WO2003066645A2 - Nucleosides et nucleotides etiquetes a la base - Google Patents

Nucleosides et nucleotides etiquetes a la base Download PDF

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WO2003066645A2
WO2003066645A2 PCT/US2003/003193 US0303193W WO03066645A2 WO 2003066645 A2 WO2003066645 A2 WO 2003066645A2 US 0303193 W US0303193 W US 0303193W WO 03066645 A2 WO03066645 A2 WO 03066645A2
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compound
triphosphate
reaction
labeled
biotin
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PCT/US2003/003193
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WO2003066645A3 (fr
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Kenneth Cruickshank
Ren Ling
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Pierce Milwaukee Llc
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Publication of WO2003066645A3 publication Critical patent/WO2003066645A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • 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

Definitions

  • the present invention relates to base-labeled nucleosides and nucleotides.
  • nucleotides labeled to permit their detection after enzymatic manipulation into other molecules are useful in many biomolecular applications. Typically, these nucleotides are referred to as "nucleotide probes," and they find particular utility where it is desired to identify nucleic acids and sequences thereof in polynucleotides such as DNA and RNA.
  • Probes fashioned with radioactive labels have been used, but their utility is limited, principally because of radiation hazards. Accordingly, polynucleotides containing other detectable moieties have become popular. In this respect, nucleotides labeled with moieties capable of detection by, for example, fluorometric, colorometric, and chemiluminescent techniques have found use as probes.
  • nucleoside components of which are N-nucleosides, C-nucleosides (base to ribose attachment through a base carbon atom) are also recognized in the literature.
  • C-nucleoside, pseudouridine is commercially available from Sigma-Aldrich (St. Louis, MO).
  • C-nucleosides capable of facile transformation into labeled derivatives, including nucleotides are not known or available.
  • R OH or mono-, di- or triphosphate
  • R' OH or H
  • R" OH or H
  • B -C(O)NH- or -C(O)NR'"-, wherein R'" - a straight chain or branched, substituted or unusubstituted, d-C 8 alkyl
  • X a detectable label, when present, and E - O, S, or NH.
  • R OH or mon-, di- or triphosphate
  • R' OH or H
  • R" OH or H
  • B -CONH- or -CONR'"-
  • R'" a straight chain or branched, substituted or unsubstituted, C ⁇ -C 8 alkyl
  • X a detectable label, when present.
  • R OH or mono-, di or triphosphate
  • R' OH or H
  • R" OH or H
  • L 2 linker comprised of A-B-C, as defined above
  • Xi and X 2 detectable label, when present
  • E O or S
  • Lj and L 2 detectable label, when present
  • E O or S
  • Lj and L 2 detectable label, when present
  • the present invention provides pyrimidine C-nucleosides in which the C5 position of the pyrimidine is attached to the Cl position of the ribose, and the base is modified to provide an active moiety for facile attachment of a detectable label.
  • Detectable labels can include those known in the art, such as those based on fluorescense, chemiluminescense, radioactivity and the like (see, e.g., the '060 patent).
  • the present invention provides compounds of the formula I:
  • R OH or mono-, di- or triphosphate
  • R' OH or H
  • R" OH or H
  • B -C(O)NH- or -C(O)NR'"-
  • R'" a straight chain or branched, substituted or unusubstituted
  • Q- alkyl such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl
  • X a detectable label
  • E O, S, or
  • X can be a fluorescent, chemiluminescent or radioactive label.
  • X can be 5-carboxytetramethyl- rhodamine (Rh), 5-carboxy-fluorescein (F), cyanine 3 (Cy3), biotin, or a binding ligand, such as an antibody.
  • the present invention provides a compound of formula II:
  • R OH or mon-, di- or triphosphate
  • R' OH or H
  • R" OH or H
  • X a detectable label, when present, such as a fluorescent, chemiluminescent or radioactive label.
  • X can be Rh, F, Cy3, biotin, or a binding ligand, such as an antibody.
  • the present invention provides a compound of formula III:
  • R OH or mono-, di or triphosphate
  • R' OH or H
  • R" OH or H
  • R triphosphate
  • R' OH
  • R" H
  • L l5 L 2 , A, B and C are as defined above
  • E O
  • Xi and X 2 a fluorophore pair capable of fluorescence resonance energy transfer (FRET) and, when X ⁇ and X 2 are different, capable of increasing effective "Stokes" shift for detection, is preferred.
  • X can be a fluorescent, chemiluminescent or radioactive label.
  • X can be Rh, F, Cy3, biotin, or a binding ligand, such as an antibody.
  • dNTPs can be introduced into nucleic acids by DNA polymerases, such as those used in nick translation (DNA polymerase I), random priming reactions (Klenow), and polymerase chain reactions (Thermus aquaticus polymerase, Taq).
  • dNTP's also can be used with DNA modifying enzymes, such as those used for labeling the 3' end of nucleic acids, e.g., terminal deoxyribonucleotidyl transferase (TdT).
  • R" OH and the corresponding ribonucleotide triphosphates (rNTPs) are obtained.
  • rNTPs ribonucleotide triphosphates
  • the compounds can be prepared in stages, starting with the nucleoside pseudouridine. As will be apparent, the elimination and addition of stages as the preparation proceeds can be used to achieve the desired labeled nucleotide. Thus, in the experimental details which follow, the preparation of the labeled 2'-dNTP is illustrated by first removing the 2' hydroxyl group from the sugar moiety of pseudouridine. By eliminating this stage, the corresponding rNTP can be obtained. Similarly, 2', 3'-dideoxyribonucleosides can be prepared by including a second deoxygenation stage. [0016] Turning specifically to the preparation of labeled dNTP, four distinct stages can be employed.
  • Stage 1 is removal of the 2'-hydroxyl group from the pseudouridine. This can be achieved by standard methods as described hereinafter and also, for example, as described by Singh et al., in Nucleic Acid Chemistry Part 4. Townsend and Tipson, eds., Wiley & Sons, NY, 1991, pp. 96-98. The resultant compound is also commercially available from Berry & Assoc, Ann Arbor, MI. Stage 2 is elaboration of the side chain at the Nl position of the pyrimidine base.
  • the key first step in this stage is selective Nl derivatization of 2'-deoxypseudouridine by Michael addition of acrylonitrile or other compounds containing activated double or triple bonds to the Nl position (Yoshida et al., Biochim. Biophys. Acta, 157, 455, 1968; Chambers, Biochemistry, 4(2), 219, 1965; Ofengand, Biochemistry, 57(6), 818, 1965; Ofengand, Biochem. Biophys. Res. Commun., 18(2), 192, 1965; and Ofengand, J. Biol. Chem., 242(21), 5034, 1967). In the event that both of the Nl and N3 positions are to be elaborated, this can be accomplished by extending the reaction.
  • the appended functionality is further modified to allow attachment of conjugatable moities.
  • the nitrile is hydrolyzed to the carboxylic acid followed by methylation of the acid with methyl iodide/potassium carbonate.
  • Reaction of the methyl ester with ethylene diamine and protection of the resulting primary amino compound with a trifluoroacetyl group is then employed to complete the side-chain synthesis.
  • Stage 3 consists of conversion of the Nl -substituted, 2'-deoxyribonucleoside into a 5 '-triphosphate using known methods (Kovacs et al., Tet. Let. 29(36), 4525-4528, 1988).
  • the trifluoroacetyl group can be removed under standard conditions, and a desired detectable ligand (fluorophore, biotin; etc.) can be conjugated to the linker by standard methods (Handbook of Fluorescent Probes and Research Chemicals. Molecular Probes Inc., Eugene, OR, 6th ed).
  • a desired detectable ligand fluorophore, biotin; etc.
  • TLC thin layer chromatography
  • System A chloroform-methanol, (9:1, v/v)
  • System B chloroform-methanol, (8:2, v/v)
  • System C chloroform-methanol, (3:2, v/v).
  • NHS 5-Carboxytetramethyl-rhodamine N-hydroxysuccinimide
  • 5-SFX NHS fluorescein succinimidyl ester
  • Pseudouridine was bought from Sigma Chemical Co., St Louis, MO. Biotin LC NHS was from Pierce Biotechnology, Inc., Rockford, IL (cat # 21336ZZ). Klenow fragment, T4 DNA polymerase, T7 DNA polymerase, Taq polymerase and Moloney murine leukemia virus (M-MuLV) reverse transcriptase were all from Pierce Milwaukee (Milwaukee, WI). Vent, Vent (exo " ), Deep Vent and Deep Vent (exo " ) DNA polymerases and lOx reaction buffers were from New England BioLabs (Beverly, MA). Tth and Tfl DNA polymerases and reaction buffers were from Promega (Madison, WI). Pfu DNA polymerase was from Stratagene (La Jolla, CA). dATP, dTTP, dCTP and dGTP were from Amersham Bioscience (AB, Piscataway, NJ).
  • High pressure liquid chromatography was carried out on a Waters system fitted with a Zorbax Bio Series oligo column (Agilent Technologies, Palo Alto, CA; cat. 820940-901) eluted with the following solvent systems.
  • Buffer A 20 mM potassium hydrogen phosphate in acetonitrile - water (1 :4, v/v)
  • Buffer B 20 mM potassium hydrogen phosphate in acetonitrile - water (1 :4, v/v) containing 1 M NaCl.
  • Gradient 0 to 30 % buffer B over 30 mins, 30-40% buffer B over the next 10 minutes, then 40 to 0 % buffer B to requilibrate.
  • Reverse-phase HPLC was carried out using a Waters NovaPak C18 column (cat. WAT086344) eluted with Buffer A: 0.1 M triethylammonium acetate (pH 7) and Buffer B, acetonitrile. Gradient: 0 to 50 % buffer B over 30 mins, 50 to 100% buffer B over the next 10 minutes, hold for 5 mins, and then 100 to 0 % buffer B over 5 mins to requilibrate.
  • Buffer A 0.1 M triethylammonium acetate (pH 7)
  • Buffer B acetonitrile
  • This example describes the preparation of 2'-deoxypseudouridine-N 1 -(N-(2-N- trifluoroacetylaminoethyl)propionamide).
  • This example describes the preparation of 2'-deoxypseudouridine-Nl -(N-(2-N- trifluoroacetylaminoethyl)propionamide)-5'-triphosphate.
  • the triphosphate was prepared according to the method of Kovacs et al., Tet. Let., 29(36), 4525-8, 1988.
  • the nucleoside, 2'-deoxypseudouridine-Nl-(N-(2-N- trifluoroacetylaminoethyl)propionamide) (0.044 g, 0.1 mmol) was suspended in dry trimethylphosphate (0.5 ml) and magnetically stirred until a solution was obtained. Powdered proton sponge (0.0322 g) was then added. The mixture was stirred and cooled in an ice-water bath for 0.5 hr.
  • Phosphorus oxychloride (0.010 ml) was then added by syringe, and the mixture was stirred at 4°C for 2 hr when TLC on silica (n-propanol/conc. ammonia/water; 11 :7:2, v/v/v) revealed only partial conversion to a lower Rf product.
  • TLC on silica n-propanol/conc. ammonia/water; 11 :7:2, v/v/v
  • the reaction mixture darkened and thickened so that the stirrer stopped; however, after 5 mins, the solution loosened and the stirrer continued.
  • a further aliquot of phosphorus oxychloride (0.004 ml) was added.
  • reaction was quenched by addition of triethylammonium acetate buffer, pH 7 (3.0 ml). After stirring at room temperature for 4-5 hrs, this intermediate was slowly converted to a low Rf compound, the linear triphosphate. The solution was evaporated to dryness, the residue was taken up in water (4.0 ml), and concentrated aqueous ammonia (4.0 ml) was added to remove the trifluoroacetyl group. After 2 hrs, the reaction was complete as judged by TLC to give a nin-hydrin positive product. The reaction was evaporated to dryness, the residue was taken up in water (5.0 mis), and the pH was adjusted to 7 by addition of several drops of triethylamine.
  • the triphosphate product was purified on a column of Q-Sepharose by eluting with a gradient of 1 M NaCl in 5 mM sodium phosphate buffer.
  • ⁇ max 272 run.
  • a degradation analysis with alkaline phosphatase in phosphate buffer revealed progressive step-wise removal of phosphate groups to give nucleoside, mono- and diphosphate compounds.
  • the reaction mixture was evaporated to dryness to remove DMF, and the product was isolated by successive preparative HPLC purifications, using analytical scale columns, first using ion-exchange on the Zorbax column, then RP using a Waters Cl 8 column as described for the carboxytetramethylrhodamine conjugate.
  • the presence of the triphosphate group was confirmed by degradation of an aliquot of the product (10 nmol) with bacterial alkaline phosphatase followed by HPLC analysis.
  • Bio-d ⁇ UTP Biotin labeled pyrimidine C5-nucleotide
  • nucleotide stock solution (2.5 ⁇ mol) was adjusted to pH 7. 5 - 8.0 with small aliquots of 0.1 N NaOH, and the pH was checked with pH paper. The aqueous layer was diluted to a final volume of 0.5 ml, and, again, the pH was confirmed. Carboxytetramethylrhodamine NHS ester (5.0 mg) in DMF (0.175 ml) was then added to the nucleotide solution, and the mixture was rotary mixed, while protected from light, for 68 hrs. The progress of the reaction was monitored by HPLC, using a Zorbax oligo series column.
  • the product was isolated by preparative HPLC on a Waters C18 column, using a gradient of acetonitrile in 0.1 M triethylammonium acetate, pH 7, as mobile phase (0 to 50 % acetonitrile over 30 mins). Multiple injections (0.15 ml) were made, and the fractions containing pure product peak (Rt « 20 min) were pooled and evaporated in vacuo.
  • the product was quantitated by ultraviolet (UV) spectrometry in 0.05 M sodium phosphate buffer, using an extinction coefficient of the dye of 60,000 at 552 nm (yield 0.398 ⁇ mol).
  • UV ultraviolet
  • This example describes the conjugation of the N 1 -substituted-2'- deoxypseudouridine-S'-triphosphate to 5-carboxyfluorescein NHS ester (Fd ⁇ UTP).
  • An aliquot of the nucleotide stock solution (5 ⁇ mol) was concentrated in vacuo to about one-fifth volume, and any precipitated salt was removed by filtration.
  • the nucleotide was treated with 5.37 ⁇ moles (3.15 mg) of fluorescein succinimidyl ester (5-SFX NHS) at room temperature for 1 hour in pH 9 carbonate buffer.
  • Rhd ⁇ UTP containing the fluorescent rhodamine label
  • This example describes the 3'-end labeling of oligonucleotide with Rhd ⁇ UTP using TdT.
  • TdT and 5X reaction buffer were obtained from Pierce Milwaukee LLC. Rhd ⁇ UTP was resuspended in sterile water to give a final concentration of 1 mM.
  • Oligo dT 15 (Promega) and Oligo dTi 6 ( from AB) were used as the templates.
  • Oligo dT 15 (0.1 ⁇ g/ ⁇ l) was incubated at 37°C for 18 hours in a 20 ⁇ l-reaction containing 45 units of TdT, IX TdT buffer, and 125 ⁇ M (2.5 nmole total) Rhd ⁇ UTP.
  • Control X was used as the positive control reaction.
  • the negative control reactions omitted either TdT or dT 15 .
  • the reactions were stopped by adding 2 ⁇ l of 0.5 M EDTA. Samples were mixed with loading dye, heated and loaded onto a 20% acrylamide gel for electrophoresis. Pictures were taken by charged coupled device (CCD) camera. UV light was used for fluorescent detection and regular light was used to observe stained gels. The gel was stained with Stains All (Sigma).
  • the Rhd ⁇ UTP was incorporated into the olignucleotide as evidenced by fluorescent detection.
  • Oligo dT 16 (0.1 ⁇ g/ ⁇ l) was incubated at 37°C for 0, 5, 10, 30 and 60 mins in a 15- ⁇ l reaction containing 9 units of TdT, IX TdT buffer, and 20 ⁇ M (0.4 nmole total) Rhd ⁇ UTP.
  • Control X was used as the positive control reaction.
  • the negative control reactions omitted dT 15 .
  • the reactions were stopped by adding 2 ⁇ l of 0.5 M EDTA. Samples were mixed with loading dye, heated and loaded onto a 20% acrylamide gel. The sample loading dye used in the rest of the experiments only contained bromophenol blue, since the xylene cyanol FF runs too close to the fluorescent-labeled oligonucleotides.
  • Rhd ⁇ UTP can be used as a substrate for TdT in 3 '-end labeling experiments.
  • Oligo dT ⁇ 5 (1 ⁇ g), 60 units of TdT and 100 ⁇ M of dye-labeled d ⁇ UTP incubated were onto at 37°C for 60 mins.
  • the reactions were loaded onto a 20% acrylamide-urea gel.
  • the fluorescent signal was observed by UV light, and nucleotide oligos were detected with Stains-All reagent.
  • Rh-, F-, Cy3- and biotin-labeled d ⁇ UTP derivatives were all utilized by TdT for the addition of a single nucleotide onto the 3' end of DNA.
  • Oct-1 specific sense or antisense oligo 22 bp; 6.7 ⁇ M
  • 60 units of TdT and 400 ⁇ M of Bio-d ⁇ UTP were incubated in a 60- ⁇ l labeling reaction at 37°C for 60 mins.
  • the labeled products were detected by acrylamide gel and Stains-All staining.
  • the gel shift assays were carried out per the instructions of the LightShiftTM Chemiluminscent EMS A kit (Pierce) using 20 femtomoles bio-d ⁇ UTP labeled Oct-1 specific DNA duplex.
  • the negative control and competition assays were performed as instructed.
  • a DNA retardation gel (Invitrogen, Carlsbad, CA) was used for separation, and the signal of biotin was detected according to the instructions of the LightShiftTM kit (Pierce).
  • All of the sense and antisense Oct-1 -specific oligos were labeled with Bio-d ⁇ UTP.
  • a band shift was observed when the HeLa nuclear extract was incubated with Bio-d ⁇ UTP labeled Oct-1 -specific DNA duplex. When unlabeled Oct-1 -specific DNA duplex was added, the shift was competed away.
  • the oligo probes which labeled with biotin-d ⁇ UTP, can be used in non- radioactive gel shift assays. They offer equivalent results to traditional radioactively labeled probes or probes labeled commercially with biotin in gel shift assays.
  • This example describes filling in the 3' recessive ends of double-stranded oligonucleotide with Rhd ⁇ UTP using DNA polymerases.
  • Annealed Oligo 13/20mer (50 ⁇ M) was incubated at 37°C for 18 hrs in a 25 ⁇ l-reaction volume containing 6 units of enzyme (T7 DNA polymerase, T4 DNA polymerase or Klenow fragments), IX reaction buffer proper for each enzyme, and 100 ⁇ M Rhd ⁇ UTP. 15.8 units of M-MuLV reverse transcriptase was used in one pair of reactions. The negative control reactions omitted the enzyme. The reactions that contained 6 units of Taq or Vent (exo-) polymerase were incubated at 45°C. All of the reactions were stopped by adding 10 ⁇ l of gel-loading buffer containing EDTA. The acrylamide gel separation, fluorescence detection, and DNA band observation were as described above.
  • T4 DNA polymerase had a very active DNase function that completely degraded 13/20mer during the incubation period.
  • T7, Taq and Vent (exo " ) DNA polymerases worked on both Rhd ⁇ UTP and control X. They even put second or third control X on the 13mer non-specifically. Klenow and M-MuLV reverse transcriptase did not work for Rhd ⁇ UTP, but worked for control X.
  • T7, Taq and Vent (exo " ) DNA polymerases were able to incorporate Rhd ⁇ UTP. Klenow and M-MuLV reverse transcriptase were not able to incorporate Rhd ⁇ UTP.
  • Annealed Oligo 13/20mer 50 ⁇ M was incubated at 45°C for 0.5, 1, 4, or 18 hrs in a 25- ⁇ l reaction volume containing 6 units of Taq or Vent (exo " ) DNA polymerase, IX reaction buffer appropriate for each enzyme, and 100 ⁇ M Rhd ⁇ UTP. Control X was used as positive control for each enzyme, and the reactions were incubated at 45°C for 0.5 hr.
  • the Vent (exo " ) DNA polymerase was further tested for 1, 5, 10 and 30 mins incubations. [0053] The Rhd ⁇ UTP incorporation was only observed at 18 hr incubation with Taq DNA polymerase. The Rhd ⁇ UTP incorporation could be detected at 1 min incubation with Vent (exo " ) DNA polymerase. Vent (exo " ) DNA polymerase worked well for incorporating Rhd ⁇ UTP.
  • This example describes Polymerase Chain Reaction (PCR) labeling with Rhd ⁇ UTP using Vent (exo " ) DNA polymerase.
  • PCR was carried out in a total volume of 25 ⁇ l.
  • the final concentration of the PCR primers was 2 ⁇ M and the concentration of each triphosphate nucleotide was 200 ⁇ M.
  • thirty-five cycles were carried out, each consisting of denaturation (30 sec at 94°C), annealing (30 sec at 56°C), and extension (2 mins at 72°C).
  • Vent (exo " ) DNA polymerase was used at a concentration of 0.04 unit ⁇ l, along with IX PCR buffer.
  • the testing PCR contained 120 ⁇ M Rhd ⁇ UTP. Control X (60 ⁇ M) was used as a positive control for PCR labeling.
  • Rhd ⁇ UTP can be used for PCR labeling with Vent (exo " ) DNA polymerase.
  • the PCR template was pGEM-T-Rafl -RBD. 3' and 5' primers of Rafl -RBD were used as PCR primers.
  • PCR was carried out in a total volume of 25 ⁇ l. Each PCR reaction contained 40 ng template DNA, 500 nM of each primer, 200 ⁇ M of dATP, dCTP and dGTP, 1-2 units
  • reaction 1 contained 60 ⁇ M Rh-d ⁇ UTP and 140 ⁇ M dTTP
  • reaction 2 contained 60 ⁇ M control X and 140 ⁇ M dTTP
  • reaction 3 contained 200 ⁇ M dTTP.
  • the PCR products were separated on 10% acrylamide gel. The fluorescent signal was visualized on UV light, and the DNA bands were detected with EtBr (0.4 ⁇ g/ ⁇ l) staining.
  • Rh-d ⁇ UTP only served as substrate for type II DNA polymerases (such as Pfu, Vent, Vent (exo-), Deep Vent, Deep Vent (exo " )).
  • the Rh-d ⁇ UTP did not serve as a substrate for type I DNA polymerases (such as Taq, Tth, Tfl), but it did not inhibit PCR reactions.
  • Vent (exo " ) was used to determine the optimal ratio of dTTP to Rh-d ⁇ UTP.
  • the final concentration of Rh-d ⁇ UTP and dTTP together was 200 ⁇ M and the ratio of Rh-d ⁇ UTP:dTTP was changed in each reaction from 0:200, 20:180; 40:160, 80:120, to 100:100.
  • the optimal ratio of dTTP to Rh-d ⁇ UTP is between 4:1 to 9:1 in PCR labeling reaction.
  • This example describes enzymatic labeling using Rh- ⁇ UTP and Bio- ⁇ UTP.
  • Titration experiments were set up as following: 500 ⁇ M GTP, ATP and CTP; 500 ng DNA template (pTRI RNA 28S) from Ambion (Austin, TX); and 10 units T7 RNA polymerase (Ambion) in each 20- ⁇ l reaction.
  • a total of 500 ⁇ M Rh- ⁇ UTP and UTP was used in the reactions, but the ratio of Rh- ⁇ UTP :UTP was changed in each reaction from 3:1, 1 :1 to 1 :3.
  • the reactions were incubated at 37°C for 2.5 hrs.
  • RNA polymerases When different RNA polymerases were tested, 125 ⁇ M Rh- ⁇ UTP and 375 ⁇ M UTP were used in the test reactions and 500 ⁇ M UTP was used in control reactions.
  • the labeled RNA probes were separated on an 8% acrylamide-urea gel. The fluorescent signal was visualized on UV light, and the RNA bands were detected with ethidium bromide (0.4 ⁇ g/ ⁇ l) staining. The pictures were taken by CCD camera using UV light.
  • Rh- ⁇ UTP was incorporated by T7, T3 and SP6 RNA polymerases in in vitro transcription with optimal labeling occurring at a 3:1 ratio of UTP: Rh- ⁇ UTP.
  • Rh- ⁇ UTP-labeled antisense and sense 15-lipoxygenase RNA probes were synthesized using SP6 or T7 RNA polymerase. The in situ hybridization was carried on overnight at 60°C using antisense RNA probe or an equal amount of sense RNA probe with lung sections from neonatal rabbit. The Rh- ⁇ UTP labeled RNA probe enabled fluorescent detection in situ.
  • RNA probes were separated on an 8% acrylamide-urea gel, and the RNA bands were detected with ethidium bromide (0.4 ⁇ g/ ⁇ l).
  • biotin labeling signals were detected according to the instruction of the SuperSignal RPA Chemiluminscent Detection kit (Pierce).
  • Bio- ⁇ UTP As the concentration of Bio- ⁇ UTP increased, the amount of RNA synthesized was decreased. Bio- ⁇ UTP can be incorporated into RNA by T7 RNA polymerase, but the incorporation rate is not as good as Biotin-CTP.
  • This example describes labeling DNA probes with biotinylated pseudouridine (Bio-d ⁇ UTP) by the random priming method.
  • Bio-d ⁇ UTP was used along with all of the components (except Bio-dCTP) from the North2South ® random prime kit (Pierce), it did not incorporate to a detectable level after a 1 hr incubation. While replacing Klenow (exo " ) with Vent (exo " ) DNA polymerase (New England Biolab) or T7 DNA polymerase (AB) in the North2South ® kit, the incorporation of Bio-d ⁇ UTP was detected after a 1 hr incubation.
  • Bio-d ⁇ UTP After 2 hrs incubation with T7 DNA polymerase, the signal from incorporated Bio-d ⁇ UTP was compatible with the signal of Bio-dCTP incorporated with Klenow (exo " ) (positive control of North2South ® random prime kit). Bio-d ⁇ UTP can be used for labeling probes with T7 DNA polymerase or Vent (exo " ) DNA polymerase by the random priming method.

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Abstract

L'invention concerne un composé de formule (I) dans laquelle R = OH ou mono-, di- ou triphosphate, R'= OH ou H, R' = OH ou H, L1 et L2, pouvant être identiques ou différents, désignent individuellement un lieur comprenant A-B-C, A = -(CH2)n, où n = 1-6 ou -(CH=CH)-, B = -C(O)NH- ou -C(O)NR' -, R' = un chaînon rectiligne ou ramifié, substitué ou non substitué, un alkyle en C1-C8, C = -(CH2)NY, où n = 1-6, et Y= -NH2, -SH, -OH, -C(O)OH ou -OP(O)2OH, X1 et X2, pouvant être identiques ou différents, représentent individuellement une étiquette pouvant être détectée, quand elle est présente et E = O, S, ou NH. Dans d'autres modes de réalisation E = NH2 et/ou L2 - X2 sont absents.
PCT/US2003/003193 2002-02-06 2003-02-04 Nucleosides et nucleotides etiquetes a la base WO2003066645A2 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996028460A1 (fr) * 1995-03-14 1996-09-19 Boehringer Mannheim Gmbh Derives de c-nucleosides et leur utilisation dans la detection d'acides nucleiques
US5728525A (en) * 1992-02-12 1998-03-17 Chromagen, Inc. Fluorescent universal nucleic acid end label
US6174998B1 (en) * 1996-03-12 2001-01-16 Roche Diagnostics Gmbh C-nucleoside derivatives and their use in the detection of nucleic acids

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5728525A (en) * 1992-02-12 1998-03-17 Chromagen, Inc. Fluorescent universal nucleic acid end label
WO1996028460A1 (fr) * 1995-03-14 1996-09-19 Boehringer Mannheim Gmbh Derives de c-nucleosides et leur utilisation dans la detection d'acides nucleiques
US6174998B1 (en) * 1996-03-12 2001-01-16 Roche Diagnostics Gmbh C-nucleoside derivatives and their use in the detection of nucleic acids

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIVAK K.J. ET AL.: 'Detection of single base differences using biotinylated nucleotides with very long linker arms' NUCLEIC ACIDS RESEARCH vol. 20, no. 18, 1992, pages 4831 - 4837, XP002021270 *
MUHLEGGER K. ET AL.: 'Non-radioactive labeling and detection of nucleic acids: IV. Synthesis and properties of digoxigenin-modified 2'-deoxyuridine-5'-triphosphates and a photoactivatable analog of digoxigenin (photodigoxigenin)' BIOLOGICAL CHEMISTRY HOPPE-SEYLER vol. 371, October 1990, pages 953 - 965, XP000575089 *

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