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WO2024110392A1 - Nouveaux composés de liaison à l'acide nucléique et leurs utilisations - Google Patents

Nouveaux composés de liaison à l'acide nucléique et leurs utilisations Download PDF

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Publication number
WO2024110392A1
WO2024110392A1 PCT/EP2023/082402 EP2023082402W WO2024110392A1 WO 2024110392 A1 WO2024110392 A1 WO 2024110392A1 EP 2023082402 W EP2023082402 W EP 2023082402W WO 2024110392 A1 WO2024110392 A1 WO 2024110392A1
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nucleic acid
preferentially
trialkylammonium
alkyl
target nucleic
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Inventor
Alain Laurent
Arnaud Burr
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Biomerieux SA
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Biomerieux SA
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Priority to EP23809218.3A priority Critical patent/EP4622974A1/fr
Priority to KR1020257019482A priority patent/KR20250107910A/ko
Priority to AU2023386451A priority patent/AU2023386451A1/en
Priority to CA3270929A priority patent/CA3270929A1/fr
Priority to CN202380080301.7A priority patent/CN120265628A/zh
Publication of WO2024110392A1 publication Critical patent/WO2024110392A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/101—Temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/113—PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence

Definitions

  • the invention is related to new compounds that can bind to nucleic acids, and in particular to DNA single strand (ssDNA) or DNA double strand (dsDNA).
  • This binding corresponds to the binding of the compound to the acid nucleic structure, illustratively by weak bonds, in particular hydrogen or ionic bonds or may correspond to any other binding by weak bonds.
  • the compounds according to the invention exhibit a change in fluorescence resulting from the binding of said compounds to the nucleic acids, typically ssDNA or dsDNA.
  • Non-specific DNA binding dyes can be used to detect and quantify DNA and RNA in a variety of environments, including solutions, cell extracts, electrophoretic gels, micro-array chips, live or fixed cells, dead cells, and environmental samples.
  • These compounds have also been used in real-time polymerase chain reaction (real-time PCR or rt-PCR), a common gene detection technique widely used in both research and diagnostics.
  • real-time PCR or rt-PCR real-time polymerase chain reaction
  • melt curve analysis a post-PCR DNA analysis technique. The melt curve analysis is specific and allows the identification of the gene, and so is useful for identifying gene mutation detection.
  • thiols can be present during PCR and some compounds described in these patents are poorly stable in the presence of thiols. Additionally, unlike other compounds described in US 7,387,887, the production of example A10 of US 7,387,887 cannot be carried out by the described process, which exclusively starts from ureas or thioureas.
  • WO 2008/052742 in the name of Roche Diagnostics Gmbh proposes a new class of fluorescent dyes which are capable of emitting fluorescence when they are excited appropriately in the status of being specifically bound to a double stranded nucleic acid.
  • This new class of fluorescent dyes comprises a pyrimidinyl ring in which the 5- and 6- positions of the pyrimidinyl ring are an integral part of a further aromatic structure, forming in particular a quinazolinyl structure.
  • This part of the fluorescent dyes is presented as mandatory in this patent application WO 2008/052742, at the end of page 6, in order to obtain interesting excitation and emission spectra.
  • the proposed compounds have improved thermal and chemical stability, but the only stability study concerns the photochemical stability.
  • this fused aromatic structure greatly influences the stability and the fluorescence of the molecules.
  • thiazole orange and SybrGreen which include a quinolinyl, and so have a structure close to the ones described in WO 2008/052742, are not satisfactorily stable, both in PCR conditions and during storage in aqueous and slightly alkaline medium.
  • the inventors propose new structures which do not include a pyrimidine forming an integral part of a further aromatic structure.
  • BIOTIUM describes numerous molecules including fluorescent nucleic acid dyes, and methods for use that includes nucleic acid detection, nucleic acid amplification reactions, and high-resolution melt curve analysis. A very broad formula is proposed for these molecules, which includes a large variety of structures.
  • the substituents of the proposed structures may comprise a positively charged moiety, which has the effect of enhancing the nucleic acid binding affinity of the molecule via electrostatic interaction between the negatively charged phosphate backbone of nucleic acid and the positive charge of the moiety.
  • This positively charged moiety may be covalently attached to the molecule by various kinds of arms starting from an N, O, S, or C atom (see the definition of L 1 and L 2 in column 15 of US 9,682,970).
  • this type of molecules including a pyrimidinium group, except molecules carrying a guanidino group, which is highly basic.
  • An example is the following one: There is no description of a method of preparation of this compound which, as a result, is not available to the public.
  • guanidino and amidino make them superior alternative to an amine side chain in the context of nucleic acid dyes”.
  • Several compounds belonging to the class of non-specific DNA binding dyes and instruments for DNA detection and/or analysis are commercially available: thiazole orange, SYBR® Green I, LCGreen® Plus (Clinical Chemistry 52:3, 2006, Mark G. Herrmann et al. “Amplicon DNA melting Analysis for Mutation Scanning and Genotyping: Cross-Platform Comparison of Instruments and Dye”) and LightCycler® 480 High Resolution Melting Dye (04909640001, Roche).
  • an object of the invention is to provide new nucleic acid binding compounds, and more specifically DNA binding compounds (and in particular dsDNA binding compounds), which exhibit, in their form bound to nucleic acid (typically DNA, and in particular dsDNA), enhanced and detectable fluorescence.
  • the purpose of the invention is to propose such new compounds with properties globally improved.
  • the purpose of the invention is to provide compounds with both properties of stability and fluorescence, which are suitable for application in nucleic acid detection, and more particularly DNA detection.
  • the compounds according to the invention have a great stability at pH 9, in a test performed at 40°C (which is an accelerated test relevant for evaluation of the stability at ambient temperature). Additionally, other results show their stability in presence of thiols in an aqueous medium. With their particularly suitable stability and fluorescence stability, the compounds according to the invention lead to high performances in nucleic acid detection, nucleic acid amplification reactions, and high-resolution melt curve analysis. Additionally, they can be formulated and stored in aqueous media and this facilitates their quick implementation in the targeted nucleic acid detection methods, typically with the use of PCR techniques.
  • - n is equal to 0, 1, 2 or 3; - Ri, Rj and Rk are identical or different and are independently selected from the group consisting of hydrogen and C 1-6 alkyl; - X is oxygen, sulfur, selenium, tellurium or C(CH 3 ) 2 , - Re is an alkyl, or a piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group bonded to the rest of the molecule by one of its carbon atom, or Re is – (CH 2 ) k1 -Y 1 , in which: o k1 is 1, 2, 3, 4, 5 or 6, and o Y 1 is a group selected from among hydroxy, C 1-6 alkoxy, amino, alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups and the groups -[N
  • the compounds of the invention are nucleic acid binding compounds.
  • the compounds according to the invention have a specific core structure which carries one or several substituents that include a function selected from among secondary amines, tertiary amines and quaternary ammoniums.
  • Secondary amines, tertiary amines and quaternary ammoniums comprise or may comprise a positively charged moiety, which has the effect to provide binding affinity site for the nucleic acid. Indeed, electrostatic interaction may occur between the negatively charged phosphate backbone of nucleic acid and the positive charge of the functions selected from among secondary amines, tertiary amines and quaternary ammoniums.
  • the positively charged functions may be a protonated amine (i.e.
  • Secondary and tertiary amines are bases whose basicity increases from secondary to tertiary. So, the fraction of their protonated form differs and increases from primary to tertiary and is a function of the pH of the medium where they are located. In targeted applications, illustratively, the pH will be in the range from 7.5-9.5, typically from 8- 9, and there will be a protonated fraction, in any case.
  • a quaternary ammonium, typically a trialkylammonium is a fully and permanently positively charged moiety independent of the pH of the medium.
  • At least two (and in particular two) of the substituents Ra, Rb, Rc, Rd, R 2 , R 3 and R 4 include a secondary amine, a tertiary amine or a quaternary ammonium; and in particular at least two (typically two) substituents Ra, Rb, Rc, Rd, R 2 , R 3 and R 4 include a quaternary ammonium, such as a trialkylammonium, typically a trimethylammonium.
  • Rc and R 2 are the two substituents that include a quaternary ammonium, such as a trialkylammonium, typically a trimethylammonium.
  • the compounds according to the invention include one of the following features or any combination of the following features, and advantageously, when they do not exclude each other, all the following features: - at least one group Y 2 , or Y 4 , and in particular only one group Y 2 or Y 4 or the two groups Y 2 and Y 4 , include(s) or is(are) a trialkylammonium, typically a trimethylammonium; - k2 is 3, 4, 5 or 6, and typically 3; - k3 is 2, 3, 4, 5 or 6; typically k3 is 2 or 3; - k4 is 4, 5 or 6, and typically 4, when Y 4 is a dialkylamino, a trialkylammonium, or -[N + R 4 ’R 4 ’’-(CH 2 ) p4 -] m4 -G 4 ’ as defined for formula (I); - Ri, Rj and Rk are hydrogen; -
  • the compounds of the invention have the formula (III): (III), wherein R 1 , R 2 , R 3 , R 4 , Ra, Rb, Rc, Rd and Re are as defined for formula (I) or (II); including their salts with at least one anion, in particular, chosen among halogenated anions, typically Cl-, Br- and I- ; trifluoroacetate, acetate, formate ; sulfonates, such as methylsulfonate, trifluoromethylsulfonate and tosylate ; sulfates, such as methylsulfate ; phosphate, pyrophosphate and triphosphate.
  • R 1 , R 2 , R 3 , R 4 , Ra, Rb, Rc, Rd and Re are as defined for formula (I) or (II); including their salts with at least one anion, in particular, chosen among halogenated anions, typically Cl-, Br- and I- ; trifluoroacetate
  • At least one (and typically one) of the groups R 2 and R 3 is –(CH 2 ) k4 -Y 4 , in which: o k4 is 1, 2, 3, 4, 5 or 6 and o Y 4 is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N + R 4 ’R 4 ’’-(CH 2 ) p4 -] m4 -G 4 ’, with m4 being 1, 2 or 3, G 4 ’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p4 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R 4 ’ and R 4 ’’’, identical or different, being a C
  • R 2 is –(CH 2 ) k4 -Y 4 as described above and R 3 is a C 1-6 alkyl, typically a methyl group.
  • Rc is a hydrogen atom, an halogen atom, in particular Br, - NHC(O)alkyl, in particular -NHCOMe, –NHCOR or –CONHR, with R being –(CH 2 ) k2 -Y 2 , in which: o k2 is 3, 4, 5 or 6; in particular k2 is 3; o Y 2 is a trialkylammonium or -[N + R 2 ’R 2 ’’-(CH 2 ) p2 -] m2 -G 2 ’, with m2 being 1, G 2 ’ being a trialkylammonium, p2 being 1, 2, 3, 4, 5 or 6, preferentially 2 or 3 and R 2 ’ and R 2 ’’’, identical or different, being a C 1-6 alkyl, preferentially a methyl or an ethyl, Y 2 being preferentially a
  • R 4 H
  • R 1 and R 3 are identical or different and are C 1-6 alkyl, and in particular methyl or ethyl
  • R 2 is –(CH 2 ) k4 -Y 4 , in which k4 is 4, 5 or 6 and Y 4 is a trialkylammonium, in particular trimethylammonium
  • Rc is hydrogen or – NHCOR or –CONHR, with R being –(CH 2 ) k2 -Y 2 , in which k2 is 3, 4, 5 or 6 and Y 2 is a trialkylammonium, in particular a trimethylammonium, or -[N + R 2 ’R 2 ’’-(CH 2 ) p2 -] m2 -G 2 ’, with m2 being 1, G’ 2 being a trialkylammonium, in particular a trimethylammonium, p2 being 2 or 3,
  • R 4 H
  • R 1 and R 3 are identical or different and are C 1-6 alkyl, and in particular methyl or ethyl
  • R 2 is hydrogen, C 1-6 alkyl, and in particular methyl or ethyl, or –(CH 2 ) k4 -Y 4 , in which k4 is 4, 5 or 6 and Y 4 is a trialkylammonium, in particular trimethylammonium and Rc is –NHCOR or –CONHR, with R being –(CH 2 ) k2 -Y 2 , in which k2 is 3, 4, 5 or 6 and Y 2 is a trialkylammonium, in particular a trimethylammonium, or -[N + R 2 ’R 2 ’’-(CH 2 ) p2 -] m2 -G 2 ’, with m2 being 1, G’ 2 being a trialkylammonium, in particular a trimethylammonium, p
  • R 4 H
  • R 1 and R 3 are identical or different and are C 1-6 alkyl, and in particular methyl or ethyl
  • R 2 is –(CH 2 ) k4 -Y 4 , in which k4 is 1, 2, 3, 4, 5 or 6 (typically k4 is 4, 5 or 6) and Y 4 is an alkylamino, dialkylamino, trialkylammonium, piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl or morpholinyl group or -[N + R 4 ’R 4 ’’-(CH 2 ) p4 -] m4 -G 4 ’, with m4 being 1, 2 or 3, G 4 ’ being H or an amino, alkylamino, dialkylamino or trialkylammonium, p4 being 1, 2, 3, 4, 5 or 6, preferentially
  • R 4 H
  • R 1 is methyl or ethyl
  • R 3 is methyl
  • the secondary amine, tertiary amine or quaternary ammonium may correspond to the following groups in the compounds of formula (I), (II), (IIa), (III) and (IIIa): piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl, morpholinyl, alkylamino, dialkylamino and trialkylammonium groups, including the ones present in the groups -[N + R 2 ’R 2 ’’-(CH 2 ) p2 -] m2 -G 2 ’ and -[N + R 4 ’R 4 ’’-(CH 2 ) p4 -] m4 -G 4 ’, as defined in the
  • Re is typically C 1-6 alkyl, in particular methyl.
  • this quaternary ammonium is trimethylammonium.
  • R 2 is –(CH 2 ) k4 -Y 4 , in which k4 is 4, 5 or 6 and Y 4 is a trialkylammonium, in particular trimethylammonium.
  • Rc is –NHCOR or –CONHR, with R being –(CH 2 ) k2 -Y 2 , in which k2 is 3, 4, 5 or 6 and Y 2 is a trialkylammonium, in particular a trimethylammonium, or -[N + R 2 ’R 2 ’’-(CH 2 ) p2 - ] m2 -G 2 ’, with m2 being 1, G’ 2 being a trialkylammonium, in particular a trimethylammonium, p2 being 2 or 3, and R 2 ’ and R 2 ’’, identical or different, being a C 1-6 alkyl, typically a methyl.
  • the compounds described in the invention include at least one quaternary ammonium, preferentially a trialkylammonium, typically a trimethylammonium. According to specific embodiments of all the compounds described in the invention, they are in the form of a trifluoroacetate salt.
  • Rc is H, an halogen atom, typically Br, or –NHCOalkyl, typically –NHCOMe, and R 2 is –(CH 2 ) k4 -Y 4 , in which k4 is 4, 5 or 6 and Y 4 is a trialkylammonium, in particular trimethylammonium, typically R 2 is – (CH 2 ) 4 -N + Me 3 ; or, ii) R 2 is an alkyl, typically methyl, and Rc is –NHCOR or –CONHR, with R being –(CH 2 ) k2 -Y 2 , in which k2 is 3, 4, 5 or 6 and Y 2 is
  • halogenated anions typically Cl-, Br- and I-
  • trifluoroacetate, acetate, formate sulfonates, such as methylsulfonate, trifluoromethylsulfonate and tosylate
  • sulfates such as methylsulfate
  • phosphate, pyrophosphate and triphosphate in particular their trifluoroacetate salt.
  • the invention also concerns the uses of these compounds in acid nucleic detection and analysis and corresponding methods, mixtures and kits, as defined in the section “Uses of the compounds according to the invention”.
  • “compound” means a compound of any formula given previously in the disclosure of the compounds or a salt of this compound.
  • compounds of formula (I), (II), (IIa), (III), (IIIa) or of any other formula given in the specification falling in the scope of formula (I) is used in the form of a salt as described in the specification, in the uses, methods, mixtures and kits of the invention.
  • the use of a compound according to this invention, for the detection of a target nucleic acid which is a single stranded or double stranded nucleic acid is another object of the invention.
  • the invention is also relating to a method for detecting a target nucleic acid which is a single stranded or double stranded nucleic acid comprising a step of mixing a compound in accordance with the invention, with a sample comprising the target nucleic acid or an amplicon of the target nucleic acid.
  • the following steps may be carried out: - amplifying the target nucleic acid to generate the amplicon, - adding a compound according to the invention to the sample comprising the target nucleic acid and/or the amplicon, before, during or after the amplifying step, - monitoring fluorescence from the compound according to the invention during or subsequent to the amplifying step.
  • the following steps are carried out: - amplifying the target nucleic acid, in the presence of the compound according to the invention, in particular by PCR, to generate the amplicon, and - during the amplification, monitoring the fluorescence of the compound according to the invention, resulting from the binding of the compound to the amplicon.
  • a step of melting the generated amplicon is carried out, while monitoring the fluorescence from the compound according to the invention, to obtain a melting curve. It is possible to obtain a melting curve when the target nucleic acid is a double stranded nucleic acid, in particular a dsDNA.
  • a melting curve can be obtained with any double stranded nucleic acid that can melt and that is capable of hybridization to a complementary nucleic acid by Watson-Crick base pairing, such as DNA, DNA-RNA hybrid, which could also include nucleotide analogs (e.g. BrdU) and/or non-phosphodiester internucleosidic linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages).
  • the use and the method according to the invention may include a step of quantifying the target nucleic acid, which is initially present in the sample.
  • the method for detecting a target nucleic acid comprises a step of mixing a compound in accordance with the invention, with a sample comprising an amplicon of the target nucleic acid
  • the quantity of target nucleic acid initially present in the sample obtained by the step of quantifying is the quantity of the target nucleic acid which is present in the sample used for obtaining the amplicon, by an amplifying step.
  • the invention also concerns a method of PCR analysis of a target nucleic acid comprising the steps of: - mixing a compound according to the invention with a sample comprising a target nucleic acid, a polymerase, and a pair of primers suitable to amplify a portion of the target nucleic acid and generate at least an amplicon, leading to a PCR mixture, - amplifying the target nucleic acid from the PCR mixture and generating at least an amplicon, and - monitoring the fluorescence from the compound according to the invention during or subsequent to the amplifying step.
  • a method of PCR analysis further comprises detecting the presence of the amplicon from the monitored fluorescence.
  • the monitoring step may occur subsequent to amplification and includes generating a melting curve.
  • the said melting curve is used to identify the genotype of the target nucleic acid, to detect or identify at least one mutation, polymorphism, preferentially single nucleotide polymorphism, and/or epigenetic variation.
  • a method of PCR analysis according to the invention may include a step of quantifying the target nucleic acid, which is initially present in the sample.
  • a method of PCR analysis of a target nucleic acid according to the invention may comprise the steps of: - mixing a compound according to the invention with a sample comprising a target nucleic acid and at least a pair of primers suitable to amplify a portion of the target nucleic acid and generate an amplicon, leading to a PCR mixture, - amplifying the target nucleic acid from the PCR mixture, and generating at least an amplicon, - during the amplifying step, monitoring the fluorescence of the compound according to the invention resulting from the binding of the compound to the amplicon, - at the end of the amplifying step, melting the generated amplicon, to obtain a melting curve, and - identifying the genotype or polymorphism of the target nucleic acid using a shape of the melting curve.
  • the amplifying step may include a plurality of temperature cycles including at least a denaturation temperature and an extension temperature, wherein each cycle has a cycle time of less than 90 seconds per cycle, and wherein the polymerase is provided at a concentration of at least 0.005 ⁇ M or 0.02 U/ ⁇ L and primers are each provided at a concentration of at least 0.1 ⁇ M.
  • the amplifying step includes a plurality of temperature cycles including at least a denaturation temperature and an extension temperature, wherein each cycle has a cycle time of less than 20 seconds per cycle, and wherein the polymerase is provided at a concentration of at least 0.5 ⁇ M or 1.9 U/ ⁇ L and primers are each provided at a concentration of at least 2 ⁇ M.
  • Another object of the invention is a PCR reaction mixture comprising: - a target nucleic acid, - a pair of primers suitable to amplify a portion of the target nucleic acid, to generate an amplicon, - a polymerase, in particular a thermostable polymerase, - a compound in accordance with the invention.
  • the PCR reaction mixture according to the invention may be in a buffer of pH from 7.5 to 9.5, preferentially from 8 to 9.
  • Another object of the invention is a kit for detecting a target nucleic acid, comprising: - a pair of primers suitable to amplify a portion of the target nucleic acid, to generate an amplicon, - a polymerase, in particular a thermostable polymerase, and - a compound in accordance with the invention.
  • the kit according to the invention typically, includes a buffer of pH from 7.5 to 9.5, preferentially from 8 to 9.
  • the compound in accordance with the invention is provided in the buffer.
  • alkyl refers to a monovalent saturated hydrocarbon moiety comprising from 1 to about 12 carbon atoms, typically 1 to 6 carbon atoms.
  • An alkyl group may be linear or branched and, illustratively includes methyl (Me), ethyl, propyl, butyl, dodecyl, 4-ethylpentyl, and the like.
  • C 1-6 alkyl refers to alkyl comprising 1, 2, 3, 4, 5 or 6 carbon atoms and typically to methyl.
  • alkenyl refers to monovalent hydrocarbon moieties comprising from 1 to about 12 carbon atoms, typically 1 to 6 carbon atoms, which contain at least one carbon-carbon double bond, wherein each double bond can have E- or Z-configuration.
  • alkynyl refers to monovalent hydrocarbon moieties comprising from 1 to about 12 carbon atoms, typically 1 to 6 carbon atoms, which contain at least one carbon-carbon triple bond.
  • the alkenyl and alkynyl groups can be linear or branched. Double bonds and triple bonds in alkenyl groups and alkynyl groups respectively can be present in any positions.
  • alkenyl and alkynyl examples are ethenyl, prop-1-enyl, prop-2-enyl, but-2-enyl, 2- methylprop-2-enyl, 3-methylbut-2-enyl, hex-3-enyl, hex-4-enyl, prop-2-ynyl, but-2- ynyl, but-3-ynyl, hex-4-ynyl or hex-5-ynyl.
  • aryl or aromatic cyclic ring (that can be mono or polycyclic and which are fused in the definition of Z) as used in the invention refers to a cyclic aromatic hydrocarbonated moiety, illustratively including but not limited to phenyl (Ph) and naphthyl. Phenyl is the illustrative aryl group and in Z is named as fused-benzo.
  • Nitrogen containing aromatic ring used in the definition of Z refers to pyrrolo, pyrazolo, isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, quinoxalinyl, quinazalinyl, and the like.
  • Z typically when fused with the other cycle ring presented in formula (I), Z can form an optionally substituted benzoxazolium or benzothiazolium ring, or an optionally substituted naphthoxazolium or naphthothiazolium ring.
  • Amino means —NH 2 .
  • Alkylamino means –NHR’ with R’ being an alkyl, in particular a C 1-6 alkyl, typically methyl or ethyl. So, alkylamino corresponds to a secondary amine.
  • Dialkylamino means –NR’R” with R’ and R” being, independently, an alkyl, in particular a C 1-6 alkyl, typically methyl or ethyl.
  • dialkylamino corresponds to a tertiary amine.
  • the most common dialkylamino groups illustrated herein are –NMe 2 and -NEt 2 .
  • Trialkylammonium means –N + R’R”R”’ with R’, R” and R’” being, independently, an alkyl, in particular a C 1-6 alkyl, typically methyl or ethyl.
  • the most common dialkylamino groups illustrated herein are –N + Me 3 and -N + Et 3 .
  • piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups can be covalently bonded to the rest of the molecules by one of their carbon atoms or by their nitrogen atom.
  • the piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups include the unsubstituted corresponding groups and the substituted corresponding groups where the nitrogen atom of the piperidinyl, piperazinyl, pyrrolidinyl or morpholinyl group is substituted in the N-position by one or two C 1-6 alkyl group(s) (typically a methyl or an ethyl), and the nitrogen atom of the pyridinyl group is substituted in the N-position by one C 1-6 alkyl group (typically a methyl or an ethyl) and is then in an ammonium form.
  • the piperidinyl, piperazinyl, pyridinyl, pyrrolidinyl and morpholinyl groups include the unsubstituted corresponding groups and the substituted corresponding groups where the nitrogen atom of the piperidinyl, piperazinyl, pyrrolidinyl or morpholinyl group is substituted in the N-position by one C 1-6 alkyl group (typically a methyl or an ethyl) and so could be in an ammonium form.
  • the compounds of the invention are cyanine derivatives, having a pyrimidinium core structure, wherein X is, in particular, oxygen or sulfur and the moiety Z represents an optionally-substituted fused benzo, forming an optionally substituted benzoxazolium or benzothiazolium ring, or an optionally-substituted fused naphtho, forming an optionally substituted naphthoxazolium or naphthothiazolium ring. It is appreciated that the compounds of formula (I), (II), (IIa), (III) or (IIIa) and their salts described herein may contain one or several chiral centers.
  • stereoisomers are understood to be included in the description of these compounds, unless otherwise indicated. Such stereoisomers include pure enantiomers, racemic mixtures, mixtures of enantiomers in any relative amount, pure diastereoisomers and mixtures of diastereoisomers containing any relative amount of one or more stereoisomeric configurations. It is also appreciated that the compounds of formula (I), (II), (IIa), (III) or (IIIa) and their salts herein may contain geometric centers. In those cases, all geometric isomers are understood to be included in the description of the compounds of formula (I), (II), (IIa), (III) or (IIIa) and their salts, unless otherwise indicated.
  • Such geometric isomers include cis, trans, E and Z isomers, either in pure form or in various mixtures of geometric configurations. It is also understood that depending upon the nature of the double bond contained in the compounds of formula (I), (II), (IIa), (III) or (IIIa) and their salts, such double bond isomers may interconvert between cis and trans, or between E and Z configurations depending upon the conditions, such as solvent composition, solvent polarity, ionic strength, and the like. So, the two forms cis/trans or E/Z are most of the time, in equilibrium.
  • the compounds according to the invention are in the form of a salt: the compound of formula (I), (II), (IIa), (III) or (IIIa) are positively charged due to the N + (Re). They may include an additional charge when they include a quaternary ammonium group.
  • the salts of the compounds of formula (I), (II), (IIa), (III) or (IIIa) include a number of anions (typically which are identical) corresponding to the number of positive charges on the compound of formula (I), (II), (IIa), (III) or (IIIa).
  • a positive charge may be formally localized on the nitrogen atom N + (Re) as depicted in Formula (I), (II), IIa), (III) and (IIIa), or alternatively, the charge may be localized on the pyrimidinyl group.
  • Nucleic acid refers to a naturally occuring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single- stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing.
  • Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non-phosphodiester internucleosidic linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages).
  • nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.
  • the pyrimidine molecule (VII) is alkylated at the N1 position by reaction with an excess of an alkylating agent (VI) (P-R 1 , with P being Cl, Br or I or a tosyl group and R 1 as defined for (I)), typically in acetonitrile at 50-90°C in a closed tube for 1-6 days to give the pyrimidinium compound (V).
  • VIP alkylating agent
  • the compound (V) is reacted with a compound (IV) (typically a benzothiazolium derivative), typically in a mixture of acetonitrile and ethanol and triethylamine at room temperature (typically 22°C), for a few minutes to yield the expected asymmetric cyanine (I), which may be purified by reverse phase chromatography.
  • a compound (IV) typically a benzothiazolium derivative
  • the compounds (IX), (VIII) and (VI) are commercially available or prepared according to common practice from commercially available compounds.
  • the compounds (IV) can also be commercially available.
  • the compounds (IV) are not commercially available, in particular, when they are substituted on the Z cycle, they can be prepared as described hereafter on Scheme 2 (routes 1 to 3 concerning the synthesis of certain compounds of formula (IV)), or according to methods similar to the described ones.
  • Scheme 2 concerns compounds (IV) where Z is a fused-benzo and Re is Me and describes different ways to introduce a substituent Rc, on this fused-benzo. In route 1, Rc is –CONH-(CH 2 ) 3 -N + (Me) 3 .
  • Rc is — NHCO-(CH 2 ) 3 -N + (Me) 3 .
  • Rc is —CONH-(CH 2 ) 3 -N + (Me) 2 -(CH 2 ) 3 -N + (Me) 3 .
  • route 1 commercial ester 4 is hydrolyzed in alkaline conditions to yield the carboxylic acid 5 which is activated under the form of a hydroxysuccinimidyl ester 6.
  • This hydroxysuccinimidyl ester 6 can be conveniently substituted by an amine, such as amine 7.
  • Peralkylation of the nitrogen atoms in the alkyl chain and in the benzothiazole ring are, advantageously, done at the same time by an alkylating agent at elevated temperature to yield expected products VI.1.
  • Route 3 is similar to route 1, but with the introduction of a more complex group Rc including two ammonium group (with one -N + (Me) 3 and –N(Me)+ 2 - in the illustrative example).
  • the commercial amino carboxylic acid 11 is activated under the form of a hydroxysuccinimidyl ester 12 and conjugated to the amino benzothiazole 13, before peralkylation of the amino atoms in the alkyl chain and in the benzothiazole ring as described in route 1 or 3 to obtain the compound VI.2.
  • Other compounds of formula (IV) can easily be prepared with other routes, adapted or close to routes 1 to 3. Depending on the wished compound, the skilled person will choose the more appropriate step to be conducted.
  • the obtained activated compounds (IX) can then react with a masked aldehyde as bis phenyl imines (XII), to yield in the presence of acetic anhydride and acetic acid, or only by fusing, the corresponding acetylated hemicyanines (XIII) which can be purified by reverse phase chromatography using a acetonitrile/water/TFA eluents.
  • the hemicyanine (XIII) can then be reacted in slightly alkaline conditions with activated pyrimidinium (V.1), to obtain the expected compounds (I).
  • the compounds according to the invention may be used for the detection of a target nucleic acid which is a single stranded or double stranded nucleic acid.
  • the compounds according to the invention have the ability to bind to nucleic acids, and in particular to double strand nucleic acids, typically to single strand DNA (ssDNA) or double strand DNA (dsDNA), typically when they are in aqueous media of pH 5-11, and as a result in aqueous medium of pH 7.5-9.5, typically of pH 8-9, generally used in PCR media.
  • the compounds according to the invention have also the ability to bind to RNA. This binding to nucleic acid corresponds to weak bonds, in particular hydrogen or ionic bonds or may correspond to any other binding by weak bond.
  • the compounds according to the invention exhibit a change in fluorescence resulting from their binding to a nucleic acid, and in particular to double strand nucleic acid, typically ssDNA or preferentially dsDNA.
  • the compounds according to the invention are able to interact with nucleic acid (i.e. RNA or DNA strands), typically with double stranded DNA, in particular the minor groove of a DNA double helix, and more generally to bind to the DNA strands by several kinds of weak binding and to lead to a change in fluorescence, which can be monitored. As a result, they are useful tools for studying nucleic acids. After their binding with nucleic acids, the compounds according to the invention behave as fluorescent dyes.
  • the invention encompasses uses and methods for detecting a target nucleic acid which is a single stranded or double stranded nucleic acid comprising a step of mixing a compound according to the invention, with a sample comprising the target nucleic acid.
  • the invention is also related to a method for detecting a target nucleic acid which is a single stranded or double stranded nucleic acid comprising a step of mixing a compound in accordance with the invention, with a sample comprising the target nucleic acid or an amplicon of the target nucleic acid.
  • the compounds according to the present invention are used for the detection of double stranded nucleic acids during a nucleic acid amplification reaction in real time and/or subsequent to amplification via a melting curve analysis or end-point analysis.
  • the compound of the invention will be a part of an amplification, preferably a PCR, reaction mixture and it can already be present at the beginning of the amplification reaction.
  • the compounds according to the invention do not significantly interfere with the efficiency of such a amplification (preferably PCR) reaction.
  • the compounds according to the invention do not significantly inhibit amplification (preferably PCR) when present at concentrations that provide high fluorescence signal for an amount of nucleic acid, and typically dsDNA generated by PCR, in the absence of the compound according to the invention.
  • amplification preferably PCR
  • the following steps may be carried out: - amplifying the target nucleic acid to generate an amplicon, - adding a compound according to the invention to the sample comprising the target nucleic acid and/or the amplicon, before, during or after the amplifying step, - monitoring fluorescence from the compound according to the invention during or subsequent to the amplifying step.
  • the following steps are carried out: -amplifying the target nucleic acid, in the presence of the compound according to the invention, in particular by PCR, to generate an amplicon, and - during the amplification, monitoring the fluorescence of the compound according to the invention, resulting from the binding of the compound to the amplicon.
  • a step of melting the generated amplicon is carried out, while monitoring the fluorescence from the compound according to the invention, to obtain a melting curve.
  • the compound of the invention and the amplicon are placed in a sample, which is suitable for their binding and the fluorescence obtaining.
  • this sample is an aqueous medium of pH 7.5-9.5, more specifically of pH 8-9.
  • the methods and uses according to the invention include a step of contacting the generated amplicon and the compound according to the invention, in conditions which enable their binding, in particular by weak bond(s).
  • the usual conditions of amplification, in particular of PCR enable this binding.
  • the binding of the compound of the invention with an amplicon occurs at a temperature lower than the melting temperature of the amplicon, when the target nucleic acid is a double stranded nucleic acid, typically dsDNA.
  • the temperature during the binding is, for instance, in the range from 20 to 60°C.
  • the sample containing the generated amplicon and the compound according to the invention often, includes one or several salts commonly used in PCR medium like NaCl and MgCl 2 .
  • the following steps may be carried out: - amplifying of the target nucleic acid, in the presence of the compound according to the invention, in particular by PCR, and typically by real time PCR, to produce an amplicon, - optionally during the amplifying step, monitoring the fluorescence of the compound according to the invention, resulting from the binding of the compound to the amplicon, and - optionally subsequent to the amplifying step, monitoring the fluorescence resulting from the binding of the compound according to the invention to the amplicon, via an end-point analysis or while melting the amplicon to obtain a melting curve.
  • the melting step allows the analysis of the targeted nucleic acid, for instance the identification of a specific genotype or polymorphism.
  • the said melting curve is used to identify the genotype of the target nucleic acid, to detect or identify at least one mutation, polymorphism, preferentially single nucleotide polymorphism, and/or epigenetic variation.
  • a melting curve (also called melt curve) is generated by slowly denaturing (melting) the generated amplicon.
  • the generation of melting curves and the use for analysis of nucleic acid are known in the art. More precisely, when a melting curve analysis is used for the detection or the quantification of a target double stranded nucleic acid (preferably dsDNA), the mixture containing the generated amplicon and the compound according to the invention is subjected to a thermal gradient.
  • the gradient is a continuous gradient, but step gradients are also possible. Most preferably, the gradient is a linear gradient.
  • the sample is subjected to a temperature increase which results in the generation of a dissociation curve.
  • the double stranded nucleic acid (preferably dsDNA) is first thermally denatured into single strands and temperature dependence of fluorescence is monitored during subsequent renaturation.
  • the amplification of the target nucleic acid, and in particular of the target DNA can be carried out by different techniques, and in particular by enzymatic amplification reaction.
  • Enzymatic amplification reaction it should be understood a process generating multiple copies of a target nucleotide fragment, by the action of at least one enzyme.
  • PCR Polymerase Chain Reaction
  • standard PCR RealTime-PCR
  • quantitative PCR digital PCR
  • multiplex PCR asymetric PCR
  • nested PCR semi-nested PCR
  • LATE-PCR Touchdown PCR
  • Hot-Start PCR COLD-PCR
  • assembly PCR LCR (Ligase Chain Reaction)
  • RCR Repair Chain Reaction
  • 3SR Self Sustained Sequence Replication
  • NASBA Nucleic Acid Sequence-Based Amplification
  • SDA Strand Displacement Amplification
  • MDA Multiple Displacement Amplification
  • RPA Recombinase Polymerase Amplification
  • HDA Helicase Dependent Amplification
  • RCA Rolling Circle Amplification
  • TMA Transcription Mediated Amplification
  • a primer is a nucleotide fragment which may consist of 5 to 100 nucleotides, preferably of 15 to 30 nucleotides, and possesses a specificity of hybridization with a target nucleic acid sequence, under conditions determined for the initiation of an enzymatic polymerization, for example in an enzymatic amplification reaction of the target nucleic acid sequence. For instance, when one reverse primer and several forward primers or alternatively one forward primer and several reverse primers are used in an amplification, they form several pairs of primers.
  • the methods, uses, kits and mixtures according to the invention will include more than one pair of primers: one for each target nucleic acid. Methods of PCR analysis using a compound according to the invention are particularly interesting.
  • the invention also concerns a method of PCR analysis of a target nucleic acid comprising the steps of: - mixing a compound according to the invention with a sample comprising a target nucleic acid, a polymerase, and a pair of primers suitable to amplify a portion of the target nucleic acid and generate at least an amplicon, leading to a PCR mixture, - amplifying the target nucleic acid from the PCR mixture and generating at least an amplicon, and - monitoring the fluorescence from the compound according to the invention during or subsequent to the amplifying step.
  • a method of PCR analysis further comprises detecting the presence of the amplicon from the monitored fluorescence.
  • the monitoring step may occur subsequent to amplification and may include generating a melting curve or end-point analysis of the fluorescence.
  • the said melting curve and in particular its shape, is used to identify the genotype of the target nucleic acid, to detect or identify at least one mutation, polymorphism, preferentially single nucleotide polymorphism (SNP), and/or epigenetic variation.
  • SNP preferentially single nucleotide polymorphism
  • a method of the invention includes the quantification of the target nucleic acid, which is initially present in the sample.
  • the quantity of target nucleic acid, which is initially present in the sample corresponds to the amount of nucleic acid which is present in the initial sample used, that means the sample used before any step of amplification.
  • Quantification of the initial amount of nucleic acid in the sample could be carried out by any method classically known by those in the art and it could be applied during or after any amplification method, preferably PCR, qPCR or LAMP.
  • One method for quantifying a target nucleic acid is by determining Cp (Crossing point – also named Ct for Cycle Threshold) and comparing the Cp to a standard or to a control.
  • Absolute quantification including amplification by qPCR, frequently uses a standard curve approach.
  • a standard curve generated from plotting the Cp values obtained from amplification, preferentially real-time PCR, against known quantities of a single reference template (also called standard or control) provides a regression line that can be used to extrapolate the quantities of the target nucleic acid in a sample of interest.
  • Serial dilutions generally 10-fold dilutions
  • Various separate reactions are run, usually one for each level of the reference target and one each for the samples of interest.
  • the method for detecting a target nucleic acid or the method of PCR analysis of a target nucleic acid may corresponds to a methods of performing quantitative amplification, preferably PCR, on a sample.
  • the methods may comprise amplifying the sample in an amplification mixture, the amplification mixture comprising a pair of target primers configured to amplify a target that may be present in the sample, the amplification mixture further comprising a plurality of quantification standard nucleic acids each provided at a different known concentration and at least one pair of quantification standard primers, the quantification standard primers configured to amplify quantification standard nucleic acids, generating a standard curve from the quantification standard amplicons, and quantifying the target nucleic acid using the standard curve.
  • either external or internal quantification standard nucleic acids maybe be used for the quantification of the target nucleic acid.
  • the standard nucleic acid is external, it is separated and not in the same reaction mixture (also called sample) as the one containing the target nucleic acid to quantify.
  • the standard nucleic acid is internal, it is in the same reaction mixture (also called sample) as the target nucleic acid to be quantified.
  • the internal standard nucleic acid(s) is(are) generally amplified at the same time as the target nucleic acid to quantify but this can also be done previously and the standard curve obtained can be stored and imported at the moment of the quantification of the target nucleic acid.
  • Quantification standard can be synthetic or natural.
  • the calibration or quantification can be performed against a known natural microorganism with known concentrations or against other naturally occurring nucleic acid templates. It could be for examples a yeast or bacteriophages for viruses and/or synthetic particles able to mimic membrane and/or capsid and/or envelope structures but also housekeeping genes.
  • the quantification of the target nucleic acid could also be done using the melting curves.
  • the target nucleic acid is a double stranded nucleic acid, preferably dsDNA
  • the quantification may imply the generation of a melting curve, and more precisely of several melting curves. Methods of quantification using a melting curve are known from those skilled in the art.
  • Livak method for example is usable. It is also possible to use the maximum of the negative first derivative of the intensity of the fluorescence and of the temperature (max of – (dIntensity of fluorescence/dTemperature) which gives the melting temperature and then the quantity of the target nucleic acid.
  • This method may further include determining a value for the melting curve, and determining a Cp by identifying the amplification cycle in which the value for the melting curve exceeds a predetermined value. The value may be determined by peak height or peak area of a negative derivative of the melting curve.
  • a set of negative derivative melting curves can be used, wherein the flattest curves represent the earliest cycles and the area under the curve increases through a number of cycles.
  • Such derivative melting curves acquired at a plurality of cycles during amplification can be used to determine Cp.
  • the height of the transition for each melting curve or the area under the negative first derivative of the melt curve can be determined for each cycle.
  • the Cp may then be assigned to the cycle at which this value exceeds a pre-determined threshold.
  • Other methods for determining Cp may be applied.
  • a melt detector may be used (see U.S. 6,387,621; US 6,730,501; and US 7,373,253, herein incorporated by reference). The detector would interrogate curve shape and background noise to determine if the produced amplicon, preferably the amplicon obtained by PCR, is present in the sample.
  • melt detector could be used to increase the sensitivity of the system (See Poritz, et al., PLos One 6(10):e2604 7).
  • additional filters could be applied to the melting curve analysis to window the melt transition to increase the specificity of the system, by analyzing only those melting curves having a melting transition, displayed as a melt peak, within a set temperature range. It is expected that such methods would result in a more accurate Cp (see WO 2014/039963).
  • Methods of continuous monitoring of temperature and fluorescence are used for relative quantification, illustratively using a compound according to the invention, as dsDNA binding dye, in a single reaction with a control or standard nucleic acid.
  • a multiplexed amplification (preferably PCR) reaction containing a control or standard nucleic acid at a known initial concentration and a target nucleic acid at an unknown concentration.
  • Primers for amplification of the control or standard nucleic acid are present at the same initial concentration as primers for amplification of the target nucleic acid.
  • the control or standard nucleic acid is selected such that its melting temperature is sufficiently well separated from the melting temperature of the target nucleic acid, so that melting of each of these nucleic acids is discernable from melting of the other.
  • the melting profile of each of the two reactions can be distinguished.
  • a corrected amplification curve for the control or standard nucleic acid at each cycle the integral of the negative first derivative of the melt curve over a pre- defined melt window can be computed and plotted as a function of the cycle number, with the Cp determined as the cycle at which each value exceeds a predetermined value.
  • a corrected amplification curve for the target nucleic acid may be generated by integrating the negative first derivative of the melting curve over the pre-defined melt window for the target as it is described in WO 2014/039963 which is incorporated by reference.
  • a method of PCR analysis may comprise the steps of mixing the compound according to the invention, with a sample comprising an unknown initial quantity of a target nucleic acid and primers configured for amplifying the target nucleic acid, to form a mixture, amplifying the target nucleic acid in the presence of the compound according to the invention to generate an amplicon, monitoring fluorescence of monitoring the fluorescence of the compound according to the invention, resulting from the binding of the compound to the amplicon throughout a temperature range during a plurality of amplification cycles to generate a plurality of melting curves, and using the melting curves to quantify the initial quantity of the target nucleic acid.
  • a method of PCR analysis of a target nucleic acid according to the invention may comprise the steps of: - mixing a compound according to the invention with a sample comprising a target nucleic acid and at least a pair of primers suitable to amplify a portion of the target nucleic acid and generate an amplicon, leading to a PCR mixture, - amplifying the target nucleic acid from the PCR mixture, and generating at least an amplicon, - during the amplifying step, monitoring the fluorescence of the compound according to the invention resulting from the binding of the compound to the amplicon, - at the end of the amplifying step, melting the generated amplicon, to obtain a melting curve, and - identifying the genotype or polymorphism of the target nucleic acid using a shape of the melting curve.
  • the amplifying step may include a plurality of temperature cycles including at least a denaturation temperature and an extension temperature, wherein each cycle has a cycle time of less than 90 seconds per cycle, and wherein the polymerase is provided at a concentration of at least 0.005 ⁇ M and primers are each provided at a concentration of at least 0.1 ⁇ M.
  • the amplifying step includes a plurality of temperature cycles including at least a denaturation temperature and an extension temperature, wherein each cycle has a cycle time of less than 20 seconds per cycle, and wherein the polymerase is provided at a concentration of at least 0.5 ⁇ M and primers are each provided at a concentration of at least 2 ⁇ M.
  • concentrations are related to the amplification mixture, in particular to the PCR mixture.
  • the PCR techniques are often classified according to the time required for the PCR and according to the quantity of primers which is used. More details are given in US 7387887 and US 9932634.
  • Classical or standard PCR are quite slow and occur in approximately 90 seconds or less per cycles, rapid PCR occur in less than 60 seconds per cycle, for example between 20 and 60 seconds per cycle, fast, ultra fast and extreme PCR occur in less than 20 seconds, preferentially less than 12 seconds for fast PCR, less than 6 seconds for ultrafast PCR and in less than 2 seconds for extreme PCR.
  • concentrations of primers and polymerase are increased. This allows maintaining PCR efficiency and yield.
  • the concentrations of primers range from at least 0.1 ⁇ M for the classical or standard PCR to at least 2 ⁇ M for extreme PCR, that is at least 0.1 ⁇ M, at least 0.2 ⁇ M, at least 0.4 ⁇ M, at least 0.6 ⁇ M, at least 0.8 ⁇ M, at least 1 ⁇ M, at least 1.2 ⁇ M, at least 1.4 ⁇ M, at least 1.6 ⁇ M, at least 1.8 ⁇ M or at least 2 ⁇ M.
  • the concentrations of polymerase range from at least 0.005 ⁇ M for classical or standard PCR to at least 0.5 ⁇ M for extreme PCR, that is at least 0.005 ⁇ M, at least 0.01 ⁇ M, at least 0.02 ⁇ M, at least 0.04 ⁇ M, at least 0.06 ⁇ M, at least 0.08 ⁇ M, at least 0.1 ⁇ M, at least 0.2 ⁇ M, at least 0.3 ⁇ M, at least 0.4 ⁇ M or at least 0.5 ⁇ M. Any kinds of these PCR can be used, according to the invention.
  • 1 ⁇ M of polymerase corresponds to 3.8 U/ ⁇ L. All these concentrations are related to the PCR mixture.
  • the invention also concerns a PCR reaction mixture, also named PCR mixture, comprising: - a target nucleic acid, - a pair of primers suitable to amplify a portion of the target nucleic acid, to generate an amplicon, - a polymerase, in particular a thermostable polymerase, - a compound according to the invention.
  • Said pair of primers is designed to amplify a specific sequence of interest in the target nucleic acid according to standard methods known in the art of molecular biology. More than one pair of primers can be used, in particular for multiplex PCR, where more than one target sequence must be amplified.
  • the target nucleic acid is typically total genomic DNA or alternatively total cellular RNA or total cellular mRNA.
  • the thermostable DNA polymerase may be a DNA polymerase or a mixture of polymerases comprising reverse transcriptase activity.
  • such a PCR reaction mixture also includes a mix of deoxynucleoside triphosphates which is usually dA, dG, dC and dT, or dA, dG, dC and dU.
  • a PCR reaction mixture classically includes a buffer.
  • the PCR reaction mixture is buffered at pH from 7.5 to 9.5, preferentially from 8 to 9.
  • Such a PCR reaction mixture may also include a thiol, typically selected from among the dithiothreitol, the beta mercaptoethanol and the thioglycerol, typically when it is dedicated to a RT-PCR analysis.
  • the concentration of the compound according to the present invention is, typically, from 1 to 20 ⁇ mol/L ( ⁇ M), and preferably from 2 to 10 ⁇ mol/L. This concentration corresponds to the concentration of the compound according to the invention in the sample which is used for monitoring the fluorescence of the compound according to the invention resulting from the binding of the compound to the amplicon.
  • a compound according to the present invention is used for detection of double stranded nucleic acids during a melting curve analysis as disclosed for other compounds known in the art.
  • a double stranded DNA fragment is subjected to a thermal gradient in the presence of a compound according to the present invention.
  • the gradient is a continuous gradient, but step gradients are also possible.
  • the gradient is a linear gradient.
  • the sample is subjected to a temperature increase which results in the generation of a dissociation curve.
  • the target double stranded nucleic acid is first thermally denatured into single strands and temperature dependence of fluorescence is monitored during subsequent renaturation. A first derivative of the melting curve may be generated and a characteristic temperature of the nucleic acid dissociation is obtained.
  • the concentration of the compound according to the present invention added before, during or after the amplification step and then used in the obtained mixture/sample used for monitoring the fluorescence is, typically, from 1 to 20 ⁇ mol/L, and preferably from 2 to 10 ⁇ mol/L.
  • This concentration corresponds to the concentration of the compound according to the invention in the sample which is used for monitoring the fluorescence of the compound according to the invention resulting from the binding of the compound to the amplicon.
  • the double stranded DNA which is analyzed is derived from a PCR amplification reaction.
  • amplification can be monitored in real time using a compound according to the invention and, in some embodiments, can be followed by subsequent melting curve analysis or end-point analysis of the fluorescence using said compound.
  • the invention also concerns a kit for detecting a target nucleic acid, comprising: ⁇ at least a pair of primers suitable to amplify a portion of the target nucleic acid, to generate an amplicon, ⁇ a polymerase, in particular a thermostable polymerase, ⁇ a compound according to the invention. If the kit is used to obtain a melting curve, the kit allows the analysis and the identification of the target nucleic acid.
  • Such a kit classically includes, also, a buffer, in particular, a buffer leading to a pH from 7.5 to 9.5, preferentially from 8 to 9.
  • a buffer for instance
  • Suitable buffers for PCR are commercially available and can be used.
  • Such a buffer may also include a thiol, for instance selected from among the dithiothreitol, the beta mercaptoethanol and the thioglycerol, typically when it is dedicated to a RT-PCR analysis.
  • the compound according to the invention can also be in a buffer, for instance in such a buffer in the kit.
  • the polymerase may, also, be stored in a buffer leading to a pH from 7.5 to 9.5, preferentially from 8 to 9.
  • this buffer may also include a thiol, typically selected from among the dithiothreitol, the beta mercaptoethanol and the thioglycerol.
  • a thiol typically selected from among the dithiothreitol, the beta mercaptoethanol and the thioglycerol.
  • Figure 4 presents the stability for the examples 1 to 4 and for a compound N7 of the prior art, in the presence of a thiol.
  • Figures 5A to 5F present the obtained fluorescence (RFU), in function of the temperature (°C) (in the left part), and the difference of fluorescence (- d(RFU)/dT(T)), in function of the temperature (°C) (in the right part), obtained in the evaluation hereafter, respectively for the examples 1 to 5 and 10 of the invention (compounds I.1 to I.5 and I.10) in or without the presence of a model DNA duplex, respectively (- DUPLEX) or (+ DUPLEX).
  • Figures 6 and 7 present respectively for example 1 and 3 of the invention (compounds I.1 and I.3): the PCR amplification curve of a biological target (panel A), the melting curve of the generated amplicon (panel B) and the first derivative of the melting curve to determine precisely the melting temperature of the amplicon (panel C).
  • Figure 8A presents the functional assays in fast real time PCR conditions of compounds described in the examples 1 to 4 of the invention (compounds I.1 to I.4) in comparison with compound N7 of the prior art.
  • Figure 8B describes with more details the maximal fluorescence (Max Fluo) obtained at the end of the PCR on panel A, the Cp (number of cycles) on panel B and the Tm (melting temperature) measurements of the amplicon on panel C given by compounds I.1 to I.4 in comparison with compound N7 of the prior art.
  • Figure 9 presents the excitation and emission maxima of the compounds I.1 to I.7 and I.10.
  • R 1 Et ;
  • R 4 H ;
  • the mixture was stirred and heated at 145°C (fusion reaction) for 2 hours. The mixture was then left to cool down to room temperature. 6 mL of acetone were added and the obtained mixture was mixed vigorously and transferred to a 50 mL tube diethyl ether. The flask was rinsed with 6 mL of acetone then diluted with 30 mL of ether, shaken vigorously and the supernatant was discarded after centrifugation at 1500 rpm. The precipitate was washed 10 times in 40 mL of acetone / ethyl ether mixture 1/9, v/v. The obtained solid was dried by evaporating the rest of solvent with a rotary evaporator and compound IV.2 was obtained.
  • the compound from the kit Resolight® from Roche diagnostic is less stable and lead to less fluorescence, than all the compounds according to the invention.
  • the interest of these various compounds relies on the possibility of obtaining absorption and emission on a broad range of wavelengths.
  • Figure 9 shows the modulation of the maximum absorption and maximum emission wavelengths, in particular by the choice of n. 3) Study of the influence of the alkyl chain in position 1 Other tests were carried out to study the influence of the presence of an alkyl chain in position 1, instead of an aryl group as in example A10 of US 7,387,887.
  • the Figure 2 compares the stability and the fluorescence obtained with the two following compounds: TFA- comparative 6 (Comp. 6) and TFA- comparative 7 (Comp. 7) It appears that methyl in position 1 (N atom corresponding to substituent R 1 in formula (I)) of the pyrimidine induces a better stability and a better fluorescence than phenyl.
  • the solutions were placed in PCR polypropylene cuvettes of 200 ⁇ L and the emitted fluorescence was recorded as a function of temperature from 20°C to 90°C (0.5°C/min) using a preliminary denaturing step during 1 minute at 95°C (CFX Maestro from Biorad Laboratories) to obtain a typical melting curve.
  • the appropriate fluorescence channel was used as a function of the fluorescence characteristics of the compound (FAM for all compounds except for I.5 where Hex channel was used).
  • the first derivative of this melting curve was calculated and plotted as a function of the temperature to precisely determine the melting temperature of the duplex.
  • FIGS. 5A to 5F show that all the compounds of the present invention can be used for the detection of the melting of a duplex (Tm) with a very good sensitivity (Tm peak height) as summarized in Table 1 hereinafter.
  • Tm duplex
  • Tm peak height very good sensitivity
  • Table 1 D Functionnal assays (PCR)
  • the compounds, I.1 of example 1 (figure 6) and I.3 of example3 (figure 7) were used at respectively 10 ⁇ M and 5 ⁇ M in a standard PCR reaction amplification using the biological model: S.cerevisiae at 10 e 5 ; 10 e 4 ; 10 e 3 and 10 e 2 cp/PCR reaction (duplicats) on the LightCycler® 480 Instrument II (Roche) equipped with a (440/488) filter.
  • the panel A, B and C of Figures 8B show respectively the details of the Max Fluo, Cp and Tm measurements showing the high reproducibility of these experiments and the ability of these compounds to detect with a great sensitivity the presence of a dedicated amplicon. is the obtained results also show that the dyes of the invention (I.1 to I.4) provide roughly twice more fluorescence (Max Fluo) than compound N7. Therefore, these dyes are much more efficient and valuable in detecting a given target with a high sensitivity.
  • both Cp and Tm are in the same order as with N7 (respectively +/- 1Cp around the Cp given by N7 and +/- 2°C around Cp given by N7), demonstrating that the dyes of the invention do not inhibit PCR.

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Abstract

L'invention concerne des composés de formule (I) et leurs sels, tels que définis dans la revendication 1 et leurs utilisations, notamment dans des applications PCR.
PCT/EP2023/082402 2022-11-22 2023-11-20 Nouveaux composés de liaison à l'acide nucléique et leurs utilisations Ceased WO2024110392A1 (fr)

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AU2023386451A AU2023386451A1 (en) 2022-11-22 2023-11-20 New nucleic acid binding compounds and uses
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