Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
  • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
  • C07D495/04—Ortho-condensed systems
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/13—Tracers or tags
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
  • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
  • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

• the present invention relates to new reagents for labeling biological molecules, a method for synthesizing said markers as well as applications for labeling biological molecules, in particular in the field of diagnostics using nucleic acid analysis.
• a first method consists in fixing the marker on the base, whether the latter is natural or modified.
• a second method proposes to fix the marker on the sugar, again whether it is natural or modified.
• a third method relates to the attachment of the marker to the phosphate. Labeling on the base has been used in particular in the approach of labeling nucleic acids by incorporating directly labeled nucleotides. The labeling on sugar is often used in the case of nucleic acid probes prepared by chemical synthesis. Labeling on phosphate has also been used to introduce functionalized arms and markers during the chemical synthesis of oligonucleotides.
• the fixing of the marker on the phosphate is a more complex technique than the technique consisting in functionalizing the base or the sugar and has been much less used in particular because of the low reactivity of the phosphate (see for example Jenc s WP et al J. Amer Chem Soc, 82, 1778- 1785, 1960).
• O'Donnel and Me Laughlin Reporter groups for the analysis of nucleic acid structure”, p 216-243, in “Bioorganic Chemistry: Nucleic Acids”, Ed Hecht SM, Oxford University Press, 1996) bearing on the methods of introducing probes into oligonucleotide fitments, effective ylation of the intemucleotide phosphodiester is considered to be impossible.
• Patent application WO-A-99/65926 describes a method for labeling a synthetic or natural ribonucleic acid (RNA) which consists in fragmenting the RNA and in labeling at the terminal phosphate.
• RNA synthetic or natural ribonucleic acid
• This document describes a certain number of functions which can be used for labeling in connection with the fragmentation such as the hydroxyl, amine, hydrazine, alkoxyamine, alkyl halide, alkyl halide of benzyl type and in particular the derivative 5- ( bromomethyl) fl.uorescéine.
• These functions make it possible to label the nucleic acids, but a fragmentation step must be associated in order to have effective labeling because this labeling occurs on the phosphate released during the fragmentation.
• this method does not work effectively on double strand
• the reagents incorporating at least one diazo function in general are unstable by themselves, which poses problems for the use of these reagents in a labeling Mt, which is prohibitive if the labeled product has the function of demonstrating the presence of a biological target molecule in a any sample.
• the reagents carrying the diazomethyl function and associated with certain markers, such as biotin are not very soluble in water, which leads to the use of organic solvents which are water-missible for coupling with biological molecules, which are only soluble in water or aqueous buffers, but these solvents, present in high concentration in the labeling reaction, slow down the reaction rate and therefore affect the efficiency of the coupling.
• the invention consists in the use of polyamine arms which, like the ethylene glycol arms, allow the biotin to be moved away from the reactive center (diazo function).
• polyamine arms which, like the ethylene glycol arms, allow the biotin to be moved away from the reactive center (diazo function).
• these new molecules allow the implementation of processes that can operate in acidic medium, which is particularly interesting for molecules incorporating diazo functions.
• selectivity of the reagents carrying a diazo function is therefore greater in an acid medium.
• the solubility provided to the polyamide chains therefore facilitates washing while reducing the background noise during subsequent detection, or even outright elimination of the purification step.
• the latter provides a labeling reagent stable at the temperature of formula (0):
• R 1 represents H or an alkyl, aryl or substituted aryl group
• R 2 represents a detectable marker or at least two detectable markers linked together by at least one multimeric structure
• L is a link arm comprising a linear chain of at least two covalent bonds and n an integer equal to 0 or 1
• A is a link arm comprising at least one covalent double bond allowing the conjugation of the diazo function with the aromatic ring and u is an integer between 0 and 2, preferably 0 or 1, • -YX- represents -CONH-, -NHCO-, -CH 2 O-, -CH 2 S-,
• m is an integer between 1 and 10, preferably between 1 and 3, and
• p is an integer between 1 and 10, preferably between 1 and 3. According to a second embodiment of the invention, it relates to a labeling reagent, according to claim 1, of formula (1):
• R 1 represents H or an alkyl, aryl or substituted aryl group
• R 2 represents a detectable marker or at least two detectable markers rehes together with at least one multimeric structure
• L is a hedge arm comprising a linear sequence of at least two covalent bonds and n an integer equal to 0 or 1,
• -YX- represents -CONH-, -NHCO-, -CH 2 O-, -CH 2 S-,
• m is an integer between 1 and 10, preferably between 1 and 3, and
• the present invention provides a reagent, according to any one of claims 1 to 4, of formula (2): in which :
• R 1 represents H or an alkyl, aryl or substituted aryl group
• R 2 represents a detectable marker or at least two detectable markers rehesed together by at least one multimeric structure
• L is a hedge arm comprising a linear sequence of at least two covalent hedges and n an integer equal to 0 or 1,
• the present invention describes a reagent, according to any one of claims 1 to 4, of formula (3):
• R 1 represents H or a substituted alkyl, aryl or aiyl group
• R 2 represents a detectable marker or at least two detectable markers rehesed together by at least one multimeric structure
• L is a hedge arm comprising a linear sequence of at least two covalent hedges and n an integer equal to 0 or 1, and
• R 2 is constituted by a D-Biotin residue of formula (4):
• R 1 is constituted by: CH 3 , and R 3 and R 4 each represent: H
• the structure - (L) n - consists of:
• R 1 represents H or an alkyl, aryl or substituted aryl group
• R 2 represents a detectable marker or at least two detectable markers rehesed together by at least one multimeric structure
• A is a hedge arm comprising at least one double covalent hedge allowing the conjugation of the diazo function with the aromatic cycle and u is an integer between 0 and 2, preferably 0 or 1,
• -YX- represents -CONH-, -NHCO-, -CH 2 O-, -CH 2 S-,
• the invention provides a reagent stable marking at the temperature of formula (7):
• R 1 represents H or an alkyl, aryl or substituted aryl group
• R 2 represents a detectable marker or at least two detectable markers rehesed together by at least one multimeric structure
• L is a hedge arm comprising a linear chain of at least two covalent hedges and n an integer equal to 0 or 1
• -YX- represents -CONH-, -NHCO-, -CH 2 O-, -CH 2 S-,
• • -Z- represents -NH-, -NHCO-, -CONH- or -O-, • m is an integer between hehe 1 and 10, preferably between 1 and 3, and
• L comprises a motif - (O-CH 2 -CH 2 ) -, repeated from 1 to 20 times, preferably from 1 to 10 times, and even more preferably from 2 to 5 times, -Z- then being represented by -NH-, -NHCO- or -CONH-.
• the invention also relates to a process for the synthesis of a labeling reagent, according to the preceding embodiments, comprising the following steps: a) a marker or a marker precursor having a reactive function R 6 is available , b) there is a hedge arm of formula (8): R * - (Z— (CH- j -) ⁇ - R. in which:
• • m is an integer between 1 and 10, preferably between 1 and 3
• • p is an integer between 1 and 10, preferably between 1 and 3
• R 7 and R 8 represent two identical or different reactive functions, c) reacting together the reactive function R 6 of said marker or marker precursor with the function R 7 of the hedge arm of formula (8) in the presence of at at least one coupling agent to form a covalent hedge, R 6 and R 7 being complementary, d) a derivative of formula (9) is available:
• R 1 represents H or an alkyl or aryl or substituted aryl group
• L is a hedge arm comprising a linear sequence of at least two covalent hedges and n an integer equal to 0 or 1
• A is a hedge arm comprising at least one double covalent hedge allowing the conjugation of the diazomethyl function with the aromatic cycle and u is an integer equal to 0 or 1, and • R 9 represents a complementary reactive function of R 8 , e) reactive function F is reacted together? of the derivative of formula (9) with the function R 8 of the hedge arm of formula (8) in the presence of at least one coupling agent to form a covalent hedge, f) reacting the hydrazine or one of its derivatives on the ketone or aldehyde function to form a hydrazone, and g) the hydrazone is transformed into a diazomethyl function using an appropriate treatment.
• the synthesis process can also include:
• the invention also relates to a method for labeling a biological molecule, in particular a nucleic acid, comprising bringing into contact in homogeneous solution, in a substantially aqueous buffer, a biological molecule and a reagent, according to the rehsation modes previously mentioned.
• the invention also relates to a labeled biological molecule capable of being obtained by the method, according to the labeling claim above.
• the labeling and fragmentation process is carried out using a labeling reagent which is chosen from the compounds of formula (3):
• R 1 represents H or an alkyl, aryl or substituted aryl group
• R 2 represents a detectable marker or at least two detectable markers rehes together with at least one multimeric structure
• L is a hedge arm comprising a linear sequence of at least two covalent hedges and n an integer equal to 0 or 1, and
• the fragmentation and the marking are carried out in two stages.
• the fragmentation and marking are carried out in one step.
• the marking is carried out in a substantially aqueous homogeneous solution.
• fragmentation takes place by enzymatic, physical or chemical route.
• the present invention also relates to any labeled nucleic acid capable of being obtained by the preceding labeling and fragmentation process.
• the present invention also relates to a t for detecting a target nucleic acid comprising a labeled nucleic acid, as defined above.
• the present invention always relates to a solid support on which is fixed at least one reagent, as defined above.
• the present invention finally relates to a method for capturing nucleic acids comprising the following steps: • there is a solid support on which is directly or indirectly attached at least one biological molecule, defined previously or a nucleic acid, also defined previously biological molecule or nucleic acid comprising a diazomethyl function, • a biological sample capable of containing hbre nucleic acids is brought into contact, and • the solid support where the molecule (s) are (are) fixed covalently to at least one nucleic acid.
• multimeric structure is meant a polymer formed from repeated units of chemical or biological synthons.
• An example is cited in example 34.2 of the description of patent application WO-A-02/090319. Those skilled in the art are invited to refer to this document if it finds the information developed below insufficient for their complete understanding on this subject. Many variants of such structures which can be used in the present invention are known, such as for example:
• detectable marker at least one marker capable of directly or indirectly generating a detectable signal. A non-exhaustive list of these markers follows:
• the enzymes which produce a detectable signal for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose-6-phosphate dehydrogenase,
• chromophores such as fluorescent, luminescent, dye compounds
• the marker is not a radioactive marker in order to avoid the security problems associated with these markers.
• the marker is electrochemically detectable and in particular the marker is a derivative of an iron complex, such as a ferrocene.
• Indirect systems can also be used, such as for example ligands capable of reacting with an anti-hgand.
• the hgand / anti-hgand couples are well known to those skilled in the art, which is the case for example of the following couples: biotin streptavidin, hapten antibody, antigen / antibody, peptide / antibody, sugar / lectin, polynucleotide / complementary to the polynucleotide.
• the ligand which carries the reactive diazomethyl function is the ligand which carries the reactive diazomethyl function.
• the anti-hgand can be directly detectable by the markers described in the preceding paragraph or be itself detectable by another hgand / anti-hgand pair.
• This stacking system is illustrated in the examples.
• Another example of indirect systems uses a specific covalent hedge between the ligand and the anti-ligand, for example methyl ketone and alkoxyamine. Examples of this system are described in patent applications WO-A-00/40590 and WO-A-98/05766.
• At least two labels are present on the labeling reagent.
• the tracer is a fluorescent compound with a small steric hindrance such as fluorescein, hexachlorofluorescein (HEX), dansyl (edans), rhodamine, tetramethyhhodamine (5 or 6-TAMRA), carbo ⁇ y-X-rhodamine (ROX), N1R-type chromophores (LI-COR Inc, Lincoln NE, USA), cyanine derivatives such as Cy5 and Cy3 (Randolph JB and al, Nucleic Acids Res., 25 (14) , p2923-2929, 1997) and in particular the derivatives of Cy5 or else the tracer is a hapten of small steric hindrance such as biotin, dinitrohenyl, or a derivative of abietane (see application WO-A-00/07982 ).
• a small steric hindrance such as fluorescein, hexachlorofluorescein (HEX),
• small steric hindrance is meant a molecular weight of less than 1000 g / mole.
• fluorophores whose excitation wavelength is greater than 450 nm, preferably greater than 600 nm.
• the detection is carried out by the recognition of an anti-hgand labeled as described above.
• biotin preferably streptavidin or an anti-biotin antibody coupled to a fluorescent compound such as fluorescein, Cy5 or phycoerythrin is used.
• a monoclonal antibody is used as described in patent application WO-A-00/07982.
• the labeling reagents of the invention are soluble in polar solvents such as DMF, DMSO, CH 3 CN, THF, DMA (dimethylacetamide), NMP (N-methylpyrrohdone), DME (dimethoxyethane).
• polar solvents such as DMF, DMSO, CH 3 CN, THF, DMA (dimethylacetamide), NMP (N-methylpyrrohdone), DME (dimethoxyethane).
• the labeling reagents are soluble in DMSO or water.
• solvent miscible with water is meant a solvent which is miscible in a proportion of at least 5% by volume with water or an aqueous buffer containing salts.
• the arm L comprises an ethylene glycol or polyethylene glycol unit to increase the solubility of the reagent in water.
• A is a hedge arm comprising at least one double ethylene type hedge allowing the conjugation of the diazomethyl function with the aromatic cycle.
• the function of the hedge arm A is to move the diazomethyl function away from the cyle to reduce the steric hindrance while retaining the stability of the diazomethyl function.
• conjugation is meant the electronic delocalization of the aromatic cycle along the carbon chain of the hedge arm AA as an example, the arm A can have the following structure:
• v is an integer between 1 and 10, preferably v is 1 or 2, and
• R 10 is H or an alkyl group, preferably R 10 is H, methyl or ethyl.
• R 10 is H or an alkyl group, preferably R 10 is H, methyl or ethyl.
• nucleic acid means a chain of at least two deoxyribonucleotides or ribonucleotides optionally comprising at least one modified nucleotide, for example at least one nucleotide comprising a modified base, such as inosine, methyl-5-deoxycytidine, dimethylamino-5-deoxyuridine, deoxyuridine, diamino-2,6-purine, bromo-5-deoxy ⁇ ridine or any other modified base allowing hybridization.
• a modified base such as inosine, methyl-5-deoxycytidine, dimethylamino-5-deoxyuridine, deoxyuridine, diamino-2,6-purine, bromo-5-deoxy ⁇ ridine or any other modified base allowing hybridization.
• This polynucleotide can also be modified at the level of the intemucleotide hedge such as for example phosphorothioates, H-phosphonates, alkyl-phosphonates, at the level of the skeleton such as for example alpha-ohgonucleotides (FR 2 607 507) or PNA (M Egholm et al., J. Am. Chem. Soc, 114, 1895-1897, 1992 or 2 'O-alkyl ribose.
• the nucleic acid can be natural or synthetic, an ohgonucleotide, a polynucleotide, a fragment of nucleic acid, a ribosomal RNA, a messenger RNA, a transfer RNA, a nucleic acid obtained by an enzymatic amplification technique such as:
• polypeptide is meant a chain of at least two amino acids.
• amino acids is meant:
• hapten designates non-immunogenic compounds, that is to say incapable by themselves of promoting an immune reaction by production of antibodies, but capable of being recognized by antibodies obtained by immunization of animals in known conditions, in particular by immunization with a hapten-protein conjugate.
• These compounds generally have a molecular mass of less than 3000 Da, and most often less than 2000 Da and can be, for example, glycosyl peptides, metabohtes, vitamins, hormones, prostaglandins, toxins or various drugs, nucleosides and nucleotides.
• antibody includes polyclonal or monoclonal antibodies, antibodies obtained by genetic recombination, and antibody fragments such as Fab or F (ab ') 2 fragments.
• antiigen denotes a compound capable of generating antibodies.
• protein includes holoproteins and heteroproteins such as nucleoproteins, lipoproteins, phosphoproteins, metalloproteins and glycoproteins, both fibrous and globular in their characteristic conformation form.
• the biological molecule has a phosphate group, that is to say having at least one motif: which is either naturally present in the biological molecule or can be introduced for example by chemical or enzymatic modification. Examples of chemical modification for proteins are given in "Chemistiy of protein conjugation and cross linking", SS Wong, CRC Press, 1991.
• the biological molecule is a nucleic acid.
• R 1 represents H or an alkyl or aryl or substituted aryl group
• R 2 represents a detectable marker or at least two detectable markers rehesed together by at least one multimeric structure
• L is a hedge arm comprising a linear chain of at least two covalent hedges
• the labeling reagent has the: a) formula (13):
• R 1 represents a methyl or phenyl group.
• L can comprise a motif - (NH-CH2-CH2) -, repeated from 1 to 20 times, preferably from 1 to 10 times, and even more preferably from 2 to 5 time.
• hydrazine derivative is meant a molecule having the NH 2 -NH- function. Tosylhydrazine is an example of such a derivative.
• the transformation of hydrazone into diazomethyl is carried out by the usual methods, in particular the oxidation with MnO 2 . Other methods are useful as described in X. Creary, Organic Syntheses, Wiley: New York, Coll. Flight. NE, p438-443, 1990; H.
• said method comprises: • an additional step of protection of the ketone or aldehyde function (in the case where R 1 is H) of compound (9), and • a step additional subsequent deprotection of said ketone or aldehyde function.
• This protection is achieved by an acetal group, for example.
• Deprotection is carried out by an appropriate means such as in acid for the acetal group.
• the precursor of the marker can have the formula (17) below.
• GPi and GP 2 represent two protective groups of identical or different a ine function and p is an integer between 1 and 10, advantageously 2 and 6 preferably 4.
• GPi and GP 2 are different in order to be able to add several patterns as is explained below.
• protective groups GP1 or GP2 which can be used in the present invention are given in TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 2 nd edition, John Wiley and Sons, New York, 1991, preferably those commonly used in peptide synthesis such as Boc (tert-butyloxycarbonyl), Fmoc (9-fluorenylmethyleneoxycarbonyl), Cbz (carboxybenzyl) or A oc (ahyloxycarbonyl).
• GP1 and GP2 are the protective groups Boc and Fmoc respectively.
• the reaction between this precursor which has a carboxyhque function and the derivative of formula (18), below takes place in the presence of a coupling agent to form the amide hedge.
• the hberated amine function is used to couple another molecule of formula (17). This process is repeated as many times as necessary to obtain a multitude of NH 2 functions protected by a protective group, for example a Boc function.
• the pattern is added between one (1) and one hundred (100) times, preferably between one (1) and twenty (20) times. Hydrazine is reacted on the ketone function originating from the phenylketone derivative to form a hydrazone and then oxidized in the presence of MnO 2 to form a diazomethyl residue.
• a tracer for example a biotin activated by an N-hydroxysuccinimide group
• a tracer for example a biotin activated by an N-hydroxysuccinimide group
• This solution preferably contains salts such as a buffer solution.
• a monophasic solution such as a DMSO water solution as opposed to a biphasic solution such as a chloroform water solution.
• the specific conditions for labeling reactions vary depending on the biological molecules and the label. With regard to nucleic acids, a pH between 5 and 8 allows efficient labeling. In particular, a pH of between 5.5 and 7.0 is preferred for all of the reagents of the invention. With the reagent of formula (11), the pH range is wider for labeling. Good labeling efficiency is obtained for a pH between 3 and 8 for this reagent.
• DNA chip is meant a sohde support of reduced size where a multitude of capture probes are fixed at predetermined positions.
• density of the nucleic acids attached to the solid support imposes significant steric constraints during hybridization and the fragmentation allows this stage of hybridization to be overcome. Examples of these DNA chips are given, for example, in the publications of G. Ramsay, Nature Biotechnology, 16, ⁇ 40-44, 1998; F. Ginot, Hu an Mutation, 10, pl-10, 1997; J.
• the fragmentation and the labeling are carried out in one step or in two steps and the labeling can be carried out either before, after or simultaneously with the fragmentation.
• the labeling and the fragmentation take place simultaneously, that is to say that the reagents necessary for these two stages are put together in substantially homogeneous solution.
• aqueous with nucleic acid for example.
• labeling and fragmentation being carried out simultaneously means that the physical means is applied to a homogeneous substantially aqueous solution containing at least the nucleic acids and the labeling reagent.
• the nucleic acid is fragmented by enzymatic, chemical or physical means.
• the enzymatic fragmentation of the nucleic acid is carried out for example by nucleases.
• Fragmentation of the nucleic acid by physical means is carried out for example by sonication or by radiation.
• Fragmentation by chemical means, if the nucleic acid is an RNA is carried out by the usual methods (see for example Chem. Rev, 98, 961-990, 1998 by Oivanen M. et al).
• Metal complexes as described in the review by G. Pratviel et al, Adv. Qrg. Chem., 45, p251-312, 1998 or the review G. Pratviel et al, Angew. Chem. Int.
• the chemical fragmentation of RNA is carried out by metal cations associated or not with a chemical catalyst.
• the metal cations are Mg + , Sr 2 *, Ba 2+ , Pb 2+ , Zn 2+ , Cd 2+ , Mn 2+ , Fe 2+ , Co 2+ , Ni 2+ , Ru ions.
• the chemical catalyst consists of imidazole, a substituted analog, for example Nmethyl-imidazole, or any chemical molecule having an affinity for RNA and carrying an imidazole nucleus or a substituted analogue.
• the conditions for fragmentation using metals are well described in patent application WD-A-99/65926.
• the metals are M + , Mn 2+ , Zn 2+ , Tb 3+ or Ce 3+ , preferably Mg 2+ , Mn 2+ , Zn 2+ .
• Effective fragmentation conditions are obtained with a concentration of metal cation such as Mn ++ between 2 and 100 mM, a concentration of imidazole between 2 and 100 mM. Especially effective conditions are obtained with a cation concentration such as Mn ++ between 3 and 15 mM, and an imidazole concentration between 20 and 50 mM, in particular 30 mM.
• the pH of the reaction should be slightly basic.
• the pH is between 8.5 and 9, which represents a very interesting compromise for achieving the combination of labeling and fragmentation with RNA.
• the chemical fragmentation of RNA is carried out by the action of a polyamine, such as spermine, putescein or cadaverine.
• RNA Concentrations of 5 to 100 mM allow fragmentation. This is total from 10 mM polyamine.
• chemical fragmentation of RNA is carried out by the action of an artificial nuclease (see G. Pratviel et al, Adv. Inorg. Chem., 45, p251-312, 1998; DS Sigman et al Chem. Rev., 93, p2295-2316, 1993), such as 1,10-phenanthroline associated with a metal cation such as iron, copper or zinc. These cations come respectively from FeSO or CuCt or ' ZnQ. in solution. Concentrations between 2 and 50 mM of 1,10-phenanthroline are used for the fragmentation of RNA in particular between 4 and 10 mM.
• the chemical fragmentation of DNA is carried out by bringing the nucleic acid into contact with a chemical means for creating an abasic site.
• the formation of an abasic site results from the cleavage of the N-glycosidic hedge which he 2-deoxyribose sugar at the nucleic base.
• DNA incorporating uracyls The abasic site obtained by depurination or depyrimidation is very unstable. The fragmentation at this site is obtained at room temperature in basic miheu. In acid miheu, the high temperature also accelerates this fragmentation. The use of molecules capable of initiating the phenomenon of ⁇ -elimination also accelerates fragmentation.
• a preferred mode of reahsation of the fragmentation is obtained by the use of an acidic pH, that is to say a pH below 5.
• the pH is 3.
• a sodium formate buffer at pH 3 makes it possible to fragment effectively according to the invention. This buffer is compatible with the one-step marking conditions as will be demonstrated in the examples. Even more advantageously an acid miheu (HCl, carbonate, H 2 SO) is used.
• the deoxyribonucleic acid contains at least one modified base capable of generating an abasic site more easily
• modified bases can be used such as N7-alkyl purines, N3-alkyl purines, O6-alkyl purines, 8-bromopurines, 8-thiopurines, 8-alkylthiopurines, 8 azidopurines or 8-alkylsulfonylpurines.
• the use of an 8-bromopurine makes it possible to have effective incorporation during the amplification, which facilitates all the more the fragmentation and labeling process according to the invention, while retaining an exceptional sensitivity for the enzymatic amplification step.
• the present invention describes a labeled biological molecule and in particular a labeled nucleic acid, capable of being obtained by any of the methods according to the invention.
• the present invention also relates to a kit for detecting a biological molecule, in particular a target nucleic acid comprising a labeling reagent according to the invention.
• a kit for detecting a biological molecule in particular a target nucleic acid comprising a labeling reagent according to the invention.
• other elements such as, for example, lysis means (microorganisms and / or cells) and / or concentration means (such as sihce or magnetic particles) and / or means of enzymatic amplification are incorporated into the kit.
• the invention relates to the use of a labeled biological molecule, in particular a labeled nucleic acid as defined above, as a probe for detecting a target biological molecule, and in particular a target nucleic acid.
• the invention also relates to the use of a nucleic acid as defined above, as a labeled target which can be fixed on a capture probe.
• the labeled biological molecule is capable of forming a complex with the target biological molecule.
• the labeled nucleic acid is sufficiently complementary to the target to hybridize specifically according to the reaction conditions, and in particular the temperature or the salinity of the reaction medium.
• the detection method is suitable for sequencing, the expression profile of messenger RNAs or the screening of mutations for research purposes as well as the screening of drugs in the pharmaceutical industry, the diagnosis of infectious or genetic diseases, the control food or industrial.
• the trend in diagnostics and especially for infectious diseases is to lower the level of sensitivity, until the detection of a single molecule in a sample which can represent several milliliters in the case of a liquid sample such as blood or urine or cerebrospinal fluid.
• This level of sensitivity can only be obtained if all the steps from sampling the sample to rendering the result are optimized.
• the various means of the invention allow this optimization without difficulty because the reagents, methods and methods of the invention can be applied very widely to different biological molecules.
• a labeling and / or fragmentation process allows not to affect the sensitivity of the amplification technique, either because it is not no need to replace the deoxyribonucleotides or ribonucleotides used in the enzymatic amplification technique, either because the ribonucleotides or deoxyribonucleotides incorporated do not alter the sensitivity.
• the grafting chemistry described in the present invention has such characteristics, from the reactivity and specificity point of view, that other applications are described below: • In a first mode of reahsation, this grafting chemistry is applied to the fixation covalent of nucleic acids on a solid support.
• a precursor of the diazomethyl function such as a ketone or hydrazine as described above, is introduced during the chemical synthesis and the diazomethyl function is introduced on the nucleic acids in a second step.
• the diazomethyl functions are introduced on the sohde support and the nucleic acids are fixed on the sohde support by means of the phosphates of the nucleic acids and in particular of the terminal phosphates (5 ′ or 3 ′) .
• the introduction of phosphate at the 3 'or 5' end of nucleic acids is well known
• a labeling reagent carrying a hgand in particular a hapten such as biotin or abietane, is fixed on the sohde support on which is fixed covalently or by adsorption a anti-ligand, such as steptavidin or an antibody for example.
• a anti-ligand such as steptavidin or an antibody for example.
• the derivatives of formula (13), (14), (15) or the PDAM derivative are examples of reagents which can be used for the manufacture of such a solid support.
• the monoclonal antibody technique makes it possible to prepare antibodies against a large number of markers such as fluorescein or a derivative of Cy5.
• the skilled person can implement a sohde support with the labeling reagents of the present invention without undue difficulty by this indirect mode of preparation of the sohde support in which a hgand / anti-hgand reaction is used to fix the diazomethyl function on the sohde support.
• a second mode of reahsation of the solid support relates to particulate supports such as latexes.
• Different modes of polymerization can be used to prepare the particles carrying a diazomethyl function from a functional polymerizable monomer carrying either a diazomethyl function or preferably a precursor function of the diazomethyl function such as an aldehyde or a ketone and in particular: • Reactor polymerization closed called “batch”: the monomers are introduced into the reactor before the start of the reaction with the other ingredients and without subsequent addition. Due to the difference in reactivity of the monomers, this process often leads to the appearance of a drift in composition. This is manifested by obtaining macromolecules having compositions which vary considerably depending on the conversion.
• a third mode of reahsation of the sohde support consists in having a sohde support comprising a first reactive nucleophilic or electrophilic function, such as for example NH 2 , SH, OH, O ⁇ NH 2 , alkylketone, aldehyde, isocyanate, isothiocyanate, maleimide, alkyl halide, N-hydroxysuccinimide ester, tosylate, then reacting a fixing intermediate, comprising a reactive function complementary to the first reactive function of the support sohde.
• This reaction between the solid support and the fixing medium is carried out in the presence, optionally of a coupling agent to form a covalent hedge.
• Such a sohde support comprising at least one diazomethyl function is also an object of the present invention as well that the sohde support comprising nucleic acids attached to the sohde support via the diazomethyl functions.
• a first application of such a sohde support is the manufacture of DNA chips. Methods exist for distributing nucleic acids on the solid support in discrete and predetermined positions. US-A-6,110,426 proposes a method for producing these DNA chips using a capillary which is brought into contact on a solid surface to deliver a controlled volume of liquid.
• US-A-6,083,763 describes a set of capillaries sliding in a device so as to compensate for the differences in height of each of them. They are brought into contact with a flat surface for the deposition by capillarity of specific ohgonucleotides.
• US-A-6,083,762 proposes a drop distribution system comprising a microdispenser coupled to a piezoelectric transducer to eject drop volumes lower than the nanohtre on a solid surface. A similar result is obtained by applying a hot source to the wall of a capillary to form a nozzle which ejects a defined volume of solution (see T.
• the diazomethyl function thus makes it possible to graft the nucleic acids covalently onto the support.
• the grafting is simple, the hedge is stable, in particular with respect to adsorption and the selectivity of the reaction with respect to the terminal phosphate makes it possible to carry out an oriented coupling of the nucleic acid on the sohde support, which facilitates all the more the subsequent hybridization steps by reducing steric hindrance.
• a second application of a sohde support according to the invention is the purification of nucleic acids.
• this purification is either direct (the sohde support carrying diazomethyl functions reacts with the nucleic acids to be purified) or indirect (capture nucleic acids are fixed on the sohde support).
• These capture nucleic acids are sufficiently complementary to the target to be captured to hybridize with the degree of specificity desired and it is the “capture nucleic acids / sohde support” complex which allows the purification of the target nucleic acids.
• the solid support is preferably in dispersed form for use in purification, such as latex particles, for example magnetic particles.
• purification step is meant in particular the separation between the nucleic acids of the microorganisms and the cellular constituents released in the lysis step which precedes the purification of the nucleic acids.
• lysis steps are well known by way of indicative example, it is possible to use the lysis methods as described in the patent applications:
• lysis methods such as thermal or osmotic shocks or treatments with chaotropic agents, such as guanidium salts (US Pat. No. 5,234,809).
• This step generally makes it possible to concentrate the nucleic acids.
• magnetic particles see on this subject the patents US-A-4,672,040 and US-A-5,750,338, and thus to purify the nucleic acids, which are fixed on these magnetic particles, by a washing step.
• This nucleic acid purification step is particularly interesting if it is desired to subsequently amplify said nucleic acids.
• a particularly interesting mode of reahsation of these magnetic particles is described in patent applications WO-A-97/45202 and WO-A-99/35500.
• sihde support includes all materials to which a nucleic acid can be attached. Synthetic materials or natural materials, optionally chemically modified can be used as a solid support, in particular polysaccharides, such as materials based on cehulose, for example paper, derivatives of cehulose such as acetate of cehulose and nitrocellulose, or dextran; polymers, copolymers, in particular based on monomers of the styrene type, natural fibers such as cotton, and synthetic fibers tehes than nylon; mineral materials such as silicon, quartz, glasses, ceramics; latexes; magnetic particles; metal derivatives, gels, etc.
• the solid support can be in the form of a microtiter plate, a membrane, a particle or a substantially flat plate of glass or silicon or derivatives.
• the invention relates to a method for capturing nucleic acids comprising the following steps: • there is a solid support on which is fixed directly or indirectly at least one molecule comprising a diazomethyl function, • a biological sample is brought into contact likely to contain hbre nucleic acids, and • the sohde support is washed where the molecule (s) are (are) covalently attached to at least one nucleic acid. Additional information can be found in another patent application of the Applicant, WO02 / 090584, filed under priority of May 4, 2001.
• FIG. 1 represents the developed formulas of various reagents used in the present invention as well as the abbreviation designating them (o- signifies ortho, m-meta and p-para).
• FIG. 2 represents the mean value of the signal and the percentage similarity of the m bio-TETA-PMDAM as a function of its concentration for rpoB.
• Biotin meta-acetophenone compound The D-biotin (1.0 gram (g), 4.1 milhmol (mmol)) is dissolved in 45 milliliters (mL) of hot anhydrous DMF. Cool to 0 ° C under argon, then add N-methylmorpholine (590 microliters ( ⁇ L), 5.33 mmol) and isobutyl chloroformate (840 ⁇ L, 6.60 mmol) successively. The mixture is left stirring for 30 minutes (min), then the 3-aminoacetophenone (824 mg, 6.10 mmol) and the N-methylmorpholine (480 ⁇ L, 4.35 mmol) are added in 10 ml of DMF.
• N-methylmorpholine 590 microliters ( ⁇ L), 5.33 mmol
• isobutyl chloroformate 840 ⁇ L, 6.60 mmol
• the D-biotin (2.80 g, 11.40 mmol) is dissolved in 30 ml of anhydrous DMF.
• the addition of carbonyldiimidazole (1.5 eq .; 2.78 g) causes a precipitate to form after a few minutes.
• the product is purified by flash chromatography on sihce gel with as eluent
• ACBA (2) (1.03 g; 4.39 mmol) is dissolved in 20 ml of anhydrous DMF under argon. The miheu is cooled in ice, and N-methylmorpholine (1.25 eq .; 725 ⁇ L) and isobutyl chloroformate (1 eq .; 690 ⁇ L) are successively added: the miheu becomes cloudy after 30 min.
• Bio-TETA (1) 0.8 eq.; 1.94 g
• Ehe is added to the activated ACBA at 0 ° C, for 30 min. The mixture is then left at room temperature overnight.
• Bio- (TETA) -Hy (4) (150 mg; 250.4 ⁇ mol) is dissolved in 1 mL of anhydrous DMSO under argon. It is left to react for 30 minutes with Mn ⁇ 2 (s) (15 eq.; 330 mg) and then filtered on frit No. 4 with celite (0.5 cm thick) and 3 A molecular sieve (0.5 cm thick). 100 ⁇ L are used for NMR with addition of 380 ⁇ L of DMSO-d 6 and 20 ⁇ L of The final volume is adjusted to 4.5 ml with anhydrous DMSO and 4% methanol. We stun in a glove box under Argon by 250 ⁇ L. The compound is fuchsia pink.
• the degree of purity (diazomethyl content) is checked by 1 H NMR and UN-vis spectrophotometry (absorbance peak of diazomethyl at 516 nm).
• RM ⁇ -1H (200 MHz, DMSO- d 6 ) ⁇ 9.93 (s, 1H, Ph-NH-CO-); 7.3 (s, 3H, EL TM - *) * ",); 6.6 (s, IH,; 6.4 (s, IH, -NH- biot); 6.3 (s, IH, -NH- biot); 4.3 (t, IH, -CH- biot) ; 3.3 (m, 12H, - ⁇ H2-NH-); 3.3 (m, 1H, -CH-S-); 2.9 (dd, 2H, -CH 2 -S-); 2.5 (4H, -CH 2 -CO-); 2.1 (s, 3H, -CH 3 ); 2.0 (m, 2H, -CH 2 -NH-CH 2 -
• DNA amphcons are generated by PCR from Mycobacterium tuberculosis 16S genomic DNA targets (10 +4 copies as starting targets) using the Roche Fast Start kit, 0.2 mM of each deoxyribonucleotide (d-ATP, d -CTP, d-GTP, d-TTP), 0.3 ⁇ M primers and 0.4 ⁇ L of enzyme.
• the parameters of the PCR are as follows:
• the transcripts are reacted from a PCR target (fragment of the 16S RNA of Mycobacterium tuberculosis) using the MEGAscript kit from Ambion: 7.5 mM of each nucleotide (ATP, CTP, GTP and UTP) and 2 ⁇ L of enzyme (RNA polymerase). The incubation time is 3 hours (h) at 37 ° C.
• the PCR amplification primers carry a T3 or T7 polymerase promoter, as described in application WO-A-99/65926 or in the article J. Clin Microbiol. 37 (1), p 49-55, 1999, which allows for transcription.
• the transcripts are analyzed by electrophoresis on agarose gel (1.5%; TBE 0.5X). The volume deposited is 5 ⁇ L and the migration takes place for 20 min at 100V. The transcripts are visualized under UV lamp after staining with ethidium bromide. Identical results from the point of view of the invention can be obtained using other amplification techniques such as NASBA or TMA, which directly generate RNA amphcons.

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  • 2004-03-26 FR FR0450600A patent/FR2868071B1/fr not_active Expired - Fee Related
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