set of synthetic oligonucleotides having said nucleobase sequences of step (a) and said oligonucleotide chemistry of step (b);
(d) robotically assaying said set of synthetic oligonucleotides of step (c) for a physical, chemical or biological activity; and
(e) selecting a subset of said set of synthetic oligonucleotides of step (c) having a desired level of physical, chemical or biological activity in order to generate said set of compounds.
17. A method of generating a set of oligonucleotides that modulate the expression of a target nucleic acid sequence via binding of said oligonucleotides with said target nucleic acid sequence, comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined criteria; (b) choosing an oligonucleotide chemistry;
(c) evaluating in silico a plurality of virtual oligonucleotides having the nucleobase sequences of (a) and the oligonucleotide chemistry of (b) according to defined criteria, and selecting those having preferred characteristics, in order to generate a set of preferred nucleobase sequences; (d) robotically synthesizing a set of synthetic oligonucleotides having said preferred nucleobase sequences of step (c) and said oligonucleotide chemistry of step (b);
(e) robotically assaying said set of synthetic oligonucleotides of step (d) for a physical, chemical or biological activity; and
(f) selecting a subset of said set of synthetic oligonucleotides of step (d) having a desired level of physical, chemical or biological activity in order to generate said set of oligonucleotides.
18. The method of claim 12, wherein said step of robotically assaying said plurality of synthetic oligonucleotide compounds is performed by computer-controlled real-time polymerase chain reaction or by computer-controlled enzyme-linked immunosorbent assay. 122
19. The method of claim 11 , wherein said target nucleic acid sequence is that of a genomic DNA, a cDNA, a product of a polymerase chain reaction, an expressed sequence tag, an mRNA or a structural RNA.
20. The method of claim 11 , wherein said target nucleic acid sequence is a human nucleic acid.
21. A method of identifying one or more nucleic acid sequences amenable to antisense binding of an oligonucleotide to said nucleic acid sequences comprising generating a library of antisense nucleobase sequences in silico according to defined criteria.
22. A method of identifying a set of compounds that modulate the expression of a target nucleic acid sequence via binding of said compounds with said target nucleic acid sequence comprising evaluating in silico a plurality of virtual oligonucleotides according to defined criteria.
23. A method of identifying one or more nucleic acid sequences amenable to antisense binding of an compound to said nucleic acid sequences comprising robotically synthesizing a plurality of synthetic antisense compounds.
24. A method of identifying one or more nucleic acid sequences amenable to antisense binding of an compound to said nucleic acid sequences comprising robotically assaying a plurality of synthetic antisense compounds for one or more desired physical, chemical or biological properties.
25. A method of identifying one or more nucleic acid sequences amenable to antisense binding of an oligonucleotide to said nucleic acid sequences comprising generating in silico a library of nucleobase sequences according to defined criteria and evaluating in silico a plurality of virtual oligonucleotides having said nucleobase sequences according to defined criteria. 123
26. A method of identifying one or more nucleic acid sequences amenable to antisense binding of an oligonucleotide to said nucleic acid sequences comprising evaluating in silico a plurality of virtual oligonucleotides according to defined criteria and robotically synthesizing a plurality of synthetic oligonucleotides corresponding to least some of said virtual oligonucleotides.
27. A method of identifying one or more nucleic acid sequences amenable to antisense binding of an oligonucleotide to said nucleic acid sequences comprising evaluating in silico a plurality of virtual oligonucleotides according to defined criteria and robotically assaying a plurality of synthetic oligonucleotides corresponding to least some of said virtual oligonucleotides for one or more desired physical, chemical or biological properties.
28. A method of identifying one or more nucleic acid sequences amenable to antisense binding of an oligonucleotide to said nucleic acid sequences comprising generating a library of nucleobase sequences in silico according to defined criteria and robotically synthesizing a plurality of synthetic oligonucleotides having said nucleobase sequences.
29. A method of identifying one or more nucleic acid sequences amenable to antisense binding of an oligonucleotide to said nucleic acid sequences comprising robotically synthesizing a plurality of synthetic oligonucleotides and robotically assaying said plurality of synthetic oligonucleotides for one or more desired physical, chemical or biological properties.
30. A method of identifying one or more nucleic acid sequences amenable to antisense binding of an oligonucleotide to said nucleic acid sequences comprising generating a library of nucleobase sequences in silico according to defined criteria and robotically assaying a plurality of synthetic oligonucleotides having said nucleobase sequences for one or more desired physical, chemical or biological properties.
31. A method of identifying one or more nucleic acid sequences amenable to antisense 124 binding of an oligonucleotide to said nucleic acid sequences comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined criteria;
(b) evaluating in silico a plurality of virtual oligonucleotides having the nucleobase sequences of (a) according to defined criteria; and
(c) robotically synthesizing a plurality of synthetic oligonucleotides corresponding to at least some of said virtual oligonucleotides.
32. A method of identifying one or more nucleic acid sequences amenable to antisense binding of an oligonucleotide to said nucleic acid sequences, comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined criteria;
(b) evaluating in silico a plurality of virtual oligonucleotides having the nucleobase sequences of (a) according to defined criteria; and (c) robotically assaying a plurality of synthetic oligonucleotides corresponding to at least some of said virtual oligonucleotides for one or more desired physical, chemical or biological properties.
33. A method of identifying one or more nucleic acid sequences amenable to antisense binding of an oligonucleotide to said nucleic acid sequences, comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined criteria;
(b) robotically synthesizing a plurality of synthetic oligonucleotides having at least some of said nucleobase sequences; and (c) robotically assaying said plurality of synthetic oligonucleotides for one or more desired physical, chemical or biological properties.
34. A method of identifying one or more nucleic acid sequences amenable to antisense binding of an oligonucleotide to said nucleic acid sequences, comprising the steps of: (a) evaluating in silico a plurality of virtual oligonucleotides according to defined criteria; 125
- (b) robotically synthesizing a plurality of synthetic oligonucleotides corresponding to at least some of said virtual oligonucleotides; and
(c) robotically assaying said plurality of synthetic oligonucleotides for one or more desired physical, chemical or biological properties.
35. A method of identifying one or more nucleic acid sequences amenable to antisense binding of an oligonucleotide to said nucleic acid sequences, comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined criteria; (b) evaluating in silico a plurality of virtual oligonucleotides having the nucleobase sequences of (a) according to defined criteria;
(c) robotically synthesizing a plurality of synthetic oligonucleotides conesponding to least some of said plurality of virtual oligonucleotides; and
(d) robotically assaying said plurality of synthetic oligonucleotides for one or more desired physical, chemical or biological properties.
36. A method of identifying one or more nucleic acid sequences amenable to antisense binding of an oligonucleotide to said nucleic acid sequences, comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined criteria;
(b) choosing an oligonucleotide chemistry;
(c) robotically synthesizing a set of synthetic oligonucleotides having said nucleobase sequences of step (a) and said oligonucleotide chemistry of step (b);
(d) robotically assaying said set of synthetic oligonucleotides of step (c) for a physical, chemical or biological activity; and
(e) selecting a subset of said set of synthetic oligonucleotides of step (c) having a desired level of physical, chemical or biological activity.
37. A method of identifying one or more nucleic acid sequences amenable to antisense binding of an oligonucleotide to said nucleic acid sequences, comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined 126 criteria;
(b) choosing an oligonucleotide chemistry;
(c) evaluating in silico a plurality of virtual oligonucleotides having the nucleobase sequences of (a) according to defined criteria, and selecting those having preferred characteristics, in order to generate a set of prefened nucleobase sequences;
(d) robotically synthesizing a set of synthetic oligonucleotides having said prefened nucleobase sequences of step (b) and said oligonucleotide chemistry of step (c);
(e) robotically assaying said set of synthetic oligonucleotides of step (d) for a physical, chemical or biological activity; and (f) selecting a subset of said set of oligonucleotides of step (d) having a desired level of physical, chemical or biological activity.
38. The method of claim 32, wherein said step of robotically assaying said plurality of synthetic antisense oligonucleotides is performed by computer-controlled real-time polymerase chain reaction or by computer-controlled enzyme-linked immunosorbent assay.
39. The method of claim 31 , wherein said nucleic acid sequence is that of a genomic DNA, a cDNA, a product of a polymerase chain reaction, an expressed sequence tag, an mRNA or a structural RNA.
40. The method of claim 31 , wherein said nucleic acid sequence is a human nucleic acid.
41. A computer formatted medium comprising computer readable instructions for identifying compounds that have one or more desired properties according to defined criteria and that bind to a genomic DNA, a cDNA, a product of a polymerase chain reaction, an expressed sequence tag, an mRNA or a structural RNA.
42. A computer formatted medium comprising computer readable instructions for performing the method of any one of claims 1 to 20. 127
43. A computer formatted medium comprising computer readable instructions for performing a method of identifying one or more nucleic acid sequences amenable to antisense binding of a compound to said nucleic acid sequences.
44. A computer formatted medium comprising computer readable instructions for performing the method of any one of claims 21 to 40.
45. A computer formatted medium comprising one or more nucleic acid sequences amenable to antisense binding of a compound to said nucleic acid sequences in computer readable form.
46. A computer formatted medium comprising one or more nucleic acid sequences amenable to antisense binding of a compound to said nucleic acid sequences in computer readable form, wherein said one or more nucleic acid sequences is identified according to the method of any one of claims 21 -40.
47. A process for validating the function of a gene or the product of said gene comprising generating in silico a library of nucleobase sequences targeted to said gene and robotically assaying a plurality of synthetic compounds having at least some of said nucleobase sequences for effects on biological function.
48. A process for validating the function of a gene or the product of said gene, comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined criteria;
(b) evaluating in silico a plurality of virtual oligonucleotides having the nucleobase sequences of (a) according to defined criteria; and
(c) robotically synthesizing a plurality of synthetic oligonucleotides conesponding to at least some of said virtual oligonucleotides.
49. A process for validating the function of a gene or the product of said gene, 128 comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined criteria;
(b) evaluating in silico a plurality of virtual oligonucleotides having the nucleobase sequences of (a) according to defined criteria; and
(c) robotically assaying a plurality of synthetic oligonucleotides conesponding to at least some of said virtual oligonucleotides for effects on biological function.
50. A process for validating the function of a gene or the product of said gene, comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined criteria;
(b) robotically synthesizing a plurality of synthetic oligonucleotides having at least some of said nucleobase sequences; and (c) robotically assaying said plurality of synthetic oligonucleotides for effects on biological function.
51. A process for validating the function of a gene or the product of said gene, comprising the steps of: (a) evaluating in silico a plurality of virtual oligonucleotides according to defined criteria;
(b) robotically synthesizing a plurality of synthetic oligonucleotides conesponding to at least some of said virtual oligonucleotides; and
(c) robotically assaying said plurality of synthetic oligonucleotides for effects on biological function.
52. A process for validating the function of a gene or the product of said gene, comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined criteria;
(b) evaluating in silico a plurality of virtual oligonucleotides having the nucleobase 129 sequences of (a) according to defined criteria;
(c) robotically synthesizing a plurality of synthetic oligonucleotides conesponding to at least some of said virtual oligonucleotides; and
(d) robotically assaying said plurality of synthetic oligonucleotides for effects on biological function.
53. A process for validating the function of a gene or the product of said gene, comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined criteria;
(b) choosing an oligonucleotide chemistry;
(c) robotically synthesizing a set of synthetic oligonucleotides having said nucleobase sequences of step (a) and said oligonucleotide chemistry of step (b);
(d) robotically assaying said set of synthetic oligonucleotides of step (c) for effects on biological function; and
(e) selecting a subset of said set of synthetic oligonucleotides of step (c) having a desired level of physical, chemical or biological activity in order to generate said set of compounds.
54. A process for validating the function of a gene or the product of said gene, comprising the steps of:
(a) generating a library of nucleobase sequences in silico according to defined criteria;
(b) choosing an oligonucleotide chemistry; (c) evaluating in silico a plurality of virtual oligonucleotides having the nucleobase sequences of (a) and the oligonucleotide chemistry of (b) according to defined criteria, and selecting those having prefened characteristics, in order to generate a set of prefened nucleobase sequences;
(d) robotically synthesizing a set of synthetic oligonucleotides having said prefened nucleobase sequences of step (c) and said oligonucleotide chemistry of step (b);
(e) robotically assaying said set of synthetic oligonucleotides of step (d) for effects 130 on biological function; and
(f) selecting a subset of said set of synthetic oligonucleotides of step (d) having a desired level of physical, chemical or biological activity in order to generate said set of oligonucleotides.
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Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1082456A1 (en) * 1998-05-01 2001-03-14 Isis Pharmaceuticals, Inc. Antisense modulation of cd40 expression
WO2001031579A2 (en) * 1999-10-27 2001-05-03 Barnhill Technologies, Llc Methods and devices for identifying patterns in biological patterns
WO2001031580A2 (en) * 1999-10-27 2001-05-03 Biowulf Technologies, Llc Methods and devices for identifying patterns in biological systems
EP1144690A1 (en) * 1998-12-03 2001-10-17 Isis Pharmaceuticals, Inc. Antisense modulation of cellular inhibitor of apoptosis-2 expression
WO2002021418A2 (en) * 2000-09-05 2002-03-14 Yeda Research And Development Co. Ltd. Method and system for identifying commercially distributed organisms
EP1414841A1 (en) * 2001-07-10 2004-05-06 Oligos Etc. Inc. Oligonucleotide-containing pharmacological compositions and their use
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US7966338B2 (en) 2001-01-19 2011-06-21 Johnson & Johnson Computer-directed assembly of a polynucleotide encoding a target polypeptide
US8916531B2 (en) 2007-11-20 2014-12-23 Isis Pharmaceuticals, Inc. Modulation of CD40 expression
US10487106B2 (en) 2010-09-01 2019-11-26 Nippon Shinyaku Co., Ltd. Antisense nucleic acids
US11185837B2 (en) 2013-08-05 2021-11-30 Twist Bioscience Corporation De novo synthesized gene libraries
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US11807956B2 (en) 2015-09-18 2023-11-07 Twist Bioscience Corporation Oligonucleic acid variant libraries and synthesis thereof

Families Citing this family (103)

* Cited by examiner, † Cited by third party
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US6766082B2 (en) * 2000-10-18 2004-07-20 Nippon Telegraph And Telephone Corporation Waveguide-type optical device and manufacturing method therefor
US20040121313A1 (en) * 2002-12-06 2004-06-24 Ecker David J. Methods for rapid detection and identification of bioagents in organs for transplantation
US20030027135A1 (en) * 2001-03-02 2003-02-06 Ecker David J. Method for rapid detection and identification of bioagents
US7226739B2 (en) 2001-03-02 2007-06-05 Isis Pharmaceuticals, Inc Methods for rapid detection and identification of bioagents in epidemiological and forensic investigations
US7666588B2 (en) 2001-03-02 2010-02-23 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
US7718354B2 (en) 2001-03-02 2010-05-18 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US8073627B2 (en) * 2001-06-26 2011-12-06 Ibis Biosciences, Inc. System for indentification of pathogens
US7217510B2 (en) 2001-06-26 2007-05-15 Isis Pharmaceuticals, Inc. Methods for providing bacterial bioagent characterizing information
JP2003331168A (en) * 2002-05-15 2003-11-21 Fujitsu Ltd Method for ordering primer synthesis for gene amplification, method for ordering reagent kit for gene amplification factor detection, and device and program for ordering primer synthesis and reagent kit
DE10238298A1 (en) * 2002-08-21 2004-03-04 Beiersdorf Ag Use of antisense oligonucleotides for the treatment of degenerative skin symptoms
DE10254214A1 (en) * 2002-11-20 2004-06-09 Beiersdorf Ag Oligoribonucleotides for the treatment of degenerative skin symptoms by RNA interference
EP1578399A4 (en) 2002-12-06 2007-11-28 Isis Pharmaceuticals Inc Methods for rapid identification of pathogens in humans and animals
US8046171B2 (en) * 2003-04-18 2011-10-25 Ibis Biosciences, Inc. Methods and apparatus for genetic evaluation
US8057993B2 (en) 2003-04-26 2011-11-15 Ibis Biosciences, Inc. Methods for identification of coronaviruses
WO2005009202A2 (en) * 2003-05-12 2005-02-03 Isis Pharmaceuticals, Inc. Automatic identification of bioagents
US8158354B2 (en) * 2003-05-13 2012-04-17 Ibis Biosciences, Inc. Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
US7964343B2 (en) * 2003-05-13 2011-06-21 Ibis Biosciences, Inc. Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
US20120122103A1 (en) * 2003-09-11 2012-05-17 Rangarajan Sampath Compositions for use in identification of bacteria
US8097416B2 (en) 2003-09-11 2012-01-17 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US20060240412A1 (en) * 2003-09-11 2006-10-26 Hall Thomas A Compositions for use in identification of adenoviruses
US8546082B2 (en) 2003-09-11 2013-10-01 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US20080138808A1 (en) * 2003-09-11 2008-06-12 Hall Thomas A Methods for identification of sepsis-causing bacteria
US20100035239A1 (en) * 2003-09-11 2010-02-11 Isis Pharmaceuticals, Inc. Compositions for use in identification of bacteria
US7480382B2 (en) * 2003-09-30 2009-01-20 Microsoft Corporation Image file container
US8163895B2 (en) 2003-12-05 2012-04-24 Ibis Biosciences, Inc. Compositions for use in identification of orthopoxviruses
US7666592B2 (en) 2004-02-18 2010-02-23 Ibis Biosciences, Inc. Methods for concurrent identification and quantification of an unknown bioagent
US8119336B2 (en) * 2004-03-03 2012-02-21 Ibis Biosciences, Inc. Compositions for use in identification of alphaviruses
ES2641832T3 (en) 2004-05-24 2017-11-14 Ibis Biosciences, Inc. Mass spectrometry with selective ion filtration by setting digital thresholds
US20100152280A1 (en) * 2004-05-24 2010-06-17 Isis Pharmaceuticals, Inc. Modulation of sid-1 expression
US20050266411A1 (en) * 2004-05-25 2005-12-01 Hofstadler Steven A Methods for rapid forensic analysis of mitochondrial DNA
EP1771250B1 (en) * 2004-06-04 2011-01-26 Abacus Diagnostica OY Temperature control of reaction vessel, system with reaction vessel and use of system
US7811753B2 (en) 2004-07-14 2010-10-12 Ibis Biosciences, Inc. Methods for repairing degraded DNA
WO2006094238A2 (en) 2005-03-03 2006-09-08 Isis Pharmaceuticals, Inc. Compositions for use in identification of adventitious viruses
US8084207B2 (en) * 2005-03-03 2011-12-27 Ibis Bioscience, Inc. Compositions for use in identification of papillomavirus
AU2006272776B2 (en) * 2005-07-21 2012-01-19 Ibis Biosciences, Inc. Methods for rapid identification and quantitation of nucleic acid variants
JP5713377B2 (en) 2005-12-28 2015-05-07 ザ スクリプス リサーチ インスティテュート Natural antisense and non-coding RNA transcripts as drug targets
KR100794705B1 (en) * 2006-06-13 2008-01-14 (주)바이오니아 Method of Inhibiting Expression of Target mRNA Using siRNA Considering Alternative Splicing of Genes
EP2064332B1 (en) * 2006-09-14 2012-07-18 Ibis Biosciences, Inc. Targeted whole genome amplification method for identification of pathogens
WO2008104002A2 (en) * 2007-02-23 2008-08-28 Ibis Biosciences, Inc. Methods for rapid forensic dna analysis
WO2008118809A1 (en) * 2007-03-23 2008-10-02 Ibis Biosciences, Inc. Compositions for use in identification of mixed populations of bioagents
WO2008151023A2 (en) 2007-06-01 2008-12-11 Ibis Biosciences, Inc. Methods and compositions for multiple displacement amplification of nucleic acids
US8534447B2 (en) 2008-09-16 2013-09-17 Ibis Biosciences, Inc. Microplate handling systems and related computer program products and methods
WO2010033599A2 (en) 2008-09-16 2010-03-25 Ibis Biosciences, Inc. Mixing cartridges, mixing stations, and related kits, systems, and methods
EP2347254A2 (en) 2008-09-16 2011-07-27 Ibis Biosciences, Inc. Sample processing units, systems, and related methods
MY188457A (en) * 2008-10-03 2021-12-10 Opko Curna Llc Treatment of apolipoprotein-a1 related diseases by inhibition of natural antisense transcript to apolipoprotein-a1
US8927511B2 (en) * 2008-12-04 2015-01-06 Curna, Inc. Treatment of vascular endothelial growth factor (VEGF) related diseases by inhibition of natural antisense transcript to VEGF
MX358603B (en) 2008-12-04 2018-08-28 Curna Inc Treatment of tumor suppressor gene related diseases by inhibition of natural antisense transcript to the gene.
CN102317458B (en) * 2008-12-04 2018-01-02 库尔纳公司 Pass through treatment of the suppression of erythropoietin(EPO) (EPO) natural antisense transcript to EPO relevant diseases
WO2010093943A1 (en) * 2009-02-12 2010-08-19 Ibis Biosciences, Inc. Ionization probe assemblies
PL2396038T3 (en) 2009-02-12 2016-05-31 Curna Inc Treatment of brain derived neurotrophic factor (bdnf) related diseases by inhibition of natural antisense transcript to bdnf
EP2408919B1 (en) 2009-03-16 2017-10-18 CuRNA, Inc. Treatment of nuclear factor (erythroid-derived 2)-like 2 (nrf2) related diseases by inhibition of natural antisense transcript to nrf2
US9708604B2 (en) 2009-03-17 2017-07-18 Curna, Inc. Treatment of delta-like 1 homolog (DLK1) related diseases by inhibition of natural antisense transcript to DLK1
US20120046236A1 (en) 2009-05-06 2012-02-23 Opko Curna Llc Treatment of tristetraproline (ttp) related diseases by inhibition of natural antisense transcript to ttp
CN106237345A (en) 2009-05-06 2016-12-21 库尔纳公司 By suppression therapy lipid transfer and the metabolic gene relevant disease of the natural antisense transcript for lipid transfer and metabolic gene
CN102459597B (en) 2009-05-08 2020-06-30 库尔纳公司 Treatment of dystrophin family-related diseases by inhibition of natural antisense transcript to DMD family
JP5922017B2 (en) 2009-05-18 2016-05-24 クルナ・インコーポレーテッド Treatment of reprogramming factor-related diseases by suppression of natural antisense transcripts against the reprogramming factor
JP2012527248A (en) 2009-05-22 2012-11-08 クルナ・インコーポレーテッド Treatment of TFE3 and insulin receptor substrate 2 (IRS2) related diseases by inhibition of natural antisense transcripts against transcription factor E3 (TFE3)
JP5960049B2 (en) 2009-05-28 2016-08-02 クルナ・インコーポレーテッド Treatment of antiviral gene-related diseases by suppression of natural antisense transcripts against antiviral genes
CN102612560B (en) 2009-06-16 2017-10-17 库尔纳公司 By suppressing to treat the related diseases of PON1 for the natural antisense transcript of PON1 (PON1)
US20120171170A1 (en) 2009-06-16 2012-07-05 Opko Curna, Llc Treatment of collagen gene related diseases by inhibition of natural antisense transcript to a collagen gene
CA2765889A1 (en) 2009-06-24 2010-12-29 Opko Curna, Llc Treatment of tumor necrosis factor receptor 2 (tnfr2) related diseases by inhibition of natural antisense transcript to tnfr2
CA2765815A1 (en) 2009-06-26 2010-12-29 Opko Curna, Llc Treatment of down syndrome gene related diseases by inhibition of natural antisense transcript to a down syndrome gene
US9194877B2 (en) 2009-07-17 2015-11-24 Ibis Biosciences, Inc. Systems for bioagent indentification
WO2011008971A1 (en) * 2009-07-17 2011-01-20 Ibis Biosciences, Inc. Lift and mount apparatus
WO2011011700A2 (en) 2009-07-24 2011-01-27 Curna, Inc. Treatment of sirtuin (sirt) related diseases by inhibition of natural antisense transcript to a sirtuin (sirt)
CN102762731B (en) 2009-08-05 2018-06-22 库尔纳公司 By inhibiting to treat insulin gene (INS) relevant disease for the natural antisense transcript of insulin gene (INS)
CN104313027B (en) 2009-08-11 2018-11-20 库尔纳公司 By inhibiting the natural antisense transcript of adiponectin (ADIPOQ) to treat adiponectin (ADIPOQ) related disease
KR101805213B1 (en) 2009-08-21 2017-12-06 큐알엔에이, 인크. Treatment of 'c terminus of hsp70-interacting protein' (chip) related diseases by inhibition of natural antisense transcript to chip
EP2470657B1 (en) 2009-08-25 2019-10-23 CuRNA, Inc. Treatment of 'iq motif containing gtpase activating protein' (iqgap) related diseases by inhibition of natural antisense transcript to iqgap
US20120295952A1 (en) 2009-09-25 2012-11-22 Curna, Inc. Treatment of filaggrin (flg) related diseases by modulation of flg expression and activity
US20110091882A1 (en) * 2009-10-02 2011-04-21 Ibis Biosciences, Inc. Determination of methylation status of polynucleotides
US9890408B2 (en) * 2009-10-15 2018-02-13 Ibis Biosciences, Inc. Multiple displacement amplification
KR101823702B1 (en) 2009-12-16 2018-01-30 큐알엔에이, 인크. Treatment of membrane bound transcription factor peptidase, site 1 (mbtps1) related diseases by inhibition of natural antisense transcript to mbtps1
JP6031356B2 (en) 2009-12-23 2016-11-24 カッパーアールエヌエー,インコーポレイテッド Treatment of uncoupling protein 2 (UCP2) -related diseases by inhibition of natural antisense transcripts against UCP2.
DK2516648T3 (en) 2009-12-23 2018-02-12 Curna Inc TREATMENT OF HEPATOCYTE GROWTH FACTOR (HGF) RELATED DISEASES BY INHIBITION OF NATURAL ANTISENSE TRANSCRIPT AGAINST HGF
EP2519633B1 (en) 2009-12-29 2017-10-25 CuRNA, Inc. Treatment of nuclear respiratory factor 1 (nrf1) related diseases by inhibition of natural antisense transcript to nrf1
CN102770540B (en) 2009-12-29 2017-06-23 库尔纳公司 P63 relevant diseases are treated by suppressing the natural antisense transcript of oncoprotein 63 (p63)
JP6083735B2 (en) 2009-12-31 2017-02-22 カッパーアールエヌエー,インコーポレイテッド Treatment of insulin receptor substrate 2 (IRS2) related diseases by inhibition of natural antisense transcripts against insulin receptor substrate 2 (IRS2) and transcription factor 3 (TFE3)
NO2521784T3 (en) 2010-01-04 2018-05-05
US8912157B2 (en) 2010-01-06 2014-12-16 Curna, Inc. Treatment of pancreatic developmental gene related diseases by inhibition of natural antisense transcript to a pancreatic developmental gene
DK2524039T3 (en) 2010-01-11 2018-03-12 Curna Inc TREATMENT OF GENDER HORMON-BINDING GLOBULIN (SHBG) RELATED DISEASES BY INHIBITION OF NATURAL ANTISENCE TRANSCRIPTS TO SHBG
KR101853510B1 (en) 2010-01-25 2018-06-20 큐알엔에이, 인크. Treatment of rnase h1 related diseases by inhibition of natural antisense transcript to rnase h1
EP2539452B1 (en) 2010-02-22 2016-07-27 CuRNA, Inc. Treatment of pyrroline-5-carboxylate reductase 1 (pycr1) related diseases by inhibition of natural antisense transcript to pycr1
CN102869777B (en) 2010-04-02 2018-11-02 库尔纳公司 CSF3 relevant diseases are treated by inhibiting the natural antisense transcript of colony stimulating factor 3 (CSF3)
EP3517613A1 (en) 2010-04-09 2019-07-31 CuRNA, Inc. Treatment of fibroblast growth factor 21 (fgf21) related diseases by inhibition of natural antisense transcript to fgf21
KR20130101442A (en) 2010-05-03 2013-09-13 큐알엔에이, 인크. Treatment of sirtuin (sirt) related diseases by inhibition of natural antisense transcript to a sirtuin (sirt)
TWI531370B (en) 2010-05-14 2016-05-01 可娜公司 Treatment of par4 related diseases by inhibition of natural antisense transcript to par4
DK2576784T3 (en) 2010-05-26 2018-02-26 Curna Inc TREATMENT OF METHIONIN SULPHOXIDE REDUCTASE A (MSRA) RELATED DISEASES BY INHIBITION OF NATURAL ANTISENCE TRANSCRIPTION TO MSRA
DK2576783T3 (en) 2010-05-26 2018-03-12 Curna Inc TREATMENT OF ATONAL HOMOLOGY 1- (ATOH1) RELATED DISEASES BY INHIBITION OF NATURAL ANTISENCE TRANSCRIPTS AT ATOH1
PL2585596T3 (en) 2010-06-23 2021-06-28 Curna, Inc. Treatment of sodium channel, voltage-gated, alpha subunit (scna) related diseases by inhibition of natural antisense transcript to scna
CN103068982B (en) 2010-07-14 2017-06-09 库尔纳公司 DLG relevant diseases are treated by suppressing the natural antisense transcript of the big homologue of plate-like (DLG)
CA2813901C (en) 2010-10-06 2019-11-12 Curna, Inc. Treatment of sialidase 4 (neu4) related diseases by inhibition of natural antisense transcript to neu4
WO2012054723A2 (en) 2010-10-22 2012-04-26 Opko Curna Llc Treatment of alpha-l-iduronidase (idua) related diseases by inhibition of natural antisense transcript to idua
WO2012068340A2 (en) 2010-11-18 2012-05-24 Opko Curna Llc Antagonat compositions and methods of use
US8987225B2 (en) 2010-11-23 2015-03-24 Curna, Inc. Treatment of NANOG related diseases by inhibition of natural antisense transcript to NANOG
US9593330B2 (en) 2011-06-09 2017-03-14 Curna, Inc. Treatment of frataxin (FXN) related diseases by inhibition of natural antisense transcript to FXN
WO2013036403A1 (en) 2011-09-06 2013-03-14 Curna, Inc. TREATMENT OF DISEASES RELATED TO ALPHA SUBUNITS OF SODIUM CHANNELS, VOLTAGE-GATED (SCNxA) WITH SMALL MOLECULES
KR101762295B1 (en) * 2012-02-10 2017-08-04 (주)바이오니아 Automatic analysis apparatus and method of biological samples
US10955291B2 (en) * 2012-03-15 2021-03-23 Amgen Inc. Methods of determining exposure to UV light
US20150031750A1 (en) 2012-03-15 2015-01-29 The Scripps Research Institute Treatment of brain derived neurotrophic factor (bdnf) related diseases by inhibition of natural antisense transcript to bdnf
TWM445178U (en) * 2012-06-25 2013-01-11 Chin-Feng Wan Microfluidic chip automatic system with optical platform
US10040048B1 (en) 2014-09-25 2018-08-07 Synthego Corporation Automated modular system and method for production of biopolymers
EP4003165B1 (en) * 2019-07-30 2024-02-14 EMD Millipore Corporation Method of synthesizing chemical compounds

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4806463A (en) * 1986-05-23 1989-02-21 Worcester Foundation For Experimental Biology Inhibition of HTLV-III by exogenous oligonucleotides
WO1989010977A1 (en) * 1988-05-03 1989-11-16 Isis Innovation Limited Analysing polynucleotide sequences
US5436327A (en) * 1988-09-21 1995-07-25 Isis Innovation Limited Support-bound oligonucleotides
US5612455A (en) * 1994-07-05 1997-03-18 Tularik, Inc. Nuclear factors and binding assay
US5700637A (en) * 1988-05-03 1997-12-23 Isis Innovation Limited Apparatus and method for analyzing polynucleotide sequences and method of generating oligonucleotide arrays
US5783431A (en) * 1996-04-24 1998-07-21 Chromaxome Corporation Methods for generating and screening novel metabolic pathways
US5824485A (en) * 1995-04-24 1998-10-20 Chromaxome Corporation Methods for generating and screening novel metabolic pathways

Family Cites Families (158)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US552538A (en) * 1896-01-07 Potato sorter and cleaner
US5023A (en) * 1847-03-20 Stocking-loom
US360966A (en) * 1887-04-12 Folding umbrella
US2122177A (en) * 1937-05-19 1938-06-28 Lincoln Eng Co Injector
US2162898A (en) * 1938-01-17 1939-06-20 Lincoln Eng Co Relief device for lubricating apparatus
US2155250A (en) * 1938-01-17 1939-04-18 Lincoln Eng Co Injector
US2283638A (en) * 1940-08-12 1942-05-19 Lincoln Eng Co Injector
US2637413A (en) * 1950-04-22 1953-05-05 Lincoln Eng Co Lubricant injector
US2627320A (en) * 1950-09-07 1953-02-03 Lincoln Eng Co Lubricating system
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
JPS4833493Y1 (en) * 1969-10-15 1973-10-11
US3664462A (en) * 1970-05-27 1972-05-23 William O Smith Sr Lubricant metering valve
US3715013A (en) * 1971-08-19 1973-02-06 Eaton Corp Lubricant injector
US3958725A (en) * 1974-10-15 1976-05-25 Auto Research Corporation Metering valve for lubrication injector
US4469863A (en) 1980-11-12 1984-09-04 Ts O Paul O P Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof
US5023243A (en) 1981-10-23 1991-06-11 Molecular Biosystems, Inc. Oligonucleotide therapeutic agent and method of making same
US4476301A (en) 1982-04-29 1984-10-09 Centre National De La Recherche Scientifique Oligonucleotides, a process for preparing the same and their application as mediators of the action of interferon
US4494913A (en) * 1982-07-08 1985-01-22 Bijur Lubricating Corp. Positive displacement injector
JPS5927900A (en) 1982-08-09 1984-02-14 Wakunaga Seiyaku Kk Oligonucleotide derivative and its preparation
FR2540122B1 (en) 1983-01-27 1985-11-29 Centre Nat Rech Scient NOVEL COMPOUNDS COMPRISING A SEQUENCE OF OLIGONUCLEOTIDE LINKED TO AN INTERCALATION AGENT, THEIR SYNTHESIS PROCESS AND THEIR APPLICATION
US4605735A (en) 1983-02-14 1986-08-12 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives
US4948882A (en) * 1983-02-22 1990-08-14 Syngene, Inc. Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis
US4824941A (en) 1983-03-10 1989-04-25 Julian Gordon Specific antibody to the native form of 2'5'-oligonucleotides, the method of preparation and the use as reagents in immunoassays or for binding 2'5'-oligonucleotides in biological systems
US4520902A (en) * 1983-04-19 1985-06-04 Lubriquip-Houdaille, Inc. Lubricant applying system and injector means
US4587044A (en) 1983-09-01 1986-05-06 The Johns Hopkins University Linkage of proteins to nucleic acids
US5118802A (en) 1983-12-20 1992-06-02 California Institute Of Technology DNA-reporter conjugates linked via the 2' or 5'-primary amino group of the 5'-terminal nucleoside
US5118800A (en) 1983-12-20 1992-06-02 California Institute Of Technology Oligonucleotides possessing a primary amino group in the terminal nucleotide
US4668476A (en) * 1984-03-23 1987-05-26 Applied Biosystems, Inc. Automated polypeptide synthesis apparatus
FR2567892B1 (en) 1984-07-19 1989-02-17 Centre Nat Rech Scient NOVEL OLIGONUCLEOTIDES, THEIR PREPARATION PROCESS AND THEIR APPLICATIONS AS MEDIATORS IN DEVELOPING THE EFFECTS OF INTERFERONS
US5258506A (en) 1984-10-16 1993-11-02 Chiron Corporation Photolabile reagents for incorporation into oligonucleotide chains
US5367066A (en) 1984-10-16 1994-11-22 Chiron Corporation Oligonucleotides with selectably cleavable and/or abasic sites
IT1196307B (en) * 1984-10-22 1988-11-16 Chiesi Farma Spa AQUEOUS PHARMACEUTICAL FORMULATIONS OF PIROXICAM MONOHYDRATE
US4828979A (en) 1984-11-08 1989-05-09 Life Technologies, Inc. Nucleotide analogs for nucleic acid labeling and detection
FR2575751B1 (en) 1985-01-08 1987-04-03 Pasteur Institut NOVEL ADENOSINE DERIVATIVE NUCLEOSIDES, THEIR PREPARATION AND THEIR BIOLOGICAL APPLICATIONS
US5185444A (en) 1985-03-15 1993-02-09 Anti-Gene Deveopment Group Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages
US5235033A (en) 1985-03-15 1993-08-10 Anti-Gene Development Group Alpha-morpholino ribonucleoside derivatives and polymers thereof
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US5405938A (en) 1989-12-20 1995-04-11 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5166315A (en) 1989-12-20 1992-11-24 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US4762779A (en) 1985-06-13 1988-08-09 Amgen Inc. Compositions and methods for functionalizing nucleic acids
US4800159A (en) * 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences
US5317098A (en) 1986-03-17 1994-05-31 Hiroaki Shizuya Non-radioisotope tagging of fragments
JPS638396A (en) 1986-06-30 1988-01-14 Wakunaga Pharmaceut Co Ltd Poly-labeled oligonucleotide derivative
EP0260032B1 (en) 1986-09-08 1994-01-26 Ajinomoto Co., Inc. Compounds for the cleavage at a specific position of RNA, oligomers employed for the formation of said compounds, and starting materials for the synthesis of said oligomers
GR871619B (en) * 1986-10-31 1988-03-03 Genetic Systems Corp Automated patient sample analysis instrument
US5276019A (en) 1987-03-25 1994-01-04 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5264423A (en) 1987-03-25 1993-11-23 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US4904582A (en) 1987-06-11 1990-02-27 Synthetic Genetics Novel amphiphilic nucleic acid conjugates
US4924624A (en) 1987-10-22 1990-05-15 Temple University-Of The Commonwealth System Of Higher Education 2,',5'-phosphorothioate oligoadenylates and plant antiviral uses thereof
US5188897A (en) 1987-10-22 1993-02-23 Temple University Of The Commonwealth System Of Higher Education Encapsulated 2',5'-phosphorothioate oligoadenylates
US5525465A (en) * 1987-10-28 1996-06-11 Howard Florey Institute Of Experimental Physiology And Medicine Oligonucleotide-polyamide conjugates and methods of production and applications of the same
DE3738460A1 (en) 1987-11-12 1989-05-24 Max Planck Gesellschaft MODIFIED OLIGONUCLEOTIDS
US5403711A (en) 1987-11-30 1995-04-04 University Of Iowa Research Foundation Nucleic acid hybridization and amplification method for detection of specific sequences in which a complementary labeled nucleic acid probe is cleaved
WO1989005358A1 (en) 1987-11-30 1989-06-15 University Of Iowa Research Foundation Dna and rna molecules stabilized by modifications of the 3'-terminal phosphodiester linkage and their use as nucleic acid probes and as therapeutic agents to block the expression of specifically targeted genes
US5082830A (en) 1988-02-26 1992-01-21 Enzo Biochem, Inc. End labeled nucleotide probe
WO1989009221A1 (en) * 1988-03-25 1989-10-05 University Of Virginia Alumni Patents Foundation Oligonucleotide n-alkylphosphoramidates
US5278302A (en) 1988-05-26 1994-01-11 University Patents, Inc. Polynucleotide phosphorodithioates
US5109124A (en) 1988-06-01 1992-04-28 Biogen, Inc. Nucleic acid probe linked to a label having a terminal cysteine
US5216141A (en) 1988-06-06 1993-06-01 Benner Steven A Oligonucleotide analogs containing sulfur linkages
US5175273A (en) 1988-07-01 1992-12-29 Genentech, Inc. Nucleic acid intercalating agents
US5262536A (en) 1988-09-15 1993-11-16 E. I. Du Pont De Nemours And Company Reagents for the preparation of 5'-tagged oligonucleotides
US5512439A (en) 1988-11-21 1996-04-30 Dynal As Oligonucleotide-linked magnetic particles and uses thereof
US5599923A (en) * 1989-03-06 1997-02-04 Board Of Regents, University Of Tx Texaphyrin metal complexes having improved functionalization
US5391723A (en) 1989-05-31 1995-02-21 Neorx Corporation Oligonucleotide conjugates
US5256775A (en) 1989-06-05 1993-10-26 Gilead Sciences, Inc. Exonuclease-resistant oligonucleotides
US4958013A (en) 1989-06-06 1990-09-18 Northwestern University Cholesteryl modified oligonucleotides
US5451463A (en) 1989-08-28 1995-09-19 Clontech Laboratories, Inc. Non-nucleoside 1,3-diol reagents for labeling synthetic oligonucleotides
US5134066A (en) 1989-08-29 1992-07-28 Monsanto Company Improved probes using nucleosides containing 3-dezauracil analogs
US5254469A (en) 1989-09-12 1993-10-19 Eastman Kodak Company Oligonucleotide-enzyme conjugate that can be used as a probe in hybridization assays and polymerase chain reaction procedures
US5591722A (en) * 1989-09-15 1997-01-07 Southern Research Institute 2'-deoxy-4'-thioribonucleosides and their antiviral activity
US5399676A (en) 1989-10-23 1995-03-21 Gilead Sciences Oligonucleotides with inverted polarity
US5264562A (en) 1989-10-24 1993-11-23 Gilead Sciences, Inc. Oligonucleotide analogs with novel linkages
DK0497875T3 (en) 1989-10-24 2000-07-03 Gilead Sciences Inc 2'-modified oligonucleotides
US5264564A (en) 1989-10-24 1993-11-23 Gilead Sciences Oligonucleotide analogs with novel linkages
US5292873A (en) 1989-11-29 1994-03-08 The Research Foundation Of State University Of New York Nucleic acids labeled with naphthoquinone probe
US5177198A (en) 1989-11-30 1993-01-05 University Of N.C. At Chapel Hill Process for preparing oligoribonucleoside and oligodeoxyribonucleoside boranophosphates
US5130302A (en) 1989-12-20 1992-07-14 Boron Bilogicals, Inc. Boronated nucleoside, nucleotide and oligonucleotide compounds, compositions and methods for using same
US5486603A (en) 1990-01-08 1996-01-23 Gilead Sciences, Inc. Oligonucleotide having enhanced binding affinity
US5459255A (en) 1990-01-11 1995-10-17 Isis Pharmaceuticals, Inc. N-2 substituted purines
US5859221A (en) * 1990-01-11 1999-01-12 Isis Pharmaceuticals, Inc. 2'-modified oligonucleotides
US5623065A (en) * 1990-08-13 1997-04-22 Isis Pharmaceuticals, Inc. Gapped 2' modified oligonucleotides
US5955589A (en) * 1991-12-24 1999-09-21 Isis Pharmaceuticals Inc. Gapped 2' modified oligonucleotides
US5149797A (en) 1990-02-15 1992-09-22 The Worcester Foundation For Experimental Biology Method of site-specific alteration of rna and production of encoded polypeptides
US5220007A (en) 1990-02-15 1993-06-15 The Worcester Foundation For Experimental Biology Method of site-specific alteration of RNA and production of encoded polypeptides
AU7579991A (en) 1990-02-20 1991-09-18 Gilead Sciences, Inc. Pseudonucleosides and pseudonucleotides and their polymers
US5214136A (en) 1990-02-20 1993-05-25 Gilead Sciences, Inc. Anthraquinone-derivatives oligonucleotides
US5321131A (en) 1990-03-08 1994-06-14 Hybridon, Inc. Site-specific functionalization of oligodeoxynucleotides for non-radioactive labelling
US5470967A (en) 1990-04-10 1995-11-28 The Dupont Merck Pharmaceutical Company Oligonucleotide analogs with sulfamate linkages
DK0455905T3 (en) * 1990-05-11 1998-12-07 Microprobe Corp Dipsticks for nucleic acid hybridization assays and method for covalent immobilization of oligonucleotides
US5707796A (en) * 1990-06-11 1998-01-13 Nexstar Pharmaceuticals, Inc. Method for selecting nucleic acids on the basis of structure
US5386023A (en) 1990-07-27 1995-01-31 Isis Pharmaceuticals Backbone modified oligonucleotide analogs and preparation thereof through reductive coupling
US5138045A (en) 1990-07-27 1992-08-11 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5623070A (en) * 1990-07-27 1997-04-22 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5608046A (en) * 1990-07-27 1997-03-04 Isis Pharmaceuticals, Inc. Conjugated 4'-desmethyl nucleoside analog compounds
US5223618A (en) 1990-08-13 1993-06-29 Isis Pharmaceuticals, Inc. 4'-desmethyl nucleoside analog compounds
US5378825A (en) 1990-07-27 1995-01-03 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs
US5610289A (en) * 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5218105A (en) 1990-07-27 1993-06-08 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5489677A (en) 1990-07-27 1996-02-06 Isis Pharmaceuticals, Inc. Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms
WO1992002258A1 (en) * 1990-07-27 1992-02-20 Isis Pharmaceuticals, Inc. Nuclease resistant, pyrimidine modified oligonucleotides that detect and modulate gene expression
US5602240A (en) * 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5618704A (en) * 1990-07-27 1997-04-08 Isis Pharmacueticals, Inc. Backbone-modified oligonucleotide analogs and preparation thereof through radical coupling
US5245022A (en) 1990-08-03 1993-09-14 Sterling Drug, Inc. Exonuclease resistant terminally substituted oligonucleotides
US5177196A (en) 1990-08-16 1993-01-05 Microprobe Corporation Oligo (α-arabinofuranosyl nucleotides) and α-arabinofuranosyl precursors thereof
US5512667A (en) 1990-08-28 1996-04-30 Reed; Michael W. Trifunctional intermediates for preparing 3'-tailed oligonucleotides
US5214134A (en) 1990-09-12 1993-05-25 Sterling Winthrop Inc. Process of linking nucleosides with a siloxane bridge
EP0549686A4 (en) * 1990-09-20 1995-01-18 Gilead Sciences Inc Modified internucleoside linkages
US5432272A (en) 1990-10-09 1995-07-11 Benner; Steven A. Method for incorporating into a DNA or RNA oligonucleotide using nucleotides bearing heterocyclic bases
EP0556301B1 (en) 1990-11-08 2001-01-10 Hybridon, Inc. Incorporation of multiple reporter groups on synthetic oligonucleotides
US5407796A (en) * 1991-01-04 1995-04-18 The Johns Hopkins University Cystic fibrosis mutation cluster
US5352775A (en) * 1991-01-16 1994-10-04 The Johns Hopkins Univ. APC gene and nucleic acid probes derived therefrom
US5719262A (en) * 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
US5714331A (en) * 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
EP0514927A1 (en) * 1991-05-24 1992-11-25 Walter Gilbert Method and apparatus for rapid nucleic acid sequencing
ES2117050T3 (en) * 1991-06-27 1998-08-01 Genelabs Tech Inc SCREENING TEST FOR THE DETECTION OF MOLECULES OF UNION TO DNA.
US5371241A (en) 1991-07-19 1994-12-06 Pharmacia P-L Biochemicals Inc. Fluorescein labelled phosphoramidites
US5639603A (en) * 1991-09-18 1997-06-17 Affymax Technologies N.V. Synthesizing and screening molecular diversity
ES2103918T3 (en) 1991-10-17 1997-10-01 Ciba Geigy Ag BICYCLE NUCLEOSIDES, OLIGONUCLEOTIDES, PROCEDURE FOR THEIR OBTAINING AND INTERMEDIATE PRODUCTS.
US5594121A (en) * 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
US5484908A (en) 1991-11-26 1996-01-16 Gilead Sciences, Inc. Oligonucleotides containing 5-propynyl pyrimidines
US5359044A (en) 1991-12-13 1994-10-25 Isis Pharmaceuticals Cyclobutyl oligonucleotide surrogates
US5595726A (en) * 1992-01-21 1997-01-21 Pharmacyclics, Inc. Chromophore probe for detection of nucleic acid
FR2687679B1 (en) * 1992-02-05 1994-10-28 Centre Nat Rech Scient OLIGOTHIONUCLEOTIDES.
US5633360A (en) * 1992-04-14 1997-05-27 Gilead Sciences, Inc. Oligonucleotide analogs capable of passive cell membrane permeation
US5434257A (en) 1992-06-01 1995-07-18 Gilead Sciences, Inc. Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages
EP0577558A2 (en) * 1992-07-01 1994-01-05 Ciba-Geigy Ag Carbocyclic nucleosides having bicyclic rings, oligonucleotides therefrom, process for their preparation, their use and intermediates
US5272250A (en) 1992-07-10 1993-12-21 Spielvogel Bernard F Boronated phosphoramidate compounds
US5293959A (en) * 1992-09-21 1994-03-15 Ingersoll-Rand Company Low pressure system lubrication for a jackhammer
US5523389A (en) * 1992-09-29 1996-06-04 Isis Pharmaceuticals, Inc. Inhibitors of human immunodeficiency virus
US5476925A (en) 1993-02-01 1995-12-19 Northwestern University Oligodeoxyribonucleotides including 3'-aminonucleoside-phosphoramidate linkages and terminal 3'-amino groups
GB9304618D0 (en) 1993-03-06 1993-04-21 Ciba Geigy Ag Chemical compounds
GB9304620D0 (en) 1993-03-06 1993-04-21 Ciba Geigy Ag Compounds
ATE208658T1 (en) * 1993-07-28 2001-11-15 Pe Corp Ny APPARATUS AND METHOD FOR NUCLEIC ACID DUPLICATION
US5502177A (en) 1993-09-17 1996-03-26 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US5463567A (en) * 1993-10-15 1995-10-31 Caterpillar Inc. Apparatus and method for providing historical data regarding machine operating parameters
US5472672A (en) * 1993-10-22 1995-12-05 The Board Of Trustees Of The Leland Stanford Junior University Apparatus and method for polymer synthesis using arrays
US5457187A (en) 1993-12-08 1995-10-10 Board Of Regents University Of Nebraska Oligonucleotides containing 5-fluorouracil
US5446137B1 (en) 1993-12-09 1998-10-06 Behringwerke Ag Oligonucleotides containing 4'-substituted nucleotides
US5519134A (en) * 1994-01-11 1996-05-21 Isis Pharmaceuticals, Inc. Pyrrolidine-containing monomers and oligomers
US5463657A (en) 1994-02-15 1995-10-31 Lockheed Missiles & Space Company, Inc. Detection of a multi-sequence spread spectrum signal
US5596091A (en) * 1994-03-18 1997-01-21 The Regents Of The University Of California Antisense oligonucleotides comprising 5-aminoalkyl pyrimidine nucleotides
US5627053A (en) * 1994-03-29 1997-05-06 Ribozyme Pharmaceuticals, Inc. 2'deoxy-2'-alkylnucleotide containing nucleic acid
US5625050A (en) * 1994-03-31 1997-04-29 Amgen Inc. Modified oligonucleotides and intermediates useful in nucleic acid therapeutics
US5507796A (en) 1994-04-28 1996-04-16 Hasson; Harrith M. Method of suspending a pelvic organ and instrument for performing the method
US5525711A (en) * 1994-05-18 1996-06-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Pteridine nucleotide analogs as fluorescent DNA probes
US5571639A (en) * 1994-05-24 1996-11-05 Affymax Technologies N.V. Computer-aided engineering system for design of sequence arrays and lithographic masks
US5597696A (en) * 1994-07-18 1997-01-28 Becton Dickinson And Company Covalent cyanine dye oligonucleotide conjugates
US5597909A (en) * 1994-08-25 1997-01-28 Chiron Corporation Polynucleotide reagents containing modified deoxyribose moieties, and associated methods of synthesis and use
US5580731A (en) * 1994-08-25 1996-12-03 Chiron Corporation N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith
US5463564A (en) * 1994-09-16 1995-10-31 3-Dimensional Pharmaceuticals, Inc. System and method of automatically generating chemical compounds with desired properties
US6015667A (en) * 1996-06-03 2000-01-18 The Perkin-Emer Corporation Multicomponent analysis method including the determination of a statistical confidence interval
US6453246B1 (en) * 1996-11-04 2002-09-17 3-Dimensional Pharmaceuticals, Inc. System, method, and computer program product for representing proximity data in a multi-dimensional space
US6571227B1 (en) * 1996-11-04 2003-05-27 3-Dimensional Pharmaceuticals, Inc. Method, system and computer program product for non-linear mapping of multi-dimensional data
CA2270527A1 (en) * 1996-11-04 1998-05-14 3-Dimensional Pharmaceuticals, Inc. System, method, and computer program product for the visualization and interactive processing and analysis of chemical data
US6016348A (en) * 1996-11-27 2000-01-18 Thomson Consumer Electronics, Inc. Decoding system and data format for processing and storing encrypted broadcast, cable or satellite video data
US6127191A (en) * 1996-12-03 2000-10-03 3-Dimensional Pharmaceuticals, Inc. Aminobenzenedicarboxylic acid-based combinatorial libraries
US6506784B1 (en) * 1999-07-01 2003-01-14 3-Dimensional Pharmaceuticals, Inc. Use of 1,3-substituted pyrazol-5-yl sulfonates as pesticides
AU6116800A (en) * 1999-07-22 2001-02-13 3-Dimensional Pharmaceuticals, Inc. 1-aryl-3-thioalkyl pyrazoles, the synthesis thereof and the use thereof as insecticides

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4806463A (en) * 1986-05-23 1989-02-21 Worcester Foundation For Experimental Biology Inhibition of HTLV-III by exogenous oligonucleotides
WO1989010977A1 (en) * 1988-05-03 1989-11-16 Isis Innovation Limited Analysing polynucleotide sequences
US5700637A (en) * 1988-05-03 1997-12-23 Isis Innovation Limited Apparatus and method for analyzing polynucleotide sequences and method of generating oligonucleotide arrays
US5436327A (en) * 1988-09-21 1995-07-25 Isis Innovation Limited Support-bound oligonucleotides
US5612455A (en) * 1994-07-05 1997-03-18 Tularik, Inc. Nuclear factors and binding assay
US5824485A (en) * 1995-04-24 1998-10-20 Chromaxome Corporation Methods for generating and screening novel metabolic pathways
US5783431A (en) * 1996-04-24 1998-07-21 Chromaxome Corporation Methods for generating and screening novel metabolic pathways

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GHOSH M. K., ET AL.: "EVALUATION OF SOME PROPERTIES OF A PHOSPHORODITHIOATE OLIGODEOXYRIBONUCLEOTIDE FOR ANTISENSE APPLICATION.", NUCLEIC ACIDS RESEARCH, INFORMATION RETRIEVAL LTD., GB, vol. 21., no. 24., 1 January 1993 (1993-01-01), GB, pages 5761 - 5766., XP002921397, ISSN: 0305-1048 *
MIRABELLI C. K., ET AL.: "IN VITRO AND IN VIVO PHARMACOLOGIC ACTIVITIES OF ANTISENSE OLIGONUCLEOTIDES.", ANTI-CANCER DRUG DESIGN, OXFORD UNIVERSITY PRESS, BASINGSTOKE, vol. 06., 1 December 1991 (1991-12-01), BASINGSTOKE, pages 647 - 661., XP002921398, ISSN: 0266-9536 *
UHLMANN E., PEYMAN A.: "ANTISENSE OLIGONUCLEOTIDES: A NEW THERAPEUTIC PRINCIPLE.", CHEMICAL REVIEWS, AMERICAN CHEMICAL SOCIETY, US, vol. 90., no. 04., 1 June 1990 (1990-06-01), US, pages 543 - 584., XP000141412, ISSN: 0009-2665, DOI: 10.1021/cr00102a001 *

Cited By (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1082456A4 (en) * 1998-05-01 2002-07-31 Isis Pharmaceuticals Inc Antisense modulation of cd40 expression
EP1082456A1 (en) * 1998-05-01 2001-03-14 Isis Pharmaceuticals, Inc. Antisense modulation of cd40 expression
EP1144690A4 (en) * 1998-12-03 2004-09-22 Isis Pharmaceuticals Inc Antisense modulation of cellular inhibitor of apoptosis-2 expression
EP1144690A1 (en) * 1998-12-03 2001-10-17 Isis Pharmaceuticals, Inc. Antisense modulation of cellular inhibitor of apoptosis-2 expression
WO2001031580A3 (en) * 1999-10-27 2002-07-11 Biowulf Technologies Llc Methods and devices for identifying patterns in biological systems
WO2001031579A3 (en) * 1999-10-27 2002-07-11 Barnhill Technologies Llc Methods and devices for identifying patterns in biological patterns
WO2001031579A2 (en) * 1999-10-27 2001-05-03 Barnhill Technologies, Llc Methods and devices for identifying patterns in biological patterns
WO2001031580A2 (en) * 1999-10-27 2001-05-03 Biowulf Technologies, Llc Methods and devices for identifying patterns in biological systems
WO2002021418A2 (en) * 2000-09-05 2002-03-14 Yeda Research And Development Co. Ltd. Method and system for identifying commercially distributed organisms
WO2002021418A3 (en) * 2000-09-05 2003-10-09 Yeda Res & Dev Method and system for identifying commercially distributed organisms
US7966338B2 (en) 2001-01-19 2011-06-21 Johnson & Johnson Computer-directed assembly of a polynucleotide encoding a target polypeptide
US8183361B2 (en) 2001-07-10 2012-05-22 Lakewood-Amedex, Inc. Oligonucleotide-containing pharmacological compositions and their use
EP1414841A4 (en) * 2001-07-10 2006-06-21 Oligos Etc Inc Oligonucleotide-containing pharmacological compositions and their use
US9567584B2 (en) 2001-07-10 2017-02-14 Lakewood Amedex, Inc. Oligonucleotide—containing pharmacological compositions and their use
US8188259B2 (en) 2001-07-10 2012-05-29 Lakewood-Amedex, Inc. Oligonucleotide-containing pharmacological compositions and their use
US8916529B2 (en) 2001-07-10 2014-12-23 Lakewood-Amedex, Inc. Oligonucleotide-containing pharmacological compositions and their use
EP1414841A1 (en) * 2001-07-10 2004-05-06 Oligos Etc. Inc. Oligonucleotide-containing pharmacological compositions and their use
EP1461674A4 (en) * 2001-12-03 2007-10-24 Dyax Corp Library screening
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USRE47320E1 (en) 2007-11-20 2019-03-26 Ionis Pharmaceuticals, Inc. Modulation of CD40 expression
US8916531B2 (en) 2007-11-20 2014-12-23 Isis Pharmaceuticals, Inc. Modulation of CD40 expression
US10487106B2 (en) 2010-09-01 2019-11-26 Nippon Shinyaku Co., Ltd. Antisense nucleic acids
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