WO2007104834A1 - Terminating substrates for dna polymerases - Google Patents

Terminating substrates for dna polymerases Download PDF

Info

Publication number
WO2007104834A1
WO2007104834A1 PCT/FI2007/050127 FI2007050127W WO2007104834A1 WO 2007104834 A1 WO2007104834 A1 WO 2007104834A1 FI 2007050127 W FI2007050127 W FI 2007050127W WO 2007104834 A1 WO2007104834 A1 WO 2007104834A1
Authority
WO
WIPO (PCT)
Prior art keywords
lanthanide
substrate according
dna
terminating
lll
Prior art date
Application number
PCT/FI2007/050127
Other languages
French (fr)
Inventor
Jari Hovinen
Original Assignee
Wallac Oy
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wallac Oy filed Critical Wallac Oy
Priority to GB0818768A priority Critical patent/GB2450454B/en
Publication of WO2007104834A1 publication Critical patent/WO2007104834A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • This invention relates to novel derivatives of labeled nucleoside triphosphates suitable for DNA sequencing.
  • DNA fragments are syn- thesised by DNA polymerase which incorporates deoxynucleotide monomers into a polymeric complementary copy of a template DNA strand.
  • An oligonucleotide primer is used to initiate the synthesis of the new DNA stand from the template DNA at a single specific location (Sanger, F., Nicken, S., Coulson, A.R., 1977, PNAS, 74, 5463).
  • ddNTP 2',3 ' -dideoxynucleoside 5 ' -triphosphate
  • dNTP 2',3 ' -dideoxynucleoside 5 ' -triphosphate
  • dNTP 2 ' -deoxynucleoside 5'- triphosphate
  • the dideoxynucleotide is not able to form a phosphodiester bond with the next incoming dNTP, and the growth of that particular DNA chain stops.
  • a series of strands is obtained, the lengths of which depend on the location of ddNTP.
  • each fragment must be labelled in some manner.
  • labelling has been accomplished with radioisotopes, such as 32 P or 35 S prior of during the polymerase reaction, i.e. using either a radioisotopically labelled primer of dNTPs.
  • radioactive detection is very sensitive, it has intrinsic hazard, expense and problems associated with the short half-lives of the radioactive isotopes commonly used.
  • a primer labelled with a detectable group such as a chemiluminescent dye
  • a detectable group such as a chemiluminescent dye
  • a chemiluminescent dye Heunkapiller, T., Kaiser, R.J., Koop, B. F., Hood, K.L, 1991 , Science, 254, 59, Smith, LM., Fung, S, Hunkapiller, M.W., Hunkapiller, T.J., Hood, L.E., 1985, Nucleic Acids Res., 13, 2399, Smith, L.M., Sanders, J.Z., Kaiser, R.J., Hughes, P., Dodd, C, Connell, C.R., Heiner, C, Kent, S.B.H., Hood, LE., 1986, Nature, 321 , 674);
  • the labeled dNTPs ought to be almost as good substrates to DNA polymerases as normal dNTPs. Since DNA polymerases are very sensitive to structural changes of their substrates, the selection of methods to attach non-radioactive markers into dNTPs are rather limited. It has already shown that the well-known terminators of DNA synthesis, 2 ' ,3 ' -dideoxy-3 ' -amino NTPs (Chidzeavadze, Z.G., Beabealashvilli, R.
  • the label is attached covalently to the nucleobase via a rigid propargylamine linker at C5 of the pyrimidine nucleosides and at C7 of 7-deazapurine nucleosides, e.g. at positions which are not involved in the formation of normal Watson-Crick base pairs.
  • these label molecules used are organic dyes which suffer from commonly known drawbacks such as Raman scattering, concentration quenching and low water solubility.
  • minisequencing A solid-phase method, called minisequencing, has been introduced for detection of point mutation of DNA (Syvanen, A.-C, Allto-Setala, K., Harju, L., S ⁇ deriund, H., 1990, Genomics, 8, 684, Jalanko, A., Kere, J., Savilahti, E., Schwartz, M., Syvanen, A.-C, S ⁇ deriund, H., 1992, Clin.
  • lanthanide(lll) chelates such as strong long-decay time luminescence make them ideal markers for numerous applications. Furthermore, large Stokes shift and very sharp emission bands enable the simultaneous use of four lanthanides (i.e. Eu, Tb, Sm, Dy) in the analysis. Time resolved fluorimetric assays based on lanthanide chelates have found increasing applications in diagnostics, research and high throughput screening.
  • the heterogenous DELFIA technique (EP 0139675 Bl; US 4,808,541; EP 0298939 Bl; US 6,127,529; US 4,565,790; WO 03/076939; Hemmila I., Dakubu, S., Mukkala, V.-M., Siitari, H., Lovgren, T., 1984, Anal. Biochem. 137, 335; Fl Pat. Appl. 20065030) is applied in assays requiring exceptional sensitivity, robustness and multi-label approach.
  • the development highly stable and luminescent lanthanide(lll) chelates (Hemmila, I.; Mukkala, V.-M. 2001 , Crit. Rev. Clin. Lab. Sci.
  • the main object of the present invention is to provide nucleoside 5 ' -triphosphates or their acylic analogues labeled with luminescent or non-luminescent lanthanide(lll) chelates to serve as terminating substrates for DNA polymerases.
  • the major advantages of the present invention are:
  • Each DNA nucleobase is detected by its own metal chelate, i.e. four bases (Ade, Gua, Cyt, Thy) and four metal chelates (Eu 3+ , Tb 3+ , Sm 3+ , Dy 3+ ). Accordingly, the base sequence can be analyzed by detecting the specific signal derived form the appropriate lanthanide chelate.
  • the lanthanide(lll) chelate can be either luminescent or non-luminescent. Accordingly, the structure of the chelate can be chosen to fulfil the requirements of the desired detection technology.
  • the sequencing procedure includes electrophoretic separation, it is desirable that the net charges of the terminating substrates do not differ from the natural 2'-deoxyribonucleoside 5 ' -triphosphates. In these cases, the use of neutral lanthanide chelates is advantageous.
  • the present invention concerns a terminating substrate for DNA polymerases of formula (I)
  • Z is triphosphate anion or its organic or inorganic salt
  • R is a recognizing moiety, which is a nucleoside comprising a base bound to a 2,3-dideoxyribose or its acyclic derivative; and Z-R has the formula (II)
  • ribose ring optionally is replaced by an acyclic derivative in which one or both of the 2- and 3- carbon atoms from the ribose ring optionally are missing;
  • -L- is a linker
  • X is a lantha ⁇ ide(lll) chelate.
  • the invention concerns the use of the terminating substrate according to claim 1 in DNA sequencing for deter- mining the base sequence wherein each base is identified by detecting the signal derived from the ianthanide chelate of said terminating substrate.
  • the base in the recognizing moiety R is adenine, guanine, cytosine, thymine or uracil. Also modifications of said bases can be used. As preferable modifications can be mentioned 7-deaza-adenine and 7- deazaguanine.
  • a preferable acyclic derivative is the dimethyl ether bridge derived from the ribose ring.
  • the salt is most preferably sodium, lithium, potassium, calcium, magnesium, ammonium tributylammonium or triethylammonium salt.
  • the recognizing moiety R is a radical selected from the following seven structures;
  • n 0 or 1.
  • the linker -L- is connected to C7 of 7-deazaadenine, C7 of 7-deazaguanine, C5 of cytosine, C5 of uracil, C3 ' of 2 ' -deoxyguanosine, C3 ' 2 ' -deoxyadenosine, C3 ' of 2'- deoxycytidine or C3 ' of thymidine.
  • the lanthanide chelate X is either luminescent or non-luminescent, and the lanthanide(lll) ion is selected in such a way that each DNA nucleobase referes to one lanthanide(lll) ion.
  • any luminescent or non-luminescent lanthanide (III) chelate is suitable as reporter group in the terminating substrate according to the present invention
  • the lanthanide chelates disclosed in the Background section above are preferable.
  • Particularly preferable lanthanide (III) chelates for this purpose are luminescent chelates based on triazacycloalkanes and nonluminescent chelates based on pyridine-2,6-diyl- bis(methylenenitrilo)tetrakis(acetic acid).
  • Biotinylated oligonucleotides and detection primer were purchased from Sigma-Genosys. Poymerase Pol B, dilution buffer, reaction buffer and acycloterminators were products of NEN. Streptavidin coated microtiter plates, assay buffer, wash buffer, enhancement solution, DELFIA Enhancer and lanthanide chelates were from PerkinElmer Life and Analytical Sciences. Minisequencing assays were analyzed with Victor multilabel oounter (Wallac Oy 1 PerkinElmer Life and Analytical Sciences).
  • Biotinylated oligonucleotides (6 pmol/weil in 40 ⁇ L) were captured on streptavidin coated microtiter plates in 30 min by shaking at RT. To remove unbound templates wells were rinsed 4 times with wash buffer. r0041l Example 3. Minisequencing reactions and analysis
  • Reaction mixture (40 ⁇ f) contained the detection primer (0.5- 5 pmol/well), polymerase Pol B (0.1 U/weil) and lanthanide chelate labeled acycloterminator (3a-d).
  • the amount of the triphosphates depended on the lanthanide chelate and was as follows.
  • the amount of Eu-GTP and Tb-UTP was 0.5 pmol/well and both Sm-ATP and Dy-CTP was 50 pmol/well.
  • the reaction was allowed to proceed with shaking 20 minutes at 68°C. After the reactions wells were rinsed 6 times with wash buffer.

Abstract

This invention concerns novel terminating substrates for DNA polymerases. The substrates are nucleoside triphosphates labeled with lanthanide chelates.

Description

TERMINATING SUBSTRATES FOR DNA POLYMERASES
FIELD OF THE INVENTION
[0001] This invention relates to novel derivatives of labeled nucleoside triphosphates suitable for DNA sequencing.
BACKGROUND OF THE INVENTION
[0002] The pubiications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
[0003] In the Sanger's sequencing method DNA fragments are syn- thesised by DNA polymerase which incorporates deoxynucleotide monomers into a polymeric complementary copy of a template DNA strand. An oligonucleotide primer is used to initiate the synthesis of the new DNA stand from the template DNA at a single specific location (Sanger, F., Nicken, S., Coulson, A.R., 1977, PNAS, 74, 5463). Four separate reactions are performed, each containing a carefully controlled ratio of one particular 2',3'-dideoxynucleoside 5'-triphosphate (ddNTP) to the corresponding 2'-deoxynucleoside 5'- triphosphate (dNTP), and the other dNTPs. Once incorporated into a growing DNA strand, the dideoxynucleotide is not able to form a phosphodiester bond with the next incoming dNTP, and the growth of that particular DNA chain stops. Thus, a series of strands is obtained, the lengths of which depend on the location of ddNTP.
[0004] In order to detect these oligomers, each fragment must be labelled in some manner. Traditionally, labelling has been accomplished with radioisotopes, such as 32P or 35S prior of during the polymerase reaction, i.e. using either a radioisotopically labelled primer of dNTPs. Although the radioactive detection is very sensitive, it has intrinsic hazard, expense and problems associated with the short half-lives of the radioactive isotopes commonly used.
[0005] Three different methods can be used to avoid radioactive detection:
[0006] (i) the use of a primer labelled with a detectable group, such as a chemiluminescent dye (Hunkapiller, T., Kaiser, R.J., Koop, B. F., Hood, K.L, 1991 , Science, 254, 59, Smith, LM., Fung, S, Hunkapiller, M.W., Hunkapiller, T.J., Hood, L.E., 1985, Nucleic Acids Res., 13, 2399, Smith, L.M., Sanders, J.Z., Kaiser, R.J., Hughes, P., Dodd, C, Connell, C.R., Heiner, C, Kent, S.B.H., Hood, LE., 1986, Nature, 321 , 674);
[0007] (ii) the use of ddNTPs tethered to detectable groups as terminators of DNA synthesis {Prober, J. M., Trainor, G. L, Dam, RJ., Hobbs, F.W., Robertson, CW. , Zagursky, R.J., Cocuzza, AJ. , Jensen, M.A., Baumeis- ter, K. 1987, Science, 238, 336, Lee, LG., Connell, C.R., Woo, S.L, Cheng, R.D., McArdle, B.F., Fuller, C.W., Hallolan, N.D., Wilson, R.K., 1992, Nucleic Acids Res., 20, 2471 );
[0008] (iii) the use mass spectrometry to analyse DNA fragments (Fizgerald, M.C., Zhu, L, Smith, LM., 1993, Rapid Commun. Mass Spectrom., 7, 895).
[0009] The labeled dNTPs ought to be almost as good substrates to DNA polymerases as normal dNTPs. Since DNA polymerases are very sensitive to structural changes of their substrates, the selection of methods to attach non-radioactive markers into dNTPs are rather limited. It has already shown that the well-known terminators of DNA synthesis, 2',3'-dideoxy-3'-amino NTPs (Chidzeavadze, Z.G., Beabealashvilli, R. Sh., Anttrazhev, A.M., Kuk- hanova, M.K., Azhayev, A., Krayevsky, A.A., 1984, Nucleic Acids Res., 12, 1671) are not substrates of DNA polymerases when bulky reporter groups are attached to their 3'-amino function (Herrlein, M. K., Konrad, R.E., Engels, J.W., Holletz, T., Cech, D., 1994, HeIv. Chim. Acta, 77, 586). Analogously, while T- amino-2'-deoxy-ara adenosine TP is an effective terminating substrate, introduction of bulky substituent to the 2'-position causes total loss of substrate properties of the molecule (Alexandriva, LA., Sharkin, Yu. A., 1993, Collect. Czech. Chem. Commun. Spec, issue, 58, 113). These problems can be solved by attaching the label to 3'-position of the carbohydrate moiety in the end of a flexible tether arm (Hovinen, J., Azhayeva, E., Azhayev, A., Guzaev, A., Lόnnberg, H., 1994, J. Chem. Soc. Perkin Trans 1 , 211). In such manner the bulky reported group may be kept distant from the catalytic centre of the polymerase enzyme, while the base residues remain unmodified. Furthermore, the flexible arm should not severely restrict the conformational motion of the sugar ring upon binding to enzyme.
[0010] A method reported by Prober et al. (Prober, J. M., Trainor, G.L., Dam, R.J., Hobbs, F.W., Robertson, C.W., Zagursky, R.J., Cocuzza, AJ. , Jensen, M.A., Baumeister, K. 1987, Science, 238, 336) consists of the preparation of ddNTPs bearing four different labels attached to their base moieties. Since their fluorescence spectra differ from each other all four terminating triphosphates can be used in a single sequencing reaction. The label is attached covalently to the nucleobase via a rigid propargylamine linker at C5 of the pyrimidine nucleosides and at C7 of 7-deazapurine nucleosides, e.g. at positions which are not involved in the formation of normal Watson-Crick base pairs. However, these label molecules used are organic dyes which suffer from commonly known drawbacks such as Raman scattering, concentration quenching and low water solubility.
[0011] A solid-phase method, called minisequencing, has been introduced for detection of point mutation of DNA (Syvanen, A.-C, Allto-Setala, K., Harju, L., Sόderiund, H., 1990, Genomics, 8, 684, Jalanko, A., Kere, J., Savilahti, E., Schwartz, M., Syvanen, A.-C, Sόderiund, H., 1992, Clin. Chem, 38, 39) The method involves hybridization of immobilized single-stranded DNA with a primer that ends immediately before the site of mutation, and elongation of the chain with a single labeled deoxyriobonucleoside 5 '-triphosphate. Parallel runs with each of the four possible nucleotides enable identification of the mutated base. Thus far, practically only radioactive detection methods have been used. Application of fluorescence techniques has been suffered from drawbacks such as low detection sensitivity and problems assosiated with the properties of the labeled triphosphates to act as good substrates for DNA polymerases.
[0012] The unique properties of lanthanide(lll) chelates such as strong long-decay time luminescence make them ideal markers for numerous applications. Furthermore, large Stokes shift and very sharp emission bands enable the simultaneous use of four lanthanides (i.e. Eu, Tb, Sm, Dy) in the analysis. Time resolved fluorimetric assays based on lanthanide chelates have found increasing applications in diagnostics, research and high throughput screening. The heterogenous DELFIA technique (EP 0139675 Bl; US 4,808,541; EP 0298939 Bl; US 6,127,529; US 4,565,790; WO 03/076939; Hemmila I., Dakubu, S., Mukkala, V.-M., Siitari, H., Lovgren, T., 1984, Anal. Biochem. 137, 335; Fl Pat. Appl. 20065030) is applied in assays requiring exceptional sensitivity, robustness and multi-label approach. The development highly stable and luminescent lanthanide(lll) chelates (Hemmila, I.; Mukkala, V.-M. 2001 , Crit. Rev. Clin. Lab. Sci. 38, 441) has enabled the use of homogenous assay technologies based on time resolution. The different photochemical properties of europium, terbium, dysprosium and samarium chelates enable development even multiparametric homogenous assays. Accordingly, a number of attempts have been made to develop new highly luminescent chelate labels suitable for time-resolved fluorometric applications. These include e.g. stabile chelates composed of derivatives of pyridines [US 4,920,195; US 4,801 ,722; US 4,761 ,481 ; PCT/FI91/00373; US 4,459,186; EP A-0770610; Remuinan et al, J. Chem. Soc. Perkin Trans 2, 1993, 1099], bipyridines [US 5,216,134], terpyri- dines [US 4,859,777; US 5,202,423; US 5,324,825} or various phenolic compounds [US 4,670,572; US 4,794,191; ltal Pat. 42508 A789] as the energy mediating groups and polycarboxylic acids as chelating parts. In addition, various dicarboxylate derivatives [US 5,032,677; US 5,055,578; US 4,772,563] macrocyclic cryptates [US 4,927,923; WO 93/5049; EP-A-493745] and macro- cyclic Schiff bases [EP-A-369000] have been disclosed. Recently, development of neutral, highly luminescent stable europium, terbium, samarium and dysprosium chelates based on azamacrocycles has been disclosed [US Pat. Appl.10/928, 143; US Pat. Appl. 11/004,061].
[0013] However, the use of lanthanide (III) chelates as reporter groups in terminating substrates for DNA polymerases has not been suggested in prior art.
SUMMARY OF THE INVENTION
[0014] The main object of the present invention is to provide nucleoside 5'-triphosphates or their acylic analogues labeled with luminescent or non-luminescent lanthanide(lll) chelates to serve as terminating substrates for DNA polymerases. The major advantages of the present invention are:
[0015] (i) Each DNA nucleobase is detected by its own metal chelate, i.e. four bases (Ade, Gua, Cyt, Thy) and four metal chelates (Eu3+, Tb3+, Sm3+, Dy3+). Accordingly, the base sequence can be analyzed by detecting the specific signal derived form the appropriate lanthanide chelate.
[0016] (ii) The lanthanide(lll) chelate can be either luminescent or non-luminescent. Accordingly, the structure of the chelate can be chosen to fulfil the requirements of the desired detection technology.
[0017] If the sequencing procedure includes electrophoretic separation, it is desirable that the net charges of the terminating substrates do not differ from the natural 2'-deoxyribonucleoside 5'-triphosphates. In these cases, the use of neutral lanthanide chelates is advantageous.
[0018] Thus, the present invention concerns a terminating substrate for DNA polymerases of formula (I)
[0019] Z-R-L-X (I)
[0020] wherein,
[0021] Z is triphosphate anion or its organic or inorganic salt;
[0022] R is a recognizing moiety, which is a nucleoside comprising a base bound to a 2,3-dideoxyribose or its acyclic derivative; and Z-R has the formula (II)
Figure imgf000006_0001
[0023] wherein the ribose ring optionally is replaced by an acyclic derivative in which one or both of the 2- and 3- carbon atoms from the ribose ring optionally are missing;
[0024] -L- is a linker; and
[0025] X is a lanthaπide(lll) chelate.
[0026] According to another aspect, the invention concerns the use of the terminating substrate according to claim 1 in DNA sequencing for deter- mining the base sequence wherein each base is identified by detecting the signal derived from the ianthanide chelate of said terminating substrate.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The base in the recognizing moiety R is adenine, guanine, cytosine, thymine or uracil. Also modifications of said bases can be used. As preferable modifications can be mentioned 7-deaza-adenine and 7- deazaguanine.
[0028] In case the ribose ring is replaced by an acyclic derivative of said ring, a preferable acyclic derivative is the dimethyl ether bridge derived from the ribose ring.
[0029] In case Z as defined above is an organic or inorganic salt of triphosphate, the salt is most preferably sodium, lithium, potassium, calcium, magnesium, ammonium tributylammonium or triethylammonium salt.
[0030] According to a preferable embodiment, the recognizing moiety R is a radical selected from the following seven structures;
Figure imgf000007_0001
[0031] where n is 0 or 1.
[0032] According to a preferable embodiment, the linker -L- is connected to C7 of 7-deazaadenine, C7 of 7-deazaguanine, C5 of cytosine, C5 of uracil, C3' of 2'-deoxyguanosine, C3' 2'-deoxyadenosine, C3' of 2'- deoxycytidine or C3'of thymidine. The linker is preferably formed from one to ten moieties, each moiety being selected from the group consisting of pheny- lene, alkyl containing 1-12 carbon atoms, ethynediyl (-C≡C-), ethylenediyl (- C=C-); ether (-O-), thioether (-S-), amide (-CO-NH- and -NH-CO- and -CO- NR' and -NR'-CO-), carbonyl (-CO-), ester (-COO- and -OOC-), disulfide (- SS-), diaza, (-N=N-), thiourea (-NH-CS-NH-) or a tertiary amine (-NR'-), where R' represents an alkyl containing less than 5 carbon atoms.
[0033] According to a preferable embodiment, the lanthanide chelate X is either luminescent or non-luminescent, and the lanthanide(lll) ion is selected in such a way that each DNA nucleobase referes to one lanthanide(lll) ion.
[0034] Although any luminescent or non-luminescent lanthanide (III) chelate is suitable as reporter group in the terminating substrate according to the present invention, the lanthanide chelates disclosed in the Background section above are preferable. Particularly preferable lanthanide (III) chelates for this purpose are luminescent chelates based on triazacycloalkanes and nonluminescent chelates based on pyridine-2,6-diyl- bis(methylenenitrilo)tetrakis(acetic acid).
EXPERIMENTAL SECTION
[0035] The invention is further elucidated by the following non- restricting examples. The structures and synthetic routes employed in the experimental part are depicted in Scheme 1. Experimental details are given in Example 1. Scheme 2 discloses an example on the synthesis procedure for the preparation of a terminating substrate for DNA polymerase labeled with a highly luminescent dysprosium chelate via a tether at the carbohydrate moiety. Syntheses of the starting materials are given by reference. Capture of the bio- tinylated oligonucleotides to the solid support is described in Example 2. Mi- nisequencing reaction and analysis is described in Example 3.
Procedures
[0036] Biotinylated oligonucleotides and detection primer were purchased from Sigma-Genosys. Poymerase Pol B, dilution buffer, reaction buffer and acycloterminators were products of NEN. Streptavidin coated microtiter plates, assay buffer, wash buffer, enhancement solution, DELFIA Enhancer and lanthanide chelates were from PerkinElmer Life and Analytical Sciences. Minisequencing assays were analyzed with Victor multilabel oounter (Wallac Oy1 PerkinElmer Life and Analytical Sciences).
IO0371 Example 1. Synthesis of the labeled triphosphates
[0038] The acycloterminators tethered to propargylamino groups, 1a-d, were allowed to react with lanthanide(lll) chelates of 2,2',2",2'"-[[4-[(4- isothiocyanatophenyl)ethyl)]pyridine-2,6- diyl]bis(methylenenitrilo)]tetrakis(acetic acid) 2a-d under slighly basic conditions (pyridine:water:triethylamine; 3:1 :0.06; v/v/v) overnight at ambient temperature. After precipitation from acetone, the crude products, 3a-d were re- dissolved in water and purified on HPLC techniques (column: lnertsil ODS-3, RP-18; Buffer A: 0.05 M ammonium acetate; Buffer B: A in 50 % (v/v) acetonit- rile; gradient: from A to 60% B in 30 min; flow rate was 1 mLmin"1). Characterization was performed on MALDI-TOF mass spectrometry. In all the cases the spectra were in accordance with the proposed structures.
F00391 Example 2. Capture of biotinylated oligonucleotides on the solid support
[0040] Biotinylated oligonucleotides (6 pmol/weil in 40 μL) were captured on streptavidin coated microtiter plates in 30 min by shaking at RT. To remove unbound templates wells were rinsed 4 times with wash buffer. r0041l Example 3. Minisequencing reactions and analysis
[0042] Reaction mixture (40 μf) contained the detection primer (0.5- 5 pmol/well), polymerase Pol B (0.1 U/weil) and lanthanide chelate labeled acycloterminator (3a-d). The amount of the triphosphates depended on the lanthanide chelate and was as follows. The amount of Eu-GTP and Tb-UTP was 0.5 pmol/well and both Sm-ATP and Dy-CTP was 50 pmol/well. The reaction was allowed to proceed with shaking 20 minutes at 68°C. After the reactions wells were rinsed 6 times with wash buffer.
[0043] Analysis of reaction was performed according to DELFIA protocol using Victor Multilabel Counter. Shortly, lanthanide ions were released to solution with enhancement solution (200 μl) and shaking 25 minutes, and in the case of Tb and Dy additional 5 min after the addition of 50 μl DELFIA Enhancer. Luminescent chelates formed during this period of time were measu- red with Victor Multilabel counter. All four acycloterminators labeled with different lanthanide ions, 3a-d, were found to incorporate specifically to primers. Totally unoptimized reactions gave 3.6, 1.4, 1.7 and 1.4 times higher specific signals over nonspecific signals for Eu, Tb, Sm and Dy, respectively.
[0044] It will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent for the expert skilled in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.
Label =
Figure imgf000011_0001
[0045] Scheme 1
Figure imgf000012_0001
Figure imgf000012_0002
pyridine, water, TEA
Hovinen βt al. J. Chem. Soc. Perkin Trans 1 , 1994, 211
Figure imgf000012_0003
[0046] Scheme 2

Claims

1. A terminating substrate for DNA polymerases of formula (I)
Z-R-L-X (I) wherein,
Z is a triphosphate anion or its organic or inorganic salt;
R is a recognizing moiety, which is a nucleoside comprising a base bound to a 2,3-dideoxyribose or its acyclic derivative; and Z-R has the formula (II)
Figure imgf000013_0001
wherein the ribose ring optionally is replaced by an acyclic derivative in which one or both of the 2- and 3- carbon atoms from the ribose ring optionally are missing;
-L- is a linker; and
X is a lanthanide(lll) chelate.
2. The substrate according to claim 1 , wherein Z is an organic or inorganic salt of triphosphate, said salt is most preferably a sodium, lithium, calcium, potassium, magnesium, ammonium tributylammonium or a triethylam- monium salt.
3. The substrate according to claim 1, wherein the recognizing moiety is a radical derived from the following seven structures;
Figure imgf000013_0002
where n is 0 or 1.
4. The substrate according to claim 1 , wherein the linker -L- is connected to C7 of 7-deazaadenine, C7 of 7-deazaguanine, C5 of cytosine, C5 of uracil, C3' of 2'-deoxyguanosine, C3' 2'-deoxyadenosine, C3' of 2'- deoxycytidine or C3 'of thymidine.
5. The substrate according to claim 1 , wherein the linker -L- is formed from one to ten moieties, each moiety being selected from the group consisting of phenylene, alkyl containing 1-12 carbon atoms, ethynediyl (-C=C- ), ethylenediyl (-C=C-); ether (-O-), thioether (-S-), amide (-CO-NH- and -NH- CO- and -CO-NR' and -NR -CO-), carbonyl (-CO-), ester (-COO- and -OOC- ), disulfide (-SS-), diaza, (-N=N-), thiourea (-NH-CS-NH-) or a tertiary amine {- NR'-), where R' represents an alkyl containing less than 5 carbon atoms.
6. The substrate according to claim 1 , wherein the lanthanide chelate X is either luminescent or non-luminescent, and the lanthanide(lll) ion is selected in such a way that each DNA nucleobase binding to the recognising moiety is detectable by one particular [anthanide(lll) ion.
7. The substrate according to claim 6 where the lanthanide is selected from europium, terbium, samarium and dysprosium.
8. Use of the terminating substrate according to claim 1 in DNA sequencing for determining the base sequence wherein each base is identified by detecting the signal derived from the lanthanide chelate of said terminating substrate.
PCT/FI2007/050127 2006-03-13 2007-03-08 Terminating substrates for dna polymerases WO2007104834A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
GB0818768A GB2450454B (en) 2006-03-13 2007-03-08 Terminating substrates for DNA polymerases

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FI20065162A FI20065162A0 (en) 2006-03-13 2006-03-13 Terminating substrates for DNA polymerases
FI20065162 2006-03-13

Publications (1)

Publication Number Publication Date
WO2007104834A1 true WO2007104834A1 (en) 2007-09-20

Family

ID=36192007

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/FI2007/050127 WO2007104834A1 (en) 2006-03-13 2007-03-08 Terminating substrates for dna polymerases

Country Status (3)

Country Link
FI (1) FI20065162A0 (en)
GB (1) GB2450454B (en)
WO (1) WO2007104834A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8119800B2 (en) 2007-12-21 2012-02-21 Korea Research Institute Of Chemical Technology Processes for preparing HIV reverse transcriptase inhibitors
US8334295B2 (en) 2007-06-29 2012-12-18 Korea Research Institute Of Chemical Technology Pyrimidine derivatives as HIV reverse transcriptase inhibitors
US8354421B2 (en) 2007-06-29 2013-01-15 Korea Research Insitute Of Chemical Technology HIV reverse transcriptase inhibitors
US9745253B2 (en) 2015-03-13 2017-08-29 Forma Therapeutics, Inc. Alpha-cinnamide compounds and compositions as HDAC8 inhibitors

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0251786A2 (en) * 1986-07-02 1988-01-07 E.I. Du Pont De Nemours And Company Alkynylamino-nucleotides
EP0340675A2 (en) * 1988-05-02 1989-11-08 The Perkin-Elmer Corporation Time-resolved fluorimetric detection of lanthanide labeled nucleotides
WO1990000623A1 (en) * 1988-07-08 1990-01-25 Wallac Oy Multi-label time-resolved fluorescence analysis of nucleic acid sequences using lanthanide chelates
US5798210A (en) * 1993-03-26 1998-08-25 Institut Pasteur Derivatives utilizable in nucleic acid sequencing
US6255475B1 (en) * 1995-01-31 2001-07-03 Marek Kwiatkowski Chain terminators, the use thereof for nucleic acid sequencing and synthesis and a method of their preparation
US20050084451A1 (en) * 2003-08-29 2005-04-21 Wallac Oy Novel chelating agents and chelates and their use
US20050181393A1 (en) * 2003-12-18 2005-08-18 Wallac Oy Novel chelating agents and highly luminescent and stable chelates and their use

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0251786A2 (en) * 1986-07-02 1988-01-07 E.I. Du Pont De Nemours And Company Alkynylamino-nucleotides
EP0340675A2 (en) * 1988-05-02 1989-11-08 The Perkin-Elmer Corporation Time-resolved fluorimetric detection of lanthanide labeled nucleotides
WO1990000623A1 (en) * 1988-07-08 1990-01-25 Wallac Oy Multi-label time-resolved fluorescence analysis of nucleic acid sequences using lanthanide chelates
US5798210A (en) * 1993-03-26 1998-08-25 Institut Pasteur Derivatives utilizable in nucleic acid sequencing
US6255475B1 (en) * 1995-01-31 2001-07-03 Marek Kwiatkowski Chain terminators, the use thereof for nucleic acid sequencing and synthesis and a method of their preparation
US20050084451A1 (en) * 2003-08-29 2005-04-21 Wallac Oy Novel chelating agents and chelates and their use
US20050181393A1 (en) * 2003-12-18 2005-08-18 Wallac Oy Novel chelating agents and highly luminescent and stable chelates and their use

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8334295B2 (en) 2007-06-29 2012-12-18 Korea Research Institute Of Chemical Technology Pyrimidine derivatives as HIV reverse transcriptase inhibitors
US8354421B2 (en) 2007-06-29 2013-01-15 Korea Research Insitute Of Chemical Technology HIV reverse transcriptase inhibitors
US8119800B2 (en) 2007-12-21 2012-02-21 Korea Research Institute Of Chemical Technology Processes for preparing HIV reverse transcriptase inhibitors
US9745253B2 (en) 2015-03-13 2017-08-29 Forma Therapeutics, Inc. Alpha-cinnamide compounds and compositions as HDAC8 inhibitors
US10266487B2 (en) 2015-03-13 2019-04-23 Forma Therapeutics, Inc. Alpha-cinnamide compounds and compositions as HDAC8 inhibitors
US10508077B2 (en) 2015-03-13 2019-12-17 Forma Therapeutics, Inc. Alpha-cinnamide compounds and compositions as HDAC8 inhibitors
US10988441B2 (en) 2015-03-13 2021-04-27 Valo Early Discovery, Inc. Alpha-cinnamide compounds and compositions as HDAC8 inhibitors
US11919839B2 (en) 2015-03-13 2024-03-05 Valo Health, Inc. Alpha-cinnamide compounds and compositions as HDAC8 inhibitors

Also Published As

Publication number Publication date
GB0818768D0 (en) 2008-11-19
GB2450454A (en) 2008-12-24
FI20065162A0 (en) 2006-03-13
GB2450454B (en) 2011-04-13

Similar Documents

Publication Publication Date Title
US9938573B2 (en) Methods and kits for nucleic acid sequencing
US5185439A (en) Acridinium ester labelling and purification of nucleotide probes
AU763671B2 (en) Detection using degradation of a tagged sequence
EP3898643B1 (en) 3' reversibly protected nucleotides for sequencing by synthesis using nanopores
KR101107315B1 (en) A DNA sequencing method using novel nucleoside triphosphates with a fluorescent 3'-O-blocking group as reversible terminators
US20040096825A1 (en) Methods and compositions for enhancing detection in determinations employing cleavable electrophoretic tag reagents
JP2003532092A (en) TAG library compounds, compositions, kits and methods of use
JPH075170A (en) Method and system for detecting radiation energy
EP0312248B1 (en) Acridinium ester labelling and purification of nucleotide probes
WO2007104834A1 (en) Terminating substrates for dna polymerases
CA2873370A1 (en) Nucleic acid probe, method for designing nucleic acid probe, and method for detecting target sequence
US6074824A (en) Method for determining DNA nucleotide sequence
US7482444B2 (en) Terminating substrates for DNA polymerases
EP1296997B1 (en) Base analogues
JPH01500353A (en) Nucleic acid detection probe containing 2'-deoxyadenosine derivative
AU619223B2 (en) Acridinium ester labelling and purification of nucleotide probes
McGall Nucleoside triphosphate analogs for nonradioactive labeling of nucleic acids
EP1161681A2 (en) Use of lna in mass spectrometry
WO2007105623A1 (en) Oligonucleotide-immobilized solid support

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07712617

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 0818768

Country of ref document: GB

Kind code of ref document: A

Free format text: PCT FILING DATE = 20070308

WWE Wipo information: entry into national phase

Ref document number: 0818768.4

Country of ref document: GB

122 Ep: pct application non-entry in european phase

Ref document number: 07712617

Country of ref document: EP

Kind code of ref document: A1