WO2010011884A2

WO2010011884A2 - Novel nucleic acid-based molecular probes

Info

Publication number: WO2010011884A2
Application number: PCT/US2009/051632
Authority: WO
Inventors: Weihong Tan; Zhiwen Tang
Original assignee: University Of Florida Research Foundation, Inc.
Priority date: 2008-07-25
Filing date: 2009-07-24
Publication date: 2010-01-28
Also published as: WO2010011884A3

Abstract

The subject invention provides a novel nucleic acid-based molecular probe design employing intramolecular signal transduction. In an embodiment specifically exemplified herein, the probe comprises four elements: an aptamer; a short, partially complementary DNA sequence; a PEG linker conjugating the aptamer with the short DNA strand; and a luminescent molecule attached to a terminus of the aptamer and a terminus of the DNA sequence.

Description

DESCRIPTION

NOVEL NUCLEIC ACID-BASED MOLECULAR PROBES

CROSS REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. Provisional Application Serial No. 61/135,927, filed July 25, 2008, which is hereby incorporated by reference herein in its entirety.

GOVERNMENT SUPPORT

The subject invention was made with government support under a research project supported by NIH National Institute of General Medical Science, Grant No. ROl GM079359. Accordingly, the government has certain rights in this invention.

BACKGROUND OF INVENTION

Aptamers are single-stranded oligonucleotides that cβru-feeognize and bind to target molecules with strong affinity and excellent specificity.¹'² Specifically, aptamers are nucleic acid molecules having specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing. Aptamers can be isolated via an in vitro selection process termed SELEX™ (System Evolution of Ligands by Exponential enrichment) against a variety of targets, including small molecules, ions, proteins and whole cells.^3"4

Since nucleic acid bases can be easily modified and molecularly engineered, aptamers can meet the stringent requirements for uses such as bioassay,^4"6 drug delivery,^7"8 signal transduction ^" and gene expression mediation.^11"12 Focusing on the development of bioassay probes, many different design principles have been advanced.¹ '^"14 While each strategy has distinct advantages, each also presents its own unique set of limitations. One design type, for example, utilizes conformation alteration during the aptamer-target binding event, providing a simple approach for aptamer biosensor design.³'^{6 15} These probe designs use the spatial change of the aptamer sequence termini to produce a fluorescent or electrochemical signal. However, for any given aptamer sequence, this method commonly encounters unpredictable structural alteration such that the spatial change may not induce perceptible signal transduction. Another strategy uses either DNA-intercalating dyes or photoactive polymers to report the recognition and binding between an aptamcr and target molecule.¹'^"19 This method, however, suffers from intrinsic limitations, such as high background, nonselectivity against specific aptamers and lack of multiplex detection capability. Besides these strategies, a competition-based probe construction also has been developed by introducing a DNA competitor to sense the binding between aptamer and target." ^" The DNA competitor hybridizes with a partial or whole aptamer sequence, but allows dehybridization when the aptamer binds to target molecule.

Similar to the methods described above, this strategy has both advantages and disadvantages. For example, while this approach offers a general means of aptamer probe fabrication, it also requires a separated oligo competitor. This limits some applications, such as in situ detection, but favors others which require a long oligo competitor to achieve steady hybridization while maintaining a low background. At the same time, the introduction of a longer oligo competitor requires careful optimization to avoid conformational changes that would hinder the recognition and affinity of aptamer toward target.

To address these shortcomings, the subject invention provides novel and versatile intramolecular signal transduction aptamer probes.

BRIEF SUMMARY The subject invention provides a novel nucleic acid-based molecular probe design employing intramolecular signal transduction. In an embodiment specifically exemplified herein, the probe comprises four elements: (a) a single stranded oligonucleotide having a first and second terminus, where the first terminus is attached to (b) a linking moiety, where the linking moiety is also attached to (c) a first terminus of a short DNA strand having a partially complementary sequence to the oligonucleotide, where the short DNA strand has a first and second terminus; and (d) luminescent molecules attached to the second termini of the oligonucleotide and the short DNA strand.

In a preferred embodiment, the oligonucleotide is an aptamer. This preferred probe is referred to herein as an ''aptamer switch probe," or ASP. The ASP design utilizes both a fluorophore and a quencher as the luminescent molecules, which are respectively attached at the two termini of the aptamer and DNA sequence of the ASP. In the absence of a target molecule, the short DNA hybridizes with the aptamer, keeping the fluorophore and quencher in close proximity, thus switching off the fluorescence. However, when the ASP meets its target, the binding between the aptamer and the target molecule disturbs the intramolecular DNA hybridization, thereby moving the quencher away from the fluorophore, and switching on the fluorescence. In specific embodiments exemplified herein, both ATP and human α-thrombin aptamers have been engineered to demonstrate this design, and both showed that fluorescence enhancement can be quantitatively mediated by the addition of various amounts of target molecules. Both of these ASP probes demonstrated excellent selectivity and prompt response towards their targets. With the advantageous characteristics arising from its intramolecular signal transduction architecture, the ASP design can be used for applications including, but not limited to, biochips and in situ imaging, which require reusability, excellent stability, prompt response, and high sensitivity.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows a scheme of an aptamer switch probe (ASP) and the design of ATP- ASP and human α-thrombin ASP (Tmb-ASP). F represents a fiuoropore and Q represents a quencher.

Figure 2A-B (A) shows the fluorescence spectra of the buffer, ATP-ASP and ATP- ASP+ATP. (B) Plot of the fluorescence of ATP-ASP as the function of ATP concentration.

Figure 3 shows the selectivity of ATP-ASP toward NTPs. The fluorescence signal enhancements were normalized to ATP sample.

Figure 4A-B shows the kinetics of ATP-ASP response. The final concentration of ATP after each addition was labeled above the curve. The peaks were caused by the stir during spiking. (A) The response at low concentration of ATP. (B) The response at high concentration of ATP.

Figure 5 The fluorescence spectra of human α-thrombin ASP (Tmb-ASP) with the addition of a series concentration of human α-thrombin (Tmb).

Figure 6 The selectivity of Tmb-ASP. The fluorescence spectra of Tmb-ASP with thrombin (Tmb), IgG, IgM and BSA.

Figure 7 shows the synthesis pathway of an ASP with Ce6 coupling. Figure 8 shows the fluorescence response selectivity of ATP-ASP. The fluorescence spectra of buffer, ATP-ASP and ATP-ASP with N lTs.

Figure 9 shows the fluorescence spectra of buffer and ATP-ASP with different concentrations of ATP.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO:1 is a synthetic oligonucleotide that preferentially binds with a high degree of specificity and affinity to ATP.

SEQ ID NO:2 is a synthetic oligonucleotide that preferentially binds with a high degree of specificity and affinity to Tmb.

DETAILED DISCLOSURE

The subject invention provides an effective molecular engineering mechanism to signal probe/target binding events. More specifically, the subject invention provides a novel nucleic acid-based molecular probe design employing intramolecular signal transduction. In an embodiment specifically exemplified herein, the probe comprises four elements: a single stranded oligonucleotide; a short, partially complementary DNA sequence to the oligonucleotide; a linking moiety that conjugates the oligonucleotide with the DNA sequence; and luminescent molecules. According to the invention, the single stranded oligonucleotide has a first and second terminus, where the first terminus is attached to the linking moiety. The linking moiety is also attached to a first terminus of a short DNA strand having a partially complementary sequence to the oligonucleotide, where the short DNA strand has a first and second terminus. Luminescent molecules are attached to the second termini of the oligonucleotide and the short DNA strand.

Oligonucleotides are well understood by the skilled artisan and methods for their preparation are well known in the art. Examples of methods for preparing desired oligonucleotides include, but are not limited to. H-phosphonate and phosphate triester methods, phosphodiester synthesis method, phosphotriester synthesis method, phosphite triester synthesis method, methods using nucleoside phosphoramidites, solid support synthesis, and oligonucleotide phosphorothioates synthesis methods. The oligonucleotides used in the present invention preferably exhibit high affinity and specificity toward a given target molecule. Preferably, the oligonucleotide of the invention ranges in size from about 10, 15. 20, 25, 30, 35. 40, 45, 50. 55, 60, 65. 70, 75, 80, 85, 90, 95, and 100 nucleotides.

In a preferred embodiment, the oligonucleotide is an aptamer. Aptamers are oligonucleic acid molecules that bind to a specific target molecule such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Aptamers are engineered through repeated rounds of in vitro selection or equivalently, SELEX™. According to the subject invention, aptamers are single-stranded oligonucleotides exhibiting high affinity and specificity toward any given target molecule. The aptamers of the invention have highly defined tertiary structures that allow them to selectively bind a target molecule to form a more stable complex than that with the short, partially complementary DNA sequence. The aptamers of the invention are single-stranded oligonucleotides that can be of any suitable size, and are preferably in the range of from about 10 to about 100 nucleotides, more preferably from about 10 to about 80 nucleotides, and more preferably from about 20 to 40 nucleotides. The precise sequence and length of the aptamer of the invention depends in part on the nature of the target molecule to which it binds. The binding location and length of the aptamer may be varied to achieve appropriate annealing and melting properties for a particular embodiment. Guidance for making such design choices can be found in many art recognized references.

As noted above, one suitable method for generating an aptamer is with the process entitled SELEX™. The SELEX™ process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in, e.g., U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, now abandoned, U.S. Pat. No. 5,475,096 entitled "Nucleic Acid Ligands", and U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled "Nucleic Acid Ligands". all of which are incorporated herein by reference. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX™ process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets.

The core SELEX™ method has been modified to achieve a number of specific objectives. For example, U.S. Pat. No. 5,707,796 (incorporated herein by reference in its entirety) describes the use of SliLEX™ in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. Pat. No. 5,763,177 (incorporated herein by reference in its entirety) describes SELEX™ based methods for selecting nucleic acid ligands containing photo reactive groups capable of binding and/or photo-crosslinking to and/or photo-inactivating a target molecule. U.S. Pat. No. 5,567,588 and U.S. Pat. No. 5,861,254 (both of which are incorporated herein by reference in their entirety) describe SELEX™ based methods which achieve highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. Pat. No. 5,496,938 (incorporated herein by reference in its entirety) describes methods for obtaining improved nucleic acid ligands after the SELEX™ process has been performed. U.S. Pat. No. 5,705,337 (incorporated herein by reference in its entirety) describes methods for covalently linking a ligand to its target.

SELEX™ can also be used to obtain nucleic acid ligands that bind to more than one site on the target molecule, and to obtain nucleic acid ligands that include non-nucleic acid species that bind to specific sites on the target. SELEX™ provides means for isolating and identifying nucleic acid ligands which bind to any envisionable target, including large and small biomolecules such as nucleic acid-binding proteins and proteins not known to bind nucleic acids as part of their biological function as well as cofactors and other small molecules. For example, U.S. Pat. No. 5,580,737 (incorporated herein by reference in its entirety) discloses nucleic acid sequences identified through SELEX™ which are capable of binding with high affinity to caffeine and the closely related analog, theophylline.

The aptamers with specificity and binding affinity to the target(s) of the present invention are typically selected by the SELEX™ process as described herein. As part of the SELEX™ process, the sequences selected to bind to the target are then optionally minimized to determine the minimal sequence having the desired binding affinity. The selected sequences and/or the minimized sequences are optionally optimized by performing random or directed mutagenesis of the sequence to increase binding affinity or alternatively to determine which positions in the sequence are essential for binding activity. Additionally, selections can be performed with sequences incorporating modified nucleotides to stabilize the aptamer molecules against degradation in vivo.

According to the subject invention, the short, partially complementary DNA sequence is a single- stranded sequence of nucleotides, where a portion of this sequence of nucleotides readily binds to a complementary sequence of the oligonucleotide sequence on the probe. Preferably, where the probe comprises an aptamer, the short DNA sequence has a portion of nucleotides that readily binds to a complementary sequence of nucleotides on the aptamer. As understood by the skilled artisan, complementarity is a property of double-stranded nucleic acids. Each strand is complementary to the other in that the base pairs between them are non-covalcntly connected via two or three hydrogen bonds. Since there is only one complementary base for any of the bases found in DNA. one can reconstruct a complementary strand for any single strand. Thus, in accordance with the subject invention, the skilled artisan can readily construct a short DNA sequence that is partially complementary to a portion of the oligonucleotide or aptamer sequence. According to the invention, a "short" DNA sequence is a sequence of nucleotides that is composed of fifty or fewer bases, preferably forty or fewer bases, even more preferably thirty or fewer bases, and most preferably twenty or fewer bases.

According to the subject invention, there is a variable linker region (1-50 residues in length) that attaches the oligonucleotide (such as an aptamer) to the short DNA sequence. According to the subject invention, the linking moiety can be a nucleic acid moiety that does not bind to cither the oligonucleotide or the short DNA sequence, a peptide nucleic acid (PNA) moiety, a peptidic moiety, a disulfide bond, a phosphodiester linkage, or a polymer such as a polyethylene glycol moiety. Suitable linkers can include modified nucleotides or modified backbone. The linker region can be double or single stranded nucleic acid and is designed to not interact with either the oligonucleotide (such as aptamer) or the short DNA strand. For example, the linker can be a random nucleic acid sequence. In another embodiment, the linker is peptidic in nature, or includes a single or PNA residues.

Typical polymers used in the invention as linking moities include polyethylene glycol ("PEG"), also known as polyethylene oxide ("PEO") and polypropylene glycol (including poly isopropylene glycol). Additionally, random or block copolymers of different alkylene oxides (such as ethylene oxide and propylene oxide) can be used. In its most common form, a polyalkylene glycol, such as PEG, is a linear polymer terminated at each end with hydroxyl groups: HO-CH₂CH₂O-(CH₂CH₂O) 11-CH₂CH₂-OH. This polymer, α-, ω- dihydroxylpolyethylene glycol, can also be represented as HO— PEG-OH, where it is understood that the— PEG-symbol represents the following structural unit: --CH₂CH₂O- (CH₂CH₂O) n-CH₂CH₂- where n typically ranges from about 4 1o about 10,000. According to the subject invention, the PEG molecule is di-functional and is sometimes referred to as "PEG diol." The terminal portions of the PEG molecule are relatively non-reactive hydroxyl moieties, the —OH groups, that can be activated, or converted to functional moieties, for attachment of the PEG to first termini of the oligonucleotide (e g., aptamer) and the short DNA sequence. Such activated PEG diols are also referred to herein as bi-activated PEGs. For example, the terminal moieties of PEG diol have been functionalized as active carbonate ester for selective reaction with amino moieties by substitution of the relatively non-reactive hydroxyl moieties, —OH, with succinimidyl active ester moieties from N-hydroxy succinimide. Poly alkylated linking moieties of the invention are typically between 500 Da and 100 kDa in size however any size can be used, the choice dependent on the nucleic acid-based probe. Other polymeric linking moieties of the invention are between 500 Da and 80 kDa in size. Still other polyalkylated linking moieties of the invention are between 500 Da and 60 kDa in size. For example, a PEG polymer may be at least 500 Da, 1 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, or 70 kDa in size. Such polymers can be linear or branched.

In an exemplary embodiment, the PEG linker serves to distance the DNA sequence from the aptamer (because if the aptamer is held too closely to the DNA sequence, it may not interact with a target molecule that comes into contact with the aptamer-DNA complex). Further, the PEG linker may assist aptamer dissociation from the DNA sequence when in close proximity to the target molecule and also enable the probe to form a structure that is more stable when the aptamer is bound to the target molecule. According to the invention, a polyalkylene glycol moiety such as PEG that is covalently bound at either end to an aptamer and short DNA sequence preferably is in a linear arrangement, nucleic acid-PAG-nucleic acid.

PEG-nucleic acid conjugates may be prepared by incorporating the PEG using the same iterative monomer synthesis described above for preparing oligonucleotides/aptamers. For example, PEGs activated by conversion to a phosphoramidite form can be incorporated into solid-phase oligonucleotide synthesis. Alternatively, oligonucleotide synthesis can be completed with site-specific incorporation of a reactive PEG attachment site. Most commonly this has been accomplished by addition of a free primary amine at the 5'-terminus (incorporated using a modifier phosphoramidite in the last coupling step of solid phase synthesis). Using this approach, a reactive PEG (e.g., one which is activated so that it will react and form a bond with an amine) is combined with the purified oligonucleotide and the coupling reaction is carried out in solution.

According to the subject invention, the luminescent molecule is any molecule that provides luminescent indication of oligonucleotide/target molecule binding. Preferably, the luminescent molecule is a fluorophore or quencher. Λ fluorophore is a molecule that has the ability to absorb energy from light, transfer this energy internally, and emit this energy as light of a characteristic wavelength. Fluorophores that can be used in accordance with this invention include, but are not limited to, Chlorin e6 (Ce6), fluorescein (FAM). letrachloro-6- carboxy fluorescein (TET). tetramethylrhodamine, CAL Fluor Gold 540 (Biosearch Technologies), HEX, JOE. VIC (Applied Biosystems). CAL Fluor Orange 560 (Biosearch Technologies), Cy3 (Λmersham Biosciences). NED (Applied Biosystems), Quasar 570 (Biosearch Technologies). Oyster 556 (Integrated DNA Technologies), TMR, CAL Fluor Red 590 (Biosearch Technologies), ROX, LC red 610 (Roche Applied Science), CAL Fluor Red 610 (Biosearch Technologies), Texas red. LC Red 640 (Roche Applied Science), CAL Fluor Red 635 (Biosearch Technologies), Cy5 (Amersham Biosciences), LC red 670 (Roche Applied Science), Quasar 670 (Biosearch Technologies), Oyster 645 (Integrated DNA Technologies), LC red 705 (Roche Applied Science), and Cy5.5 (Amersham Biosciences). Preferred embodiments of the invention use any one of the following fluorophores: Ce6, FAM. Cy3, or Cy5.

According to the subject invention, a quencher is a substance that absorbs excitation energy from a fluorophore. As taught herein, suppression of emission from a fluorophore (or "quenching^") occurs as a result of the formation of a complex between the fluorophore and the quencher, where the absorption spectra of the two molecules change upon formation of the complex (for example, when the fluorophore and quencher are in close proximity with each other). When the two molecules are not in close proximity with each other, fluorescence emission is restored. The quencher can be another fluorphore or a non- fluorescent molecule. Quenchers that can be used in accordance with the invention include, but are not limited to, Deep Dark Quencher I (DDQ-I; Eurogentec), Dabcyl, Eclipse (Epock Biosciences). Iowa Black FQ (Integrated DNA Technologies), Black Hole Quencher 1 (BHQ-I ; Biosearch Technologies), QSY-7 (Molecular Probes). Black Hole Quencher 2 (BHQ-2; Biosearch Technologies), Deep Dark Quencher II (DDQ-II; Eurogentec), Iowa Black RQ (Integrated DNA Technologies), QSY-21 (Molecular Probes), Black Hole Quencher 2 (BIIQ-2; Biosearch Technologies), and Black Hole Quencher 3 (BHQ-3; Biosearch Technologies). In preferred embodiments. BHQ-I and and BHQ-2 quenchers are used. Target molecules that arc detected using the probes of the invention include a range of different molecules such as ligands, natural or synthetic peptides, antibodies, amino acids, proteins, cells, bacteria, whole viruses, and small biomolecules such as ATP, sugars, and the like.

As shown in Figure 1, in a preferred embodiment, the probe comprises three elements: an aptamer, a short DNA sequence complementary to part of the aptamer and a PEG linker connecting these two. A fluorophore and a quencher are covalently attached at the two termini of the conjugated DNA-aptamer sequences. Upon target binding, the conformation of the probe can then be '"switched on," where the fluorophore and quencher are separated from each other to allow emission from the fluorophore to be detected. In the absence of a target molecule, the short DNA sequence hybridizes with a small section of the aptamer, keeping the fluorophore and quencher in close proximity, thus quenching the emission from the fluorophore. Conversely, when the probe meets its target molecule, the binding between aptamer and target molecule disturbs the intramolecular DNA hybridization thereby moving the quencher away from the fluorophore, resulting in the restoration of fluorescence.

Compared with other aptamer probe designs, the ASP strategy provides a robust probe construction by integrating aptamer, competitor and signaling moieties into one molecule. This design utilizes intramolecular hybridization/dehybridization and therefore requires a much shorter competitor, which reduces the impact on aptamer spatial conformation and simplifies probe optimization. The ASP design can be used for any aptamers.

To demonstrate the feasibility of this principle, two aptamers were selected with which to construct ASP models: one targets the small biomolecule. ATP, and the other binds to human α-thrombin (Tmb). Chlorin e6 (Ce6) and FAM were chosen as fluorophores, while Black Hole Quencher 2 (BHQ-2) was used to quench the fluorescence of Ce6 and FAM. The Ce6 was chosen in order to use the same design for singlet oxygen generation in target- controlled phototherapy. Five to six PEG Spacer 18s were added as the linker to connect the aptamer and the partially complementary DNA sequence. The synthesis of ASPs was conducted on a DNA synthesizer sequentially followed with an in-tube coupling of Ce6. The purification of probes was carried out on a HPLC, and only the product with all three absorptions of DNA, Ce6/FAM and BHQ2 was collected. UV spectra of products further confirmed the quality of the probe synthesis. To test the fluorescence signaling of the ATP-ASP with the addition of ATP, three samples were prepared in 10 mJVl Tris-HCl buffer with 6 mM MgCl₂ and irradiated at a wavelength of 404 nm, which is the maximum absorption of Ce6.

The ATP-ASP showed significant fluorescence increase upon target binding. As shown in Figure 2, the ATP-ASP presents up to a 30-fold enhancement of fluorescence immediately after the addition of 3.5 mM ATP. The response of ATP-ASP toward a series concentration of ATP was also investigated, and the results present a linear relationship between the fluorescence enhancement and the concentration, demonstrating that the fluorescence of ATP-ASP can be quantitatively mediated by ATP concentration. The ATP-ASP also shows excellent specific response against ATP analogs. As shown in Figure 3, the fluorescence signal does not show much change after the addition of GTP, UTP or CTP at the concentration of 1.0 mM, but it does show significant enhancement after the introduction of 1.0 mM ATP. The excellent selectivity of ATP -ASP is similar to the original ATP aptamer.²³ This result clearly confirms the fact that the ASP design does not affect the selectivity of the ATP aptamer.

The response kinetics of ATP-ASP was then tested by real-time monitoring of the sample with several successive additions of ATP. As shown in Figure 4, the response of ATP-ASP toward the introduction of ATP is prompt. The ATP-ASP delivered over 90% response and reached equilibrium within 5 seconds regardless of the concentration level of ATP. The fast kinetics, which derives from the short oligo, is an advantage of the intramolecular hybridization design. Apparently, the shorter complementary oligo introduces less effect on the aptamer's spatial folding, keeping, as a result, higher recognition affinity, better selectivity and faster hybridization/dehybridization rate, which are all required for optimal aptamer probe design and application.^21"25 Compared with previous designs, the ASP is simpler and efficient in signaling aptamer-target binding process for both analytical applications and binding mechanism studies. Next, to demonstrate the versatility of the ASP design, the same principle and molecular scheme was used to design and synthesize a Tmb-ASP targeting human α- thrombin, which is an important protein in human blood. In this design, the rluorophore was conjugated at the competitor side to verify the effect of fluorophore position on fluorescence restoration. The length of the competitor is only 5 bases, which is much shorter than the intermolecular hybridization design.

The fluorescence of Tmb-ASP was enhanced up to 17.6 times after the addition of 300 nM thrombin (Figure 5). The fluorescence signal of Tmb-ASP could also be quantitatively mediated by the addition of various thrombin concentrations. Furthermore, the Tmb-ASP presented selective response toward thrombin. In contrast, when tested with IgG, IgM and BSA at the concentration of 200 nM, the Tmb-ASP did not produce fluorescence enhancement compared with the sample containing 200 nM thrombin (Figure 6).

The success of ATP-ASP and Tmb-ASP shows that ASP design can be used for targeting proteins and small biornolecules. The ASP design has several significant advantages over conventional designs. First, compared to intermolecular DNA hybridization, the intramolecular DNA hybridization requires a shorter oligo to achieve the same melting temperature. The shorter competitor hybridizes to a smaller partial aptamer and leaves more aptamer sequence free, thus increasing the binding affinity between aptamer and target, as well as improving the sensitivity. Second, the equilibrium of intramolecular hybridization and dehybridization between oligos is more stable and faster than intermolecular events, enabling faster response and lower background, which are important for signaling transduction in probe construction. Third, and perhaps most importantly, this design conjugates the aptamer, competitor oligo and signaling groups into one molecule, preventing the dissociation of probe during applications and making ASP a robust molecular probe. The ASP strategy can be used for many applications, including but not limited to biochips and in situ imaging, which require reusability, excellent stability, prompt response and high sensitivity.

The following example illustrates a procedure for practicing the invention. This example should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Materials and Methods for Synthesis of ATP-ASP and Tmb-ASP Aptamer switch probe (ASP) can be synthesized with different fluorescence dyes. We synthesized ASP using chlorine e6 (Ce6. Frontier Scientific, Inc.) and fluorescein (FAM) as fluorophore. The ASP probes conjugated with FAM can be synthesized on a DNA synthesizer using on-machine standard protocol, but the Ce6 conjugation requires in-tube reaction. However, the HPLC purification determines the quality of probes. Benefiting from the higher hydrophobicity of Ce6, the Ce6-linked ASP probes can be purified better than FAM-labeled probes; therefore, we used Ce6-coupled ASP for most experiments.

The synthesis of Ce6-conjugated ASP is shown in Figure 7. Ce6 linked ASP synthesis includes two major steps: the on machine DNA synthesis and the off machine coupling of Ce6.

1 he DNA product was synthesized from Black Hole Quencher 2 CPG (Glen Research Corp.) on an ABI 3400 DNA synthesizer. The DNA sequence was uploaded into a DNA synthesizer on line. Except for normal DNA bases, the Spacer Phosphoramidite 18 (Glen Research Corp.) and 5 '-Amino group (Glen Research Corp.) were also synthesized on machine. The synthesis protocol was set up according to the requirements specified by the reagents^" manufacturers. Following on machine synthesis, the DNA product was deprotected and cleaved from CPG by incubating with 2.5 ml ammonium hydroxide (Fisher Scientific, Inc.) for 17 hours at 40 ⁰C in water bath. The cleaved DNA product was transferred into a 15 ml centrifuge tube and mixed with 250 μl 3.0 M NaCl (Fisher Scientific, Inc.) and 5.0 ml ethanol. after which the sample was placed into a freezer at -20 ⁰C for ethanol precipitation. Afterwards, the DNA product was spun at 4000 rpm under 3 ⁰C for 20 minutes. The supernatant was removed, and the precipitated DNA product was dissolved in 500 μl 0.2 M trithylamine acetate (TEAA, Glen Research Corp.) for HPLC purification. The HPLC purification was performed with a cleaned Alltech Cl 8 column on a Varian Prostar FIPLC machine. The collected DNA product was dried and detritylation processed by dissolving and incubating in 200 μl 80% acetic acid (Fisher Scientific Inc.) for 20 minutes. The detritylation DNA product was mixed with 400 μl ethanol and dried by a vacuum dryer. The DNA product was then ready for off machine coupling of Ce6.

Each Ce6 molecule has three carboxyl groups, which might be conjugated with the amino group at the 3' end of the DNA product. To improve the coupling efficiency and reduce the multiple coupling products, the amount of Ce6 was 10 times more than DNA product in the coupling reaction. 10 μmole Ce6 was mixed with an equal molecular amount of NJsT-Dicyclohexylcarbodiimide (Dec, Sigma-Λldrich, Inc.) and N-Hydroxysuccinimide (NHS. Sigma-Aldrich, Inc.) and dissolved in 250 μl N.N-Dimethylformamide (DMF, Acros) for activation reaction with 1 hour stirring. The purified DNA product was dissolved in 250 μl 0.1 M pH 7.5 NaHCO₃ solution and mixed with the activated Ce6 for coupling. The coupling reaction was performed with strong stirring for at least 8 hours before ethanol precipitation. To remove the uncoupled reagents, the ΛSP product was precipitated three times with the addition of 100 μl 3.0 M NaCl and 2.0 ml ethanol. The precipitated ASP was dissolved in 500 μl 0.1 M TEAA buffer for HPLC purification. To eliminate the residual free unconjugated chemicals, the reaction product was HPLC- purified twice to obtain the pure ASP.

The purified ASP was quantified by determining the UV absorption at 260 nm, after which the ASP was dissolved in DNA grade water and stored in the freezer at -20 ⁰C for future experiments.

Fluorescence experiment

All fluorescence measurements were performed using Fluorolog (Jobin Yvon Horiba) or microplate reader (TECAN). A 100 μl macro cuvette was used for the experiments on Fluorolog, and the sampling volume was 120 μl to ensure that the measure window was completely filled with solution. A 96-well plate (Nunc) containing the samples in microplate reader was used.

The fluorescence emission of ASP was scanned from 600 nm to 800 nm with excitation at 404 nm, which is the maximum absorption of Ce6.

Test the fluorescence response of ATP-ASP

The ATP-ASP samples were prepared in 10 mM Tris-HCl buffer containing 6 mM

MgCl₂. The ATP-ASP concentration in all samples was 1.0 μM. The ATP concentration range in the titration experiment was from 0.5 mM to 4.5 mM. In the selectivity experiment, the concentration for all NTPs was 1.0 mM. The fluorescence spectra for all samples were measured at 20 ⁰C. See Figures 8 and 9. Determine the fluorescence response of Tmb-ΛSP

The Tmb-ASP samples were prepared in 10 mM Tris-HCl buffer containing 5 mM KCl and ImM MgCb. The Tmb-ASP concentration in all samples was 0.20 μM. The thrombin concentration range in samples was from 20 nM to 0.3 μM. The fluorescence spectra of all samples were measured at 25 ⁰C.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

REFERENCES

(I ) Ellington, A. D.; Szostak. J. W. Nature 1990, 346 (6287), 818-822. (2) Tuerk. C; Gold, L. Science 1990, 249 (4968), 505-510.

(3) Shangguan, D.: Li, Y.; Tang, Z.; Cao, Z.; Chen, H.; Mallikaratchy, P.; Sefah, K.; Yang, C; Tan, W.. PNAS 2006, 103 (32), 11838-11843. (4) Navani. N. K.; Li, Y. F. Curr. Opin. Chem. Biol. 2006, 10 (3), 272-281.

(5) Yang, C. J.; Jockusch, S.; Viccns, M.; Turro. N. J.; Tan, W. H. Proc. Natl. Acad. ScL USA 2005, 702 (48), 17278-17283. (6) Hamaguchi, N.; Ellington, A.; Stanton, M. Anal. Biochem. 2001, 294 (2), 126-131.

(7) McNamara, J. Q.; Andrechek, E. R.; Wang, Y.; Viles, D.; Rempel, R. E.; Gilboa, E.; Sullenger, B. A.; Giangrande, P. II. Nat. Biotech. 2006, 24 (8), 1005-1015. (8) Lee, J. F.; Stovall, G. M.; Ellington, A. D. Curr. Opin. Chem. Biol. 2006, 10 (3), 282-289.

(9) Cho, E. J.; Yang, L.; Levy, M.; Ellington, A. D. J. Am. Chem. Soc. 2005, 127 (7), 2022- 2023. (10) Shlyahovsky, B.; Li, D.; Weizmann, Y.; Nowarski, R.; Kotler, M.; Willner, I. J. Am. Chem. Soc. 2007, 129 (13), 3814-3815.

II 1) Bayer, T. S.; Smolke, C. D. Nat. Biotech. 2005, 23 (3), 337-343. (12) Friedrichs, E.; Simmel, F. C. Chembiochem 2007, 8 (14), 1662-1666.

(13) Mairal, T.; Ozalp, V. C: Sanchez, P. L.; Mir, M.; Katakis, L; O'Sullivan, C. K. Anal Bioanal. Chem. 2008, 390 (4), 989-1007. (14) Willner, I.; Zayats, M. Angew. Chem. Int. Edit. 2007, 46 (34), 6408-6418.

(15) Stojanovic, M. N.; de Prada, P.; Landry, D. W. J Am. Chem. Soc. 2001, 123 (21), 4928-4931. (16) Lin, C. H.; Patel, D. J. Chem. Biol. 1997, 4 (11), 817-832.

(17) Li, B. L.; Wei, H.; Dong, S. J. Chem. Commun. 2007, (1), 73-75.

(18) Jiang. Y. X.; Fang, X. H.; Bai, C. L. Anal. Chem. 2004, 76 (17), 5230-5235.

(19) Ho. H. A.; Leclerc, M. J. Am. Chem. Soc. 2004, 126 (5). 1384-1387. (20) Li, N.; Ho, C. M. J. Am. Chem. Soc. 2008, 130 (8). 2380-2381.

(21) Rupcich, N.; Nutiu. R.; Li, Y. F.; Brennan, J. D. Angew. Chem. Int. Edit. 2006, 45 (20), 3295-3299.

(22) Nutiu, R.; Li. Y. F. J. Am. Chem. Soc. 2003, 125 (16). 4771-4778.

(23) Huizenga, D. E.; Szostak, J. W. Biochemistry 1995, 34 (2), 656-665. (24) Bonnet, G.; Krichevsky, O.; Libchaber, A. Proc. Natl. Acad. Set USA 1998, 95 (15). 8602-8606.

(25) Yang, C; Martinez. K.; Lin, H.; Tan, W. J. Am. Chem. Soc. 2006, 128 (31), 9986- 9987.

Claims

CLAIMS We claim:

1. A nucleic acid-based molecular probe comprising:

(a) an oligonucleotide that selectively binds to a target molecule, said nucleotide having a first and second terminus of the oligonucleotide;

(b) a DNA sequence partially complementary to the oligonucleotide, said DNA sequence having a first and second terminus; and

(c) a linking moiety attached to the first terminus of the oligonucleotide and to the first terminus of the DNA sequence, -wherein the linking moiety conjugates the oligonucleotide to the DNA sequence; and

(d) a first luminescent molecule attached to the second terminus of the oligonucleotide and a second luminescent molecule attached to the second terminus of the DNA sequence.

2. The probe of claim 1, wherein the oligonucleotide is an aptamer.

3. The probe of claim 1, wherein the linking moiety is selected from the group consisting of: a nucleic acid moiety that does not bind to either the oligonucleotide or the short DNA sequence, a peptide nucleic acid (PNA) moiety, a peptidic moiety, a disulfide bond, a phosphodiester linkage, or a polymer.

4. The probe of claim 3, wherein the polymer is polyethylene glycol (PEG).

5. The probe of claim 1, wherein first and second luminescent molecules are interchangeably a fluorophore and quencher, wherein the fluorophore emits fluorescence emission and the quencher inhibits fluorescence emission by the fluorophore when the quencher is in close proximity with the fluorophore.

6. The probe of claim 5, wherein when the target molecule is absent, the DNA sequence partially hybridizes with the oligonucleotide, such that the fluorophore/quencher located at the termini of the aptamer and DNA sequence are in close proximity to inhibit fluorescence emission from the fluorophorc.

7. The probe of claim 6, wherein the fiuorophore is selected from the group consisting of: Chlorin e6 (Ce6). fluorescein (FAM). tetrachloro-ό-carboxyfluorescein (TET), tetramethylrhodamine. CAL Fluor Gold 540. HEX. JOE, VIC. CAL Fluor Orange 560, Cy3, NED, Quasar 570, Oyster 556. TMR, CAL Fluor Red 590, ROX, LC red 610, CAL Fluor Red 610, Texas red, LC Red 640, CAL Fluor Red 635, Cy5, LC red 670, Quasar 670, Oyster 645, LC red 705, and Cy5.5 and wherein the quencher is selected from the group consisting of: Deep Dark Quencher I. Dabcyl, Eclipse, Iowa Black FQ, Black Hole Quencher 1, QSY- 7, Black Hole Quencher 2, Deep Dark Quencher II. Iowa Black RQ, QSY-21, Black Hole Quencher 2, and Black Hole Quencher 3.

8. The probe of claim 7, wherein the fiuorophore is selected from the group consisting of: Ce6 and FAM and wherein the quencher is Black Hole Quencher 2.

9. The probe of claim 1, wherein the target molecule is selected from the group consisting of: ligands, natural or synthetic peptides, antibodies, amino acids, proteins, cells, bacteria, whole viruses, small biomolecules, ATP. and sugar molecules.

10. The probe of claim 1 , wherein the target molecule is ATP.

1 1. The probe of claim 1 , wherein the target molecule is thrombin.

12. A method for identifying the presence of a target molecule in a sample comprising the steps of:

(a) introducing to the sample, at least one nucleic acid-based molecular probe comprising:

(i) an oligonucleotide that selectivel} binds to a target molecule, said oligonucleotide having a first and second terminus;

(ii) a DNA sequence partially complementary to the oligonucleotide, said

DNA sequence having a first and second terminus: (iii) a linking moiety attached to the first terminus of the oligonucleotide and to the first terminus of the DNA sequence, wherein the linking moiet} conjugates the oligonucleotide to the DNA sequence, and (iv) a first luminescent molecule attached to the second terminus of the oligonucleotide and a second luminescent molecule attached to the second terminus of the DNA sequence; and (b) measuring any luminescent emission from the luminescent molecules.

13. The method of claim 12, wherein the oligonucleotide is an aptamer.

14. The method of claim 12, wherein the linking moiety is selected from the group consisting of: a nucleic acid moiety that does not bind to either the oligonucleotide or the short DNA sequence, a peptide nucleic acid (PNA) moiety, a peptidic moiety, a disulfide bond, a phosphodiester linkage, or a polymer.

15. The method of claim 14, wherein the polymer is polyethylene glycol (PEG).

16. The probe of claim 12, wherein first and second luminescent molecules are interchangeably a fluorophore and quencher, wherein the fluorophore emits fluorescence emission and the quencher inhibits fluorescence emission by the fluorophore when the quencher is in close proximity with the fluorophore.

17. The method of claim 16, wherein when the target molecule is absent, the DNA sequence partially hybridizes with the oligonucleotide, such that the fluorophore/quencher located at the termini of the aptamer and DNA sequence are in close proximity to inhibit fluorescence emission from the fluorophore.

18. The method of claim 16, wherein the fluorophore is selected from the group consisting of: Chlorin e6 (Ce6). fluorescein (FAM), tetrachloro-ό-carboxyfluorescein (TET), tetramethylrhodamine, CAL Fluor Gold 540, HEX, JOE, VlC, CAL Fluor Orange 560. Cy3, NED, Quasar 570, Oyster 556, TMR, CAL Fluor Red 590, ROX, LC red 610, CAL Fluor Red 610, Texas red, LC Red 640, CAL Fluor Red 635, Cy5, LC red 670, Quasar 670. Oyster 645, LC red 705, and Cy5.5 and wherein the quencher is selected from the group consisting of: Deep Dark Quencher I, Dabcyl, Eclipse, Iowa Black FQ, Black Hole Quencher 1, QSY- 7, Black Hole Quencher 2, Deep Dark Quencher II, Iowa Black RQ, QSY-2L Black Hole Quencher 2, and Black Hole Quencher 3.

19. The method of claim 18, wherein the fluorophore is selected from the group consisting of: Ce6 and FAM and wherein the quencher is Black Hole Quencher 2.