Application of heteroaromatic ring compound in nucleic acid detection

Abstract

Use of heteroaromatic ring compounds in nucleic acid detection is provided. The invention relates to application of a heteroaromatic ring compound, in particular to application of the heteroaromatic ring compound as a nucleic acid protective agent in nucleic acid detection, a method for inhibiting nucleic acid degradation in nucleic acid detection, a method for detecting target nucleic acid molecules and a method for determining a target single-stranded polynucleotide sequence, and further relates to a reagent and a kit containing the heteroaromatic ring compound.

Description

Application of heteroaromatic compounds in nucleic acid detection Technical Field

[0001] The present invention relates to the field of nucleic acid detection, and to the use of heteroaromatic compounds in nucleic acid detection. In particular, the present invention relates to the use of heteroaromatic compounds as nucleic acid protectants in nucleic acid detection, a method for inhibiting nucleic acid degradation, a method for detecting target nucleic acid molecules, a sequencing method, and reagents and kits containing the heteroaromatic compounds. Background Art

[0002] With the vigorous development of gene sequencing technology, second-generation high-throughput sequencing technology and single-molecule sequencing technology have overcome the shortcomings of Sanger sequencing such as high cost and long time consumption, and have greatly promoted the application of gene sequencing technology in various research fields.

[0003] However, during sequencing and other nucleic acid sample processing, nucleic acids are continuously exposed to various harmful environmental conditions, causing them to be damaged and degraded, affecting their further processing and detection. For example, during sequencing, nucleic acid damage and degradation hinder the extension of the DNA duplex, leading to an increase in sequencing error rates. This cumulative damage becomes more pronounced as sequencing read lengths increase.

[0004] Numerous studies have shown that the primary environmental damage to nucleic acids during processing, such as sequencing, comes from laser irradiation during fluorescence detection. The sustained, intense excitation light source, along with the shorter-wavelength, higher-energy EM radiation from fluorophores, generates reactive oxygen species (ROS) in aqueous solutions, such as singlet oxygen, superoxide anions, and iron-promoted hydroxyl radicals. These highly reactive free radicals directly or indirectly lead to pyrimidine dimerization, oxidative modification of nucleotides, and even cleavage of phosphate bonds, ultimately hindering further processing and detection of nucleic acid samples, manifesting as a loss of fluorescence signal intensity and high error rates.

[0005] In order to ensure high-quality sequencing of long nucleotide sequences, it is particularly important to provide an efficient and low-cost nucleic acid protection solution.

[0006] The current main approach to nucleic acid protection is to inhibit light-induced degradation of nucleic acid sequences by adding antioxidants. Commonly used antioxidants include vitamin C derivatives and polyphenols. These antioxidants interact with oxygen free radicals in the environment through their highly reactive hydroxyl groups, quenching free radicals and thus protecting nucleic acid sequences. However, due to the high reactivity of these antioxidants, they gradually interact with oxygen in the air during storage and use, causing degradation and deterioration, affecting the final use effect. In addition, the poor water solubility of most polyphenols also limits their application in aqueous systems.

[0007] Summary of the Invention

[0008] One of the objectives of the present invention is to provide a method for reducing damage to nucleic acid samples. The technical solution of the present invention involves introducing an additive during the handling, processing, detection, and reaction of nucleic acid samples. This additive is in full contact with the nucleic acid sample and protects the nucleic acid sample by quenching harmful factors such as free radicals in the sample environment and absorbing short-wavelength, high-energy, intense EM radiation.

[0009] To achieve this effect, the additive used preferably has one or more of the following characteristics: good water solubility to ensure sufficient contact with the nucleic acid sample in the water medium; chemical inertness, no reaction with the active groups in the nucleic acid, and the ability to maintain the normal functional form of the sample; strong interaction with free radicals to quench harmful factors; good absorption of short-wavelength, high-energy, strong EM radiation; its aqueous solution is colorless and transparent, and does not affect the detection of fluorescent luminescence signals, etc.

[0010] The inventors have discovered that a class of heteroaromatic compounds can achieve the above effects, thereby providing the following invention:

[0011] Use of heteroaromatic compounds as nucleic acid protective agents in nucleic acid detection

[0012] In one aspect, the present application provides the use of a heteroaromatic ring compound as a nucleic acid protectant in nucleic acid detection, wherein the heteroaromatic ring compound is selected from a compound as represented by formula (I) or an isomer, salt or nitrogen oxide thereof, or a solvate (e.g., hydrate) of a compound as represented by formula (I) or an isomer, salt or nitrogen oxide thereof;

[0013]

[0014] wherein X1, X2, X3 are each independently selected from C or N; X4 is selected from N or S; and at least one of X1, X2, X3, X4 is N;

[0015] m is 1 or 2;

[0016] R1 is absent or selected from H, C 1-6 Alkyl, amino, hydroxyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 alkoxy;

[0017] R2 is absent or selected from -(CH2) n -COOH or -(CH2) n -CONH2; n is 0, 1, 2, 3 or 4; optionally, -(CH2) n - is substituted with an amino group; optionally, the amino group is replaced with -(C=O)-(CH2)q -NH2 substituted; q is 0, 1, 2, 3 or 4;

[0018] R3 does not exist or is H;

[0019] Alternatively, R2 and R3 form a 5-6 membered heteroaryl group containing 1-2 nitrogen atoms, wherein the heteroaryl group is optionally substituted by 1 or 2 substituents, each of which is independently selected from the group consisting of: hydroxyl, amino, carbonyl, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 alkoxy;

[0020] R4 is absent or H, or is selected from the following structures:

[0021]

[0022] In certain embodiments, one, two, or three of X1, X2, X3, and X4 are N.

[0023] In certain embodiments, one or two of X1, X2, X3, and X4 are N.

[0024] In certain embodiments, X1 is N. In certain embodiments, X1 is C. In certain embodiments, X2 is C. In certain embodiments, X3 is C. In certain embodiments, X3 is N. In certain embodiments, X4 is N. In certain embodiments, X4 is S.

[0025] In certain embodiments, m is 1. In certain embodiments, m is 2.

[0026] In certain embodiments, R1 is absent. In certain embodiments, R1 is H. In certain embodiments, R1 is C 1-6 alkyl.

[0027] In certain embodiments, R2 is absent. In certain embodiments, R2 is selected from -(CH2) n -COOH or -(CH2) n -CONH2. In certain embodiments, n is 0, 1 or 2. In certain embodiments, the -(CH2) n - is substituted with an amino group. In certain embodiments, the amino group is substituted with -(C=O)-(CH2) q In certain embodiments, q is 2.

[0028] In certain embodiments, R3 is absent. In certain embodiments, R3 is H.

[0029] In certain embodiments, R4 is absent. In certain embodiments, R4 is H.

[0030] In certain embodiments, R4 has one of the structures shown in formula (1), (2), (3), or (4).

[0031] In certain embodiments, the compound of formula (I) has one of the following structures:

[0032]

[0033] In certain embodiments, the compound of formula (I) has a structure as shown in formula (II), (III) or (V).

[0034] In certain embodiments, R2 is selected from -(CH2) n -COOH or -(CH2) n -CONH2; n is 0, 1 or 2; optionally, -(CH2) n - is substituted with an amino group; optionally, the amino group is replaced with -(C=O)-(CH2) q preferably, q is 0, 1 or 2. In certain embodiments, R4 is absent.

[0035] In certain embodiments, the compound represented by formula (I) has one of the structures represented by formula (II), (III), (IV), and (V); R2 and R3 form a 6-membered heteroaryl group containing 1 or 2 nitrogen atoms (e.g., pyrimidinyl); the heteroaryl group is optionally substituted with 1 or 2 substituents, each of which is independently selected from the group consisting of: hydroxyl, amino, carbonyl, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1- 6 alkoxy.

[0036] In certain embodiments, the compound of formula (I) has one of the following structures:

[0037]

[0038] Wherein, Q and M are independently selected from hydrogen, hydroxyl, amino, carbonyl, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 In certain embodiments, Q and M are each independently selected from hydrogen, hydroxy, amino, and carbonyl.

[0039] In certain embodiments, Q is selected from the group consisting of: hydrogen, amino, hydroxy, carbonyl. In certain embodiments, Q is amino.

[0040] In certain embodiments, M is selected from the group consisting of: hydrogen, amino. In certain embodiments, M is hydrogen.

[0041] In certain embodiments, R4 is absent or has a structure as shown in formula (1), (2), (3), or (4). In certain embodiments, R4 is absent or has a structure as shown in formula (2).

[0042] In certain embodiments, the compound represented by formula (I) includes but is not limited to the following compounds:

[0043]

[0044] In certain embodiments, the compound represented by formula (I) is selected from the following compounds:

[0045]

[0046] In certain embodiments, the compound represented by formula (I) is the following compound:

[0047]

[0048] In certain embodiments, the compound of formula (I) is monosodium adenosine 5'-monophosphate (IR41).

[0049] As used herein, the term "alkyl" includes 1-6 carbon atoms, or 1-4 carbon atoms, or 1-3 carbon atoms, or 1-2 carbon atoms, saturated linear or branched monovalent hydrocarbon groups, wherein the alkyl groups may be independently optionally substituted with one or more substituents described herein. Further examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), n-propyl (n-Pr, -CH2CH2CH3), isopropyl (i-Pr, -CH(CH3)2), n-butyl (n-Bu, -CH2CH2CH2CH3), isobutyl (i-Bu, -CH2CH(CH3)2), sec-butyl (s-Bu, -CH(CH3)CH2CH3), tert-butyl butyl (-C(CH3)3), n-pentyl (-CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2) , 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), n-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(C H3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3).

[0050] The term "alkenyl" refers to a linear or branched monovalent hydrocarbon group of 2-6 carbon atoms, or 2-4 carbon atoms, wherein at least one position is unsaturated, i.e., a carbon-carbon sp2 double bond, wherein the alkenyl group can be independently and optionally substituted with one or more substituents described in the present invention, including groups with "anti", "normal" or "E" or "Z" orientations, specific examples of which include, but are not limited to, vinyl (-CH=CH2), allyl (-CH2CH=CH2) and butylene (-CH2CH2CH=CH2) and the like.

[0051] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon group of 2 to 6 carbon atoms, or 2 to 4 carbon atoms, wherein at least one position is unsaturated, i.e., a carbon-carbon triple bond is sp, wherein the alkynyl group may be independently and optionally substituted with one or more substituents described herein, specific examples of which include, but are not limited to, ethynyl (-C≡CH) and propargyl (-CH2C≡CH).

[0052] The term "alkoxy" as used herein refers to an alkyl group, as defined herein, connected to the rest of the compound molecule via an oxygen atom. In some embodiments, the alkoxy group is C 1-4 Alkoxy groups; examples thereof include, but are not limited to, methoxy, ethoxy, propoxy, and butoxy, etc., and the alkoxy groups may be independently unsubstituted or substituted with one or more substituents described herein.

[0053] The term "heteroaryl" refers to monocyclic, bicyclic, and tricyclic ring systems containing 5-12 ring atoms, or 5-10 ring atoms, or 5-6 ring atoms, wherein at least one ring system is aromatic and at least one ring system contains one or more heteroatoms, wherein each ring contains 5-7 ring atoms and has one or more points of attachment to the rest of the molecule. The term "heteroaryl" can be used interchangeably with the terms "heteroaromatic ring" or "heteroaromatic compound". The heteroaryl group is optionally substituted with one or more substituents described herein. In one embodiment, the 5-10 heteroaryl group contains 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N, wherein the nitrogen atom can be further oxidized.

[0054] Examples of heteroaryl groups include, but are not limited to, furanyl, imidazolyl (e.g., N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), isoxazolyl, oxazolyl (e.g., 2-oxazolyl, 4-oxazolyl, 5-oxazolyl), pyrrolyl (e.g., N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), pyridinyl, pyrimidinyl (e.g., 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl), pyridazinyl, thiazole 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiodiazolyl, 1,3,4-thiodiazolyl, 1,2,5-thiodiazolyl, 1,2,3 ... oxadiazole, pyrazinyl, 1,3,5-triazinyl; also include the following bicyclic rings, but are in no way limited to these bicyclic rings: benzimidazolyl, benzofuranyl, benzothiophenyl, indolyl (such as 2-indolyl), purinyl, quinolyl (such as 2-quinolyl, 3-quinolyl, 4-quinolyl), 1,2,3,4-tetrahydroisoquinolyl, 1,3-benzodioxolyl, indolinyl, isoquinolyl (such as 1-isoquinolyl), [1,2,4]triazolo[4,3-b]pyridazinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl and [1,2,4]triazolo[1,5-a]pyridinyl, and the like.

[0055] The term "isomer" includes all isomeric forms (such as enantiomers, diastereomers, geometric isomers or conformational isomers), for example, R and S configurations containing asymmetric centers, (Z) and (E) isomers of double bonds, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers, geometric isomers or conformational isomers are all within the scope of the present invention.

[0056] The term "salt" includes both organic and inorganic salts of the compounds used in the present invention. Salts formed from acids include, but are not limited to, inorganic salts formed by reaction with amino groups, such as hydrochlorides, hydrobromides, phosphates, sulfates, and perchlorates; organic salts, such as acetates, oxalates, maleates, tartrates, citrates, succinates, and malonates; or salts obtained by other methods described in the literature, such as ion exchange. Other acceptable salts include adipate, malate, 2-hydroxypropionate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1- 4 alkyl) 4. The present invention also contemplates quaternary ammonium salts formed by any compound containing a N group. Water-soluble or oil-soluble or dispersible products can be obtained by quaternization. Alkali metals or alkaline earth metals that can form salts include sodium, lithium, potassium, calcium, magnesium, etc. Acceptable salts further include appropriate, non-toxic ammonium, quaternary ammonium salts and amine cations formed by counter ions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C 1-8 Sulfonates and aromatic sulfonates.

[0057] The term "solvate" refers to an association formed between one or more solvent molecules and the compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol.

[0058] The term "hydrate" refers to an association in which the solvent molecule is water.

[0059] The term "nitrogen oxide" refers to when a compound contains several amine functional groups, where one or more nitrogen atoms can be oxidized to form an N-oxide. Specific examples of N-oxides are N-oxides of tertiary amines or N-oxides of nitrogen-containing heterocyclic nitrogen atoms. Available oxidants such as hydrogen peroxide or peracids (e.g., peroxycarboxylic acids) can be used to treat the corresponding amines to form N-oxides (see Advanced Organic Chemistry, Wiley Interscience, 4th edition, Jerry March, pages). In particular, N-oxides can be prepared by the method of LW Deady (Syn. Comm. 1977, 7, 509-514), for example, by reacting an amine compound with meta-chloroperoxybenzoic acid (MCPBA) in an inert solvent (e.g., dichloromethane).

[0060] In certain embodiments, the nucleic acid protectant is used to protect nucleic acids to reduce or avoid photodamage (photoinduced damage) or oxidative damage.

[0061] In certain embodiments, the nucleic acid detection involves detecting a fluorescent signal. In certain embodiments, the fluorescent signal can be generated by a light-induced reaction or a non-light-induced reaction (e.g., a bioluminescent reaction).

[0062] In certain embodiments, the bioluminescent reaction refers to a reaction in which luciferase catalyzes its substrate to produce a fluorescent signal. As used herein, the term "illumination reaction" refers to a reaction that is exposed to an optical energy source. Typically, in such reactions, an optical energy source is provided to observe the production and / or consumption of reactants or products, which have specific optical characteristics indicative of their presence, such as changes in the absorption and / or emission spectra (intensity, wavelength, etc.) of the reaction mixture or its components.

[0063] As used herein, the term "non-illuminated reaction" refers to a reaction that occurs without the aid of an optical energy source. In a non-illuminated reaction, an optical signal can be generated, such as through bioluminescence, thereby allowing the production and / or consumption of reactants or products to be observed.

[0064] As used herein, the term "bioluminescence," also known as bioluminescence, refers to the phenomenon of light emission generated by energy from a natural enzymatic chemical reaction. It is a type of chemiluminescence. Bioluminescence occurs naturally in many organisms, such as fireflies, deep-sea jellyfish, and some bacteria.

[0065] In certain embodiments, the illumination reaction includes the generation of an optical signal (eg, a fluorescent signal) caused by base extension. In certain embodiments, the optical characteristic in the illumination reaction of the present invention is preferably fluorescence.

[0066] In certain embodiments, the nucleic acid detection is nucleic acid sequence determination (sequencing) or other scenarios (eg, quantitative PCR).

[0067] In certain embodiments, the nucleic acid detection is sequencing, such as high-throughput sequencing, such as sequencing by synthesis (SBS sequencing), ligation sequencing, hybridization sequencing, nanopore sequencing, or combined probe-anchor ligation (cPAL) sequencing.

[0068] In certain embodiments, the nucleic acid detection is quantitative PCR.

[0069] Reagents, kits and their uses

[0070] In one aspect, the present application provides a reagent comprising one or more heteroaromatic ring compounds as defined above and a Tris buffer solution.

[0071] In the present invention, the Tris buffer solution refers to a buffer solution using tris (hydroxymethyl)aminomethane (Tris) as a buffer system.

[0072] Tris is widely used in the preparation of buffer solutions in biochemistry and molecular biology. Tris is a weak base, and its aqueous solution has a pH of approximately 10.5. By adding hydrochloric acid to adjust the pH to the desired value, a buffer solution of that pH is obtained. Buffer solutions can also be prepared using Tris and its hydrochloride (Tris-HCl).

[0073] The effective buffering range of Tris buffer solution is between pH 7.0 and 9.2. Tris buffer solution can be used as a solvent for dissolving nucleic acids.

[0074] In certain embodiments, the concentration of the heteroaromatic compound in the reagent is 0.1-500 mM, such as 0.1-0.5 mM, 0.5-1 mM, 1-5 mM, 5-10 mM, 10-50 mM, 50-100 mM, 100-200 mM or 200-500 mM.

[0075] In certain embodiments, the reagent is a scanning reagent. Preferably, the heteroaromatic compound is used as a nucleic acid protecting agent in nucleic acid sequencing.

[0076] In certain embodiments, the reagent further comprises sodium chloride and / or a DNA stabilizer (e.g., Tween-20). The role of sodium chloride is to provide a salt solution background to protect the primers during detection.

[0077] In another aspect, the present application provides a kit comprising the reagents of the present invention.

[0078] In certain embodiments, the kit of the present invention may further comprise one or more other reagents required for nucleic acid detection, for example, primers, polymerase, buffer solution, washing solution, or any combination thereof.

[0079] In certain embodiments, the kits of the present invention are used for sequencing.

[0080] In certain embodiments, the kit of the present invention may further comprise: a reagent for fixing the nucleic acid molecule to be sequenced to a support (e.g., by covalent or non-covalent attachment); a primer for initiating nucleotide polymerization; a polymerase for carrying out nucleotide polymerization; one or more buffer solutions; one or more washing solutions; or any combination thereof.

[0081] In certain embodiments, the test kit of the present invention may also include reagents and / or devices for extracting nucleic acid molecules from a sample. Methods for extracting nucleic acid molecules from a sample are well known in the art. Therefore, various reagents and / or devices for extracting nucleic acid molecules may be configured as needed in the test kit of the present invention, such as reagents for crushing cells, reagents for precipitating DNA, reagents for washing DNA, reagents for dissolving DNA, reagents for precipitating RNA, reagents for washing RNA, reagents for dissolving RNA, reagents for removing protein, reagents for removing DNA (for example, when the target nucleic acid molecule is RNA), reagents for removing RNA (for example, when the target nucleic acid molecule is DNA), and any combination thereof.

[0082] In certain embodiments, kit of the present invention also comprises, and is used for the reagent of pre-treatment nucleic acid molecule.In kit of the present invention, The reagent for pre-treatment nucleic acid molecule is not subject to additional restriction, and can be selected according to actual needs.The described reagent for pre-treatment nucleic acid molecule for example comprises, and is used for the reagent (for example DNA enzyme I) of nucleic acid molecule fragmentation, and is used for the reagent (for example archaeal dna polymerase of polishing nucleic acid molecule end, for example T4DNA polymerase, Pfu archaeal dna polymerase, Klenow archaeal dna polymerase), joint molecule, label molecule, and is used for the reagent (for example ligase, for example T4DNA ligase) that joint molecule is connected with target nucleic acid molecule, and is used for the reagent (for example, losing 3'-5' exonuclease activity but showing 5'-3' exonuclease activity) of repairing nucleic acid end, and is used for the reagent (for example, archaeal dna polymerase, primer, dNTP) of amplification nucleic acid molecule, and is used for the reagent (for example chromatography column) of separation and purification nucleic acid molecule, and its any combination.

[0083] In certain embodiments, the kit of the present invention further comprises a support for fixing the nucleic acid molecules to be sequenced. Typically, the support for fixing the nucleic acid molecules to be sequenced is in a solid phase for ease of operation. Therefore, in this disclosure, "support" is sometimes also referred to as "solid support" or "solid phase support". However, it should be understood that the "support" mentioned herein is not limited to solid, and it can also be a semi-solid (e.g., gel).

[0084] As used herein, the terms "loaded," "fixed," and "attached" when used in reference to nucleic acids, mean attached directly or indirectly to a solid support via covalent or non-covalent bonds. In certain embodiments of the present disclosure, the methods of the present invention include immobilizing nucleic acids on a solid support via covalent attachment. Generally, however, it is only necessary that the nucleic acids remain fixed or attached to the solid support under conditions where the solid support is desired to be used (e.g., in applications where nucleic acid amplification and / or sequencing is required). In certain embodiments, immobilizing nucleic acids on a solid support can include immobilizing an oligonucleotide to be used as a capture primer or an amplification primer on a solid support such that the 3' end is available for enzymatic extension and at least a portion of the primer sequence is capable of hybridizing to a complementary nucleic acid sequence; the nucleic acid to be immobilized is then hybridized to the oligonucleotide, in which case the immobilized oligonucleotide or polynucleotide can be in a 3'-5' direction. In certain embodiments, immobilizing nucleic acids on a solid support can include binding a nucleic acid binding protein to the solid support by amino modification, and capturing nucleic acid molecules by the nucleic acid binding protein. Alternatively, loading can occur by other means besides base pair hybridization, such as covalent attachment as described above. Non-limiting examples of nucleic acid attachment to a solid support include nucleic acid hybridization, biotin-streptavidin binding, sulfhydryl binding, photoactivated binding, covalent binding, antibody-antigen, physical confinement via a hydrogel or other porous polymer, and the like. Various exemplary methods for immobilizing nucleic acids on solid supports can be found in, for example, G. Steinberg-Tatman et al., Bioconjugate Chemistry 2006, 17, 841-848; Xu X. et al. Journal of the American Chemical Society 128 (2006) 9286-9287; U.S. patent applications US 5639603, US 5641658, US2010248991; international patent applications WO 2001062982, WO 2001012862, WO 2007111937, WO0006770, all of which are incorporated herein by reference in their entirety for all purposes, in particular for all teachings relating to the preparation of solid supports having nucleic acids immobilized thereon.

[0085] In the present invention, the support can be made of various suitable materials. Such materials include, for example, inorganic substances, natural polymers, synthetic polymers, and any combination thereof. Specific examples include, but are not limited to, cellulose, cellulose derivatives (e.g., nitrocellulose), acrylic resins, glass, silica gel, silicon dioxide, polystyrene, gelatin, polyvinyl pyrrolidone, copolymers of vinyl and acrylamide, cross-linked polystyrenes such as divinylbenzene (see, for example, Merrifield Biochemistry 1964, 3, 1385-1390), polyacrylamide, latex, dextran, rubber, silicon, plastics, natural sponges, metal plastics, cross-linked dextran (e.g., Sephadex TM ), agarose gel (Sepharose TM ), and other supports known to those skilled in the art.

[0086] In certain preferred embodiments, the support used to immobilize the nucleic acid molecules to be sequenced can be a solid support comprising an inert substrate or matrix (e.g., a glass slide, polymer beads, etc.) that has been functionalized, for example, by applying an intermediate material containing reactive groups that allow for covalent attachment of biomolecules such as polynucleotides. Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass, particularly the polyacrylamide hydrogels described in WO 2005 / 065814 and US 2008 / 0280773, the contents of which are incorporated herein by reference in their entirety. In such embodiments, the biomolecules (e.g., polynucleotides) can be directly covalently attached to the intermediate material (e.g., hydrogel), while the intermediate material itself can be non-covalently attached to the substrate or matrix (e.g., a glass substrate). In certain preferred embodiments, the support is a glass or silicon wafer whose surface is modified with a layer of avidin, amino, acrylamide silane, or aldehyde chemical groups.

[0087] In the present invention, the support or solid support is not limited to its size, shape and configuration. In some embodiments, the support or solid support is a planar structure, such as a slide, chip, microchip and / or array. The surface of such a support can be in the form of a planar layer.

[0088] In certain preferred embodiments, the support for immobilizing the nucleic acid molecules to be sequenced is an array of beads or wells (also referred to as a chip). The array can be prepared using any of the materials outlined herein for preparing solid supports, and preferably, the surface of the beads or wells on the array is functionalized to facilitate the immobilization of nucleic acid molecules. The number of beads or wells on the array is not limited. For example, each array may contain 10-10 2 , 10 2-10 3 , 10 3 -10 4 , 10 4 -10 5 , 10 5 -10 6 , 10 6 -10 7 , 10 7 -10 8 , 10 8 -10 9 , 10 10 -10 11 , 10 11 -10 12 In certain exemplary embodiments, the surface of each bead or hole can be fixed with one or more nucleic acid molecules. Accordingly, each array can be fixed with 10-10 2 , 10 2 -10 3 , 10 3 -10 4 , 10 4 -10 5 , 10 5 -10 6 , 10 6 -10 7 , 10 7 -10 8 , 10 8 -10 9 , 10 10 -10 11 , 10 11 -10 12 or more nucleic acid molecules. Therefore, such arrays can be particularly advantageously used for high-throughput sequencing of nucleic acid molecules.

[0089] In certain preferred embodiments, the kit of the present invention further comprises a reagent for fixing the nucleic acid molecule to be sequenced to the support (e.g., by covalent or non-covalent attachment). Such reagents include, for example, reagents for activating or modifying nucleic acid molecules (e.g., their 5' ends), such as phosphoric acid, thiol, amine, carboxylic acid, or aldehyde; reagents for activating or modifying the surface of the support, such as amino-alkoxysilanes (e.g., aminopropyltrimethoxysilane, aminopropyltriethoxysilane, 4-aminobutyltriethoxysilane, etc.); cross-linking agents, such as succinic anhydride, phenyl diisocyanate (Guo et al., 1994), maleic anhydride (Yang et al., 1998), 1-ethyl-3-(3-dimethoxysilane)-1-ol, ... methylaminopropyl)-carbodiimide hydrochloride (EDC), m-maleimidobenzoic acid-N-hydroxysuccinimide ester (MBS), N-succinimidyl[4-iodoacetyl]aminobenzoic acid (SIAB), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide (SMCC), N-γ-maleimidobutyryloxy-succinimide ester (GMBS), 4-(p-maleimidophenyl)butyric acid succinimide (SMPB); and any combination thereof.

[0090] In certain preferred embodiments, the kit of the present invention also includes a primer for initiating nucleotide polymerization. In the present invention, the primer is not subject to additional restrictions, as long as it can specifically anneal to a region of the target nucleic acid molecule. In some exemplary embodiments, the length of the primer can be 5-50bp, such as 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50bp. In some exemplary embodiments, the primer can include naturally occurring or non-naturally occurring nucleotides. In some exemplary embodiments, the primer includes naturally occurring nucleotides or is composed of naturally occurring nucleotides. In some exemplary embodiments, the primer includes modified nucleotides, such as locked nucleic acid (LNA). In certain preferred embodiments, the primer includes a universal primer sequence.

[0091] In some preferred embodiments, test kit of the present invention also comprises the polymerase for carrying out nucleotide polymerization reaction.In the present invention, various suitable polymerases can be used to carry out polyreaction.In some exemplary embodiments, described polymerase can be the new DNA chain (such as DNA polymerase) synthesized with DNA as a template.In some exemplary embodiments, described polymerase can be the new DNA chain (such as reverse transcriptase) synthesized with RNA as a template.In some exemplary embodiments, described polymerase can be the new RNA chain (such as RNA polymerase) synthesized with DNA or RNA as a template.Therefore, in some preferred embodiments, described polymerase is selected from DNA polymerase, RNA polymerase, and reverse transcriptase.

[0092] In certain preferred embodiments, the kits of the present invention further comprise one or more excision reagents.

[0093] The term "excision reagent" as used herein refers to a reagent that can remove blocking groups and / or detectable labels on nucleic acid intermediates (i.e., growing nucleic acid chains) during nucleic acid detection, thereby exposing the 3'OH end of the ribose sugar for use in the next round of detection. In certain embodiments, the excision reagent is tris(3-hydroxypropyl)phosphine (THPP), which can be used to excise disulfide-containing blocking groups. In certain embodiments, the excision reagent is a sulfhydryl reagent, such as dimercaprol, which can be used to excise azide-containing blocking groups. In certain embodiments, the excision reagent is tris(2-carboxyethyl)phosphine (TCEP), which can be used to excise detectable labels containing fluorescent groups.

[0094] In certain preferred embodiments, the kit of the present invention further comprises one or more buffer solutions. Such buffer solutions include, but are not limited to, buffer solutions for DNA enzyme I, buffer solutions for DNA polymerase, buffer solutions for ligase, buffer solutions for eluting nucleic acid molecules, buffer solutions for dissolving nucleic acid molecules, buffer solutions for carrying out nucleotide polymerization reactions (e.g., PCR), and buffer solutions for carrying out ligation reactions. The kit of the present invention may comprise any one or more of the above-mentioned buffer solutions.

[0095] In certain embodiments, the buffer solution for DNA polymerase comprises monovalent salt ions (e.g., sodium ions, chloride ions) and / or divalent salt ions (e.g., magnesium ions, sulfate ions, manganese ions). In certain embodiments, the concentration of the monovalent salt ions or divalent salt ions in the buffer solution is 10 μM-200 mM, e.g., 10 μM, 50 μM, 100 μM, 200 μM, 500 μM, 1 mM, 3 mM, 10 mM, 20 mM, 50 mM, 100 mM, 150 mM, or 200 mM.

[0096] In certain embodiments, the buffer solution for DNA polymerase comprises tris(hydroxymethylaminomethane) (Tris).In certain embodiments, the concentration of Tris in the buffer solution is 10mM-200mM, such as 10mM, 20mM, 50mM, 100mM, 150mM or 200mM.

[0097] In certain embodiments, the buffer solution for DNA polymerase comprises an organic solvent, such as DMSO or glycerol (glycerol). In certain embodiments, the mass content of the organic solvent in the buffer solution is 0.01%-10%, such as 0.01%, 0.02%, 0.05%, 1%, 2%, 5% or 10%.

[0098] In certain embodiments, the pH of the buffer solution for DNA polymerase is 7.0-9.0, e.g., 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

[0099] In certain embodiments, the buffer solution for DNA polymerase comprises: monovalent salt ions (e.g., sodium ions, chloride ions), divalent salt ions (e.g., magnesium ions, sulfate ions, manganese ions), Tris, and an organic solvent (e.g., DMSO or glycerol). In certain embodiments, the pH of the buffer solution is 8.8.

[0100] In certain preferred embodiments, the kit of the present invention further comprises one or more washing solutions. Examples of such washing solutions include, but are not limited to, phosphate buffer, citrate buffer, Tris-HCl buffer, acetate buffer, carbonate buffer, and the like. The kit of the present invention may comprise any one or more of the above-mentioned washing solutions.

[0101] In another aspect, the present application provides use of the reagent or kit of the present invention for nucleic acid detection.

[0102] In certain embodiments, the nucleic acid detection involves detecting a fluorescent signal.

[0103] In certain embodiments, the fluorescent signal can be generated by a light-induced reaction or a non-light-induced reaction (eg, a bioluminescent reaction).

[0104] In certain embodiments, the bioluminescence refers to a reaction in which luciferase catalyzes its substrate to produce a fluorescent signal.

[0105] In certain embodiments, the nucleic acid detection involves a light-induced reaction or a non-light-induced reaction (eg, a bioluminescent reaction).

[0106] In certain embodiments, the illumination reaction includes the generation of an optical signal (eg, a fluorescent signal) caused by base extension.

[0107] In certain embodiments, the optical characteristic in the illumination reaction of the present invention is preferably fluorescence.

[0108] In certain embodiments, the nucleic acid detection can be used for nucleic acid sequence determination (sequencing) or other scenarios (eg, quantitative PCR).

[0109] In certain embodiments, the nucleic acid detection is sequencing, such as high-throughput sequencing, such as sequencing by synthesis (SBS), sequencing by ligation, sequencing by hybridization, nanopore sequencing, or cPAL sequencing.

[0110] In certain embodiments, the nucleic acid detection is quantitative PCR.

[0111] In another aspect, the present application provides a method for preparing the reagent of the present invention, comprising dissolving the components of the reagent in ultrapure water to form a transparent and uniform solution, and then filtering the solution to obtain the reagent of the present invention. Ultrasound can be used to assist the dissolution.

[0112] Method for inhibiting nucleic acid degradation and method for detecting target nucleic acid molecules

[0113] In another aspect, the present application provides a method for inhibiting nucleic acid degradation in nucleic acid detection, which comprises adding one or more heteroaromatic compounds as defined above to inhibit the degradation of the nucleic acid to be detected in the reaction mixture.

[0114] In certain embodiments, the detection involves detecting a fluorescent signal, and the reaction mixture comprises a reactant that can generate a fluorescent signal, a nucleic acid to be detected, and a buffer.

[0115] In certain embodiments, the reactants capable of generating a fluorescent signal include: labeled or unlabeled nucleotides (eg, dNTPs), and optionally further include other reagents that allow the reactants to generate a fluorescent signal, such as a polymerase, primers, and / or fluorescent probes.

[0116] In certain embodiments, the fluorescent signal is generated by a light-induced reaction or a non-light-induced reaction (eg, bioluminescence).

[0117] In another aspect, the present application provides a method for detecting a target nucleic acid molecule, which comprises detecting the target nucleic acid molecule in a reaction mixture in the presence of one or more heteroaromatic compounds as defined above.

[0118] In certain embodiments, the detecting involves detecting a fluorescent signal, and the reaction mixture comprises a reactant that can generate a fluorescent signal, a target nucleic acid molecule, and a buffer.

[0119] In certain embodiments, the reactants capable of generating a fluorescent signal include: labeled or unlabeled nucleotides (eg, dNTPs), and optionally further include other reagents that allow the reactants to generate a fluorescent signal, such as a polymerase, primers, and / or fluorescent probes.

[0120] In certain embodiments, the fluorescent signal is generated by a light-induced reaction or a non-light-induced reaction (eg, bioluminescence).

[0121] In certain embodiments, the method is sequencing or quantitative PCR.

[0122] In certain embodiments, the sequencing is SBS sequencing, ligation sequencing, hybridization sequencing, nanopore sequencing, or cPAL sequencing.

[0123] In another aspect, the present application provides a method for determining the sequence of a target single-stranded polynucleotide, comprising:

[0124] (a) incorporating one or more nucleotides into the complementary strand of a target single-stranded polynucleotide sequence;

[0125] (b) in the presence of one or more heteroaromatic compounds as defined above, subjecting the incorporated nucleotide to conditions whereby a fluorescent signal is generated and detecting the fluorescent signal to determine the type of the incorporated nucleotide.

[0126] In certain embodiments, the method further comprises, (c) repeating steps (a)-(b) at least once.

[0127] In certain embodiments, the method further comprises, (d) determining the sequence of the target single-stranded polynucleotide.

[0128] In certain embodiments, the concentration of the heteroaromatic compound is greater than 0.1 mM, preferably 0.1-500 mM (e.g., 0.1-0.5 mM, 0.5-1 mM, 1-5 mM, 5-10 mM, 10-50 mM, 50-100 mM, 100-200 mM, or 200-500 mM), more preferably 5-20 mM, and most preferably 10 mM. In the present invention, the incorporated nucleotides are natural or modified nucleotide analogs. Nucleotides typically contain a sugar, a nucleobase, and at least one phosphate group. Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include, for example, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidylic acid (CMP), cytidylic acid diphosphate (CDP), cytidylic acid triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenylic acid (dAMP), Deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP) and deoxyuridine triphosphate (dUTP). Nucleotide analogs comprising modified nucleobases can also be used in the methods described herein.Exemplary modified nucleobases that can be included in polynucleotides, whether having a natural backbone or an analogous structure, include, for example, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynyl Cytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-halogenated adenine or guanine, 8-aminoadenine or guanine, 8-thioadenine or guanine, 8-sulfanyladenine or guanine, 8-hydroxyadenine or guanine, 5-halogen-substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, etc.

[0129] In certain embodiments, the incorporated nucleotide includes a sugar unit, a nucleobase, a phosphate group, and a cleavable blocking group (eg, a 3' blocking group) to reversibly prevent further extension of the complementary strand of the target single-stranded polynucleotide sequence.

[0130] In certain embodiments, the nucleobase is selected from adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U).

[0131] In certain embodiments, each type of nucleotide can produce a fluorescent signal that is distinguishable from one another to identify the type of nucleotide incorporated. For example, the four deoxyribonucleotides containing adenine (A), guanine (G), cytosine (C), and thymine (T), respectively, can produce different fluorescent signals, making them easy to distinguish from one another.

[0132] In certain embodiments, the fluorescent signal is generated by a photoluminescence reaction or a bioluminescence reaction.

[0133] In certain embodiments, the method further comprises, between (b) and (c): (b') removing the moiety directly or indirectly linked thereto that can generate a fluorescent signal from the incorporated nucleotide; and / or, washing to remove unincorporated nucleotides.

[0134] In certain embodiments, the method further comprises, between (b) and (c): (b") removing the cleavable blocking group from the incorporated nucleotide to allow further extension of the complementary strand of the target single-stranded polynucleotide sequence.

[0135] In certain embodiments, the target single-stranded polynucleotide is present on a nucleic acid array.

[0136] In certain embodiments, each site on the array comprises multiple copies of a single target single-stranded polynucleotide.

[0137] In certain embodiments, the nucleic acid array is immobilized on a solid support (eg, a chip).

[0138] Light-induced sequencing

[0139] In certain embodiments, the fluorescent signal is generated by a light-induced reaction.

[0140] In certain embodiments, the incorporated nucleotides are fluorescently labeled nucleotides (e.g., dNTPs). In certain embodiments, the conditions for producing a fluorescent signal from the incorporated nucleotides include irradiating a reaction mixture containing fluorescently labeled nucleotides (e.g., dNTPs) to produce a fluorescent signal. In certain embodiments, the fluorescently labeled nucleotides can be linked to a nucleotide (e.g., a base) via a linker. The linker can be acid-labile, photolabile, or contain a disulfide bond.

[0141] In certain embodiments, the method comprises: incorporating one or more fluorescently labeled nucleotides into a complementary strand of a target single-stranded polynucleotide sequence; irradiating the reaction mixture in the presence of one or more heteroaromatic compounds as defined above to generate a fluorescent signal, and detecting the fluorescent signal to determine the type of incorporated nucleotides. In this case, those skilled in the art will appreciate that the reaction mixture comprises reactants capable of generating a fluorescent signal (e.g., fluorescently labeled nucleotides, polymerase, primers, etc.), a target single-stranded polynucleotide molecule, and a buffer (e.g., Tris buffer, etc.).

[0142] In certain embodiments, the method further comprises: removing the fluorescent label attached to the incorporated nucleotides; and / or, washing to remove unincorporated nucleotides. In certain embodiments, the method further comprises: removing the cleavable blocking group from the incorporated nucleotides to allow further extension of the complementary strand of the target single-stranded polynucleotide sequence.

[0143] In certain embodiments, the conditions for giving the incorporated nucleotides to produce fluorescent signals include: the reaction mixture contains unlabeled nucleotides (e.g., dNTPs) and fluorescently labeled affinity reagents (e.g., antibodies), and the affinity reagents are capable of specifically binding to the unlabeled nucleotides, and the reaction mixture is irradiated to produce fluorescent signals. Such embodiments may be referred to as Cool MPS sequencing, and detailed teachings thereof may refer to PCT International Application WO2018129214A1. In such embodiments, based on the SBS sequencing principle, the fluorescent group is not directly labeled on the incorporated nucleotides, but is labeled on an affinity reagent (e.g., an antibody, an aptamer, an Affimer, a Knottin, etc.), and the affinity reagent can specifically bind to bases, sugars, cleavable blocking groups, or a combination of these components incorporated into the nucleotides, so that the type of incorporated nucleotides can be identified by the affinity reagent.

[0144] In certain embodiments, the method comprises: incorporating one or more unlabeled nucleotides into the complementary strand of the target single-stranded polynucleotide sequence; providing a fluorescently labeled affinity reagent and indirectly linking the fluorescent label to the incorporated nucleotide through specific binding between the affinity reagent and the nucleotide; irradiating the reaction mixture in the presence of one or more heteroaromatic compounds as defined above to generate a fluorescent signal and detecting the fluorescent signal to determine the type of incorporated nucleotide.

[0145] In certain embodiments, the method further comprises: removing the affinity reagent attached to the incorporated nucleotides; and / or, washing to remove unincorporated nucleotides. In certain embodiments, the method further comprises: removing the cleavable blocking group from the incorporated nucleotides to allow further extension of the complementary strand of the target single-stranded polynucleotide sequence.

[0146] Sequencing based on bioluminescence reactions

[0147] In certain embodiments, the fluorescent signal is generated by a bioluminescent reaction. The detection principle of bioluminescence includes that the incorporated nucleotides are not directly labeled with fluorescent groups, but are labeled with affinity substances such as biotin or digoxin. After the polymerization reaction, a pairing member of the above affinity substance with luciferase is added, thereby indirectly binding the luciferase to the incorporated nucleotides, and then adding the reaction substrate to generate a fluorescent signal, thereby determining the type of incorporated nucleotides. This process does not require excitation light irradiation. Detailed teachings on bioluminescent reactions can be found in, for example, PCT International Application WO2020227953A1.

[0148] In certain embodiments, the incorporated nucleotides are tagged nucleotides (e.g., dNTPs). In certain embodiments, the tags carried by the nucleotides are members of any molecular pairing that can specifically bind to each other. Specific binding between pairing members enables the connection of the nucleotide to the luciferase. Exemplary pairing members include, but are not limited to: (a) haptens or antigenic compounds in combination with corresponding antibodies or their binding portions or fragments, such as digoxin-digoxin antibodies, N3G-N3G antibodies, FITC-FITC antibodies; (b) nucleic acid aptamers and proteins; (c) non-immune binding pairs (e.g., biotin-avidin, biotin-streptavidin, biotin-neutravidin); (d) hormone-hormone binding proteins; (e) receptor-receptor agonists or antagonists; (f) lectins-carbohydrates; (g) enzyme-enzyme cofactors; (h) enzyme-enzyme inhibitors; and (i) complementary oligonucleotides or polynucleotides capable of forming nucleic acid duplexes.

[0149] In certain embodiments, the tag carried by the nucleotide is a small molecule marker selected from biotin, digoxigenin, N3G or FITC, and the luciferase carries a pairing member corresponding to the small molecule marker. For example, in a specific embodiment, if the tag carried by the nucleotide is biotin, the luciferase can be a luciferase labeled with streptavidin; if the tag carried by the nucleotide is digoxigenin, the luciferase can be a luciferase labeled with a digoxigenin antibody. The sources of the luciferase include but are not limited to organisms such as firefly, gaussia, and Renilla. For example, the luciferase labeled with streptavidin can be SA-Gluc:Streptavidin-Gaussia princeps luciferase from Adivity. The luciferase labeled with a digoxigenin antibody can be digoxin antibody-Gluc or digoxin antibody-Nluc.

[0150] In certain embodiments, the conditions for administering the incorporated nucleotides to generate a fluorescent signal include: the reaction mixture contains a nucleotide with a label (e.g., dNTP), a luciferase that can specifically bind to the label, and a substrate for the luciferase, and the reaction mixture generates a fluorescent signal through a bioluminescent reaction.

[0151] In certain embodiments, the method comprises: incorporating one or more nucleotides with a tag into the complementary strand of the target single-stranded polynucleotide sequence; providing a luciferase linked to a pairing member capable of specifically binding to the tag, and indirectly linking the luciferase to the incorporated nucleotide through specific binding between the pairing members; providing a substrate for the luciferase in the presence of one or more heteroaromatic compounds as defined above to generate a fluorescent signal, and detecting the fluorescent signal to determine the type of the incorporated nucleotide.

[0152] In certain embodiments, the method further comprises: removing the luciferase linked thereto from the incorporated nucleotides; and / or, washing to remove unincorporated nucleotides. In certain embodiments, the method further comprises: removing a cleavable blocking group from the incorporated nucleotides to allow further extension of the complementary strand of the target single-stranded polynucleotide sequence.

[0153] The following discussion will use sequencing by synthesis as an example, which is not meant to be limiting. All uses and methods of using the reagents of the present invention in a nucleic acid detection step involving an illumination reaction are within the scope of the present invention.

[0154] In certain embodiments, the method for determining the sequence of a target single-stranded polynucleotide comprises:

[0155] (a) providing a duplex, nucleotides, a polymerase, and an excision reagent; the duplex comprises a growing nucleic acid chain and a nucleic acid molecule to be sequenced;

[0156] (b) performing a reaction cycle comprising the following steps (i), (ii) and (iii):

[0157] Step (i): Under the action of a polymerase, nucleotides are incorporated into the growing nucleic acid chain to form a nucleic acid intermediate containing a blocking group and a detectable label;

[0158] Step (ii): detecting the detectable label on the nucleic acid intermediate in the presence of a reagent of the present invention;

[0159] Step (iii): using a cleavage reagent to remove the blocking group on the nucleic acid intermediate.

[0160] In certain embodiments, the reaction cycle further comprises step (iv): removing the detectable label on the nucleic acid intermediate using a cleavage reagent.

[0161] In certain embodiments, the method comprises the steps of:

[0162] The first step is to load DNA nanoballs (DNBs) onto the prepared sequencing chip;

[0163] In the second step, the prepared dNTP molecule mixture solution is pumped into the chip, and under the action of DNA polymerase, the dNTP is added to the complementary strand of the DNA to be tested;

[0164] The third step is photographing and scanning. Since the dNTPs are modified and have fluorescent group molecules, laser is used as the excitation wavelength for photographing. Since laser can damage DNA, a scanning reagent containing a nucleic acid protectant is added during this photographing step. The nucleic acid protectant is one or more heteroaromatic compounds as defined above.

[0165] In the fourth step, the fluorescent group at the base end of the dNTP and the blocking group at the 3'OH end of the ribose are removed and washed away by a removal reagent, leaving the 3'-OH group exposed for the next round of reaction.

[0166] Step 5. Repeat "Step 2, Step 3, Step 4" at least once;

[0167] The sixth step is to determine the nucleotide sequence of the nucleic acid molecule to be tested by analyzing the photographic results.

[0168] In the method of the present invention, the target single-stranded polynucleotide is sometimes also referred to as a "target single-stranded polynucleotide molecule", "test nucleic acid", "test nucleic acid molecule", or "target nucleic acid molecule", which have the same meaning and can be used interchangeably.

[0169] In the method for the present invention, nucleic acid molecule to be tested is not limited by length.In some preferred embodiments, the length of nucleic acid molecule to be tested can be at least 10bp, at least 20bp, at least 30bp, at least 40bp, at least 50bp, at least 100bp, at least 200bp, at least 300bp, at least 400bp, at least 500bp, at least 1000bp, or at least 2000bp.In some preferred embodiments, the length of nucleic acid molecule to be tested can be 10-20bp, 20-30bp, 30-40bp, 40-50bp, 50-100bp, 100-200bp, 200-300bp, 300-400bp, 400-500bp, 500-1000bp, 1000-2000bp, or more than 2000bp.In some preferred embodiments, nucleic acid molecule to be tested can have a length of 10-10000bp, to facilitate high-throughput sequencing.

[0170] In certain preferred embodiments, the nucleic acid molecules to be tested may be pretreated before being fixed to the support. Such pretreatment includes, but is not limited to, fragmentation of the nucleic acid molecules, end-padding, addition of adapters, addition of tags, amplification of the nucleic acid molecules, separation and purification of the nucleic acid molecules, and any combination thereof.

[0171] The term "nanosphere" as used herein generally refers to a macromolecule or complex having a compact, (approximately) spherical shape, eg, with an inner diameter typically ranging between about 1 nm and about 1000 nm, preferably between about 50 nm and about 500 nm.

[0172] As used herein, the term "nucleic acid nanoball" is generally a concatemer comprising multiple copies of a target nucleic acid molecule. These nucleic acid copies are typically arranged one after another in a continuous linear chain of nucleotides, but the nucleic acid nanoballs of the present invention can also be made from any nucleic acid molecule using the methods described herein. This tandem repeat structure, together with the single-stranded nature of DNA, causes the nanoball to fold (folding) configuration. Generally speaking, the multiple copies of the target nucleic acid molecules in the nucleic acid nanoball each contain a linker sequence with a known sequence to facilitate amplification or sequencing thereof. The linker sequences of each target nucleic acid molecule are generally the same, but may also be different. Nucleic acid nanoballs generally include DNA nanoballs, also referred to herein as DNBs (DNA nanoballs).

[0173] Nucleic acid nanospheres can be produced using, for example, rolling circle replication (RCA). The RCA process has been used to prepare multiple continuous copies of the M13 genome (Blanco et al., (1989) J Biol Chem 264:8935-8940). In this method, nucleic acids are replicated through linear concatemerization. Those skilled in the art can find guidance on the conditions and reagents for selecting RCA reactions in many references, including U.S. Patents US5,426,180, US5,854,033, US6,143,495 and US5,871,921, for all purposes, particularly for all teachings related to the preparation of nucleic acid nanospheres using RCA or other methods, which are incorporated herein by reference in their entirety.

[0174] The nucleic acid nanospheres can be loaded onto the surface of a solid support as described herein. The nanospheres can be attached to the surface of the solid support by any suitable method, non-limiting examples of which include nucleic acid hybridization, biotin-streptavidin binding, sulfhydryl binding, photoactivated binding, covalent binding, antibody-antigen, physical confinement via a hydrogel or other porous polymer, or a combination thereof. In some cases, the nanospheres can be digested with a nuclease (e.g., a DNA nuclease) to generate smaller nanospheres or fragments from the nanospheres.

[0175] In certain embodiments, the surface of the solid support may have reactive functional groups that react with complementary functional groups on the target single-stranded polynucleotide molecule to form covalent bonds, for example, using the same techniques used to attach cDNA to a microarray, for example, see Smirnov et al. (2004), Genes, Chromosomes & Cancer, 40: 72-77 and Beaucage (2001), Current Medicinal Chemistry, 8: 1213-1244, both of which are incorporated herein by reference. DNB can also be effectively attached to hydrophobic surfaces, such as clean glass surfaces with low concentrations of various reactive functional groups (e.g., -OH groups). Attachment via covalent bonds formed between the target single-stranded polynucleotide molecule and the reactive functional groups on the surface is also referred to herein as "chemical attachment."

[0176] In other embodiments, the target single-stranded polynucleotide molecule can be adsorbed onto a surface. In this embodiment, the target single-stranded polynucleotide molecule is immobilized by non-specific interactions with the surface, or by non-covalent interactions such as hydrogen bonds, van der Waals forces, etc. Beneficial effects

[0177] The present invention can achieve the following beneficial effects:

[0178] The present invention incorporates heteroaromatic compounds during the nucleic acid detection process, allowing them to come into contact with the nucleic acid sample, thereby inhibiting damage and degradation caused by nucleic acid sample processing. Heteroaromatic compounds are not only highly water-soluble but also chemically stable, resisting oxygen in the air and resistant to degradation during storage and use. This overcomes the oxidative degradation issue inherent in existing nucleic acid protection solutions. DETAILED DESCRIPTION

[0179] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0180] The following description provides more details on using the heteroaromatic ring compounds of the present invention for massively parallel nucleic acid sequencing. The method may include performing at least 5 nucleic acid sequencing cycles using a sequencing slide.

[0181] Each cycle in the sequencing process includes:

[0182] a) incorporating a nucleotide molecule carrying a cleavable blocking group and labeled with a fluorescent dye into the complementary chain of the nucleic acid to be detected;

[0183] b) contacting the nucleotide chain with a scanning reagent comprising 0.1 mM to 200 mM of an additive, wherein the pH of the scanning reagent is about 6.0-9.0;

[0184] c) laser excitation of fluorescent dye molecules to emit fluorescence and detecting the fluorescence value to obtain information about the incorporated nucleotide molecules, thereby inferring the sequence information of the nucleic acid to be tested;

[0185] d) Removal of the fluorescent dye group and the cleavable blocking group using a regeneration reagent.

[0186] The sequencing slide is loaded with single-stranded DNA nanoballs (DNBs), for example, containing E. coli genomic DNA. In the following examples, imaging tests were performed using an MGI sequencing slide (MGISEQ-2000RS Sequencing Slide); the kit used was the MGISEQ2000RS High-Throughput (Rapid) Sequencing Reagent Set (FCL PE100), suitable for PE150 sequencing; and the library used was the Standard Library Reagent V3.0 (26 ng / tube, E320, barcode 97-104).

[0187] 1. Experimental Design

[0188] Sequencing system: MGISEQ-2000

[0189] Sequencing library: Enterprise reference, standard library V3.0

[0190] Sequencing reagents: HotMPS High-throughput Sequencing Set (G400HM FCL PE100), catalog number: 940-000091-00

[0191] Add different concentrations of the heteroaromatic compound of interest to the scanning reagent in the kit. Also prepare a control scanning reagent without the additive. Perform PE150 sequencing on the same MGISEQ-2000 sequencer. Compare the sequencing quality of each scanning reagent after the sequence is removed from the instrument. Compare the data from the various scanning reagents, particularly regarding second-strand Q30 (%), error rate (%), and signal rebound value.

[0192] 2. Evaluation indicators and standards

[0193] Evaluation index qualification standard Q30 (%) is based on the blank control, the higher the value, the better

[0194] The Q30 (%) of the first chain is based on the blank control, the higher the value, the better. The Q30 (%) of the second chain is based on the blank control, the higher the value, the better. The error rate (%) of the first chain is based on the blank control, the lower the value, the better. The error rate (%) of the second chain is based on the blank control, the lower the value, the better. The average error rate (%) is based on the blank control, the lower the value, the better. The signal recovery value is based on the blank control, the higher the value, the better.

[0195] Note: 1. Q30 refers to the percentage of bases with an error rate of less than 0.001 (accuracy greater than 99.9%) in basecall results.

[0196] 2. From the perspective of sensitivity, the sensitivity of two-chain Q30(%) is higher than that of Q30(%) or one-chain Q30(%).

[0197] 3. Experimental steps

[0198] a. Preparation of DNB

[0199] DNB preparation was performed using the MGISEQ2000RS High-Throughput (Rapid) Sequencing Kit.

[0200] 1) Remove DNB Preparation Buffer, DNB Polymerase Mix I, TE Buffer, and DNB Stop Buffer and place on ice for approximately 0.5 h. Once thawed, vortex and mix for 5 seconds. Briefly centrifuge and place on ice until ready for use.

[0201] 2) Using a 0.2 mL eight-tube strip or PCR tube, prepare the reaction mixture on ice as follows:

[0202] Component volume (μL) Standard library 3 TE buffer 17 DNB preparation buffer 20 Total volume 40

[0203] 3) Vortex the reaction mixture and centrifuge for 5 seconds. Place the mixture in a PCR instrument for primer hybridization. The reaction conditions are shown in the table below:

[0204] Temperature Time Hot cover (105℃) On 95℃ 1min 65℃ 1min 40℃ 1min 4℃ Hold

[0205] 4) Remove the DNB polymerase mixture II (LC) and place it on ice. Centrifuge briefly for 5 seconds and place it on ice until ready to use.

[0206] 5) When the PCR instrument reaches 4°C, remove the PCR tube and centrifuge for 5 seconds. Then, add the following components on ice:

[0207] Components 100 μL System Addition Volume (μL) DNB Polymerase Mix I 40 DNB Polymerase Mix II (LC) 4

[0208] 6) The reaction mixture was vortexed and centrifuged for 5 seconds. The reaction mixture was immediately placed in a PCR instrument. The reaction conditions were as follows:

[0209] Temperature Time Hot Cover (35℃) On 30℃ 25min 4℃ Maintain

[0210] 7) When the PCR instrument reaches 4°C, remove the PCR tube, add 20 μL of stop buffer, and mix slowly 5-8 times with a wide-mouth pipette tip.

[0211] 8) After DNB preparation, take 2 μL DNB and use ssDNA Assay Kit and Fluorometer instrument was used for concentration detection.

[0212] b. Prepare the slide and sequencing reagent tank

[0213] 1) Remove the slide from the -25°C to -15°C freezer and place it at room temperature for at least 60 minutes (no more than 24 hours).

[0214] 2) Remove DNB Loading Buffer II and place on ice for approximately 0.5 h to thaw. Vortex mix for 5 seconds, centrifuge briefly, and place on ice until ready for use.

[0215] 3) Take out a new 1.5 mL centrifuge tube and prepare the DNB loading system as shown in the table below:

[0216] Component FCL Addition volume (μL) DNB loading buffer II 33DNB99 Total volume 132

[0217] 4) Mix the DNB loading system slowly 5-8 times using a wide-mouth pipette tip.

[0218] 5) Take out the MGIDL-200H portable injector and install the sealing gasket into the sealing gasket groove. Install the chips into the injector.

[0219] 6) Use a flared pipette tip to load 27 μL of DNB at L01-L04 respectively and let it stand at room temperature for more than half an hour.

[0220] 7) Prepare a PE150 sequencing reagent tank and take out the required HotMPS dNTP Mix and HotMPS dNTP Mix II 1 hour in advance. Thaw them at room temperature and set aside.

[0221] 8) Before use, remove Adv Seqenzyme II and place on ice.

[0222] 9) Add samples according to the following table:

[0223] Reagents: Well 1, Well 2, Well 15: HotMP SdNTP Mix 1500μL / HotMPS dNTP Mix II 1500μL / Adv Seqenzyme II 1000μL / MDA Reagent + MDA Enzyme / / 3875μL + 125μL

[0224] 10) Mix the reagents and set aside.

[0225] c. Sequencing

[0226] Place the reagent tank into the instrument, edit the script, and start PE150 sequencing.

[0227] Experimental design:

[0228] Example No. Test Conditions 1 Scanning reagent added 10mM IR132 Scanning reagent added 10mM IR203 Scanning reagent added 10mM IR224 Scanning reagent added 10mM IR34

[0229] 5 Scanning Reagent: Add 5mM IR416 Scanning Reagent: Add 10mM IR417 Scanning Reagent: Add 20mM IR418 Scanning Reagent: Add 10mM IR45

[0230] In the above examples, a scanning reagent without heteroaromatic compounds was used as a blank control, wherein the scanning reagent was the Tris buffer solution pre-filled in well 10 of the sequencing reagent reservoir in the MGISEQ2000RS high-throughput (rapid) sequencing reagent set.

[0231] PE150 sequencing was performed using the prepared DNBs and fluorescent dye-labeled nucleotides with cleavable blocking groups (from the FCL PE100 reagent set): first sequencing the first strand, then the second strand. The nucleotides with cleavable blocking groups have a cleavable structure located at the base-linked fluorescent group and at the 3'-OH position of the ribose sugar. In each cycle, after the fluorescent dye-labeled nucleotide was incorporated into the complementary strand of the nucleic acid to be tested, a scanning reagent was pumped in to take a picture. The imaging instrument used during this process was the MGISEQ-2000RS. The fluorescent dye was illuminated at excitation wavelengths of approximately 532 nm and 660 nm, with exposure times of 30 milliseconds for cycles 1-100, 40 milliseconds for cycles 100-200, and 50 milliseconds for cycles 200-300. After the picture was taken, the blocking groups at the fluorescent group and the 3'-OH position of the ribose sugar were removed using THPP. The sequencing results are shown in Tables 1-6.

[0232] Table 1

[0233]

[0234] Table 2

[0235]

[0236] Table 3

[0237]

[0238] Table 4

[0239]

[0240] Table 5

[0241]

[0242] Table 6

[0243]

[0244]

[0245] As shown in Tables 1-6, all tested heteroaromatic ring additives were able to varying degrees to improve sequencing quality degradation caused by DNA template damage (reduced Q30, increased error rate, and low signal rebound values ​​are considered to be poor sequencing quality). IR41, adenosine 5'-monophosphate monosodium, showed the greatest improvement in sequencing quality.

[0246] As shown in Table 5, PE150 sequencing was performed using the addition of 5'-adenosine monophosphate (AMP) to the scanning reagent. Compared to the control scanning reagent, the second-strand Q30 (%) of the samples supplemented with different concentrations of 5'-AMP ranged from 92.82 to 94.27, all higher than the 91.97 of the blank control. The second-strand error rate (%) ranged from 0.22 to 0.35, all lower than the 0.4 of the blank control. The signal recovery value ranged from 1.85 to 1.89, all higher than the 1.75 of the blank control.

[0247] By comparing the sequencing quality of different concentrations of 5'-adenosine monophosphate monosodium, it was found that 10mM 5'-adenosine monophosphate monosodium had the best improvement effect.

[0248] The above results show that including different concentrations of the above-mentioned heteroaromatic ring compounds (such as monosodium 5'-adenosine monophosphate) in the scanning reagent can reduce the decline in sequencing quality caused by damage to the DNA template and / or extended chain. The above-mentioned heteroaromatic ring compounds have a protective effect on nucleic acid chains such as DNA templates or extended chains during nucleic acid detection.

[0249] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention. Any modification or equivalent replacement of the technical solutions of the present invention without departing from the purpose and scope of the technical solutions of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of a heteroaromatic compound as a nucleic acid protectant in nucleic acid detection, wherein the heteroaromatic compound is selected from a compound represented by formula (I) or an isomer, salt or nitrogen oxide thereof, or a solvate (e.g., a hydrate) of a compound represented by formula (I) or an isomer, salt or nitrogen oxide thereof; in, X1, X2, X3 are each independently selected from C or N; X4 is selected from N or S; and at least one of X1, X2, X3, X4 is N; m is 1 or 2; R1 is absent or selected from H, C 1-6 Alkyl, amino, hydroxyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 alkoxy; R2 is absent or selected from -(CH2) n -COOH or -(CH2) n -CONH2; n is 0, 1, 2, 3 or 4; optionally, -(CH2) n - is substituted with an amino group; optionally, the amino group is replaced with -(C=O)-(CH2) q -NH2 substituted; q is 0, 1, 2, 3 or 4; R3 does not exist or is H; Alternatively, R2 and R3 form a 5-6 membered heteroaryl group containing 1-2 nitrogen atoms, wherein the heteroaryl group is optionally substituted by 1 or 2 substituents, each of which is independently selected from the group consisting of: hydroxyl, amino, carbonyl, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 alkoxy; R4 is absent or H, or is selected from the following structures:

2. The use according to claim 1, wherein The compound represented by formula (I) has one of the following structures: Preferably, R2 is selected from -(CH2) n -COOH or -(CH2) n -CONH2; n is 0, 1 or 2; optionally, -(CH2) n - is substituted with an amino group; optionally, the amino group is substituted with -(C=O)-(CH2) q -NH2 substituted; preferably, q is 0, 1 or 2; Preferably, R4 is absent; Preferably, R2 and R3 form a 6-membered heteroaryl group containing 1 or 2 nitrogen atoms (e.g., pyrimidinyl); the heteroaryl group is optionally substituted by 1 or 2 substituents, each of which is independently selected from the group consisting of: hydroxyl, amino, carbonyl, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1- 6 alkoxy.

3. The use according to claim 1, wherein The compound represented by formula (I) has one of the following structures: Wherein, Q and M are independently selected from hydrogen, hydroxyl, amino, carbonyl, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy; preferably selected from hydrogen, hydroxy, amino, carbonyl; Preferably, R4 does not exist, or R4 has one of the structures shown in formula (1), (2), (3), and (4).

4. The use according to any one of claims 1 to 3, wherein the compound represented by formula (I) is selected from the following compounds: Preferably, More preferably, 5. The use according to any one of claims 1 to 4, wherein The nucleic acid detection involves detecting a fluorescent signal; preferably, the fluorescent signal is generated by a light reaction or a bioluminescent reaction.

6. The use according to claim 5, wherein The nucleic acid detection is sequencing, such as high-throughput sequencing, such as SBS sequencing, ligation sequencing, hybridization sequencing, nanopore sequencing, or cPAL sequencing; Optionally, the nucleic acid detection is quantitative PCR.

7. A reagent comprising one or more heteroaromatic compounds as defined in any one of claims 1 to 4, and further comprising a Tris buffer solution; Preferably, in the reagent, the concentration of the heteroaromatic compound is 0.1-500 mM, more preferably 10 mM; Preferably, the reagent is a scanning reagent; Preferably, the heteroaromatic compound is used as a nucleic acid protectant in nucleic acid sequencing.

8. A kit comprising the reagent of claim 7; Preferably, the kit further comprises one or more other reagents required for nucleic acid detection, such as primers, polymerase, buffer solution, washing solution, or any combination thereof; Preferably, the kit is used for sequencing; More preferably, the kit further comprises: A support for immobilizing the nucleic acid molecule to be sequenced, a reagent for immobilizing the nucleic acid molecule to be sequenced to the support, and / or one or more excision reagents.

9. Use of the reagent according to claim 7 or the kit according to claim 8 for nucleic acid detection; Preferably, the nucleic acid detection is sequencing, such as high-throughput sequencing, such as SBS sequencing, ligation sequencing, hybridization sequencing, nanopore sequencing, or cPAL sequencing; Preferably, the nucleic acid detection is quantitative PCR.

10. A method for inhibiting nucleic acid degradation in nucleic acid detection, comprising adding one or more heteroaromatic compounds as defined in any one of claims 1 to 4 to a reaction mixture during detection of a nucleic acid to be detected, so as to inhibit degradation of the nucleic acid to be detected; Preferably, the detection involves detecting a fluorescent signal, and the reaction mixture comprises a reactant capable of generating a fluorescent signal, a nucleic acid to be detected, and a buffer; Preferably, the fluorescent signal is generated by a light reaction or a bioluminescent reaction.

11. A method for detecting a target nucleic acid molecule, comprising detecting the target nucleic acid molecule in a reaction mixture in the presence of one or more heteroaromatic compounds as defined in any one of claims 1 to 4; Preferably, the detection involves detecting a fluorescent signal, and the reaction mixture comprises a reactant capable of generating a fluorescent signal, a target nucleic acid molecule, and a buffer; Preferably, the fluorescent signal is generated by a light reaction or a bioluminescent reaction.

12. A method for determining a target single-stranded polynucleotide sequence, comprising: (a) incorporating one or more nucleotides into the complementary strand of a target single-stranded polynucleotide sequence; (b) in the presence of one or more heteroaromatic compounds as defined in any one of claims 1 to 4, providing conditions such that the incorporated nucleotide generates a fluorescent signal and detecting the fluorescent signal to determine the type of the incorporated nucleotide; Preferably, the method further comprises, (c) repeating steps (a)-(b) at least once; Preferably, the method further comprises, (d) determining the sequence of the target single-stranded polynucleotide; Preferably, the concentration of the heteroaromatic compound is greater than 0.1 mM, preferably 0.1-500 mM, more preferably 5-20 mM, and most preferably 10 mM.

13. The method of claim 12, wherein the incorporated nucleotide comprises a sugar unit, a nucleobase, a phosphate group, and a cleavable blocking group to reversibly prevent further extension of the complementary strand of the target single-stranded polynucleotide sequence; Preferably, the nucleobase is selected from adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U); Preferably, each type of nucleotide is capable of generating mutually distinguishable fluorescent signals to identify the type of incorporated nucleotide; Preferably, the fluorescent signal is generated by a light reaction or a bioluminescent reaction; Preferably, the method further comprises, between (b) and (c): (b') removing from the incorporated nucleotide a portion that is directly or indirectly linked thereto and that can generate a fluorescent signal; and / or, washing to remove unincorporated nucleotides; Preferably, the method further comprises, between (b) and (c): (b″) removing the cleavable blocking group from the incorporated nucleotide to allow further extension of the complementary strand of the target single-stranded polynucleotide sequence; Preferably, the target single-stranded polynucleotide is present in a nucleic acid array; Preferably, each site on the array comprises multiple copies of a single target single-stranded polynucleotide; Preferably, the nucleic acid array is immobilized on a solid support.

14. The method of claim 12 or 13, wherein The conditions for providing the incorporated nucleotides with a fluorescent signal include: irradiating a reaction mixture to generate a fluorescent signal, wherein the reaction mixture contains fluorescently labeled nucleotides; preferably, the fluorescently labeled nucleotides are linked to the nucleotides via a linker; Preferably, the linker is acid-labile, photolabile or contains a disulfide bond.

15. The method of claim 14, wherein The method comprises: incorporating one or more fluorescently labeled nucleotides into the complementary strand of a target single-stranded polynucleotide sequence; irradiating the reaction mixture in the presence of one or more heteroaromatic compounds as defined in any one of claims 1 to 4 to generate a fluorescent signal and detecting the fluorescent signal to determine the type of the incorporated nucleotides; Preferably, the method further comprises: removing the fluorescent label attached to the incorporated nucleotide; and / or, washing to remove unincorporated nucleotides; Preferably, the method further comprises: removing the cleavable blocking group from the incorporated nucleotide to allow further extension of the complementary strand of the target single-stranded polynucleotide sequence.

16. The method of claim 12 or 13, wherein The conditions for generating a fluorescent signal by administering the incorporated nucleotides include: a reaction mixture containing unlabeled nucleotides and a fluorescently labeled affinity reagent, wherein the affinity reagent is capable of specifically binding to the unlabeled nucleotides, and irradiating the reaction mixture to generate a fluorescent signal; Preferably, the affinity reagent specifically binds to a base, a sugar, a cleavable blocking group, or a combination of these components incorporated into the nucleotide; Preferably, the affinity reagent is an antibody, an aptamer, an affimer or a knottin; preferably, the affinity reagent is an antibody.

17. The method of claim 16, wherein The method comprises: incorporating one or more unlabeled nucleotides into the complementary strand of a target single-stranded polynucleotide sequence; providing a fluorescently labeled affinity reagent, and indirectly linking the fluorescent label to the incorporated nucleotide through specific binding between the affinity reagent and the nucleotide; irradiating the reaction mixture in the presence of one or more heteroaromatic compounds as defined in any one of claims 1 to 4 to generate a fluorescent signal, and detecting the fluorescent signal to determine the type of the incorporated nucleotide; Preferably, the method further comprises: removing the affinity reagent attached to the incorporated nucleotide; and / or, washing to remove unincorporated nucleotides; Preferably, the method further comprises: removing the cleavable blocking group from the incorporated nucleotide to allow further extension of the complementary strand of the target single-stranded polynucleotide sequence.

18. The method of claim 12 or 13, wherein The conditions for generating a fluorescent signal by administering the incorporated nucleotide include: a reaction mixture containing a nucleotide with a label, a luciferase capable of specifically binding to the label, and a substrate of the luciferase, and the reaction mixture generating a fluorescent signal through a bioluminescent reaction; Preferably, the label carried by the nucleotide is a small molecule marker, for example, selected from biotin, digoxigenin, N3G or FITC; and the luciferase carries a pairing member corresponding to the small molecule marker.

19. The method of claim 18, wherein The method comprises: incorporating one or more nucleotides with a tag into the complementary strand of a target single-stranded polynucleotide sequence; providing a luciferase linked to a pairing member capable of specifically binding to the tag, and indirectly linking the luciferase to the incorporated nucleotide through specific binding between the pairing members; providing a substrate for the luciferase in the presence of one or more heteroaromatic compounds as defined in any one of claims 1 to 4 to generate a fluorescent signal, and detecting the fluorescent signal to determine the type of the incorporated nucleotide; Preferably, the method further comprises: removing the luciferase linked thereto from the incorporated nucleotides; and / or, washing to remove unincorporated nucleotides; Preferably, the method further comprises: removing the cleavable blocking group from the incorporated nucleotide to allow further extension of the complementary strand of the target single-stranded polynucleotide sequence.