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  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/30—Nucleotides
  • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
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  • 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
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
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  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/30—Nucleotides
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  • 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
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

• the present invention relates to a method for amplifying a sequence of a target nucleic acid.
• a method for treating a nucleotide which consists in introducing onto one of the constituent elements of said nucleotide, namely sugar, the purine or pyrimidine base, and the phosphate groups, a functional group having various uses and in particular those of marking.
• the nucleotide thus treated obtained could be incorporated into a polynucleotide, in particular in the double-stranded form without the double helix being destabilized by the presence of this nucleotide.
• the functional groups attached to the nucleotide according to this prior art exhibit various phenomena such as, for example, steric hindrance, hydrophobic interactions or complexation phenomena, which prevent recognition of the polynucleotide having incorporated said treated nucleotide, by most enzymes, which would recognize the corresponding polynucleotide, in which said treated nucleotide has not been incorporated.
• an amplification method which overcomes the drawbacks mentioned above and which in particular does not disturb the incorporation of the nucleotides and consequently does not significantly influence the yield and / or the sensitivity in a reaction. of target amplification and which also makes it possible to obtain an excellent labeling of the amplification products while avoiding phenomena of instability of the marker linked to a prior incorporation of the latter.
• the subject of the present invention is a method for amplifying a sequence of a target nucleic acid, according to which:
• the sequence of a target nucleic acid at least one oligonucleotide primer specific for the target sequence, for one or more enzymatic activities, for nucleotides,
• the target sequence is amplified, under conditions adapted in particular to enzymatic activity or activities, process according to which at least one of the nucleotides is a pre-functionalized nucleotide, differing from the other nucleotides at least by the presence of at least one function covalent reactive, unprotected, arranged in at least one predetermined site of the base of said nucleotide, to obtain a pre-functionalized amplification product comprising at least one said pre-functionalized nucleotide.
• a reagent comprising an anti-reactive covalent function, specific to the reactive function of the prefunctionalized nucleotide, and a functional group, is available, and
• the prefunctionalized amplification product is reacted, directly or indirectly, with the reagent, to obtain a functionalized amplification product.
• nucleotide By nucleotide according to the invention is meant a natural or modified nucleotide monomer as defined below.
• the nucleotide monomer can be a natural nucleotide of nucleic acid whose constituent elements are a sugar, a phosphate group and a nitrogenous base; in RNA the sugar is ribose, in DNA the sugar is 2 '-deoxy-ribose; depending on whether it is DNA or RNA, the nitrogen base is chosen from Adenine, guanme, uracil, cytosine, thymine; or a nucleotide modified in at least one of the three constituent elements, - for example, the modification can take place at the level of the bases, generating modified products such as mosine, methyl- 5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2, 6-purine, bromo-5-deoxyuridine and any other modified base allowing hybridization, at the sugar level, namely for example the replacement at least one deoxyribose with an analog (example: PE Nielsen et al, Science
• protective group is meant the groups conventionally used in the chemical synthesis of nucleosides, nucleotides and oligonucleotides (see for example: Chemistry of Nucleosides and Nucleotides, Edited by Leroy B. To nsend, Plénum Press, New York and London and Protocols for Oligonucleotides and Analogs, Synthesis and Properties, Edited by Sudhir Agrawal, Humana Press, Totowa, New Jersey).
• a functional labeling group of the invention is a molecule capable of directly or indirectly generating a detectable signal. It is in particular chosen from radioactive isotopes, enzymes notably chosen from peroxidase, alkaline phosphatase, b-galactosidase, and those capable of hydrolyzing a chromogenic, fluorigenic or luminescent substrate, chemical compounds chromophores, chromogens, fluorophores, fluorigenes or luminescent, analogs of nucleotide bases, and ligands such as biotin.
• enzymes notably chosen from peroxidase, alkaline phosphatase, b-galactosidase, and those capable of hydrolyzing a chromogenic, fluorigenic or luminescent substrate, chemical compounds chromophores, chromogens, fluorophores, fluorigenes or luminescent, analogs of nucleotide bases, and ligands such as biotin.
• the marker cannot directly generate a signal
• the enzyme it is necessary to add a developer, for example a substrate corresponding to the enzyme, the enzyme / substrate reaction generating a detectable complex, for example a chromogenic or luminescent compound.
• the revealing reagent can be ortho-phenylene diamine, 4-methyl-umbelliferyl-phosphate.
• the reactive covalent function of the prefunctionalized nucleotide and the anti-reactive function of the reagent are respectively electrophilic and nucleophilic organic chemical functions, or vice versa.
• the electrophilic organic chemical function is advantageously chosen from the aldehyde, activated ester, carboxylic acid, isothiocyanate, haloacyl derivatives and sulfonyl chloride functions.
• the nucleophilic organic chemical function is advantageously chosen from the amine, thiol, oxyamine, hydrazine and hydrazide functions, preferably it is the alkoxyamine function.
• the reactive covalent function of the modified nucleotide is grafted onto the base via a coupling arm and / or the anti-reactive covalent function of the reagent is grafted onto the functional group by through a coupling arm.
• the coupling arm is in particular chosen from the hydrocarbon chains, saturated or unsaturated, optionally interrupted by amine, amide and oxy functions.
• the reactive covalent function is the oxyamine function
• the anti-reactive covalent function of the reagent is the aldehyde function
• the latter is linked to a functional labeling group such as a fluorescent or luminescent group.
• the aldehyde function is linked to the functional group by the coupling arm -NH-CS-NH- (CH2) 3- and the functional group of the reagent is fluore ⁇ cein.
• the prefunctionalized nucleotide product can comprise one or more reactive covalent functions, identical or different, introduced by one or more nucleotides. Said reactive covalent functions can react with one or more reagents, identical or different, simultaneously or sequentially. The detection of the functional labeling groups of the functionalized product can be simultaneous or sequential.
• the labeled functionalized amplification product can be detected qualitatively and / or quantitatively in the homogeneous or heterogeneous phase.
• the reagent and the prefunctionalized nucleotide product interact in the same liquid medium.
• the pre-functionalized product can be treated with the reagent before or after capture on a solid support, that is to say directly or indirectly. Capture on the solid support can be carried out by known means, such as adsorption, covalence, in particular by using anti-reactive covalent functions available on the surface of the solid support or by hybridization with a polynucleotide compound.
• the solid support in all appropriate forms such as tube, cone, well, microtiter plate, sheet, chip or soluble polymer, is chosen from polystyrenes, styrene-butadiene copolymers, ⁇ tyrene-butadiene copolymers in admixture with polystyrenes, polypropylenes, polycarbonates, polystyrene-acrylonitrile copolymers, styrene-methyl methacrylate copolymers, among synthetic and natural fibers, among polysaccharides and cellulose derivatives, glass, silicon and their derivatives.
• the target nucleic acid sequence is a DNA or RNA sequence
• the enzymatic activities include the DNA polymerase activities RNA- and / or DNA-dependent
• the enzymatic activities can also include ribonuclease H activity and DNA-dependent RNA polymerase activity, to amplify the target nucleic acid sequence according to a sequence of reverse transcription, transcription and digestion reactions.
• the enzymatic activities ribonuclease H and DNA polymerase can be provided by a single enzyme or else each by a different enzyme.
• the method of the invention can be used for the amplification of a target nucleic acid in a sample according to techniques well known to those skilled in the art, such as PCR (Polymerase Chain Reaction ), RT-PCR (Reverse Transcription- Polymerase Chain Reaction), TMA (Transcription Mediated Amplification) or the NASBA technique (Nucleic Acid Sequence Based Amplification), or any other enzymatic amplification technique.
• PCR Polymerase Chain Reaction
• RT-PCR reverse Transcription- Polymerase Chain Reaction
• TMA Transcription Mediated Amplification
• NASBA Nucleic Acid Sequence Based Amplification
• the invention also relates to a nucleotide analog or a nucleotide, prefunctionalized, capable of being subjected to an enzymatic treatment.
• enzymatic treatment of a nucleotide analog or of a nucleotide all reactions are included, in vivo or in vi tro, during which at least one enzyme intervenes, the activity of which is linked to a nucleotide.
• reactions are chosen from those used in molecular biology techniques such as transcription, ligation, elongation, cutting, and more particularly in amplification techniques, (WINN-DEEN, Journal of Clinical Assay, vol 19, p21-26 (1996).
• the enzymes whose activities are related to the nucleotide may in particular be selected from the following non-exhaustive list • DNA dependent DNA polymerases such as Klenow fragment of DNA polymerase I of E. coli, Taq polymerase, the T7, T4 or T5 DNA polymerases, eukaryotic cellular or viral polymerases a, b, g, - DNA RNA dependent polymerases such as AMV (Avian Myoblastosi ⁇ Virus), MMLV (Moloney Murine Leukemia Virus) polymerases; RNA polymerases such as T7, T3, SP6, N4, PBSII RNA polymerases, RNA polymerase from E. col i; enzymes with nuclease activity such as restriction endonucleases, RNAse H; or else polyA polymerases, replicases, such as Q-beta-replicase, terminal transferases, or ligases.
• the TMA technique for the amplification of a target nucleic acid sequence RNA or the amplification of a target nucleic acid sequence DNA after a reverse transcription step said technique being described in international application WO 91/01384 incorporated by reference.
• a nucleotide analog, prefunctionalized according to the invention corresponds to the general formula (I)
• Z represents a coupling arm
• n is an integer equal to 0 or 1
• X represents a reactive covalent function attached to at least one site of the nucleic base B
• R 1 represents H or OH
• R 2 represents H, OH, a mono-di- or tri-phosphate group, or an OR group in which R represents a protective group
• R 3 represents H, a protective group, or a mono-, di- or tri-phosphate group.
• R 1 and R 2 each independently of one another represent H, OH and R3 represent a mono-, di- or tri-phosphate group.
• a prefunctionalized nucleotide of the invention is chosen from the following nucleotides:
• nucleotide whose nucleic base is derived from cytosine and comprises, at least on the amino function in position 4 of the pyrimidine cycle, at least one reactive function of nucleophilic covalence, unprotected, and which does not significantly influence, the enzymatic treatment of said nucleotide, the reactive covalence function being chosen from the functions ⁇ 2, 0-NH2, SH, hydrazine and hydrazide and the reactive covalence function is linked to said amino function in position 4 of the pyrimidine cycle by an arm of coupling chosen from (-CH 2 -) n 1 , (-0-CH 2 -) n 1 in which n- ⁇ is an integer between 1 and 12; (-CH 2 -0-CH 2 -) n 2 , (-CH 2 -CH 2 -0-CH 2 -
• n 2 is an integer between 1 and 6; and -NH-CH 2 -
• the reactive covalent function is linked to said amino function via a coupling arm chosen from -CH 2 -, -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -, - CH 2 - CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -.
• a coupling arm chosen from -CH 2 -, -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -, - CH 2 - CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -, and NH-CH 2 -0-CH 2 -CH 2 .
• nucleotide the base of which is derived from adenine and comprises at least on the amino function in position 6 of the pyrimidine cycle, at least one reactive covalent function
• RECTIFIED SHEET (RULE 91) ISA / EP nucleophile, unprotected, and not significantly influencing the enzymatic treatment, characterized in that the reactive covalent function is chosen from the NH 2 functions ( CH 2 -0-NH 2 / SH / hydrazine and hydrazide • the reactive covalent function is linked to said amino function via a coupling arm chosen from (-CH 2 -
• n ⁇ _ represents an integer between 1 and 12, - (-CH 2 -0-CH 2 -) n 2 , (-CH 2 -CH 2 -0-CH 2 -CH 2 -) n 2 , (-CH 2 -CH 2 -) n 2 , (-CH 2 -CH 2 -)
• n 2 is an integer between 1 and 6, - and NH- ⁇ CH 2 -0-CH -CH 2 .
• the reactive covalent function is linked to said amino function via a coupling arm chosen from -CH 2 -, -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -, - CH 2 -CH 2 -CH 2 -
• the invention also relates to the use of a prefunctionalized nucleotide as defined above, in an enzymatic amplification treatment.
• the pre-functionalized nucleotide analogue or nucleotide has, with respect to the enzymatic treatment, a behavior substantially identical to that of the nucleotide analogue or the corresponding nucleotide.
• the function introduced into a site of the base of said analogue or nucleotide thanks to its biological and chemical inertness with respect to enzymes, does not significantly modify either the affinity or the specificity of the enzyme to with respect to its substrate.
• FIG. 1 represents a general diagram of synthesis of cytidine nucleotides carrying an amino arm in position 4;
• FIG. 2 represents a diagram of synthesis of the cytidine nucleotide carrying an alkyloxyamine arm in position 4;
• FIG. 3 represents a synthesis scheme by transamination of nucleotides
• FIG. 4 shows the effect of the incorporation of CTP- (N4) -C 6 0 2 -NH 2 (27a) on the sensitivity of TMA;
• FIG. 5 shows the effect of the incorporation of CTP- (N4) -Cault-NH 2 (27b) on the sensitivity of TMA.
• Figures • and b respectively show the results of semi-quantification by the ELOSA technique (described in PCT patent application WO 92/19812) of the number of amplicons produced from a target range of 16S RNA from Mycobact Umi um tubercul osi s in the presence of different ratios of nucleotides 21 a and 27b relative to their respective natural nucleotides.
• the strategy generally used consists in the synthesis of protected nucleosides carrying a reactive function also protected. Deprotection of the hydroxyl function at position 5 'releases the modified nucleoside, which is finally triphosphorylated at this position according to the method of Lud ig-Eckstein (J. Lud ig, F. Eckstein, J. Org. Chem., 1989, 54 , 631-635).
• Triazolo (2) (Samano, Miles, Robins, J. Am. Chem. Soc, 1994, 116, 9331-9332)
• Adenosine-isopropylidene (1) (lg, 3.2 mmol) and amidine (1.4 g, 6.5 mmol) are stirred in pyridine (15 ml) at 100 ° C under argon for 48 h .
• the pyridine is then evaporated and co-evaporated with toluene.
• the oil obtained is then taken up in ethyl acetate and this organic phase is washed with water saturated with NaCl. After drying over Na2SO and evaporation, the product (2) is obtained in the form of a white powder with a yield of 60% (700 mg, 1.9 mmol).
• Product 2 was characterized by mass spectrometry and proton NMR.
• the product (2) (500 mg, 1.4 mmol) is dissolved in 20 ml of acetonitrile.
• Compound (3) (1.31 g, 7 mmol) is then added.
• the reaction medium is heated to 50 ° C for 2 days and analyzed by HPLC.
• the residue After evaporation of the acetonitrile, the residue is taken up in ethyl acetate and this organic phase is washed with water saturated with NaCl. After drying and evaporation of the ethyl acetate, the residue is chromatographed on silica gel (eluent: CH2Cl2, then CH Cl / MeOH f 98/2, 95/5 (v / v)). After evaporation and precipitation in hexane, the product (4) is obtained in the form of a white powder with a yield of 80% (563 mg, 1.12 mmol). The nucleoside (4) was characterized by mass spectrometry and by proton NMR.
• the nucleoside (4) (48 mg, 100 mmol) is dissolved in anhydrous pyridine and is evaporated 2 times. Then added under argon 100 ml of pyridine, 300 ml of dioxane and a freshly prepared solution of 2-chloro-4H-1,2,3-dioxaphosphorin-4-one in dioxane (130 ml, 130 mmol). The stirring is left for 20 minutes and then a 0.5 M solution of tributylammonium pyrophosphate in anhydrous DMF (320 ml) and simultaneously 130 ml of tributylamine are added.
• the ditertbutyldicarbonate (1.2 eq., 0.019 mole, 4.18 g) dissolved in 20 ml of dioxane is then added dropwise, in ice, under argon. After 3 hours of reaction at 0 ° C., the dioxane is evaporated and then the pH is brought back to 3 with an aqueous solution of IN HCl and extraction is carried out with ether (3 ⁇ 50 ml). The organic phases are then washed with an aqueous solution of IN HCl then with a saturated aqueous NaCl solution. After drying over Na 2 SO 2 and evaporation, the product (8a) is obtained in the form of a yellow oil (2.56 g, 0.013 mol, 80%). It was characterized by proton and carbon 13 NMR.
• the product (2) (246 mg, 0.68 mmol) is dissolved in 20 ml of 1 acetonitrile.
• the chain (8b) (700 mg, 3.43 mmol) is then added.
• the mixture is brought to 50 ° C.
• the reaction is followed by HPLC: it progresses very slowly. After one week, the solvent is evaporated.
• the residue obtained is dissolved in ethyl acetate and the organic phase is washed with water saturated with NaCl. After drying over Na 2 S0 4 and evaporation, a yellow oil is obtained which is chromatographed on silica gel (eluent: CH2Cl2 / MeOH: 97/3, v / v).
• the product (9) is obtained in the form of a white powder (120 mg, 0.24 mmol, 35%).
• the product (9) is characterized by proton NMR and by mass spectrometry.
• nucleoside (9) into nucleotide (10) is carried out according to the protocol described in the ex € ⁇ mple I (preparation of nucleotide 5).
• Ethyl orthoformate (166 ml, 148 mg, 1 mmol) is added under argon at room temperature. suspension of product (12) (200 mg, 0.5 mmol) in acetone (2 ml) containing APTS (9.5 mg, 0.05 mmol). The reaction is followed by TLC (CH 2 C1 2 / MeOH 90/10). After 2 hours, dichloromethane is added and the organic phase is washed with a 10% aqueous NaHCO 3 solution and then with a saturated aqueous NaCl solution. After drying and evaporation, the product (13) is obtained in the form of a yellow powder (167 mg, 0.38 mmol, 76%).
• Nucleoside 13 (44 mg, 100 mmol) is dissolved in anhydrous pyridine and is evaporated 2 times. Then added under argon 100 ml of pyridine, 300 ml of dioxane and a freshly prepared solution of 2-chloro-4H-1,2,3-dioxaphosphorin-4-one in dioxane (130 ml, 130 mmol). Stirring is left for 20 minutes and then a 0.5 M solution of tributylammonium pyrophosphate in anhydrous DMF (320 ml) and 130 ml of tributylamine are added.
• Carboxymethoxylamine hydrochloride (6.6 g, 60.4 mmol) is dissolved in 132 ml of water in the presence of sodium hydroxide (2 g, 50 mmol) and dioxane (66 ml). The solution is cooled in an ice bath. D-tert-butyldicarbonate (13.97 g, 64.1 mmol) dissolved in dioxane is added dropwise
• the product (16) is obtained in the form of a white powder (10 g, 52 mmol, 86%).
• the product (16) was characterized by proton NMR.
• the product (16) (2 g, 10.4 mmol) is dissolved in freshly distilled THF. After the medium has cooled in ice, DCC is added (2.15 g, 10.4 mmol). The mixture is stirred cold for 15 minutes. The propargylic amine is then added. (714 ml, 10.4 mmol) The stirring is maintained at room temperature under argon for 2 hours. After filtration, the solvent is evaporated. The residue thus obtained is taken up in dichloromethane and is washed with an aqueous solution of IN HCl, then with a solution of NaHCO 3 at 5% and finally with an aqueous solution saturated with NaCl.
• the nucleoside (19) (102 mg, 200 mmol) is dissolved in anhydrous pyridine and is evaporated 2 times. However, then add 200 ml of pyridine under argon, 600 ml of dioxane and a freshly prepared solution of 2-chloro-4H-1, 2, 3-dioxaphosphorin-4-one in dioxane (260 ml, 260 mmol). We leave the stirring for 20 minutes then add a 0.5 M solution of tributylammonium pyrophosphate in anhydrous DMF (640 ml) and simultaneously 260 ml of tributylamme
• a 1% iodine solution is added in a pyridme / water mixture (98/2, v / v) (4 ml, 314 mmol). After 20 minutes of stirring, the excess iodine is destroyed with an aqueous solution of NaHS0 3 at 5% and the stirring is continued for 10 minutes. It is evaporated to dryness and then a water / dichloromethane extraction is carried out. The formation is checked.
• the nucleophilic substitution of the tosyl group with a diamined alkyl chain followed by protection with ethyl trifluoroacetate of the free amino function of the introduced chain is carried out in one step.
• the compound (24) (1 mmol) below in THF (5 ml) is treated with a molar solution of tetrabutylammonium fluoride (1.2 mmol, 1.2 eq) in THF. After 3 to 4 hours of stirring at room temperature and under argon, the reaction is neutralized with acetic acid (1 eq.) And then evaporated to dryness.
• the reaction is oxidized by a 1% iodine solution in the pyridine-water mixture (98: 2, v: v) (4 ml) and stirring maintained for 30 min.
• the excess iodine is then destroyed by an aqueous solution of sodium bisulfite at 5% and the reaction medium evaporated to dryness.
• the residue obtained is dissolved in water (20 ml) and treated by washing with dichloromethane.
• aqueous phase is recovered and analyzed by HPLC (DEAE 8 column HR Millipore 5x100 mm, buffer A: Tris HCl 20 mM pH 7.6, buffer B: Tris HCl 20 mM, 0.5 M NaCl pH 7.6, flow rate: 0.5 ml / min, gradient: 0 to 35% B in 40 min). Retention time: 24 to 29 min.
• TLC: Rf 0.5, eluent: propanol: water: NH40H (6: 3: 1, V: v: v)
• reaction medium is again evaporated and then co-evaporated to dryness with water.
• desired compound is purified and desalted by
• the fully protected nucleotide (32) is completely deprotected in 2 ′, 3 ′ by trifluoroacetic acid according to the protocol described in Example 10.
• the deprotection of the alkoxyamine function is carried out in an aqueous solution of hydrazine for one overnight at room temperature.
• the nucleotide (32) is then purified by HPLC under the same conditions as those described in Example 10.
• Cytidine triphosphate (0.1 mmol) is added to the solution composed of sodium bisulfite (12 mmol, 120 eq.) And 2-2 'oxy-bis-ethylamine hydrochloride (7.5 mmol, 75 eq.) In the water (2.5 ml), the pH of which was previously adjusted to 7.0 with a 10 M sodium hydroxide solution, stirring being maintained at 37 ° C for 3 days.
• the compound (33) is purified and desalted by HPLC (see HPLC conditions in Example 10).
• EXAMPLE 17 Synthesis of [4-N- (adipic acid-hydrazide) -5 '-O-triphosphate] -cytidine (34) by transamination.
• the nucleotide (34) was prepared by transamination according to the protocol described in Example 16, using adipic acid dihydrazide (83 mg, 0.44 mol) for the introduction of the hydrazide function. Its purification and its desalting were also carried out according to Example 10, its structure was confirmed by proton NMR.
• the fluoresceem isothiocyanate (91 mg, 2.35 mmol, Aldrich, F 250-2) is dissolved in anhydrous DMF (10 ml), under argon. Aminobutylaldehyde diethylacetal (421 mg, 392 ml, 235 mmol) is then added. After one hour, the DMF was evaporated and the residue is chromatographed on silica gel (eluent: CH C1 2 / MeOH: 90/10, v / v). After evaporation in solvent, the product (35) is obtained in the form of an orange powder (1.21 g, 2.2 mmol, 94%). It has been characterized pa. Proton NMR and mass spectrometry.
• the protected product (35) (342 mg, 0.62 mmol) is placed in 20 ml of a 30% aqueous solution of acetic acid. After one hour of reaction, the solvent is evaporated off and co-evaporated with acetonitrile. The residue obtained is chromatographed on silica gel (eluent: CH 2 C1, / MeOH 90/10, v / v). After evaporation of the solvent, the product (36) is obtained in the form of an orange powder (145 mg, 0.30 mmol, 48%). It was characterized by proton NMR and mass spectrometry.
• EXAMPLE 19 Chemical synthesis of a prefunctionalized polynucleotide.
• the synthesis of the starting nucleoside was carried out using the 2 '-deoxy-8-mercaptoadenosine precursor (37) obtained according to the strategy described by A. LAAYOUN, J.-L. DECOUT and J. LHOMME in Tetrahedron Lett., 1994 , 35, 4989-4990.
• the starting nucleotide thus obtained is characterized by the usual spectroscopic methods.
• exocyclic amine of adenine is protected by a benzoyl group, the 5 'hydroxyl is protected by dimethoxytrityl and the 3' hydroxyl by the group N, N-diisopropyl-2-cyanoethylphosphoramidite.
• the purification of the synthesized polynucleotide is carried out by reverse phase HPLC (semi-preparative Macherey Nagel column 10 mm x 25 cm, - C18; 5 ⁇ m of porosity; eluent: 20 min gradient from 0 to 30% acetonitrile mixed with 0.1 M aqueous ammonium acetate solution at pH 6).
• the fractions containing the polynucleotide are collected and lyophilized.
• the nucleotide L incorporated in the preceding step is activated so as to obtain the polynucleotide diol 5 'CGCACMCACGC 3', in which
• the labeling reaction is carried out using the reagent (40) consisting of a dansyl nucleus as a fluorescent functional group linked via a diethylene glycol chain to an oxyamine (nucleophilic) function.
• This reagent was prepared according to the described method D. BOOTURYN et al. In Tetrahedron, 1997, 53, 5485-5492.
• the labeling reaction is carried out in water in the presence of a slight excess of reagent (40) (1.5 equivalent) relative to the oligonucleotide.
• reagent 40
• HPLC HPLC indicates a rapid disappearance of the starting products and the formation of a new product corresponding to the functionalized polynucleotide (41).
• Analysis of the proton NMR spectrum also confirms the attachment of the dansyl marker.
• EXAMPLE 21 Enzymatic synthesis of a prefunctionalized polynucleotide by transcription.
• a PCR is carried out on the HIVgag gene carried in a linearized pGEM plasmid, using a conventional primer, and a primer carrying the sense sequence of a promoter for ⁇ RNA polymerase of phage T7 upstream of a conventional primer sequence .
• a product of 158 base pairs is obtained, which is purified by phenol / chloroform extraction and passage through microcon 30
• the transcription reactions are carried out in Eppendorf tubes in a final volume of 25 ⁇ l, in a 40 mM buffer of Tris / HCl, pH 8.1, 20 mM of MgCl 2 , 5 mM of Dithiotreitol, 1 mM of spermid, 8 % of polyethylene glycol, 0.01% of Triton X 100, 50 ⁇ g / ml of bovine serum albumin
• the pre-functionalized nucleotide (43) and the four ribonucleotides (CTP, GTP, ATP, UTP) are added respectively to the concentration 1.33 mM and 4 mM each.
• the PCR matrix is added at the concentration of 10 9 copies / ⁇ l, and the phage T7 RNA polymerase (Patent Application FR 97 04166) at the concentration of 1 u / ⁇ l
• the reaction is incubated for 60 mm at 37 ° C. .
• 0.5 u / ⁇ l of DNAse 1 are added to the medium in order to destroy the DNA matrix.
• the sample is incubated at 37 ° C. continuously for 15 mm.
• the RNAs obtained are purified by passing through Sephadex G50.
• a first control is carried out with a reaction without addition of T7 RNA polymerase
• a second control is carried out without activated nucleotide, as a control of transcription
• the purified transcripts are marked according to the experimental mode described in Example 2 and visualized under an ultra violet lamp after electrophoretic migration in agarose gel.
• EXAMPLE 22 Synthesis of a prefunctionalized polynucleotide during a TMA amplification reaction.
• prefunctionalized nucleotides 27a CTP- (N4) -C 6 0 2 -NH 2
• 27b CTP- (N4) -C4 - NH 2
• 32 CTP- (N4) -C 6 -0NH 2
• Amplification reactions are carried out in parallel in the presence of a labeled nucleotide carrying a fluorescein, UTP-12-fluorescein (Boerhinger, ref 1 427 857.
• the prefunctionalized nucleotides 27a, 27b, 32, and for comparison, the labeled nucleotide of Boerhinger, ref 1 427 857, are tested for their incorporation in the products of the TMA amplification reaction, according to the Gen-Probe amplified MTD2 kits. (amplified ⁇ yco-ac erium tuberculo ⁇ is direct test).
• This reaction allows the amplification of a 136 base fragment of the 16S RNA of mycobacteria. It uses your enzymatic activities (DNA dependent RNA polymerase, DNA dependent DNA polymerase, DNA dependent RNA polymerase, and ribonucleaseH) and two enzymes, T7 RNA polymerase, and AMV reverse transcriptase.
• RNA which resemble a cycle of replication of RNA viruses
• this reaction allows the specific amplification of '' a nucleic acid sequence recognized by two primers (a single primer and a primer containing the T7 RNA polymerase promoter).
• the amplification factor is very important (10) and the sensitivity very good (1 to 10 copies).
• the products obtained are for 90% of single-stranded RNA, and for 10% of double-stranded DNA.
• the amplification reactions are carried out from
• 16S RNA 10 copies of 16S RNA from Mycobacter iu tuberculosis.
• This target RNA is previously obtained and quantified according to the following method: the 16S RNA gene is cloned into a p asmid, under the control of a promoter. By transcription m vi tro, 16S RNA is obtained. This is purified by extraction with phenol-chloroform, and filtration on microcon 30 (Amicon).
• Classic TMA reactions contain 4 mM of each of the natural rNTPs (ATP, CTP, GTP and UTP).
• the reaction is carried out by including one of these modified nucleotides in the reaction buffer.
• the reaction is studied in the presence of different concentration ratios between the modified nucleotide and the natural nucleotide, while keeping at 4 mM the total concentration of each nucleotide of the same series, modified or natural.
• the ratios studied are 0%, 10%, 30%, 50% and 70% (except for UTP-fluorescein, for which we could not test 70%).
• a negative control is carried out, consisting of a reaction comprising all the reagents, including the highest modified nucleotide ratio used (70 or 50%), except the enzymes (replaced by the buffer for taking up the lyophilized enzymes).
• the amplification reactions are analyzed by electrophoresis of 5 ⁇ l of reaction on denaturing polyac trylamide gel (6% acrylamide, 7M urea, IX TBE, apparatus bioRad electrophoresis, 170 volts, 45 minutes) and staining with ethidium bromide, then by Northern.
• the nucleic acids are transferred onto a membrane (Nylon N, semi-dry bioRad transfer device, 0.5 X TBE, 25V, 15 minutes) and hybridized with nucleic probes specific for Myco acté ium tuberculosis -.
• the amplification products on gel are also visualized before staining with ethidium bromide by excitation of fluorescein on an ultraviolet table.
• the expected amplification products are visualized in large quantities after staining with ethidium bromide. After Northern, these products hybridize with the specific probe.
• Example 22 The amplification products obtained during the production of Example 22 are separated from the excess nucleotides in the reaction by filtration on Microcon 30 TM (from Amicon), and taken up in 50 ⁇ l of water.
• Microcon 30 TM from Amicon
• the purification is carried out on sephadex G50 (absorption of fluorescein on Microcon).
• a step of digestion of the nucleic acids is then carried out up to the nucleoside stage:
• nuclease PI Boerhinger, ref 236225, l ⁇ g / ⁇ l, 0.3u / ⁇ l. The reaction is incubated at 37 ° C for 30 minutes.
• nucleoside composition is then determined by analysis on high pressure liquid chromatography (HPLC, Beckman, Gold system), and by comparison with nucleoside standards (injection of 50 ng of each nucleoside). 20 ⁇ l of the digestion are injected onto column C18 (Ultrasphere), heated to 45 ° C. The separation is carried out in 50 mM sodium phosphate buffer pH 7, comprising a methanol gradient (after 10 minutes, passage in 15 minutes from 0% to 30% of 95% methanol). The peaks are visualized by absorption at 254 nm, and the areas of the peaks are calculated by integration.
• the ratio between the area of the peak of the prefunctionalized nucleotide and that of its natural analogue makes it possible to determine the incorporation efficiency, that is to say the ratio between the amount of prefunctionalized nucleotide incorporated and the total amount of nucleotide of the same incorporated series.
• control without enzyme should not give rise to the appearance of peak. This control demonstrates the good efficiency of the step of removing excess nucleotides.
• the quantity of fluorescein nucleotide incorporated is measured by the fluorescence intensity: the fluorescence intensity of a standard fluorescein range is read on a Perkin LS spectrofluorimeter 50. The fluorescence intensity of the different amplification reactions is measured. By comparison with the calibration curve, the quantity of fluorescein incorporated is determined. The incorporation yield is calculated relative to the amplicon concentration measured by absorption at 260 nm.
• the HPLC analysis makes it possible to separate the eight natural nucleosides, as well as the three nucleosides analogous to the three pre-functionalized nucleotides tested.
• TMA was performed from decreasing amounts of target
• reaction products are studied inter alia by the method described in Example 22.
• reaction products are also analyzed in quantitative fashion by ELOSA (PCT WO 92/19812), and comparison of the intensity of the signals with those of standard ranges.
• pre-functionalized nucleotide (s) are analyzed by electrophoresis and staining, Northern, and ELOSA.
• ELOSA which are representative of the different analytical methods used, are represented in FIGS. 4 and 5.
• a concentration of prefunctionalized nucleotide (s) can be determined such that the sensitivity of the TMA reaction is not significantly affected, even with a very low number of copies of initial target.
• the nucleotide 27b can replace the natural nucleotide.
• the marking thus obtained is compared with that obtained in the presence of the nucleotide labeled UTP-12 -fluorescein (Boerhinger, ref 1 427 857).
• the amplicons are obtained by the method described in Example 22 from 10 ⁇ copies of 16 ⁇ RNA target of Mycobac er iu tuberculosis, and in the presence of 50% of prefunctionalized nucleotide 27b.
• the amplicons obtained are coupled to fluorescein-NHS (Boerhinger 8370042128), according to the following method: 30 ⁇ l of TMA amplification products are mixed with 30 ⁇ l of 0.2M carbonate buffer, 0.15 M NaCl, at pH8.8 , and 40 ⁇ l (approximately 200 equivalents) of NHS fluorescein (3.5 mg / ml in DMSO). After stirring for one hour at room temperature, the labeling is analyzed.
• the labeled amplicons (15 ⁇ l) are analyzed by gel electrophoresis as described in Example 22, and viewed under ultraviolet, before and after staining with ethidium bromide. The profiles are compared with those obtained by labeling in the presence of 50% of UTP-12-fluorescein.
• the signals obtained without coloring are more intense than those obtained by direct labeling with fluorescein.
• the oxyamine function as carried by the nucleotide 32, such a good result will be possible after coupling with the fluorophore 36.
• the reactivity of the oxyamine aldehyde couple allows reduced coupling times as shown in example 20.

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