Use of accelerated 5-formylcytosine in marking and nucleic acid methylation sequencing

Abstract

This disclosure provides compositions for use in the preparation of target nucleic acid molecules, comprising one or more 5-formylcytosine bases or adducts of 5-formylcytosine. Also disclosed herein is a method for efficiently synthesizing nucleic acid molecules comprising one or more 5-formylcytosine bases from target nucleic acid molecules comprising one or more 5-hydroxymethylcytosine bases. This disclosure also provides a method for detecting epigenetic modifications in target nucleic acid molecules, such as epigenetic modifications characterized by methylation of cytosine at position 5 (e.g., 5-methylcytosine; 5-hydroxymethylcytosine).

Description

[Technical Field]

[0001] This disclosure relates to the field of nucleic acid-based diagnostics. More specifically, the present invention relates to a method for detecting epigenetic modifications in nucleic acid molecules, which may have biological and clinical significance. [Background technology]

[0002] DNA methylation plays a major role in regulating various physiological and pathological processes in mammals. It is a crucial epigenetic event in regulating embryonic development, genomic imprinting, X-inactivation, cell differentiation, and proliferation. While abnormal patterns of DNA methylation correlate with DNA instability and ultimately trigger subsequent genetic or acquired diseases such as cancer, DNA methylation is increasingly being reported as a potential biomarker for other mental and metabolic disorders. DNA methylation primarily occurs at the C5 position within the cytosine ring of cytosine-guanine (CpG) dinucleotides and is often clustered in gene regulatory sites such as promoter regions. High-density methylation of CpG within gene promoter regions is associated with condensed chromatin structures that lead to transcriptional silencing of related genes. Hypermethylation of DNA in the promoter regions of certain important cancer-associated genes can lead to silencing of tumor suppressor genes and ultimately to tumorigenesis. Alterations in DNA methylation are thought to be present and detectable in tumors and blood. Therefore, abnormal DNA methylation of specific oncogenes can sometimes be considered a biomarker for the early diagnosis of cancer.

[0003] Bisulfite genome sequencing provides a qualitative, quantitative, and efficient method for identifying 5-methylcytosine at single-base-pair resolution. This method is based on the discovery that the deamination reaction between cytosine and 5-methylcytosine (5-mC) proceeds with very different results after treatment with sodium bisulfite. In bisulfite sequencing (also known as bisulfite conversion), the target nucleic acid is first treated with a bisulfite reagent. This specifically converts unmethylated cytosine to uracil residues while remaining unaffected by methylated cytosine. The resulting uracil residues are recognized as thymine in subsequent PCR amplification and sequencing, while 5-mC remains unaffected by this conversion and remains as cytosine, thus allowing 5-mC to be distinguished from unmethylated cytosine. A subsequent PCR process is required to determine the methylation status of the target locus by using specific methylation primers after bisulfite treatment. The actual methylation state can be determined by either direct sequencing of PCR products (e.g., detection of mean methylation state) or subcloning sequencing (e.g., detection of single-molecule distribution of methylation patterns). Furthermore, bisulfite sequencing analysis can not only identify DNA methylation states along single-strand DNA, but also detect DNA methylation patterns on double-strand DNA, since the converted DNA strands are no longer self-complementary and the amplified products can be measured individually.

[0004] Unfortunately, bisulfite treatment results in the degradation of a significant portion of the sample DNA. For example, one undesirable consequence of bisulfite conversion is the disruption of the original target's double-stranded structure due to loss of sequence complementarity. Furthermore, bisulfite conversion can be incomplete unless performed at high temperatures for extended periods, which can often degrade up to approximately 95% of the DNA input. Moreover, as mentioned above, the bisulfite method does not distinguish between 5-mC and the closely related 5-hydroxymethylcytosine (5-hmC), which is another potential epigenetic biomarker.

[0005] Less arduous alternatives for detecting methylated cytosine include enzymatic treatment with 10-11 translocation (TET) dioxygenase and detection of either the enzymatic treatment or the oxidation product. A specific method called TAPS (TET-assisted pyridine-borane sequencing) involves oxidizing methylated cytosine in nucleic acids using TET and a co-catalyst (e.g., Fe(II) ions and alpha-ketoglutaric acid), and then treating the oxidation product with a borane derivative to form dihydrouracil (DHU), which is read as T during sequencing. See Liu, Y., et al. (2019) Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution. Nat. Biotechnol. 37, 424-429.

[0006] Other detection methods also utilize TET, but offer an alternative to borane reduction. For example, the oxidation product can react with malononitrile to form an adduct that can also be read as T during sequencing. See Zhu C., et al., (2017) Single-Cell 5-Formylcytosine Landscapes of Mammalian Early Embryos and ESCs at Single-Base Resolution, Cell Stem Cell, 20:720-731.e5. Malononitrile reacts exclusively with 5-formylcytosine (5-fC). See also U.S. Patent No. 10,519,184. The malononitrile adduct of 5-formylcytosine acts like a thymidine nucleotide and can be distinguished from cytosine. However, the formation of the malononitrile adduct of 5-formylcytosine is time-consuming, taking 12–36 hours, thus increasing the processing time required to prepare sequencing samples.

[0007] There is a need for a rapid and convenient methylation detection assay that can be deployed in clinical laboratories. [Overview of the Initiative]

[0008] The applicant discovered that copper salts can be used to facilitate the addition of malononitrile or similar compounds to 5-formylcytosine. The applicant unexpectedly discovered that a combination of copper salts and a chelating agent selected from bipyridine or phenanthroline can efficiently form 5-formylcytosine adducts in both single-stranded and double-stranded nucleic acid molecules at temperatures lower than those required by prior art methods, in less than approximately one hour. The applicant also discovered that the conversion of 5-formylcytosine to 5-formylcytosine adducts can be efficiently carried out using highly basic and denatured samples (e.g., those containing approximately 10–20 mM NaOH) or slightly alkaline samples containing buffers (e.g., those containing approximately 10 mM TRIS at approximately pH 8). Furthermore, the applicant surprisingly discovered that 5-formylcytosine adducts can be formed from 5-hydroxymethylcytosine in a "one-pot" synthesis within approximately three hours, for example, within approximately two hours, without requiring the purification of intermediates. These and other aspects of the present disclosure are further described herein.

[0009] A first aspect of this disclosure is (a) one or more nucleic acid molecules each having one or more 5-formylcytosine bases, (b) a copper salt, (c) a chelating agent selected from one of bipyridine or phenanthroline, and (d) a compound having formula (I): [ka]

[0010] (In the formula,

[0011] R is an electron-withdrawing group selected from cyano, nitro, C1-C6 alkyl, carboxylic acid ester, unsubstituted carboxamide, C1-C6 alkyl monosubstituted carboxamide, C1-C6 alkyl disubstituted carboxamide, substituted carbonyl moiety, and substituted sulfonyl moiety, wherein the substitution is selected from C1-C6 linear or branched alkyl groups, C4-C6 cycloalkyl groups, phenyl, 5-membered or 6-membered heteroaryl groups, and 5-membered or 6-membered heteroaryl groups with a fused benzene ring.

[0012] The pH of the aforementioned composition is in the range of approximately 7 to approximately 12.

[0013] In some embodiments, R is a cyano, nitro, C1-C6 alkyl, or carboxylic acid ester. In some embodiments, R is cyano or C1-C6 alkyl. In some embodiments, the compound of formula (I) is a malononitrile.

[0014] In some embodiments, the chelating agent is phenanthroline. In some embodiments, the chelating agent is bipyridine.

[0015] In some embodiments, the copper salt forms a complex with the chelating agent. In some embodiments, the ratio of copper salt to chelating agent in the composition is in the range of about 1:3 to about 1:1.75. In some embodiments, the ratio of copper salt to chelating agent in the composition is about 1:2.75. In some embodiments, the ratio of copper salt to chelating agent in the composition is about 1:2.5. In some embodiments, the ratio of copper salt to chelating agent in the composition is about 1:2.25.

[0016] In some embodiments, the chelating agent is 2,2'-bipyridine or a derivative thereof. In some embodiments, the derivative of 2,2'-bipyridine is selected from the group consisting of 4,4'-dimethyl-2,2'-bipyridine, 5,5'-dimethyl-2,2'-bipyridine, 4,4'-diethyl-2,2'-bipyridine, 5,5'-diethyl-2,2'-bipyridine, and 4,4'-dimethoxy-2,2'-bipyridine.

[0017] In some embodiments, the copper salt is selected from the group consisting of Cu(ClO4)2, CuSO4, Cu(ACN)4 triflate, and Cu(OAc)2. In some embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine.

[0018] In some embodiments, one or more nucleic acid molecules are single-stranded. In some embodiments, one or more nucleic acid molecules are double-stranded. In some embodiments, the pH is in the range of about 8 to about 12. In some embodiments, the composition further comprises an N-oxide reagent.

[0019] A second aspect of the present disclosure is a composition comprising (a) one or more nucleic acid molecules each having one or more 5-hydroxymethylcytosine bases, (b) a copper salt, (c) a chelating agent selected from the group consisting of bipyridine or phenanthroline, and (d) an N-oxide reagent, wherein the pH of the composition is in the range of about 7 to about 12, and the N-oxide reagent is selected from the group consisting of ABNO, AZADO, and Me-AZADO.

[0020] In some embodiments, the copper salt forms a complex with the chelating agent. In some embodiments, the ratio of copper salt to chelating agent in the composition is in the range of about 1:3 to about 1:1. In some embodiments, the ratio of copper salt to chelating agent in the composition is about 1:2.5. In some embodiments, the ratio of copper salt to chelating agent in the composition is about 1:2.2. In some embodiments, the ratio of copper salt to chelating agent in the composition is about 1:2.

[0021] In some embodiments, the chelating agent is 2,2'-bipyridine or a derivative thereof. In some embodiments, the bipyridine is selected from the group consisting of 4,4'-dimethyl-2,2'-bipyridine, 5,5'-dimethyl-2,2'-bipyridine, 4,4'-diethyl-2,2'-bipyridine, 5,5'-diethyl-2,2'-bipyridine, 4,4'-dimethoxy-2,2'-bipyridine, and 5,5'-dimethoxy-2,2'-bipyridine.

[0022] In some embodiments, the copper salt is selected from the group consisting of Cu(ClO4)2, CuSO4, Cu(ACN)4 triflate, and Cu(OAc)2. In some embodiments, the N-oxide reagent is AZADO. In some embodiments, the N-oxide reagent is Me-AZADO. In some embodiments, the N-oxide reagent includes ABNO. In some embodiments, the ratio of copper salt to N-oxide in the composition ranges from about 1:0.5 to about 1:0.1. In some embodiments, the ratio of copper salt to N-oxide in the composition is about 1:0.2.

[0023] In some embodiments, this composition further includes a solvent. In some embodiments, the solvent is acetonitrile.

[0024] In some embodiments, the composition includes a base selected from the group consisting of NaOH, KOH, and LiOH. In some embodiments, the composition includes TRIS, TAPSO, TEA, EPPS, tricine, Gly-Gly, Bicine, TABS, AMPSO, CHES, CAPSO, AMP, and CAPS. In some embodiments, the pH of the composition ranges from about 8 to about 12.

[0025] In some embodiments, one or more nucleic acid molecules are single-stranded. In some embodiments, one or more nucleic acid molecules are double-stranded.

[0026] A third aspect of the present disclosure is a method for preparing one or more nucleic acid molecules each having one or more 5-formylcytosine bases, comprising: (a) obtaining a sample comprising one or more nucleic acid molecules each having one or more 5-hydroxymethylcytosine bases; and (b) reacting the obtained sample with a first composition comprising a chelating agent selected from one of a copper salt, bipyridine, or phenanthroline, and an N-oxide reagent, wherein the N-oxide reagent is selected from the group consisting of ABNO, AZADO, and Me-AZADO. In some embodiments, the first composition further comprises at least one base or buffer.

[0027] In some embodiments, the reaction is carried out at a pH in the range of about 7 to about 12.5. In some embodiments, the reaction is carried out at a pH in the range of about 8 to about 12. In some embodiments, the reaction is carried out at a temperature in the range of about 20°C to about 35°C. In some embodiments, the temperature is in the range of about 20°C to about 30°C. In some embodiments, the temperature is in the range of about 25°C to about 30°C. In some embodiments, the reaction is carried out over a period of time in the range of about 30 minutes to about 90 minutes. In some embodiments, this period is about 60 minutes.

[0028] In some embodiments, the ratio of copper salt to chelating agent in the first composition is about 1:2.75. In some embodiments, the ratio of copper salt to chelating agent in the first composition is about 1:2.5. In some embodiments, the ratio of copper salt to chelating agent in the first composition is about 1:2.25. In some embodiments, the chelating agent is 2,2'-bipyridine or a derivative thereof. In some embodiments, the bipyridine is selected from the group consisting of 4,4'-dimethyl-2,2'-bipyridine, 5,5'-dimethyl-2,2'-bipyridine, 4,4'-diethyl-2,2'-bipyridine, 5,5'-diethyl-2,2'-bipyridine, 4,4'-dimethoxy-2,2'-bipyridine, and 5,5'-dimethoxy-2,2'-bipyridine.

[0029] In some embodiments, the copper salt is selected from the group consisting of Cu(ClO4)2, CuSO4, Cu(ACN)4 triflate, and Cu(OAc)2. In some embodiments, the N-oxide is ABNO. In some embodiments, the copper salt is Cu(ACN)4 triflate or Cu(OAc)2, the chelating agent is 2,2'-bipyridine, and the N-oxide is ABNO.

[0030] In some embodiments, the method further includes monitoring the reaction by liquid chromatography and / or mass spectrometry. In some embodiments, the method further includes performing at least one additional downstream reaction after the reaction between the acquired sample and the first composition. In some embodiments, the at least one additional downstream reaction includes producing one or more nucleic acid molecules having one or more adducts of 5-formylcytosine by converting 5-formylcytosine bases in one or more nucleic acid molecules to adducts of 5-formylcytosine. In some embodiments, the adducts of 5-formylcytosine are malononitrile adducts of 5-formylcytosine.

[0031] In some embodiments, the method further comprises contacting one or more nucleic acid molecules having one or more adducts of 5-formylcytosine with a polymerase to obtain one or more amplified nucleic acid molecules, the one or more amplified nucleic acid molecules having a thymine base at a position corresponding to the position of the 5-formylcytosine adduct in each of the one or more nucleic acid molecules having one or more adducts of 5-formylcytosine.

[0032] In some embodiments, the method further includes sequencing one or more amplified nucleic acid molecules. In some embodiments, the sequencing includes next-generation sequencing.

[0033] A fourth aspect of the present disclosure is a method for preparing one or more nucleic acid molecules, each having one or more adducts of 5-formylcytosine, the method comprising: (a) obtaining a sample comprising one or more nucleic acid molecules each having one or more 5-formylcytosine bases; and (b) chelating the obtained sample with a chelating agent selected from one of copper salts, bipyridine or phenanthroline, and a compound having formula (I): [ka]

[0034] (In the formula,

[0035] R is an electron-withdrawing group selected from cyano, nitro, C1-C6 alkyl, carboxylic acid ester, unsubstituted carboxamide, C1-C6 alkyl monosubstituted carboxamide, C1-C6 alkyl disubstituted carboxamide, substituted carbonyl moiety, and substituted sulfonyl moiety, wherein the substitution is selected from C1-C6 linear or branched alkyl groups, C4-C6 cycloalkyl groups, phenyl, 5-membered or 6-membered heteroaryl groups, and 5-membered or 6-membered heteroaryl groups fused with a benzene ring). The method involves reacting the compound with a composition comprising: R is cyano or C1-C6 alkyl. In some embodiments, the compound of formula (I) is malononitrile.

[0036] In some embodiments, the reaction is carried out at a pH in the range of about 7 to about 12.5. In some embodiments, the reaction is carried out at a pH in the range of about 8 to about 12. In some embodiments, the reaction is carried out at a temperature in the range of about 20°C to about 35°C. In some embodiments, the temperature is in the range of about 20°C to about 30°C. In some embodiments, the temperature is in the range of about 25°C to about 30°C. In some embodiments, the reaction is carried out over a period of time in the range of about 30 minutes to about 90 minutes. In some embodiments, this period is about 60 minutes.

[0037] In some embodiments, the chelating agent is phenanthroline. In some embodiments, the chelating agent is bipyridine. In some embodiments, the ratio of copper salt to chelating agent in the first composition is about 1:2.75. In some embodiments, the ratio of copper salt to chelating agent in the first composition is about 1:2.5. In some embodiments, the ratio of copper salt to chelating agent in the first composition is about 1:2.25. In some embodiments, the chelating agent is 2,2'-bipyridine or a derivative thereof. In some embodiments, bipyridine is selected from the group consisting of 4,4'-dimethyl-2,2'-bipyridine, 5,5'-dimethyl-2,2'-bipyridine, 4,4'-diethyl-2,2'-bipyridine, 5,5'-diethyl-2,2'-bipyridine, 4,4'-dimethoxy-2,2'-bipyridine, and 5,5'-dimethoxy-2,2'-bipyridine.

[0038] In some embodiments, the copper salt is selected from the group consisting of Cu(ClO4)2, CuSO4, Cu(ACN)4 triflate, and Cu(OAc)2. In some embodiments, the method further includes monitoring the reaction by liquid chromatography and / or mass spectrometry.

[0039] In some embodiments, the method further comprises contacting one or more nucleic acid molecules having one or more adducts of 5-formylcytosine with a polymerase to obtain one or more amplified nucleic acid molecules, the one or more amplified nucleic acid molecules having a thymine base at a position corresponding to the position of the 5-formylcytosine adduct in each of the one or more nucleic acid molecules having one or more adducts of 5-formylcytosine.

[0040] In some embodiments, the method further includes sequencing one or more amplified nucleic acid molecules. In some embodiments, the sequencing includes next-generation sequencing.

[0041] In some embodiments, the sample obtained is prepared by (i) obtaining a solution containing one or more nucleic acid molecules each having one or more 5-hydroxymethylcytosine bases, and (ii) oxidizing the 5-hydroxymethylcytosine bases of the one or more nucleic acid molecules in the solution to obtain the sample containing the one or more nucleic acid molecules each having one or more 5-formylcytosine bases. In some embodiments, the oxidation involves exposing the solution to a formulation containing a chelating agent selected from one of a copper salt, bipyridine, or phenanthroline, and an N-oxide.

[0042] A fifth aspect of the present disclosure is a method for synthesizing one or more nucleic acid molecules, each comprising one or more adducts of 5-formylcytosine, the method comprising: (a) obtaining a sample comprising one or more nucleic acid molecules each comprising one or more 5-hydroxymethylcytosine bases; (b) contacting the obtained sample with a first composition at a first temperature and for a first period of time to obtain a mixture comprising one or more nucleic acid molecules each comprising one or more 5-formylcytosine bases, wherein the first composition comprises a chelating agent selected from one of a copper salt, bipyridine or phenanthroline, and an N-oxide reagent; and (c) contacting the obtained mixture with a second composition at a second temperature and for a second period of time, wherein the second composition comprises a compound having formula (I): [ka]

[0043] (In the formula,

[0044] The process involves contacting a mixture containing R, which is an electron-withdrawing group selected from cyano, nitro, C1-C6 alkyl, carboxylic acid ester, unsubstituted carboxamide, C1-C6 alkyl monosubstituted carboxamide, C1-C6 alkyl disubstituted carboxamide, substituted carbonyl moiety, and substituted sulfonyl moiety, wherein the substitution is selected from C1-C6 linear or branched alkyl groups, C4-C6 cycloalkyl groups, phenyl, 5-membered or 6-membered heteroaryl groups, and 5-membered or 6-membered heteroaryl groups with a fused benzene ring.

[0045] This yields one or more nucleic acid molecules, each containing one or more adducts of 5-formylcytosine.

[0046] In some embodiments, the obtained mixture is brought into contact with a second composition without first purifying it. In some embodiments, the obtained sample is brought into contact with the first composition at a pH in the range of about 8 to about 12. In some embodiments, the obtained sample is brought into contact with the second composition at a pH in the range of about 8 to about 12.

[0047] In some embodiments, the first temperature is in the range of about 20°C to about 35°C. In some embodiments, the second temperature is in the range of about 20°C to about 35°C. In some embodiments, the first and second temperatures are approximately the same. In some embodiments, the first duration is about 30 minutes to about 90 minutes, and the second duration is about 30 minutes to about 90 minutes.

[0048] In some embodiments, the copper salt is Cu(ACN)4 triflate or Cu(OAc)2, and the chelating agent is 2,2'-bipyridine. In some embodiments, the ratio of the amount of Cu(ACN)4 triflate or Cu(OAc)2 to the amount of 2,2'-bipyridine present in the first composition is in the range of about 1:1 to about 1:3.

[0049] In some embodiments, the compound of formula (I) is malononitrile.

[0050] In some embodiments, the bipyridine is selected from the group consisting of 4,4'-dimethyl-2,2'-bipyridine, 5,5'-dimethyl-2,2'-bipyridine, 4,4'-diethyl-2,2'-bipyridine, 5,5'-diethyl-2,2'-bipyridine, 4,4'-dimethoxy-2,2'-bipyridine, and 5,5'-dimethoxy-2,2'-bipyridine.

[0051] In some embodiments, one or more nucleic acid molecules in the acquired sample are single-stranded nucleic acid molecules. In some embodiments, one or more nucleic acid molecules in the acquired sample are double-stranded nucleic acid molecules. In some embodiments, one or more nucleic acid molecules in the acquired sample are ligated to one or more adapters. In some embodiments, one or more nucleic acid molecules in the acquired sample include one or more barcodes. In some embodiments, one or more barcodes are unique molecular identifiers.

[0052] In some embodiments, the sample obtained is prepared by (i) obtaining a solution containing one or more nucleic acid molecules each having one or more 5-methylcytosine bases, and (ii) oxidizing the 5-methylcytosine bases of the one or more nucleic acid molecules in the solution to obtain the sample containing one or more nucleic acid molecules each having one or more 5-hydroxymethylcytosine bases.

[0053] In some embodiments, the method further comprises contacting one or more nucleic acid molecules having one or more adducts of 5-formylcytosine with a polymerase to obtain one or more amplified nucleic acid molecules, the one or more amplified nucleic acid molecules having a thymine base at a position corresponding to the position of the 5-formylcytosine adduct in each of the one or more nucleic acid molecules having one or more adducts of 5-formylcytosine. In some embodiments, the 5-formylcytosine adduct is a malononitrile adduct of 5-formylcytosine.

[0054] In some embodiments, the method further comprises ligating one or more adapters to one or more nucleic acid molecules having one or more adducts of 5-formylcytosine before contacting the nucleic acid molecules having one or more adducts of 5-formylcytosine with the polymerase. In some embodiments, the polymerase is a DNA polymerase. In some embodiments, the DNA polymerase is a uracil-resistant polymerase.

[0055] In some embodiments, the method further includes sequencing one or more amplified nucleic acid molecules. In some embodiments, the sequencing includes next-generation sequencing.

[0056] In some embodiments, the acquired sample is obtained from a tumor. In some embodiments, the acquired sample is obtained from a specimen suspected of having a tumor.

[0057] A sixth aspect of the present disclosure is a kit comprising (i) a first container containing a copper salt and a chelating agent selected from bipyridine or phenanthroline, and (ii) a second container containing a polymerase. In some embodiments, the kit comprises an adduct-forming reagent having formula (I). [ka]

[0058] (In the formula,

[0059] The third vessel further comprises a third vessel containing R, which is an electron-withdrawing group selected from cyano, nitro, C1-C6 alkyl, carboxylic acid ester, unsubstituted carboxamide, C1-C6 alkyl monosubstituted carboxamide, C1-C6 alkyl disubstituted carboxamide, substituted carbonyl moiety, and substituted sulfonyl moiety, wherein the substitution is selected from C1-C6 linear or branched alkyl groups, C4-C6 cycloalkyl groups, phenyl, 5-membered or 6-membered heteroaryl groups, and 5-membered or 6-membered heteroaryl groups fused with a benzene ring.

[0060] In some embodiments, the ratio of the amount of chelating agent to the amount of copper salt in the first container is in the range of about 2:1. In some embodiments, the polymerase is a heat-stable polymerase.

[0061] In some embodiments, the kit further comprises at least one buffer solution or a strong base.

[0062] A seventh aspect of the present disclosure is a method for detecting one or more epigenetic changes in a target nucleic acid molecule, comprising: (a) obtaining a sample comprising one or more nucleic acid molecules each having one or more 5-hydroxymethylcytosine bases; (b) contacting the obtained sample with a first composition at a first temperature and for a first period of time to obtain a mixture comprising one or more nucleic acid molecules each having one or more 5-formylcytosine bases, wherein the first composition comprises a chelating agent selected from one of a copper salt, bipyridine or phenanthroline, and an N-oxide reagent; and (c) contacting the obtained mixture with a second composition at a second temperature and for a second period of time, wherein the second composition comprises a compound having formula (I): [ka]

[0063] (In the formula,

[0064] R is an electron-withdrawing group selected from cyano, nitro, C1-C6 alkyl, carboxylic acid ester, unsubstituted carboxamide, C1-C6 alkyl monosubstituted carboxamide, C1-C6 alkyl disubstituted carboxamide, substituted carbonyl moiety, and substituted sulfonyl moiety, wherein the substitution is selected from C1-C6 linear or branched alkyl groups, C4-C6 cycloalkyl groups, phenyl, 5-membered or 6-membered heteroaryl groups, and 5-membered or 6-membered heteroaryl groups fused with a benzene ring.

[0065] (d) obtaining one or more nucleic acid molecules each containing one or more adducts of 5-formylcytosine, (d) contacting one or more nucleic acid molecules having one or more adducts of 5-formylcytosine with a polymerase to obtain one or more amplified nucleic acid molecules, wherein one or more amplified nucleic acid molecules have a thymine base at a position corresponding to the position of the 5-formylcytosine adduct in each of the one or more nucleic acid molecules having one or more adducts of 5-formylcytosine, and (e) sequencing the one or more amplified nucleic acid molecules.

[0066] In some embodiments, the adduct of 5-formylcytosine is a malononitrile adduct of 5-formylcytosine.

[0067] In some embodiments, the method further comprises ligating one or more adapters to one or more nucleic acid molecules having one or more adducts of 5-formylcytosine before contacting the nucleic acid molecules having one or more adducts of 5-formylcytosine with the polymerase. In some embodiments, the polymerase is a DNA polymerase. In some embodiments, the DNA polymerase is a uracil-resistant polymerase. In some embodiments, the sequencing includes next-generation sequencing. [Brief explanation of the drawing]

[0068] A patent or application file must include at least one drawing created in color. A copy of the published patent or patent application containing the color drawing will be provided by the Patent Office upon request and payment of the required fees.

[0069] For a general understanding of the features of this disclosure, refer to the drawings. Throughout the drawings, the same reference numbers are used to identify identical elements.

[0070] [Figure 1]Figure showing LC-MS traces of the conversion of 5-formylcytosine nucleotides in nucleic acid molecules to their respective malononitrile adducts under different reaction conditions.

[0071] [Figure 2] Figure showing LC-MS traces of the conversion of 5-formylcytosine nucleotides in nucleic acid molecules to their respective malononitrile adducts under different reaction conditions.

[0072] [Figure 3] Figure showing LC-MS traces of the conversion of 5-formylcytosine nucleotides in nucleic acid molecules to their respective malononitrile adducts under different reaction conditions.

[0073] [Figure 4] Figure showing LC-MS traces of the conversion of 5-formylcytosine nucleotides in nucleic acid molecules to their respective malononitrile adducts under different reaction conditions.

[0074] [Figure 5] Sequencing data comparing the conversion rate of a single 5-formylcytosine base-containing oligonucleotide treated with standard malononitrile in Tris to that of a Cu2+ / 2,2'-bipyridine complex treated with 100 mM malononitrile.

[0075] [Figure 6] This figure shows LC-MS traces of the conversion of 5-hydroxymethylcytosine nucleotides in nucleic acid molecules to their respective malononitrile adducts in a tandem reaction. [Modes for carrying out the invention]

[0076] Unless otherwise explicitly stated, in any method described in the claims herein that includes multiple steps or actions, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described.

[0077] As used herein, the singular forms “a,” “an,” and “the” include multiple referents unless the context explicitly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context explicitly indicates otherwise. The term “includes” is defined inclusively, and “includes A or B” means to include A, B, or A and B.

[0078] Where used herein and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” is interpreted as inclusive; that is, it includes at least one of the number of elements or list of elements, but also includes multiple elements, and optionally any additional items not listed. Only terms that are explicitly indicated as not being such, such as “one of” or “exactly one of” or “consisting of” as used in the claims, refer to including exactly one element of the number of elements or list of elements. In general, the term “or” as used herein shall be interpreted as indicating an exclusive alternative (i.e., “either one or the other but not both”) only when preceded by a term indicating exclusivity, such as “either,” “one of,” “one of” or “exactly one of”. “Consisting of essentially” shall have the usual meaning as used in the field of patent law where used in the claims.

[0079] Terms such as “comprising,” “including,” and “having” are used interchangeably and have the same meaning. Similarly, terms such as “comprises,” “includes,” and “has” are used interchangeably and have the same meaning. Specifically, each term is defined in accordance with the general U.S. Patent Law definition of “comprising,” and is therefore interpreted as an open term meaning “at least the following,” and not to exclude additional features, limitations, aspects, etc. Thus, for example, “a device having components a, b, and c” means that the device includes at least components a, b, and c. Similarly, the wording: “a method including steps a, b, and c” means that the method includes at least steps a, b, and c. Furthermore, while steps and processes may be outlined here in a specific order, those skilled in the art will recognize that the ordering of steps and processes may vary.

[0080] As used herein and in the claims, the phrase “at least one” relating to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of every element specifically listed in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows for the existence of elements other than those specifically identified in the list of elements referred to by the phrase “at least one,” whether or not they are related to those specifically identified elements. Therefore, as a non-restrictive example, “at least one of A and B” (or equivalently, “at least one of A or B,” or equivalently, “at least one of A and / or B”) may, in one embodiment, mean including at least one, optionally more than one A, and no B (and optionally including elements other than B); in another embodiment, mean including at least one, optionally more than one B, and no A (and optionally including elements other than A); and in yet another embodiment, mean including at least one, optionally more than one A, and including at least one, optionally more than one B (and optionally including other elements).

[0081] As used herein, the term “adapter” refers to a nucleotide sequence that can be added to another sequence to impart additional properties to that sequence. An adapter may be single-stranded, double-stranded, or may have both single-stranded and double-stranded portions.

[0082] As used herein, “amplification” refers to a process in which the copy number increases. Amplification can be a process in which replication occurs repeatedly over time, forming multiple copies of a template. Amplification can result in an exponential or linear increase in the copy number as amplification progresses. Exemplary amplification strategies include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), rolling circle replication (RCA), cascade RCA, and nucleic acid-based amplification (NASBA). Amplification can also utilize linear or cyclic templates. Amplification can be carried out under any suitable temperature conditions, such as using thermal cycling or isothermal conditions. Furthermore, amplification can be carried out in an amplification mixture (or reagent mixture), which is any composition capable of amplifying a nucleic acid target if the nucleic acid target is present in the mixture. PCR amplification relies on repeated heating and cooling cycles (i.e., thermal cycling) to achieve consecutive replication rounds. PCR can be performed by thermal cycling between two or more temperature setpoints, such as a higher denaturation temperature and a lower annealing / extension temperature, or, in particular, between three or more temperature setpoints, such as a higher denaturation temperature, a lower annealing temperature, and an intermediate extension temperature. PCR can be performed using a thermally stable polymerase, such as Taq DNA polymerase. PCR results in an exponential increase in the amount of product amplicon over a series of cycles. PCR is described, for example, in U.S. Patent Nos. 4,683,202, 4,683,195, 4,000,159, 4,965,188, and 5,176,995, the respective disclosures of which are incorporated herein by reference in their entirety.

[0083] As used herein, terms such as “biological sample,” “tissue sample,” and “specimen” refer to any sample containing biomolecules (such as proteins, peptides, nucleic acids, lipids, carbohydrates, or combinations thereof) obtained from any organism, including viruses. Examples of other organisms include mammals (veterinary animals such as humans, cats, dogs, horses, cattle, and pigs, as well as laboratory animals such as mice, rats, and primates), insects, annelids, arachnids, marsupials, reptiles, amphibians, bacteria, and fungi. Biological samples include tissue samples (such as tissue sections and needle biopsies), cell samples (such as cytological smears like Pap smears or blood smears, or cell samples obtained by microdissection), or cell fractions, fragments, or organelles (such as those obtained by lysing cells and separating their components by centrifugation). Other examples of biological samples include blood, serum, urine, semen, feces, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (e.g., obtained by surgical biopsy or needle biopsy), nipple aspirate, earwax, milk, vaginal fluid, saliva, swabs (such as oral swabs), or any material containing biomolecules derived from the initial biological sample. In certain embodiments, the term “biological sample” as used herein refers to a sample (such as a homogenized or liquefied sample) prepared from a tumor or a portion thereof obtained from the subject.

[0084] As used herein, "a" and "b" are integers. a -C b"C1-C4 alkyl" refers to the number of carbon atoms in an alkyl, alkenyl, or alkynyl group, or the number of carbon atoms in a cycloalkyl, cycloalkenyl, cycloalkynyl, or aryl ring, or the total number of carbon atoms and heteroatoms in a heteroalkyl, heterocyclyl, heteroaryl, or heteroalicyl group. That is, an alkyl, alkenyl, alkynyl, cycloalkyl ring, cycloalkenyl ring, cycloalkynyl ring, aryl ring, heteroaryl ring, or heteroalicyl ring can contain "a" to "b" carbon atoms. Therefore, for example, a "C1-C4 alkyl" group refers to all alkyl groups having 1 to 4 carbon atoms, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2, CH3CH2CH(CH3)-, and (CH3)3C-. If "a" and "b" are not specified with respect to alkyl, alkenyl, alkynyl, cycloalkylcycloalkenyl, cycloalkynyl, aryl, heteroaryl, or heteroalicyl group, the broadest range described in those definitions is assumed.

[0085] As used herein, the term “alkyl” includes linear alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched alkyl groups (e.g., isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl-substituted cycloalkyl groups, and saturated aliphatic groups including cycloalkyl-substituted alkyl groups. The term alkyl further includes alkyl groups which may further include oxygen, nitrogen, sulfur, or phosphorus atoms substituting one or more carbon atoms in the hydrocarbon backbone. In certain embodiments, linear or branched alkyl groups have 50 or fewer carbon atoms in their backbone (e.g., C1-C1 in the case of linear groups). 50 In the case of a branched chain, C1-C 50 ).

[0086] As used herein, the terms “cycloalkyl” and “heterocycloalkyl,” either by themselves or in combination with other terms, mean the cyclic versions of “alkyl” and “heteroalkyl,” respectively, unless otherwise specified. In some embodiments, “heterocycloalkyl” is also referred to as “heterocyclic” group or moiety. Cycloalkyl and heterocycloalkyl are non-aromatic. Cycloalkyl and heterocycloalkyl may be further substituted with any of the substituents described herein, for example.

[0087] As used herein, “derivative” is used in accordance with its obvious and ordinary meaning in chemistry and biology, and refers to a compound that is structurally similar to another compound (i.e., a so-called “reference” compound), but differs in composition, for example, by substituting one atom with an atom of a different element, or in the presence of a particular functional group, or by substituting one functional group with another, or in the absolute stereochemistry of one or more chiral centers of the reference compound. Thus, a derivative is a compound that is similar or equivalent to a reference compound in function and appearance, but is neither similar nor equivalent in structure or origin.

[0088] As used herein, the term “next-generation sequencing” refers to sequencing technologies that offer high-throughput sequencing compared to conventional Sanger electrophoresis and capillary electrophoresis-based approaches, where the sequencing process is carried out in parallel, for example, generating thousands or millions of relatively small sequence reads at once. Some examples of next-generation sequencing technologies include, but are not limited to, synthesis sequencing, ligation sequencing, and hybridization sequencing. These technologies produce shorter reads (ranging from approximately 25 to 500 bp) but generate hundreds of thousands or millions of reads in a relatively short time. Examples of such sequencing devices available from Illumina (San Diego, California) include, but are not limited to, iSEQ, MiniSEQ, MiSEQ, NextSEQ, and NoveSEQ.

[0089] Illumina's next-generation sequencing technology is expected to enable rapid sequencing using clonal amplification and synthesis sequencing (SBS) chemistry. This process identifies DNA bases while simultaneously incorporating them into the nucleic acid strand. Each base emits a unique fluorescent signal when added to the growing strand, which is used to determine the order of the DNA sequence. A non-exclusive example of a sequencing device available from ThermoFisher Scientific (Waltham, Massachusetts) is the Ion Personal Genome Machine (PGM) system.

[0090] Ion Torrent sequencing is thought to measure the direct release of H+ (protons) from the incorporation of individual bases by DNA polymerase. A non-limiting example of a sequencing device available from Pacific Biosciences (Menlo Park, California) is the PacBio Sequel System. A non-limiting example of a sequencing device available from Roche (Pleasanton, California) is the Roche 454. Next-generation sequencing methods sometimes include nanopore sequencing. Generally, three nanopore sequencing approaches have been pursued: strand sequencing, where DNA bases are identified as they sequentially pass through nanopores; exonuclease-based nanopore sequencing, where nucleotides are enzymatically cleaved one by one from the DNA molecule and monitored as they are captured and pass through nanopores; and synthetic nanopore sequencing (SBS) approaches, where identifiable polymer tags are attached to nucleotides and registered in nanopores during enzymatic DNA synthesis. What all these methods have in common is the need to precisely control the reaction rate so that each base is determined in order.

[0091] Strand sequencing requires a method to slow the passage of DNA through nanopores and decode multiple bases within the channel; for this purpose, a ratchet approach utilizing molecular motors has been developed. Exonuclease-based sequencing requires the release of each nucleotide close enough to the pore to ensure its capture and passage through the pore, at a rate slow enough to obtain a valid ionic current signal. Furthermore, both methods rely on the distinction between four native bases, two relatively similar purines, and two similar pyrimidines.

[0092] The nanopore SBS approach utilizes synthetic polymer tags attached to nucleotides that are specifically designed to produce unique and readily distinguishable ion current blockage signatures for sequencing. In some embodiments, sequencing of nucleic acid molecules by nanopore sequencing involves preparing a nanopore sequencing complex and determining a polynucleotide sequence. Methods for preparing nanopores and nanopore sequencing are described in U.S. Patent Application Publication 2017 / 0268052, as well as International Publications 2014 / 074727, 2006 / 028508, 2012 / 083249, and 2014 / 074727, which are incorporated herein by reference in their entirety. In some embodiments, tagged nucleotides may be used in determining polynucleotide sequences (see, for example, International Publication No. 2020 / 131759, International Publication No. 2013 / 191793, and International Publication No. 2015 / 148402, which are incorporated herein by reference in their entirety).

[0093] Analysis of sequencing-generated data is generally performed using software and / or statistical algorithms that perform various data transformations, such as the conversion of signal emissions to base calls, and the conversion of base calls to consensus sequences of nucleic acid templates. Such software, statistical algorithms, and the use of such algorithms are described in detail in U.S. Patent Application Publications 2009 / 0024331, 2017 / 0044606, and International Publication 2018 / 034745, which are incorporated herein by reference in their entirety.

[0094] As used herein, the terms “nucleic acid” or “nucleic acid molecule” refer to high molecular weight biochemical macromolecules consisting of nucleotide chains that transmit genetic information. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The monomers that make up nucleic acids are called nucleotides. Each nucleotide consists of three components: a nitrogen-containing heterocyclic base, either a purine or pyrimidine (also known as a nucleic acid base), and a pentose sugar. The structure of the sugar in the nucleotide differs depending on the type of nucleic acid. DNA contains 2-deoxyribose, and RNA contains ribose.

[0095] In this specification, “polymerase” refers to an enzyme that catalyzes the process of nucleic acid replication. More specifically, DNA polymerase catalyzes the polymerization of deoxyribonucleotides along a DNA strand, “reading” the DNA strand and using it as a template. The newly polymerized molecule is complementary to the template strand and identical to the template partner strand.

[0096] As used herein, the term “sequencing” refers to the determination of the order and position of bases in a nucleic acid molecule. More specifically, the term “sequencing” refers to a biochemical method for determining the order of nucleotide bases, adenine, guanine, cytosine, and thymine, in DNA oligonucleotides. Sequencing, as used herein, may include, but is not limited to, parallel sequencing or any other sequencing method known to those skilled in the art, such as strand termination, rapid DNA sequencing, wandering-spot analysis, Maxam-Gilbert sequencing, dye-terminator sequencing, or any other modern automated DNA sequencing apparatus.

[0097] overview

[0098] This disclosure provides compositions for converting 5-hydroxymethylcytosine bases in target nucleic acid molecules to 5-formylcytosine bases. This disclosure also provides compositions for converting 5-formylcytosine bases in target nucleic acid molecules to various adducts of 5-formylcytosine, such as malononitrile adducts of 5-formylcytosine. Each composition of this disclosure utilizes a mixture of a copper salt and a chelating agent selected from bipyridine or phenanthroline.

[0099] This disclosure provides a method for efficiently synthesizing nucleic acid molecules containing one or more 5-formylcytosine bases from target nucleic acid molecules containing one or more 5-hydroxymethylcytosine bases. This disclosure also provides a method for efficiently synthesizing nucleic acid molecules containing one or more 5-formylcytosine adducts from target nucleic acid molecules containing one or more 5-formylcytosine bases. Furthermore, this disclosure provides an efficient "one-pot" synthesis method for forming nucleic acid molecules containing one or more 5-formylcytosine adducts from target nucleic acid molecules containing one or more 5-hydroxymethylcytosine bases. Advantageously, the "one-pot" synthesis method of this disclosure does not require the purification of any intermediates and enables the rapid construction of nucleic acid libraries. Furthermore, the "one-pot" synthesis method is rapid, enabling the formation of 5-formylcytosine adducts within approximately 3 hours, for example, within approximately 2 hours, for example, within approximately 90 minutes.

[0100] This disclosure also provides a method for detecting epigenetic modifications in target nucleic acid molecules, such as epigenetic modifications characterized by methylation of cytosine at position 5 (e.g., 5-methylcytosine; 5-hydroxymethylcytosine). In some embodiments, the method for detecting epigenetic modifications includes converting the cytosine bases in a nucleic acid molecule characterized by methylation at position 5 to adducts of 5-formylcytosine, and subsequently amplifying the nucleic acid molecule containing the adducts of 5-formylcytosine in the presence of a polymerase, which reads one or more adducts of 5-formylcytosine as thymine during amplification. The amplified product may then be sequenced by next-generation sequencing or the like. A diagnostic decision may then be made based on the data from the sequencing.

[0101] This disclosure also provides a method for amplifying and / or sequencing a target nucleic acid molecule containing one or more epigenetic modifications. In some embodiments, the amplification method first requires converting the target nucleic acid molecule containing one or more epigenetic modifications into a nucleic acid molecule containing one or more 5-formylcytosine bases. In some embodiments, the amplification method then requires converting one or more 5-formylcytosine bases of the nucleic acid molecule into adducts of 5-formylcytosine. In some embodiments, one or more adapters are ligated to the nucleic acid molecule before amplification, and the adapters may include one or more molecular barcodes, one or more multiple identifiers (MIDs), and / or one or more unique molecular identifiers (UIDs). By amplifying the nucleic acid molecule containing one or more adducts of 5-formylcytosine in the presence of polymerase, the polymerase reads one or more adducts of 5-formylcytosine as thymine. The amplified target nucleic acid molecule may then be sequenced using next-generation sequencing technology or the like.

[0102] These and other embodiments are described herein.

[0103] composition

[0104] As described above, this disclosure relates to compositions for use in the preparation of target nucleic acid molecules comprising one or more 5-formylcytosine bases or 5-formylcytosine adducts. Generally, the compositions of this disclosure comprise a target nucleic acid molecule, a copper salt, a chelating agent selected from one of bipyridine or phenanthroline, an N-oxide reagent, an adduct-forming reagent, a base, a buffer, a solvent, and the like.

[0105] target nucleic acid molecule

[0106] In some embodiments, the compositions of the present disclosure include one or more target nucleic acid molecules derived from a biological sample. In some embodiments, the target nucleic acid molecule includes one or more 5-hydroxymethylcytosine bases. In some embodiments, the target nucleic acid molecule includes one or more 5-formylcytosine bases.

[0107] In some embodiments, the target nucleic acid molecule is single-stranded. In some embodiments, the target nucleic acid molecule is single-stranded and contains one or more 5-hydroxymethylcytosine (5-hmC) bases, for example, one 5-hydroxymethylcytosine base, for example, two 5-hydroxymethylcytosine bases, for example, three 5-hydroxymethylcytosine bases, for example, four 5-hydroxymethylcytosine bases, for example, five or more 5-hydroxymethylcytosine bases. In some embodiments, the target nucleic acid molecule is single-stranded and contains one or more 5-formylcytosine (5-fC) bases, for example, one 5-formylcytosine base, for example, two 5-hydroxymethylcytosine bases, for example, three 5-formylcytosine bases, for example, four 5-formylcytosine bases, for example, five or more 5-formylcytosine bases.

[0108] In some embodiments, the target nucleic acid molecule is double-stranded. In some embodiments, the target nucleic acid molecule is double-stranded and contains one or more 5-hydroxymethylcytosine bases, e.g., one 5-hydroxymethylcytosine base, e.g., two 5-hydroxymethylcytosine bases, e.g., three 5-hydroxymethylcytosine bases, e.g., four 5-hydroxymethylcytosine bases, e.g., five or more 5-hydroxymethylcytosine bases. In some embodiments, the target nucleic acid molecule is double-stranded and contains one or more 5-formylcytosine bases, e.g., one 5-formylcytosine base, e.g., two 5-hydroxymethylcytosine bases, e.g., three 5-formylcytosine bases, e.g., four 5-formylcytosine bases, e.g., five or more 5-formylcytosine bases. In some embodiments, the 5-hydroxymethylcytosine bases are on one or both strands of the double-stranded target nucleic acid molecule.

[0109] In some embodiments, the sample may be obtained from any source containing a target nucleic acid sequence having one or more cytosine residues of interest, such as tissue (including tumor tissue or FFPE tissue), blood, skin, swab (e.g., buccal, vaginal), urine, saliva, etc. In some embodiments, the sample is derived from the subject or patient. In some embodiments, the sample may include a fragment of solid tissue or a tumor sample derived from the subject or patient, for example, by biopsy. In this specification, the term “tumor sample” includes a sample prepared from a tumor, or a sample prepared from a sample that may contain or is suspected of containing cancer cells, or a sample being tested for the potential presence of cancer cells, such as a lymph node. In this specification, “tumor” means a mass or neoplasm. A neoplasm itself is defined as an abnormal new cell growth that usually grows more rapidly than normal cells, continues to grow if left untreated, and sometimes causes damage to adjacent structures. Tumor size can vary greatly. Tumors may be solid or filled with fluid. Tumors can refer to benign (not malignant, generally harmless) or malignant (metastatic) growths. Some tumors may contain benign tumor cells (such as carcinoma in situ) and simultaneously malignant cancer cells (such as adenocarcinoma). This should be understood as including neoplasms present in multiple locations throughout the body. Therefore, for the purposes of this disclosure, tumors include primary tumors, lymph nodes, lymphoid tissue, and metastatic tumors.

[0110] Methods for isolating nucleic acids from biological samples and / or purifying samples are known, for example, as described by Sambrook, and several kits are commercially available (e.g., high-purity RNA isolation kits, high-purity viral nucleic acid kits, and MagNA pure LC whole nucleic acid isolation kits, DNA isolation kits for cells and tissues, DNA isolation kits for mammalian blood, high-purity FFPET DNA isolation kits, available from Roche). In the context of the methods of this disclosure, genomic DNA can be collected, purified, and / or isolated.

[0111] It is understood that nucleic acid molecules can be isolated from biological samples using any of the various procedures known in the art, such as the MagMAX® DNA Multi-Sample Ultra Kit (Applied Biosystems, Thermo Fisher Scientific), the MagMAX® Express-96 Magnetic Particle Processor and the KingFisher® Flex Magnetic Particle Processor (Thermo Fisher Scientific), the RecoverAll® Total Nucleic Acid Isolation Kit for FFPE and the PureLink® FFPE RNA Isolation Kit (Ambion®, Thermo Fisher Scientific), the ABI Prism® 6100 Nucleic Acid PrepStation and the ABI Prism® 6700 Automated Nucleic Acid Workstation (Applied Biosystems, Thermo Fisher Scientific). It will be understood that nucleic acid molecules derived from biological samples may be cleaved or sheared before analysis, including the use of mechanical force, sonication, restriction endonuclease cleavage, or any other method known in the art.

[0112] In some embodiments, the length of the target nucleic acid molecule is in the range of about 10 mer to about 5000 mer. In some embodiments, the length of the target nucleic acid molecule is in the range of about 10 mer to about 2500 mer. In some embodiments, the length of the target nucleic acid molecule is in the range of about 10 mer to about 2000 mer. In some embodiments, the length of the target nucleic acid molecule is in the range of about 10 mer to about 1000 mer. In some embodiments, the length of the target nucleic acid molecule is in the range of about 10 mer to about 500 mer. In some embodiments, the length of the target nucleic acid molecule is in the range of about 10 mer to about 250 mer. In other embodiments, the length of the target nucleic acid molecule is in the range of about 15 mer to about 150 mer. In yet another embodiment, the length of the target nucleic acid molecule is in the range of about 15 mer to about 100 mer. In yet another embodiment, the length of the target nucleic acid molecule is in the range of about 15 mer to about 60 mer.

[0113] In some embodiments, the target nucleic acid molecule contains at least one cytosine analog per chain, for example, one or more 5-hydroxymethylcytosine or one or more 5-formylcytosine. In some embodiments, the target nucleic acid molecule contains at least two cytosine analogs per chain. In some embodiments, the target nucleic acid molecule contains at least three cytosine analogs per chain. In some embodiments, the target nucleic acid molecule contains at least four cytosine analogs per chain.

[0114] copper salt

[0115] In some embodiments, the compositions of the present disclosure comprise one or more copper salts. In some embodiments, the copper salt may be any salt of copper in any of the common oxidation states of copper, including monovalent copper salts, Cu(I), and divalent copper salts, Cu(II). In some embodiments, the copper salt is selected from copper halides, copper nitrate, copper acetate, copper sulfate, copper formate, and copper oxide. Examples of copper salts include copper(I) oxide, copper(I) chloride, copper(I) iodide, copper(I) cyanide, copper(I) thiocyanate, copper(I) sulfate, copper(I) sulfide, copper(I) acetylide, copper(I) bromide, copper(I) fluoride, Cu(ACN)4 triflate, copper(I) hydroxide, copper(I) hydride, copper(I) nitrate, copper(I) phosphide, copper(I) thiophene-2-carboxylate, copper(I) t-butoxide, copper(II) sulfate, copper(II) chloride, copper(II) hydroxide, copper(II) sulfate, copper(II) oxide, copper(II) acetate, copper(II) fluoride, copper(II) bromide, copper(II) carbonate, copper(II) hydroxide, and copper(II) chlorate. Examples include, but are not limited to, copper(II) arsenate, copper(II) azide, copper(II) acetylacetone, copper(II) aspirate, copper(II) cyanurate, copper(II) glycinate, copper(II) phosphate, copper(II) perchlorate, copper(II) selenite, copper(II) lenite, copper(II) sulfide, copper(II) thiocyanate, copper(II) triflate, copper(II) tetrafluoroborate, copper(II) acetate, triarsenite, copper(II) benzoate, copper(II) arsenite, copper(II) chromate, copper(II) gluconate, copper(II) peroxide, copper(II) usnate, and copper(II) oxychloride.

[0116] In some embodiments, the copper salt is copper(II) perchlorate (Cu(ClO4)2), copper(II)CuSO4, copper(II)Cu(OAc)2, copper(II)CuCl2, copper(I)Cu(ACN)4 triflate, copper(I)CuBr, or copper(I)CuCl chloride.

[0117] Oxidizing agent

[0118] In some embodiments, the compositions of the present disclosure also include one or more oxidizing agents. Oxidation of alcohols to corresponding carbonyl compounds is a fundamental transformation in organic chemistry. The present disclosure converts the alcohol portion of a 5-hydroxymethylcytosine base to its respective carbonyl portion, thereby providing a 5-formylcytosine base. In some embodiments, the oxidizing agent used to achieve this transformation is an amine oxide, also known as an amine N-oxide or N-oxide.

[0119] In some embodiments, the N-oxide is 2,2,6,6-tetramethylpiperidinyl oxy or 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). In other embodiments, the N-oxide is 9-azabicyclo[3.3.1]nonane N-oxyl (ABNO). In yet another embodiment, the N-oxide is 2-azadamantane N-oxyl (AZADO) or its derivatives or analogues (e.g., 1-Me-AZADO, 2-azadamantane N-oxyl-AZADO, 1,3-dimethyl-azado, 1,3-dimethyl-azado, 9-azabicyclo[3.3.1]nonane N-oxyl, and 9-azanoradamatane N-oxyl-azado).

[0120] Adductor formation reagents

[0121] In some embodiments, the compositions of the present disclosure include a compound having formula (I) (referred to herein as “adduct-forming reagent”) [ka]

[0122] (In the formula,

[0123] R is an electron-withdrawing group selected from cyano, nitro, C1-C6 alkyl, carboxylic acid ester, unsubstituted carboxamide, C1-C6 alkyl-monosubstituted carboxamide, C1-C6 alkyl-disubstituted carboxamide, substituted carbonyl moiety, and substituted sulfonyl moiety, and the substitution is selected from C1-C6 straight or branched alkyl group, C4-C6 cycloalkyl group, phenyl, 5- or 6-membered heteroaryl, and 5- or 6-membered heteroaryl fused with a benzene ring).

[0124] In some embodiments, R is cyano. In some embodiments, R is methyl or ethyl. In still other embodiments, R is a carboxylic acid ester.

[0125] Further suitable "adduct-forming reagents" are described in U.S. Patent No. 11,293,050, the disclosure of which is incorporated herein by reference in its entirety.

[0126] Chelating agent

[0127] In some embodiments, the compositions of the present disclosure utilize a chelating agent selected from one of bipyridine or phenanthroline. Suitable bipyridines include 2,2'-bipyridine and its derivatives. In some embodiments, suitable bipyridines contain one substituent or two substituents. In other embodiments, suitable bipyridines contain more than two substituents. In some embodiments, the disubstituted bipyridine may be symmetric or asymmetric and has the formula (IIA):

Chemical formula

[0128] wherein R1, R2, R3, and R4 are independently H, methyl, -CHR 7 , -OH, -OMe, -OCH2R 7 , -NH2, -NHR 7 , -NR 7 R 7, and -SO3, each R 7 These are independently C1-C4 alkyl groups.

[0129] In some embodiments, the chelating agent is phenanthroline having formula (IIB): [ka]

[0130] In the formula, R1, R2, R3, R4, R5, and R6 are independently H, methyl, and -CHR. 7 -OH, -OMe, -OCH2R 7 -NH2, -NHR 7 , -NR 7 R 7 , and -SO3, each R 7 These are independently C1-C4 alkyl groups.

[0131] Non-limiting examples of derivatives of 2,2'-bipyridine include, but are not limited to, 4,4'-dimethyl-2,2'-bipyridine, 5,5'-dimethyl-2,2'-bipyridine, 4,4'-diethyl-2,2'-bipyridine, 5,5'-diethyl-2,2'-bipyridine, 4,4'-dimethoxy-2,2'-bipyridine, and 5,5'-dimethoxy-2,2'-bipyridine.

[0132] In some embodiments, phenanthroline is 1,10-phenanthroline.

[0133] buffer solution

[0134] In some embodiments, the compositions of the present disclosure include one or more buffers. Non-limiting examples of preferred buffers include TRIS((tris(hydroxymethyl)aminomethane), or Examples include 2-amino-2-(hydroxymethyl)propane-1,3-diol; HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); MOPS (3-(N-morpholino)propanesulfonic acid); TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid); TEST (2-[[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino]ethanesulfonic acid); and phosphates.

[0135] base

[0136] Suitable bases include, but are not limited to, strong bases such as NaOH, LiOH, KOH, RbOH, CsOH, Ca(OH)2, Sr(OH)2, and Ba(OH)2. In some embodiments, the base is NaOH.

[0137] Composition for converting 5-fC bases into their respective adducts

[0138] A first composition of this disclosure (referred to herein as the “5-fC adduct forming composition”) is adapted for preparing nucleic acid molecules containing one or more adducts of 5-formylcytosine (hereinafter referred to as the “5-formylcytosine adduct” or “5-fC adduct”) from nucleic acid molecules containing one or more 5-formylcytosine bases.

[0139] In some embodiments, the 5-fC adduct-forming composition comprises one or more nucleic acid molecules each having one or more 5-formylcytosine bases, a chelating agent selected from one of a copper salt, bipyridine, or phenanthroline, and an adduct-forming reagent having formula (I), wherein the copper salt, chelating agent, and adduct-forming reagent having formula (I) may be selected from any of those enumerated herein. In some embodiments, the copper salt forms a complex with the chelating agent. In some embodiments, the 5-fC adduct-forming composition comprises a solvent such as acetonitrile.

[0140] In some embodiments, the target nucleic acid molecule in the 5-fC adduct-forming composition is single-stranded or double-stranded and includes one 5-formylcytosine base, at least two 5-formylcytosine bases, at least three 5-formylcytosine bases, at least four 5-formylcytosine bases, at least five 5-formylcytosine bases, at least six 5-formylcytosine bases, and so on.

[0141] In some embodiments, the 5-fC adduct-forming composition has a pH in the range of about 7 to about 12.5. In some embodiments, the 5-fC adduct-forming composition has a pH in the range of about 8 to about 12.0. In some embodiments, the 5-fC adduct-forming composition has a pH of about 8. In some embodiments, the 5-fC adduct-forming composition has a pH of about 8.5. In some embodiments, the 5-fC adduct-forming composition has a pH of about 9. In some embodiments, the 5-fC adduct-forming composition has a pH of about 9.5. In some embodiments, the 5-fC adduct-forming composition has a pH of about 10. In some embodiments, the 5-fC adduct-forming composition has a pH of about 10.5. In some embodiments, the 5-fC adduct-forming composition has a pH of about 11. In some embodiments, the 5-fC adduct-forming composition has a pH of about 11.5. In some embodiments, the 5-fC adduct-forming composition has a pH of about 12. In some embodiments, the 5-fC adduct-forming composition has a pH of about 12.5.

[0142] In some embodiments, the 5-fC adduct-forming composition contains a strong base. In some embodiments, the strong base is selected from the group consisting of NaOH, KOH, and LiOH. In some embodiments, an amount of base is added such that the pH of the composition is about 7 to about 12.5, for example, about 8 to about 12.5. As an example, a preferred composition for converting one or more 5-fC bases to a 5-formylcytosine base contains about 2 mM to about 20 mM NaOH. In some embodiments, a preferred composition for converting one or more 5-fC bases to a 5-fC adduct contains about 2 mM to about 10 mM NaOH.

[0143] In other embodiments, the 5-fC adduct-forming composition comprises a buffer. In some embodiments, the buffer is TRIS. For example, a preferred composition for converting one or more 5-hydroxymethylcytosine bases to 5-formylcytosine bases comprises about 0 mM to about 50 mM of TRIS. Another example is a preferred composition for converting one or more 5-hydroxymethylcytosine bases to 5-formylcytosine bases comprising about 1 mM to about 50 mM of TRIS. Yet another example is a preferred composition for converting one or more 5-hydroxymethylcytosine bases to 5-formylcytosine bases comprising about 10 mM to about 50 mM of TRIS. Yet another example is a preferred composition for converting one or more 5-hydroxymethylcytosine bases to 5-formylcytosine bases comprising about 20 mM to about 50 mM of TRIS.

[0144] In some embodiments, the amount of copper salt in the 5-fC adduct-forming composition is in the range of about 0.5 mM to about 8 mM. In some embodiments, the amount of copper salt in the 5-fC adduct-forming composition is in the range of about 0.5 mM to about 6 mM. In some embodiments, the amount of copper salt in the 5-fC adduct-forming composition is in the range of about 0.5 mM to about 4 mM. In some embodiments, the amount of copper salt in the 5-fC adduct-forming composition is in the range of about 1 mM to about 3 mM. In some embodiments, the amount of copper salt in the 5-fC adduct-forming composition is about 1 mM. In some embodiments, the amount of copper salt in the 5-fC adduct-forming composition is about 1.5 mM. In some embodiments, the amount of copper salt in the 5-fC adduct-forming composition is about 1.75 mM. In some embodiments, the amount of copper salt in the 5-fC adduct-forming composition is about 2 mM. In some embodiments, the amount of copper salt in the 5-fC adduct-forming composition is about 2.25 mM. In some embodiments, the amount of copper salt in the 5-fC adduct-forming composition is about 2.5 mM. In some embodiments, the amount of copper salt in the 5-fC adduct-forming composition is about 3 mM.

[0145] In some embodiments, the amount of chelating agent in the 5-fC adduct-forming composition is in the range of about 1 mM to about 20 mM. In some embodiments, the amount of chelating agent in the 5-fC adduct-forming composition is in the range of about 1 mM to about 15 mM. In some embodiments, the amount of chelating agent in the 5-fC adduct-forming composition is in the range of about 1 mM to about 10 mM. In some embodiments, the amount of chelating agent in the 5-fC adduct-forming composition is in the range of about 2 mM to about 8 mM. In some embodiments, the amount of chelating agent in the 5-fC adduct-forming composition is in the range of about 2 mM to about 6 mM. In some embodiments, the amount of chelating agent in the 5-fC adduct-forming composition is about 1 mM. In some embodiments, the amount of chelating agent in the 5-fC adduct-forming composition is about 2 mM. In some embodiments, the amount of chelating agent in the 5-fC adduct-forming composition is about 2.5 mM. In some embodiments, the amount of chelating agent in the 5-fC adduct-forming composition is about 3 mM. In some embodiments, the amount of chelating agent in the 5-fC adduct-forming composition is about 3.5 mM. In some embodiments, the amount of chelating agent in the 5-fC adduct-forming composition is about 4 mM. In some embodiments, the amount of chelating agent in the 5-fC adduct-forming composition is about 4.5 mM. In some embodiments, the amount of chelating agent in the 5-fC adduct-forming composition is about 5 mM. In some embodiments, the amount of chelating agent in the 5-fC adduct-forming composition is about 5.5 mM. In some embodiments, the amount of chelating agent in the 5-fC adduct-forming composition is about 6 mM.

[0146] In some embodiments, the ratio of copper salt to chelating agent in the 5-fC adduct-forming composition is in the range of about 1:5 to about 1:1. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC adduct-forming composition is in the range of about 1:4 to about 1:1. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC adduct-forming composition is in the range of about 1:3 to about 1:1. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC adduct-forming composition is about 1:2.8. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC adduct-forming composition is about 1:2.7. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC adduct-forming composition is about 1:2.6. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC adduct-forming composition is about 1:2.5. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC adduct-forming composition is about 1:2.4. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC adduct-forming composition is about 1:2.3. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC adduct-forming composition is about 1:2.2. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC adduct-forming composition is about 1:2. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC adduct-forming composition is about 1:1.8. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC adduct-forming composition is about 1:1.6. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC adduct-forming composition is about 1:1.4. In some embodiments, the ratio of the amount of copper salt to the amount of chelating agent in the 5-fC adduct-forming composition is about 1:1.2. In some embodiments, the ratio of the amount of copper salt to the amount of chelating agent in the conversion composition is about 1:1.

[0147] In some embodiments, the copper salt in the 5-fC adduct-forming composition is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the chelating agent is 2,2'-bipyridine or a derivative thereof. In other embodiments, the copper salt is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the chelating agent is 2,2'-bipyridine or a derivative thereof, with a ratio of copper salt to chelating agent of about 1:2.2. In other embodiments, the copper salt in the 5-fC adduct-forming composition is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, the chelating agent is 2,2'-bipyridine or a derivative thereof, and the ratio of the amount of copper salt to the amount of chelating agent is about 1:1.8.

[0148] In some embodiments, the copper salt in the 5-fC adduct-forming composition is Cu(ClO4)2, and the chelating agent is 2,2'-bipyridine. In other embodiments, the copper salt in the 5-fC adduct-forming composition is Cu(ClO4)2, and the chelating agent is 2,2'-bipyridine or a derivative thereof, with a ratio of copper salt to chelating agent of about 1:2.2. In other embodiments, the copper salt in the 5-fC adduct-forming composition is Cu(ClO4)2, and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of about 1:2. In other embodiments, the copper salt in the 5-fC adduct-forming composition is Cu(ClO4)2, and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of about 1:1.8.

[0149] In some embodiments, the copper salt in the 5-fC adduct-forming composition is Cu(ACN)4 triflate, and the chelating agent is 2,2'-bipyridine. In other embodiments, the copper salt in the 5-fC adduct-forming composition is Cu(ACN)4 triflate, and the chelating agent is 2,2'-bipyridine or a derivative thereof, with a ratio of copper salt to chelating agent of about 1:2.2. In other embodiments, the copper salt in the 5-fC adduct-forming composition is Cu(ACN)4 triflate, and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of about 1:2. In other embodiments, the copper salt in the 5-fC adduct-forming composition is Cu(ACN)4 triflate, and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of about 1:1.8.

[0150] In some embodiments, the copper salt in the 5-fC adduct-forming composition is Cu(OAc)2, and the chelating agent is 2,2'-bipyridine. In other embodiments, the copper salt in the 5-fC adduct-forming composition is Cu(OAc)2, and the chelating agent is 2,2'-bipyridine or a derivative thereof, with a ratio of copper salt to chelating agent of about 1:2.2. In other embodiments, the copper salt in the 5-fC adduct-forming composition is Cu(OAc)2, and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of about 1:2. In other embodiments, the copper salt in the 5-fC adduct-forming composition is Cu(OAc)2, and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of about 1:1.8.

[0151] As described above, in some embodiments, a composition for converting one or more 5-fC bases into one or more 5-fC adducts is an adduct-forming reagent having formula (I), [ka]

[0152] (In the formula,

[0153] R is an electron-withdrawing group selected from cyano, nitro, C1-C6 alkyl, carboxylic acid ester, unsubstituted carboxamide, C1-C6 alkyl monosubstituted carboxamide, C1-C6 alkyl disubstituted carboxamide, substituted carbonyl moiety, and substituted sulfonyl moiety, wherein the substitution is selected from C1-C6 linear or branched alkyl groups, C4-C6 cycloalkyl groups, phenyl, 5-membered or 6-membered heteroaryl groups, and 5-membered or 6-membered heteroaryl groups fused with a benzene ring.

[0154] In some embodiments, the amount of adduct-forming reagent having formula (I) in the composition for converting one or more 5-fC bases to one or more 5-fC adducts is in the range of about 50 mM to about 2500 mM. In some embodiments, the amount of adduct-forming reagent having formula (I) in the composition for converting one or more 5-fC bases to one or more 5-fC adducts is in the range of about 50 mM to about 2000 mM. In some embodiments, the amount of adduct-forming reagent having formula (I) in the composition for converting one or more 5-fC bases to one or more 5-fC adducts is in the range of about 50 mM to about 1000 mM. In some embodiments, the amount of adduct-forming reagent having formula (I) in the composition for converting one or more 5-fC bases to one or more 5-fC adducts is in the range of about 50 mM to about 500 mM. In some embodiments, the amount of adduct-forming reagent having formula (I) in the composition for converting one or more 5-fC bases to one or more 5-fC adducts is in the range of about 50 mM to about 200 mM. In other embodiments, the amount of adduct-forming reagent having formula (I) in the composition for converting one or more 5-fC bases to one or more 5-fC adducts is in the range of about 75 mM to about 100 mM.

[0155] In some embodiments, the adduct-forming reagent in the composition for converting one or more 5-fC bases to one or more 5-fC adducts is malononitrile (i.e., R is cyano). In some embodiments, the amount of malononitrile in the composition for converting one or more 5-fC bases to one or more 5-fC adducts is in the range of about 50 mM to about 200 mM. In other embodiments, the amount of malononitrile in the composition for converting one or more 5-fC bases to one or more 5-fC adducts is in the range of about 75 mM to about 100 mM.

[0156] The following are exemplary compositions for converting 5-fC bases in single-stranded or double-stranded nucleic acids into 5-fC adducts. [Table 1]

[0157] Composition for converting 5-hmC base to 5-fC base

[0158] The second composition of this disclosure is adapted for preparing nucleic acid molecules having one or more 5-formylcytosine bases (such as single-stranded and double-stranded nucleic acid molecules) from nucleic acid molecules containing one or more 5-hydroxymethylcytosine bases (referred to herein as “5-fC-forming composition”).

[0159] In some embodiments, the 5-fC forming composition comprises one or more nucleic acid molecules (such as single-stranded and double-stranded nucleic acid molecules) each having one or more 5-hydroxymethylcytosine bases, a chelating agent selected from one of bipyridine or phenanthroline, and an N-oxide reagent, the copper salt, chelating agent, and N-oxide reagent may be selected from any of those listed herein. In some embodiments, the N-oxide reagent is not TEMPO. In some embodiments, the N-oxide reagent is selected from ABNO, AZADO, and Me-AZADO. In some embodiments, the 5-fC forming composition comprises a solvent such as acetonitrile.

[0160] In some embodiments, the target nucleic acid molecule in the 5-fC-forming composition is single-stranded or double-stranded and includes one 5-hydroxymethylcytosine base, at least two 5-hydroxymethylcytosine bases, at least three 5-hydroxymethylcytosine bases, at least four 5-hydroxymethylcytosine bases, at least five 5-hydroxymethylcytosine bases, at least six 5-hydroxymethylcytosine bases, and so on.

[0161] In some embodiments, the pH of the 5-fC forming composition is in the range of about 7 to about 12.5. In some embodiments, the pH of the 5-fC forming composition is in the range of about 8 to about 12.0. In some embodiments, the pH of the 5-fC forming composition is about 8. In some embodiments, the pH of the 5-fC forming composition is about 8.5. In some embodiments, the pH of the 5-fC forming composition is about 9. In some embodiments, the pH of the 5-fC forming composition is about 9.5. In some embodiments, the pH of the 5-fC forming composition is about 10. In some embodiments, the pH of the 5-fC forming composition is about 10.5. In some embodiments, the pH of the 5-fC forming composition is about 11. In some embodiments, the pH of the 5-fC forming composition is about 11.5. In some embodiments, the pH of the 5-fC forming composition is about 12. In some embodiments, the pH of the 5-fC forming composition is about 12.5.

[0162] In some embodiments, the 5-fC forming composition includes a strong base. In some embodiments, the strong base is selected from the group consisting of NaOH, KOH, and LiOH. In some embodiments, an amount of base is added such that the pH of the composition is about 7 to about 12, for example, about 8 to about 12. As an example, a suitable composition for converting one or more 5-hydroxymethylcytosine bases to 5-formylcytosine bases includes about 5 mM to about 20 mM NaOH. In some embodiments, a suitable composition for converting one or more 5-hydroxymethylcytosine bases to 5-formylcytosine bases includes about 10 mM NaOH.

[0163] In other embodiments, the 5-fC forming composition includes a buffer. In some embodiments, the buffer is TRIS. For example, a preferred composition for converting one or more 5-hydroxymethylcytosine bases to 5-formylcytosine bases contains about 0 mM to about 50 mM of TRIS. Another example is a preferred composition for converting one or more 5-hydroxymethylcytosine bases to 5-formylcytosine bases containing about 1 mM to about 50 mM of TRIS. Yet another example is a preferred composition for converting one or more 5-hydroxymethylcytosine bases to 5-formylcytosine bases containing about 5 mM to about 50 mM of TRIS. Yet another example is a preferred composition for converting one or more 5-hydroxymethylcytosine bases to 5-formylcytosine bases containing about 10 mM to about 50 mM of TRIS.

[0164] In some embodiments, the amount of copper salt in the 5-fC forming composition is in the range of about 0.5 mM to about 8 mM. In some embodiments, the amount of copper salt in the 5-fC forming composition is in the range of about 0.5 mM to about 6 mM. In some embodiments, the amount of copper salt in the 5-fC forming composition is in the range of about 0.5 mM to about 4 mM. In some embodiments, the amount of copper salt in the 5-fC forming composition is in the range of about 1 mM to about 3 mM. In some embodiments, the amount of copper salt in the 5-fC forming composition is about 1 mM. In some embodiments, the amount of copper salt in the 5-fC forming composition is about 1.5 mM. In some embodiments, the amount of copper salt in the 5-fC forming composition is about 1.75 mM. In some embodiments, the amount of copper salt in the 5-fC forming composition is about 2 mM. In some embodiments, the amount of copper salt in the 5-fC forming composition is about 2.25 mM. In some embodiments, the amount of copper salt in the 5-fC forming composition is about 2.5 mM. In some embodiments, the amount of copper salt in the 5-fC forming composition is about 3 mM.

[0165] In some embodiments, the amount of chelating agent in the 5-fC forming composition is in the range of about 1 mM to 20 mM. In some embodiments, the amount of chelating agent in the 5-fC forming composition is in the range of about 1 mM to 15 mM. In some embodiments, the amount of chelating agent in the 5-fC forming composition is in the range of about 1 mM to 10 mM. In some embodiments, the amount of chelating agent in the 5-fC forming composition is in the range of about 2 mM to 8 mM. In some embodiments, the amount of chelating agent in the 5-fC forming composition is in the range of about 2 mM to 6 mM. In some embodiments, the amount of chelating agent in the 5-fC forming composition is about 1 mM. In some embodiments, the amount of chelating agent in the 5-fC forming composition is about 2 mM. In some embodiments, the amount of chelating agent in the 5-fC forming composition is about 2.5 mM. In some embodiments, the amount of chelating agent in the 5-fC forming composition is about 3 mM. In some embodiments, the amount of chelating agent in the 5-fC forming composition is about 3.5 mM. In some embodiments, the amount of chelating agent in the 5-fC forming composition is about 4 mM. In some embodiments, the amount of chelating agent in the 5-fC forming composition is about 4.5 mM. In some embodiments, the amount of chelating agent in the 5-fC forming composition is about 5 mM. In some embodiments, the amount of chelating agent in the 5-fC forming composition is about 5.5 mM. In some embodiments, the amount of chelating agent in the 5-fC forming composition is about 6 mM.

[0166] In some embodiments, the ratio of copper salt to chelating agent in the 5-fC forming composition is in the range of about 1:5 to about 1:1. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC forming composition is in the range of about 1:4 to about 1:1. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC forming composition is in the range of about 1:3 to about 1:1. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC forming composition is about 1:2.8. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC forming composition is about 1:2.7. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC forming composition is about 1:2.6. In some embodiments, the ratio of copper salt to chelating agent in the 5-fC forming composition is about 1:2.5. In some embodiments, the ratio of the amount of copper salt to the amount of chelating agent in the 5-fC forming composition is about 1:2.4. In some embodiments, the ratio of the amount of copper salt to the amount of chelating agent in the 5-fC forming composition is about 1:2.3. In some embodiments, the ratio of the amount of copper salt to the amount of chelating agent in the 5-fC forming composition is about 1:2.2. In some embodiments, the ratio of the amount of copper salt to the amount of chelating agent in the 5-fC forming composition is about 1:2. In some embodiments, the ratio of the amount of copper salt to the amount of chelating agent in the 5-fC forming composition is about 1:1.8. In some embodiments, the ratio of the amount of copper salt to the amount of chelating agent in the 5-fC forming composition is about 1:1.6. In some embodiments, the ratio of the amount of copper salt to the amount of chelating agent in the 5-fC forming composition is about 1:1.4. In some embodiments, the ratio of the amount of copper salt to the amount of chelating agent in the 5-fC forming composition is about 1:1.2. In some embodiments, the ratio of the amount of copper salt to the amount of chelating agent in the 5-fC forming composition is about 1:1.

[0167] In some embodiments, the copper salt is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the chelating agent is 2,2'-bipyridine or a derivative thereof. In other embodiments, the copper salt is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the chelating agent is 2,2'-bipyridine or a derivative thereof, with the ratio of the amount of copper salt to the amount of chelating agent being about 1:2.2. In other embodiments, the copper salt is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, the chelating agent is 2,2'-bipyridine or a derivative thereof, and the ratio of the amount of copper salt to the amount of chelating agent is approximately 1:1.8.

[0168] In some embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine. In other embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine or a derivative thereof, with a ratio of copper salt to chelating agent of approximately 1:2.2. In other embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:2. In other embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:1.8.

[0169] In some embodiments, the copper salt is Cu(ACN)4 triflate and the chelating agent is 2,2'-bipyridine. In other embodiments, the copper salt is Cu(ACN)4 triflate and the chelating agent is 2,2'-bipyridine or a derivative thereof, with a ratio of copper salt to chelating agent of approximately 1:2.2. In other embodiments, the copper salt is Cu(ACN)4 triflate and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:2. In other embodiments, the copper salt is Cu(ACN)4 triflate and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:1.8.

[0170] In some embodiments, the copper salt is Cu(OAc)2 and the chelating agent is 2,2'-bipyridine. In other embodiments, the copper salt is Cu(OAc)2 and the chelating agent is 2,2'-bipyridine or a derivative thereof, with a ratio of copper salt to chelating agent of approximately 1:2.2. In other embodiments, the copper salt is Cu(OAc)2 and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:2. In other embodiments, the copper salt is Cu(OAc)2 and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:1.8.

[0171] In some embodiments, the ratio of the amount of copper salt to the amount of N-oxide (e.g., ABNO, AZADO, and Me-AZADO) in the 5-fC forming composition is in the range of about 1:1 to about 1:0.1. In some embodiments, the ratio of the amount of copper salt to the amount of N-oxide (e.g., ABNO, AZADO, and Me-AZADO) in the 5-fC forming composition is in the range of about 1:0.5 to about 1:0.1. In some embodiments, the ratio of the amount of copper salt to the amount of N-oxide (e.g., ABNO, AZADO, and Me-AZADO) in the 5-fC forming composition is about 1:0.5. In some embodiments, the ratio of the amount of copper salt to the amount of N-oxide (e.g., ABNO, AZADO, and Me-AZADO) in the 5-fC forming composition is about 1:0.4. In some embodiments, the ratio of the amount of copper salt to the amount of N-oxide (e.g., ABNO, AZADO, and Me-AZADO) in the 5-fC forming composition is about 1:0.3. In some embodiments, the ratio of the amount of copper salt to the amount of N-oxide (e.g., ABNO, AZADO, and Me-AZADO) in the 5-fC forming composition is about 1:0.2. In some embodiments, the ratio of the amount of copper salt to the amount of N-oxide (e.g., ABNO, AZADO, and Me-AZADO) in the 5-fC forming composition is about 1:0.1.

[0172] In some embodiments, the copper salt in the 5-fC forming composition is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the N-oxide reagent is ABNO or AZADO or a derivative or analog thereof, and the ratio of the amount of copper salt to the amount of N-oxide in the composition is about 1:0.4. In some embodiments, the copper salt in the 5-fC forming composition is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the N-oxide reagent is ABNO or AZADO or a derivative or analog thereof, with the ratio of the amount of copper salt to the amount of N-oxide in the composition being about 1:0.3. In some embodiments, the copper salt in the 5-fC forming composition is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the N-oxide reagent is ABNO or AZADO or a derivative or analog thereof, with the ratio of the amount of copper salt to the amount of N-oxide in the composition being about 1:0.2. In some embodiments, the copper salt in the 5-fC forming composition is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, the N-oxide reagent is ABNO or AZADO or its derivatives or analogs, and the ratio of the amount of copper salt to the amount of N-oxide in the composition is about 1:0.1.

[0173] The following are exemplary compositions for converting 5-hmC bases to 5-fC bases in single-stranded or double-stranded nucleic acids. [Table 2]

[0174] Synthesis method

[0175] This disclosure also relates to methods for preparing target nucleic acid molecules comprising one or more 5-formylcytosine bases or adducts thereof. In some embodiments, this disclosure provides a “one-pot” synthesis method for forming 5-formylcytosine base adducts.

[0176] Method for preparing one or more nucleic acid molecules containing one or more 5-fC adducts.

[0177] In some embodiments, the methods of the present disclosure include converting one or more 5-fC bases in one or more nucleic acid molecules into one or more 5-fC adducts. The conversion of one or more 5-fC bases to one or more 5-fC adducts is carried out in the presence of a chelating agent selected from one of copper salts, bipyridine, or phenanthroline, an adduct-forming reagent (such as an adduct-forming reagent having formula (I) as described in Scheme 1 below). In some embodiments, the product of this reaction, i.e., one or more 5-fC adducts, can be used in one or more downstream processes, such as one or more amplification steps and / or one or more sequencing steps (such as to detect epigenetic changes in one or more target nucleic acid molecules), but is not limited to these. [ka] Scheme 1

[0178] In some embodiments, the conversion of one or more 5-fC bases to their respective adducts is carried out using any one of the 5-fC adduct-forming compositions described herein, including but not limited to any of those described in Composition Examples 1 to 3. In this regard, in some embodiments, the method involves contacting a sample containing one or more nucleic acid molecules, each having one or more 5-fC bases, with any one of the 5-fC adduct-forming compositions described herein for a predetermined time and at a predetermined temperature, etc. In some embodiments, the sample is contacted with any one of the 5-fC adduct-forming compositions described herein, where the pH of the 5-fC adduct-forming composition is in the range of about 7 to about 12.5, for example, in the range of about 8 to about 12.0.

[0179] In some embodiments, the conversion may proceed at a temperature in the range of about 20°C to about 35°C, for example, in the range of about 25°C to about 30°C. In some embodiments, the conversion may proceed over a period of about 1 minute to about 120 minutes. In some embodiments, the conversion may proceed over a period of about 1 minute to about 90 minutes. In some embodiments, the conversion may proceed over a period of about 1 minute to about 60 minutes. In some embodiments, the conversion may proceed over a period of about 1 minute to about 45 minutes. In some embodiments, the conversion may proceed over a period of about 1 minute to about 30 minutes. In some embodiments, the conversion may proceed over a period of about 1 minute to about 15 minutes.

[0180] In some embodiments, the Disclosure provides a method for preparing one or more nucleic acid molecules, each having one or more 5-fC adducts, the method comprising (a) obtaining a sample comprising one or more nucleic acid molecules, each having one or more 5-formylcytosine bases, and (b) reacting the obtained sample with a composition comprising a chelating agent selected from one of copper salts, bipyridine, or phenanthroline, and an adduct-forming agent, for a predetermined amount of time and at a predetermined temperature. In some embodiments, the adduct-forming agent is any compound capable of forming adducts of 5-fC bases incorporated within the nucleic acid molecules. In other embodiments, the adduct-forming agent has formula (I) as described herein. In yet another embodiment, the adduct-forming agent is malononitrile.

[0181] In some embodiments, the reaction is carried out at a pH in the range of about 7 to about 12.5, for example, at a pH in the range of about 8 to about 12. In some embodiments, the reaction is carried out at room temperature. In other embodiments, the reaction is carried out at a temperature in the range of about 20°C to about 35°C, for example, at about 25°C to about 30°C. In some embodiments, the reaction may proceed over a period of time ranging from about 1 minute to about 120 minutes. In some embodiments, the reaction may proceed over a period of time ranging from about 1 minute to about 90 minutes. In some embodiments, the reaction may proceed over a period of time ranging from about 1 minute to about 60 minutes. In some embodiments, the reaction may proceed over a period of time ranging from about 1 minute to about 45 minutes. In some embodiments, the reaction may proceed over a period of time ranging from about 1 minute to about 30 minutes. In some embodiments, the reaction may proceed over a period of time ranging from about 1 minute to about 15 minutes.

[0182] In some embodiments, the copper salt is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the chelating agent is 2,2'-bipyridine or a derivative thereof. In other embodiments, the copper salt is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the chelating agent is 2,2'-bipyridine or a derivative thereof, with the ratio of the amount of copper salt to the amount of chelating agent being about 1:2.2. In other embodiments, the copper salt is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, the chelating agent is 2,2'-bipyridine or a derivative thereof, and the ratio of the amount of copper salt to the amount of chelating agent is approximately 1:1.8.

[0183] In some embodiments, the copper salt is Cu(ACN)4 triflate and the chelating agent is 2,2'-bipyridine. In other embodiments, the copper salt is Cu(ACN)4 triflate and the chelating agent is 2,2'-bipyridine or a derivative thereof, with a ratio of copper salt to chelating agent of approximately 1:2.2. In other embodiments, the copper salt is Cu(ACN)4 triflate and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:2. In other embodiments, the copper salt is Cu(ACN)4 triflate and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to bipyridine of approximately 1:1.8.

[0184] In some embodiments, the copper salt is Cu(OAc)2 and the chelating agent is 2,2'-bipyridine. In other embodiments, the copper salt is Cu(OAc)2 and the chelating agent is 2,2'-bipyridine or a derivative thereof, with a ratio of copper salt to chelating agent of approximately 1:2.2. In other embodiments, the copper salt is Cu(OAc)2 and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:2. In other embodiments, the copper salt is Cu(OAc)2 and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to bipyridine of approximately 1:1.8.

[0185] In some embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine. In other embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine or a derivative thereof, with a ratio of copper salt to chelating agent of approximately 1:2.2. In other embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:2. In other embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to bipyridine of approximately 1:1.8.

[0186] In some embodiments, the amount of adduct-forming reagent is in the range of about 50 mM to about 200 mM. In other embodiments, the amount of adduct-forming reagent is in the range of about 75 mM to about 100 mM. In some embodiments, the amount of adduct-forming reagent is about 100 mM. In other embodiments, the amount of adduct-forming reagent is about 125 mM. In yet another embodiment, the amount of adduct-forming reagent is about 150 mM.

[0187] In some embodiments, the adduct-forming reagent is malononitrile (i.e., R is cyano). In some embodiments, the amount of malononitrile is in the range of about 50 mM to about 200 mM. In other embodiments, the amount of malononitrile is about 100 mM. In other embodiments, the amount of malononitrile is about 125 mM. In other embodiments, the amount of malononitrile is about 150 mM. In other embodiments, the amount of malononitrile is about 175 mM. In other embodiments, the amount of malononitrile is about 200 mM. When using malononitrile in the range of about 125 mM to about 150 mM and copper salt in an amount of about 2 mM, the reaction is expected to be completed within about 45 to about 60 minutes. Also, when using malononitrile in an amount of about 100 mM, the reaction is expected to be completed within 70 minutes.

[0188] In some embodiments, the copper salt is Cu(ClO4)2, the chelating agent is 2,2'-bipyridine, and the adduct-forming reagent is malononitrile. In other embodiments, the copper salt is Cu(ClO4)2, the chelating agent is 2,2'-bipyridine, and the adduct-forming reagent is malononitrile, with a ratio of copper salt to chelating agent of approximately 1:2.2. In other embodiments, the copper salt is Cu(ClO4)2, the chelating agent is 2,2'-bipyridine, and the adduct-forming reagent is malononitrile, with a ratio of copper salt to chelating agent of approximately 1:2. In other embodiments, the copper salt is Cu(ClO4)2, the chelating agent is 2,2'-bipyridine, and the adduct-forming reagent is malononitrile, with a ratio of copper salt to chelating agent of approximately 1:1.8.

[0189] In some embodiments, the copper salt is Cu(OAc)2, the chelating agent is 2,2'-bipyridine, and the adduct-forming reagent is malononitrile. In other embodiments, the copper salt is Cu(OAc)2, the chelating agent is 2,2'-bipyridine, and the adduct-forming reagent is malononitrile, with a ratio of copper salt to chelating agent of approximately 1:2.2. In other embodiments, the copper salt is Cu(OAc)2, the chelating agent is 2,2'-bipyridine, and the adduct-forming reagent is malononitrile, with a ratio of copper salt to chelating agent of approximately 1:2. In other embodiments, the copper salt is Cu(OAc)2, the chelating agent is 2,2'-bipyridine, and the adduct-forming reagent is malononitrile, with a ratio of copper salt to chelating agent of approximately 1:1.8.

[0190] In some embodiments, the copper salt is Cu(ACN)4 triflate, the chelating agent is 2,2'-bipyridine, and the adduct-forming reagent is malononitrile. In other embodiments, the copper salt is Cu(ACN)4 triflate, the chelating agent is 2,2'-bipyridine, and the adduct-forming reagent is malononitrile, with a ratio of copper salt to chelating agent of approximately 1:2.2. In other embodiments, the copper salt is Cu(ACN)4 triflate, the chelating agent is 2,2'-bipyridine, and the adduct-forming reagent is malononitrile, with a ratio of copper salt to chelating agent of approximately 1:2. In other embodiments, the copper salt is Cu(ACN)4 triflate, the chelating agent is 2,2'-bipyridine, and the adduct-forming reagent is malononitrile, with a ratio of copper salt to chelating agent of approximately 1:1.8.

[0191] The applicant surprisingly discovered that both single-stranded and double-stranded nucleic acid molecules containing one or more 5-fC bases were modified with high efficiency in less than 1 hour at about 25°C in the presence of about 2 mM copper salt, about 3 mM to about 4 mM bipyridine, and 150 mM adduct-forming reagent (e.g., malononitrile). The reaction is thought to be possible in a highly basic and denatured sample (e.g., about 10 to about 20 mM NaOH) or in a slightly alkaline sample with a pH of about 8 and a buffer such as about 10 mM TRIS (see Example 2, Figure 1). The presence of both the copper salt and bipyridine is thought to be important for “speeding up” the reaction (see Example 3, Figure 2). The reaction rate is thought to be proportional to the concentration of copper (see Example 4, Figure 3). For example, a reaction using approximately 4 mM copper is about twice as fast as a reaction using only approximately 2 mM copper and adduct-forming reagent (see Examples 4 and 5, Figures 3 and 4). A reaction using approximately 150 mM malononitrile is about twice as fast as a reaction using only approximately 75 mM malononitrile. It is thought that the reaction can be further accelerated (e.g., up to a few minutes) by introducing additional amounts of copper (see Figure 3). The applicant also confirmed the formation of 5-fC adducts and their conversion to thymine during PCR (see Example 6 and Figure 5 of this specification).

[0192] Method for preparing one or more nucleic acid molecules containing one or more 5-fC bases

[0193] In some embodiments, the methods of the present disclosure include converting one or more 5-hmC bases in one or more nucleic acid molecules to one or more 5-fC bases. The conversion from one or more 5-hmC bases to one or more 5-fC bases is carried out in the presence of a chelating agent selected from one of copper salts, bipyridine, or phenanthroline, and an oxidizing agent (N-oxide, preferably ABNO, AZADO, and Me-AZADO, etc.), as described in Scheme 2 below. In some embodiments, the conversion from one or more 5-hmC bases to one or more 5-fC bases does not involve TEMPO. [ka] Scheme 2

[0194] In some embodiments, the conversion of one or more 5-hmC bases to one or more 5-fC bases is carried out using any one of the 5-fC forming compositions described herein, including but not limited to any of the compositions described in Examples 4 to 6. In this regard, in some embodiments, the method involves contacting a sample containing one or more nucleic acid molecules, each having one or more 5-hmC bases, with any one of the 5-fC forming compositions described herein (for example, for a predetermined time and at a predetermined temperature). In some embodiments, the sample is contacted with any one of the 5-fC forming compositions described herein, where the pH of the 5-fC forming composition is in the range of about 7 to about 12.5, for example, in the range of about 8 to about 12.5. In some embodiments, the conversion may proceed at a temperature in the range of about 20°C to about 35°C, for example, in the range of about 25°C to about 30°C.

[0195] In some embodiments, the conversion may proceed over a period ranging from about 10 minutes to about 200 minutes. In some embodiments, the conversion may proceed over a period ranging from about 10 minutes to about 120 minutes. In some embodiments, the conversion may proceed over a period ranging from about 10 minutes to about 90 minutes. In some embodiments, the conversion may proceed over a period ranging from about 10 minutes to about 60 minutes. In some embodiments, the conversion may proceed over a period ranging from about 10 minutes to about 45 minutes. In some embodiments, the conversion may proceed over a period ranging from about 10 minutes to about 30 minutes.

[0196] In some embodiments, the reaction product (one or more 5-fC bases formed) may be used as a starting material for further downstream reactions, such as one-pot synthesis.

[0197] In some embodiments, the Disclosure provides a method for preparing one or more nucleic acid molecules, each having one or more 5-fC bases, the method comprising (a) obtaining a sample comprising one or more nucleic acid molecules, each having one or more 5-hydroxymethylcytosine bases, and (b) reacting the obtained sample with a composition comprising a chelating agent selected from one of copper salts, bipyridine, or phenanthroline, and an N-oxide (e.g., preferably ABNO, AZADO, and Me-AZADO) for a predetermined amount of time and at a predetermined temperature. In some embodiments, the composition comprising the N-oxide does not contain TEMPO. In some embodiments, the reaction is carried out at a pH in the range of about 7 to about 12.5, for example, at a pH in the range of about 8 to about 12.0.

[0198] In some embodiments, the reaction takes place at room temperature. In other embodiments, the reaction takes place at a temperature in the range of about 20°C to about 35°C, for example, about 25°C to about 30°C. In some embodiments, the reaction may proceed over a period of time ranging from about 1 minute to about 120 minutes. In some embodiments, the reaction may proceed over a period of time ranging from about 1 minute to about 90 minutes. In some embodiments, the reaction may proceed over a period of time ranging from about 1 minute to about 60 minutes. In some embodiments, the reaction may proceed over a period of time ranging from about 1 minute to about 45 minutes. In some embodiments, the reaction may proceed over a period of time ranging from about 1 minute to about 30 minutes. In some embodiments, the reaction may proceed over a period of time ranging from about 1 minute to about 15 minutes.

[0199] In some embodiments, the copper salt is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the chelating agent is 2,2'-bipyridine or a derivative thereof. In other embodiments, the copper salt is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the chelating agent is 2,2'-bipyridine or a derivative thereof, with the ratio of the amount of copper salt to the amount of chelating agent being about 1:2.2. In other embodiments, the copper salt is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, the chelating agent is 2,2'-bipyridine or a derivative thereof, and the ratio of the amount of copper salt to the amount of chelating agent is approximately 1:1.8.

[0200] In some embodiments, the copper salt is Cu(ACN)4 triflate and the chelating agent is 2,2'-bipyridine. In other embodiments, the copper salt is Cu(ACN)4 triflate and the chelating agent is 2,2'-bipyridine or a derivative thereof, with a ratio of copper salt to chelating agent of approximately 1:2.2. In other embodiments, the copper salt is Cu(ACN)4 triflate and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:2. In other embodiments, the copper salt is Cu(ACN)4 triflate and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:1.8.

[0201] In some embodiments, the copper salt is Cu(OAc)2 and the chelating agent is 2,2'-bipyridine. In other embodiments, the copper salt is Cu(OAc)2 and the chelating agent is 2,2'-bipyridine or a derivative thereof, with a ratio of copper salt to chelating agent of approximately 1:2.2. In other embodiments, the copper salt is Cu(OAc)2 and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:2. In other embodiments, the copper salt is Cu(OAc)2 and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:1.8.

[0202] In some embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine. In other embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine or a derivative thereof, with a ratio of copper salt to chelating agent of approximately 1:2.2. In other embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:2. In other embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:1.8.

[0203] In some embodiments, the ratio of the amount of copper salt to the amount of N-oxide (e.g., ABNO, AZADO, and Me-AZADO) in the 5-fC forming composition is in the range of about 1:0.5 to about 1:0.1. In some embodiments, the ratio of the amount of copper salt to the amount of N-oxide (e.g., ABNO, AZADO, and Me-AZADO) in the 5-fC forming composition is about 1:0.5. In some embodiments, the ratio of the amount of copper salt to the amount of N-oxide (e.g., ABNO, AZADO, and Me-AZADO) in the 5-fC forming composition is about 1:0.4. In some embodiments, the ratio of the amount of copper salt to the amount of N-oxide (e.g., ABNO, AZADO, and Me-AZADO) in the 5-fC forming composition is about 1:0.3. In some embodiments, the ratio of the amount of copper salt to the amount of N-oxide (e.g., ABNO, AZADO, and Me-AZADO) in the 5-fC forming composition is about 1:0.2. In some embodiments, the ratio of the amount of copper salt to the amount of N-oxide (e.g., ABNO, AZADO, and Me-AZADO) in the 5-fC forming composition is about 1:0.1.

[0204] In some embodiments, the copper salt in the 5-fC forming composition is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the N-oxide reagent is ABNO or AZADO or a derivative or analog thereof, and the ratio of the amount of copper salt to the amount of N-oxide in the composition is about 1:0.4. In some embodiments, the copper salt in the 5-fC forming composition is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the N-oxide reagent is ABNO or AZADO or a derivative or analog thereof, with the ratio of the amount of copper salt to the amount of N-oxide in the composition being about 1:0.3. In some embodiments, the copper salt in the 5-fC forming composition is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the N-oxide reagent is ABNO or AZADO or a derivative or analog thereof, with the ratio of the amount of copper salt to the amount of N-oxide in the composition being about 1:0.2. In some embodiments, the copper salt in the 5-fC forming composition is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, the N-oxide reagent is ABNO or AZADO or its derivatives or analogs, and the ratio of the amount of copper salt to the amount of N-oxide in the composition is about 1:0.1.

[0205] In some embodiments, the reaction product, i.e., one or more target nucleic acid molecules each having one or more 5-fC bases, is used as a starting material for a downstream reaction. In some embodiments, the reaction product is used as a starting material for a downstream reaction without first purifying the product. For example, the reaction product, i.e., one or more target nucleic acid molecules each having one or more 5-fC bases, may be further reacted with an adduct-forming reagent, such as an adduct-forming reagent having formula (I) (as described herein), with or without first purifying the product.

[0206] "One-pot" synthesis for preparing one or more nucleic acid molecules containing one or more 5-fC adducts.

[0207] In some embodiments, the methods of the present disclosure include a “one-pot” method for converting one or more 5-hmC bases of one or more nucleic acid molecules to one or more 5-fC adducts. “One-pot” synthesis is thought to enable tandem oxidation from 5-hmC bases to 5-fC adducts via a 5-fC intermediate in less than about 3 hours, e.g., less than 2 hours. This is considered extremely valuable, particularly for reducing sample preparation time for nucleic acid methylation sequencing reactions.

[0208] In some embodiments, the reaction products, i.e., one or more 5-fC adducts, may be used in one or more downstream processing steps, including, but not limited to, one or more amplification and / or one or more sequencing processes (for example, to detect epigenetic changes in one or more target nucleic acid molecules).

[0209] The "one-pot" conversion of one or more 5-hmC bases to one or more 5-fC adducts is carried out in a two-step reaction. In the first step, one or more 5-hmC bases are reacted in the presence of a chelating agent selected from one of copper salts, bipyridine, or phenanthroline, and an oxidizing agent (such as an N-oxide) to obtain a reaction mixture containing one or more nucleic acid molecules having one or more 5-fC bases. In the second step, an adduct-forming reagent (such as an adduct-forming reagent having formula (I)) is introduced into the mixture, which then contains one or more nucleic acid molecules containing one or more 5-fC bases, to obtain one or more nucleic acid molecules containing one or more 5-fC adducts. In some embodiments, the second step is carried out without first purifying the reaction mixture generated after the first step.

[0210] The two-step process is thought to be able to produce one or more target nucleic acid molecules containing one or more 5-fC adducts in a period of less than approximately 3 hours, less than approximately 2 hours, or for example, less than approximately 2 hours (e.g., less than approximately 90 minutes, less than approximately 60 minutes, etc.). In some embodiments, the “one-pot” synthesis follows the synthesis of scheme 3 below (wherein the group R is the group of formula (I) herein). [ka] Scheme 3

[0211] In some embodiments, the “one-pot” synthesis first involves contacting a sample containing one or more nucleic acid molecules having one or more 5-hmC bases with a 5-fC forming composition containing one of the 5-fC forming compositions disclosed herein to obtain a reaction mixture containing one or more nucleic acid molecules having one or more 5-fC bases. In some embodiments, the first step may, of course, proceed for about 90 minutes, for example, about 60 minutes, or for example, about 30 minutes, depending on the reaction conditions and the concentrations of the reagents used (as described herein). Subsequently, in some embodiments, the obtained reaction mixture is contacted with a 5-fC adduct forming composition containing one of the 5-fC adduct forming compositions disclosed herein to obtain one or more nucleic acid molecules containing one or more 5-fC adducts. In some embodiments, the second step may, of course, proceed for about 90 minutes, for example, about 60 minutes, or for example, about 30 minutes, depending on the reaction conditions and the concentrations of the reagents used (as described herein). In some embodiments, the reaction mixture is not purified before the second step.

[0212] In some embodiments, the Disclosure provides a method for synthesizing one or more nucleic acid molecules, each comprising: (a) obtaining a sample comprising one or more nucleic acid molecules each comprising one or more 5-formylcytosine bases; (b) contacting the obtained sample with a first composition at a first temperature and for a first period, wherein the first composition comprises a chelating agent selected from one of copper salts, bipyridine, or phenanthroline, and an N-oxide reagent (e.g., TEMPO, ABNO, AZADO, and Me-AZADO) in the sample to obtain a mixture comprising one or more nucleic acid molecules each comprising one or more 5-formylcytosine bases; and (c) contacting the obtained mixture with a second composition at a second temperature and for a second period, wherein the second composition comprises an adduct-forming reagent. In some embodiments, the adduct-forming reagent is any compound capable of forming adducts of 5-fC bases incorporated within the nucleic acid molecules. In other embodiments, the adduct-forming agent has formula (I) as described herein. In yet another embodiment, the adduct-forming agent is malononitrile.

[0213] In some embodiments, the first predetermined period is in the range of about 10 minutes to about 48 hours (when using N-oxide TEMPO). In some embodiments, the first predetermined period is in the range of about 10 minutes to about 24 hours (when using N-oxide TEMPO). In other embodiments, when using an N-oxide reagent selected from ABNO, AZADO, and Me-AZADO, the first predetermined period is in the range of about 10 minutes to about 4 hours, for example, about 10 minutes to about 3 hours, for example, about 10 minutes to about 2 hours, for example, about 10 minutes to about 1 hour.

[0214] In some embodiments, the second predetermined period is in the range of about 1 minute to about 120 minutes, for example, about 5 minutes to about 90 minutes, for example, about 5 minutes to about 60 minutes, for example, about 50 minutes, for example, about 40 minutes, for example, about 30 minutes.

[0215] In some embodiments, the first and second predetermined temperatures are independently in the range of about 20°C to about 35°C, for example, in the range of about 25°C to about 30°C.

[0216] In some embodiments, the copper salt is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the chelating agent is 2,2'-bipyridine or a derivative thereof. In other embodiments, the copper salt is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the chelating agent is 2,2'-bipyridine or a derivative thereof, with the ratio of the amount of copper salt to the amount of chelating agent being about 1:2.2. In other embodiments, the copper salt is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, the chelating agent is 2,2'-bipyridine or a derivative thereof, and the ratio of the amount of copper salt to the amount of chelating agent is approximately 1:1.8.

[0217] In some embodiments, the copper salt is Cu(ACN)4 triflate and the chelating agent is 2,2'-bipyridine. In other embodiments, the copper salt is Cu(ACN)4 triflate and the chelating agent is 2,2'-bipyridine or a derivative thereof, with a ratio of copper salt to chelating agent of approximately 1:2.2. In other embodiments, the copper salt is Cu(ACN)4 triflate and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:2. In other embodiments, the copper salt is Cu(ACN)4 triflate and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:1.8.

[0218] In some embodiments, the copper salt is Cu(OAc)2 and the chelating agent is 2,2'-bipyridine. In other embodiments, the copper salt is Cu(OAc)2 and the chelating agent is 2,2'-bipyridine or a derivative thereof, with a ratio of copper salt to chelating agent of approximately 1:2.2. In other embodiments, the copper salt is Cu(OAc)2 and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:2. In other embodiments, the copper salt is Cu(OAc)2 and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:1.8.

[0219] In some embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine. In other embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine or a derivative thereof, with a ratio of copper salt to chelating agent of approximately 1:2.2. In other embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:2. In other embodiments, the copper salt is Cu(ClO4)2 and the chelating agent is 2,2'-bipyridine, with a ratio of copper salt to chelating agent of approximately 1:1.8.

[0220] In some embodiments, the ratio of copper salt to N-oxide reagent (e.g., TEMPO, ABNO, AZADO, and Me-AZADO) is in the range of about 1:0.5 to about 1:0.1. In some embodiments, the ratio of copper salt to N-oxide reagent (e.g., TEMPO, ABNO, AZADO, and Me-AZADO) is about 1:0.5. In some embodiments, the ratio of copper salt to N-oxide reagent (e.g., TEMPO, ABNO, AZADO, and Me-AZADO) is about 1:0.4. In some embodiments, the ratio of copper salt to N-oxide reagent (e.g., TEMPO, ABNO, AZADO, and Me-AZADO) is about 1:0.3. In some embodiments, the ratio of copper salt to N-oxide reagent (e.g., TEMPO, ABNO, AZADO, and Me-AZADO) is about 1:0.2. In some embodiments, the ratio of the amount of copper salt to the amount of N-oxide reagent (e.g., TEMPO, ABNO, AZADO, and Me-AZADO) is about 1:0.1. In some embodiments, the copper salt in the 5-fC forming composition is selected from Cu(ClO4)2, Cu(OAc)2, CuSO4, and Cu(ACN)4 triflate, and the N-oxide reagent is ABNO or AZADO or its derivatives or analogs.

[0221] In some embodiments, the amount of adduct-forming reagent is in the range of about 50 mM to about 200 mM. In other embodiments, the amount of adduct-forming reagent is in the range of about 75 mM to about 100 mM. In some embodiments, the amount of adduct-forming reagent is about 100 mM. In other embodiments, the amount of adduct-forming reagent is about 125 mM. In yet another embodiment, the amount of adduct-forming reagent is about 150 mM.

[0222] In some embodiments, the adduct-forming reagent is malononitrile (i.e., R is cyano). In some embodiments, the amount of malononitrile is in the range of about 50 mM to about 200 mM. In other embodiments, the amount of malononitrile is about 100 mM. In other embodiments, the amount of malononitrile is about 125 mM. In other embodiments, the amount of malononitrile is about 150 mM. In other embodiments, the amount of malononitrile is about 175 mM. In other embodiments, the amount of malononitrile is about 200 mM. When malononitrile is used in amounts ranging from about 125 mM to about 150 mM, the reaction is expected to be completed within about 45 to about 60 minutes. Also, when an amount of about 100 mM of malononitrile is used, the reaction is expected to be completed within 70 minutes.

[0223] In some embodiments, the copper salt is Cu(ClO4)2, the chelating agent is 2,2'-bipyridine, and the adduct-forming reagent is malononitrile. In other embodiments, the copper salt is Cu(ClO4)2, the chelating agent is 2,2'-bipyridine, and the adduct-forming reagent is malononitrile, with a ratio of copper salt to chelating agent of approximately 1:2.2. In other embodiments, the copper salt is Cu(ClO4)2, the chelating agent is 2,2'-bipyridine, and the adduct-forming reagent is malononitrile, with a ratio of copper salt to chelating agent of approximately 1:2. In other embodiments, the copper salt is Cu(ClO4)2, the chelating agent is 2,2'-bipyridine, and the adduct-forming reagent is malononitrile, with a ratio of copper salt to chelating agent of approximately 1:1.8.

[0224] A method for amplifying and / or sequencing a target nucleic acid sequence containing at least one modified cystine.

[0225] This disclosure also provides methods for amplifying and / or sequencing nucleic acid sequences containing at least one modified cysteine ​​base. In some embodiments, this disclosure provides methods for detecting epigenetic modifications of nucleic acids. In some embodiments, the method includes obtaining one or more target nucleic acid molecules containing one or more 5-hmC bases or one or more 5-fC bases, and converting one or more 5-hmC bases or one or more 5-fC bases into one or more 5-fC adducts. Methods for converting one or more 5-hmC bases or one or more 5-fC bases into one or more 5-fC adducts are described herein. In some embodiments, the 5-fC adducts are 5-fC malononitrile adducts.

[0226] Once a sample containing one or more nucleic acid molecules having one or more 5-fC adducts is obtained, the sample is brought into contact with polymerase to obtain one or more amplified nucleic acid molecules. The polymerase is thought to read the 5-fC adducts as thymine during amplification. Therefore, in some embodiments, one or more amplified nucleic acid molecules have thymine bases at positions corresponding to the positions of the 5-fC adducts in each of the one or more nucleic acid molecules having one or more 5-formylcytosine adducts.

[0227] In some embodiments, the polymerase can copy a strand containing a 5-fC adduct by recognizing the adduct as T (i.e., incorporating A on the opposite side of the adduct). Examples of polymerases capable of accommodating 5-fC adducts described herein include DNA polymerases known to accommodate uracil (U) in DNA strands. The polymerase may be a naturally occurring polymerase or an engineered polymerase.

[0228] Non-exclusive examples of polymerases include prokaryotic DNA polymerases (e.g., Pol I, Pol II, Pol III, Pol IV, and Pol V), eukaryotic DNA polymerases, archaeal DNA polymerases, telomerases, reverse transcriptases, and RNA polymerases. Reverse transcriptases are RNA-dependent DNA polymerases that synthesize DNA from an RNA template. The reverse transcriptase family possesses both the functionality of DNA polymerases and the functionality of RNAseHs, which degrade RNA that has been base-paired with DNA. RNA polymerases are enzymes that synthesize RNA using DNA as a template in the process of gene transcription. RNA polymerases polymerize ribonucleotides at the 3' end of the RNA transcript.

[0229] Its active ingredient is the genus Thermococcus litoralis(Vent、GenBank:AAA72101)、Pyrococcus furiosus(Pfu,GenBank:D12983、BAA02362)、Pyrococcus woesii、Pyrococcus GB-D(Deep Vent、GenBank:AAA67131)、Thermococcus kodakaraensis CODE(CODE、GenBank:BD175553、BAA06142;Thermococcus sp.strain CODE(Pfx、GenBank:AAE68738)), Thermococcus gorgonarius(Tgo、Pdb:4699806), Sulfolobus solataricus (GenBank:NC002754, P26811), Aeropyrum pernix (GenBank:BAA81109), Archaeglobus fulgidus (GenBank:029753), Pyrobaculum aerophilum (GenBank:AAL63952), Pyrodictium occultum (GenBank:BAA07579, BAA07580), Thermococcus 9 degrees Nm (GenBank:AAA88769, Q56366), Thermococcus fumicolans (GenBank:CAA93738, P74918), Thermococcus hydrothermalis (GenBank:CAC18555), Thermococcus sp.GE8(GenBank:CAC12850), Thermococcus sp.JDF-3(GenBank:AX135456;WO0132887); sp.TY(GenBank:CAA73475)、Pyrococcus abyssi(GenBank:P77916)、Pyrococcus glycovorans(GenBank:CAC12849)、Pyrococcus horikoshii(GenBank:NP 143776)、Pyrococcus sp.GE23(GenBank:CAA90887) Pyrococcus sp.ST700 (GenBank:CAC 12847), Thermococcus pacificus (GenBank:AX411312.1), Thermococcus zilligii (GenBank:DQ3366890), Thermococcus aggregans, Thermococcus barossii, Thermococcus celer (GenBank:DD259850.1), Thermococcus profundus (GenBankE14137), Thermococcus siculi (GenBank:DD259857.1), Thermococcus thioreducens, Thermococcus onnurineus NA1, Sulfolobus acidocaldarium, Sulfolobus tokodaii, Pyrobaculum calidifontis, Pyrobaculum islandicum (GenBank:AAF27815), Methanococcus jannaschii (GenBank:Q58295), Desulforococcus species TOK, Desulforococcus, Pyrolobus, Pyrodictium, Staphylothermus, Vulcanisaetta, Methanococcus (GenBank:P52025) and other archaeal B polymerases, e.g., GenBank AAC62712, P956901, BAAA07579), species of thermophilic bacteria of the genus Thermos (e.g., Flabus, Rubers, Thermophilus, Lacteus, Rubens, Aquaticus), Bacillus stearothermophilus, Thermotoga maritima, Methanothermus fervidus, KOD polymerase, TNA1 polymerase, Thermococcus sp. 9 degrees N-7, T4, T7, phi29, Pyrococcus furiosus, P. abyssi, T. gorgonarius, T. litoralis, T. zilligii, T. sp. GT, P. sp. GB-D, KOD, Pfu, T. gorgonarius, T. zilligii, T. litoralis and Thermococcus sp.It may originate from 9N-7 polymerase.

[0230] In some embodiments, the amplification and / or sequencing (or detection of epigenetic changes) method further includes ligating one or more adapters to one or more nucleic acid molecules having one or more adducts of 5-formylcytosine before contacting the nucleic acid molecules having one or more adducts of 5-formylcytosine with a polymerase. Adapters of various shapes and functions are known in the art (see, for example, PCT / EP2019 / 05515, U.S. Patent No. 8822150 and U.S. Patent No. 845193, filed February 28, 2019; these disclosures are incorporated herein by reference in their entirety). In some embodiments, the function of the adapter is to introduce a desired element into the nucleic acid.

[0231] In some embodiments, the elements carried by the adapter include at least one of a nucleic acid barcode, a multiple identifier, a unique molecular identifier, a primer binding site, or a ligation site. As used herein, the term “barcode” refers to a nucleic acid sequence that can be detected and identified. In some embodiments, the barcode comprises about 5 to about 20 nucleotides, and in a sample, nucleic acids incorporating the barcode can be distinguished or grouped according to the barcode. In some embodiments, the barcode comprises about 5 to about 15 nucleotides. In some embodiments, the barcode comprises about 5 to about 10 nucleotides. In some embodiments, the barcode comprises about 10 to about 15 nucleotides. In some embodiments, the barcode comprises about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 nucleotides.

[0232] In some embodiments, the barcode has the following formula:

[0233] (W)(N)(N)(N)(N)(N)(W)(N)(N)(N)(N)(N)(W), or

[0234] (N)(W)(N)(N)(N)(W)(N)(N)(N),

[0235] In the formula, N contains (in aggregates) approximately 25% adenosine, approximately 25% guanine, approximately 25% cytosine, and approximately 25% thymine, and W contains (in aggregates) approximately 50% adenosine and approximately 50% thymine.

[0236] In this specification, the term “multiple identifier” or “MID” refers to a barcode that identifies the source of the target nucleic acid (e.g., the sample from which the nucleic acid originates). In some embodiments, all or substantially all target nucleic acids from the same sample will share the same MID. In some embodiments, nucleic acids from different sources or samples can be mixed and sequenced simultaneously. In some embodiments, by using the MID, sequence reads (obtained during a sequencing step as described herein) can be assigned to the individual samples from which the target nucleic acid originated.

[0237] As used herein, the term “unique molecular identifier” or “UID” refers to a barcode that identifies an attached nucleic acid. In some embodiments, all or substantially all target nucleic acids from the same sample will have different UIDs. In some embodiments, all or substantially all offspring (e.g., amplicons) derived from the same original target nucleic acid will share the same UID.

[0238] Examples of barcodes, MIDs, and UIDs are described in U.S. Patent Application Publication No. 2020 / 0032244, U.S. Patents No. 7,393,665, No. 8,168,385, No. 8,481,292, No. 8,685,678, and No. 8,722,368, and International Publication No. 2018 / 138237, which are incorporated herein by reference in their entirety.

[0239] After preparing one or more amplified nucleic acid molecules (with or without the adapter ligation step), the one or more amplified nucleic acid molecules are sequenced. In some embodiments, the sequencing includes next-generation sequencing.

[0240] In some embodiments, the sequencing step includes sequence alignment. In some embodiments, alignment is used to obtain a consensus sequence from multiple sequences, for example, multiple sequences having the same unique molecular ID (UID). The molecular ID is a barcode that can be attached to each molecule before sequencing, or before the amplification step if an amplification step is included. In some embodiments, the UID is located in the 5' portion of the RT primer. Similarly, the UID may be located at the 5' end of the final barcode subunit attached to the compound barcode. In other embodiments, the UID is located within an adapter and is attached to one or both ends of the target nucleic acid by ligation.

[0241] In some embodiments, a consensus sequence is obtained from multiple sequences, all having the same UID. Sequences with the same UID are presumed to originate from the same original molecule through amplification. In other embodiments, UIDs are used to eliminate artifacts, i.e., variations present in the descendants of a single molecule (characterized by a particular UID). Such artifacts resulting from PCR errors or sequencing errors can be eliminated using UIDs.

[0242] In some embodiments, the number of each sequence in a sample can be quantified by quantifying the relative number of sequences with each UID within a population having the same MID. In some embodiments, each UID represents a single molecule in the original sample, and the proportion of each sequence variant in the original sample in which all molecules share the same MID can be determined by counting the different UIDs associated with each sequence variant. Those skilled in the art will be able to determine the number of sequence reads required to determine a consensus sequence. In some embodiments, the number in question is the number of reads per UID required for accurate quantification results ("sequence depth"). In some embodiments, the desired depth is 5 to 50 reads per UID.

[0243] kit

[0244] In some embodiments, the disclosure provides a kit comprising components for performing the detection and / or amplification of epigenetic changes. In some embodiments, the kit comprises components for detecting cytosine methylation in nucleic acids by detecting 5-fC.

[0245] In some embodiments, the kit comprises (i) a copper salt, (ii) a chelating agent selected from bipyridine or phenanthroline, (iii) an adduct-forming reagent, (iv) a polymerase, and (v) optionally one or more PCR reagents. In some embodiments, the copper salt and chelating agent are contained in a single container and mixed in a predetermined ratio. In some embodiments, the predetermined ratio is about 1:2.5 (chelating agent:copper). In some embodiments, the predetermined ratio is about 1:2.25 (chelating agent:copper). In some embodiments, the predetermined ratio is about 1:2 (chelating agent:copper). In some embodiments, the predetermined ratio is about 1:1.75 (chelating agent:copper). In some embodiments, the predetermined ratio is about 1:1.5 (chelating agent:copper). In some embodiments, the adduct-forming reagent is malononitrile.

[0246] In some embodiments, the polymerase is selected from those described herein. An example of a polymerase is Taq or Taq-derived polymerase (e.g., KAPA2G polymerase from KAPA BIOSYSTEMS). Another example of a polymerase is a B-family DNA polymerase (e.g., KAPA HIFI polymerase from KAPA BIOSYSTEMS).

[0247] In some embodiments, the PCR reagent contains nucleotides. In some embodiments, the PCR reagent contains deoxyribonucleoside triphosphates (dNTPs), i.e., all four naturally occurring deoxyribonucleoside triphosphates (dNTPs). In some embodiments, the PCR reagent contains deoxyribonucleoside triphosphate molecules, including all of dATP, dCTP, dGTP, and dTTP. In some embodiments, the PCR reagent also contains compounds useful for assisting the activity of nucleic acid polymerases. For example, in some embodiments, the PCR reagent contains a divalent cation, such as a magnesium ion. In some embodiments, the magnesium ion is provided in the form of magnesium chloride, magnesium acetate, or magnesium sulfate. In some embodiments, the PCR reagent further includes a buffer or buffer solution. In some embodiments, each of the PCR reagents is provided individually. In other embodiments, each of the PCR reagents is provided in a mixture.

[0248] In some embodiments, the kit includes one or more adapter molecules and / or ligases.

[0249] In some embodiments, the kit further includes one or more bases, buffers, and / or oxidizing agents.

[0250] In some embodiments, a kit is provided comprising a chelating agent and a copper salt pre-mixed in a first container and an N-oxide reagent (e.g., TEMPO, ABNO, AZADO, and Me-AZADO) in a second container. In some embodiments, the kit further comprises one or more buffers and / or bases.

[0251] In some embodiments, a kit is provided comprising a chelating agent and a copper salt pre-mixed in a first container and an adduct-forming reagent (such as one of formula (I)) in a second container. In some embodiments, the kit further comprises one or more buffers and / or bases.

[0252] Examples

[0253] Example 1 - General procedure for promoting Mal-fC formation with copper / 2,2'-bipyridine complex [ka]

[0254] The complex solution was prepared as follows: Approximately 100 mM Cu(OAc)2 in 25 μL of water or approximately 100 mM Cu(ACN)4 triflate in acetonitrile was added to approximately 100 mM 2,2'-bipyridine (Bpy) in approximately 50 μL of acetonitrile, followed by the addition of approximately 15 μL of water and approximately 10 μL of approximately 100 mM NaOH aqueous solution (approximately 25 mM Cu / approximately 50 mM Bpy). This mixture was vortexed for approximately 30 seconds. In an Eppendorf tube, approximately 4 μL of the complex was added to approximately 50 to 2000 ng of fC oligo in approximately 39 μL of approximately 10 to 20 mM approximately 100 mM NaOH aqueous solution, and the mixture was vortexed for approximately 10 seconds. Approximately 1 M malononitrile solution in approximately 7.5 μL of acetonitrile was added to the mixture, and the reaction was shaken at approximately 25°C for approximately 60 minutes. (2 mM Cu / 4 mM Bpy / 150 mM Malononitrile). The reaction product was then purified using a Zymo DNA clean filter according to the corresponding instructions.

[0255] Example 2 - Comparison of Mal-fC formation rates with and without Cu / 2,2'-bipyridine complex

[0256] We conducted studies comparing the conditions for forming malononitrile adducts on FC oligos using a Cu / 2,2'-bipyridine (Bpy) complex with other known conditions. In these experiments, 500 ng of fC oligo (SEQ ID NO: 1 5'-Phos-CACGTCCAGATCAAT(fC)GACTATGAGCAGTACA) was reacted with 150 mM malononitrile at 25°C for 1 hour. These reactions contained 15-20% acetonitrile, with a total volume of 50 μL. The reaction products were purified using Zymo DNA Clean, and the obtained products were analyzed by LC-MS to compare the reaction progress. The mass of the fC oligo was 9894, and the mass of the reaction product was 9942. Figure 1 (Trace S0) shows the mass of the unreacted oligo. Figure 1 (Trace S1) shows the reaction in 10 mM Tris pH 8. Figure 1 (Trace S2) shows the reaction in 10 mM NaOH. Figure 1 (Trace S3) shows the reaction in the presence of approximately 2 mM Cu(OAc)2, approximately 4 mM Bpy, and approximately 10 mM NaOH, while Figure 1 (Trace S4) shows the reaction in the presence of approximately 2 mM Cu(OAc)2, approximately 4 mM Bpy, and approximately 10 mM Tris at pH approximately 8. As seen in Figure 1, the reaction was only partially completed without the Cu / Bpy complex, but completed within 1 hour with the complex.

[0257] Example 3 - Effects of different additives on Mal-fC formation rate

[0258] This study investigated the effects of various additives commonly used in Cu / TEMPO oxidation from hmC oligos to fC on the progression of malononitrile adduct formation on fC oligos. In these experiments, approximately 500 ng of 30-mer ds-DNA double-stranded DNA oligos, each containing one fC nucleotide (SEQ ID NO: 2 GTACTGCTCATAGT(fC)GATTGATCTGGACGTGA) and one complementary strand (SEQ ID NO: 3 5'-Phos-TCACGTCCAGATCAATCGACTATGAGCAGTAC), were treated with approximately 150 mM malononitrile for approximately 1 hour at approximately 25°C in the presence of Cu(II) salt, 2,2'-bipyridine (Bpy), Cu(II) / Bpy complex, or Cu(II) / Bpy / TEMPO. The reaction mixture contained 20 mM NaOH aqueous solution and 15-20% acetonitrile, with a total volume of 50 μL. The reaction products were purified using Zymo DNA Clean, and the obtained products were analyzed by LC-MS to compare the progress of the reactions. The molecular weights of the starting fC oligo, complementary oligo, and Mal-fC oligo were 9978, 9866, and 10026 Da, respectively.

[0259] Figure 2 (Trace S1) shows the reaction with approximately 2 mM Cu(ClO4)2. Figure 2 (Trace S2) shows the reaction with approximately 3 mM Bpy. Figure 2 (Trace S3) shows the reaction with approximately 2 mM Cu(ClO4)2 and approximately 3 mM Bpy. Figure 2 (Trace S4) shows the reaction with approximately 2 mM Cu(ClO4)2, approximately 3 mM Bpy, and 2 mM TEMPO. As seen in Figure 2, only the conditions containing at least both the Cu(II) salt and Bpy were completed in approximately 1 hour, indicating that the Cu / Bpy complex is more effective in catalyzing the formation of the malononitrile adduct than the individual components.

[0260] Example 4: Effect of Cu / 2,2-bipyridine complex concentration on Mal-fC formation rate

[0261] Studies were conducted to determine the effect of the concentration of Cu(I or II) / 2,2'-bipyridine (Bpy) complex on the progress of the formation of malononitrile adducts on fC oligos and to select the optimal concentration. In these experiments, 350 ng of a 30-mer ds-DNA oligonucleotide with one strand containing one fC nucleotide (SEQ ID NO: 2 GTACTGCTCATAGT(fC)GATTGATCTGGACGTGA) and the complementary strand (SEQ ID NO: 3 5'-Phos-TCACGTCCAGATCAATCGACTATGAGCAGTAC) was treated with about 150 mM malononitrile at about 25 °C in the presence of about 2 - about 5 mM Cu(ACN)4 triflate and about 1.25 equivalents of Bpy. The reaction mixture contained about 10 mM aqueous NaOH and about 15 - about 20% acetonitrile, and the total volume was about 50 μL. The reaction time decreased as the concentration of the copper complex increased. The reaction products were purified using Zymo DNA Clean and the obtained products were analyzed by LC-MS to compare the progress of the reaction. The molecular weights of the starting fC oligo, complementary oligo, and Mal-fC oligo were 9978, 9866, and 10026 Da, respectively.

[0262] Figure 3 (Trace S1) shows the formation of adducts completed within 45 minutes with 2 mM Cu(ACN)4 triflate and 2.5 mM Bpy. Figure 3 (Trace S2) shows the reaction completed within 30 minutes with 3 mM Cu(ACN)4 triflate and 3.75 mM Bpy. Figure 3 (Trace S3) shows the reaction completed within 22 minutes with 4 mM Cu(ACN)4 triflate and 5 mM Bpy. Figure 3 (Trace S4) shows the reaction completed within about 15 minutes with 6 mM Cu(ACN) triflate and about 7.5 mM Bpy. As seen in Figure 3, the time required for the competition of adduct formation is inversely correlated with the concentration of the copper / Bpy complex. On the other hand, the rate of adduct formation is proportional to the concentration of the copper complex. However, when the copper concentration is high, precipitates are formed during the reaction, resulting in a decrease in the recovery rate of DNA oligos. It was determined that about 2 mM of copper is sufficient to complete the formation of adducts within about 1 hour without causing significant loss of DNA oligos during the reaction.

[0263] Example 5 - Influence of Malononitrile Concentration on the Formation Rate of Mal-fC

[0264] Studies were conducted to determine the influence of the concentration of malononitrile on the progress of the formation of malononitrile adducts on fC oligomers. In these experiments, 350 ng of a 30-mer double-stranded DNA oligo having one strand containing one fC nucleotide (SEQ ID NO: 2 GTACTGCTCATAGT(fC)GATTGATCTGGACGTGA) and the complementary strand (SEQ ID NO: 3 5’-Phos-TCACGTCCAGATCAATCGACTATGAGCAGTAC) was treated with 75 - 150 mM malononitrile at about 25 °C in the presence of about 2 - about 4 mM Cu(ACN)4 triflate / 1.25 equivalents of Bpy. The reaction contained about 10 mM aqueous NaOH solution and about 15 - about 20% acetonitrile, and the total volume was 50 μL. In one experiment, as shown in Figure 4 (Trace S1), the ds-DNA oligo was treated with about 150 mM malononitrile and about 2 mM Cu(I) as normal. In other experiments, as shown in Figure 4 (Trace S2), the oligo was treated with half the amount of malononitrile (about 75 mM), but the concentration of Cu(I) was doubled (about 4 mM). Both reactions took about 45 minutes to complete. The molecular weights of the starting fC oligo, complementary oligo, and Mal-fC oligo were 9978, 9866, and 10026 Da, respectively.

[0265] As seen in Figure 4, based on the results of Experiment 4, when the malononitrile concentration was reduced by half, the time required for the competition of adduct formation increased by about 2-fold. The formation rate of the malononitrile adduct is proportional to the concentration of malononitrile.

[0266] Example 6 - Conversion Rates of Oligonucleotides Containing One fC Base When Treated with Standard 150 mM Malononitrile in TRIS and with a Cu / 2,2'-Bipyridine Complex Added with 100 mM Malononitrile, Investigated by Sequencing<000091​​Sequencing studies were conducted to compare the formation of malononitrile adducts with a single fC base oligo containing a Cu(I or II) / 2,2'-bipyridine (Bpy) complex catalyzed by the current procedure in Tris buffer. Approximately 50 ng of adapter-linked 5-fC modified oligo (SEQ ID NO: 1 5'-Phos-CACGTCCAGATCAAT(fC)GACTATGAGCAGTACA) was treated for approximately 20 hours at approximately 37°C in approximately 10 mM Tris with approximately 150 mM malononitrile, pH approximately 8.0, or under accelerated conditions (10 mM NaOH, approximately 2 mM Cu(ClO4)2 and approximately 4 mM Bpy, approximately 100 mM malononitrile, approximately 15% acetonitrile) at room temperature for approximately 70 minutes. The product was purified and amplified by PCR with KAPA2G for approximately 30 seconds or extension for approximately 2 minutes. The sample was then sequenced. The results shown in Figure 5 indicate that the conversion rate under accelerated conditions was equivalent to that under standard overnight conditions.

[0268] Example 7-5 Tandem oxidation of hmC-containing oligonucleotide to fC oligonucleotide and subsequent formation of a malononitrile adduct

[0269] This example describes a general procedure for forming a tandem oxidation / malononitrile adduct with an hmC oligo using a copper / 2,2'-bipyridine (Bpy) complex. The complex solution was prepared as follows: 25 μL of 100 mM Cu(ClO4)2 in water was added to approximately 50 μL of approximately 100 mM Bpy in acetonitrile, followed by the addition of approximately 25 μL of water (approximately 25 mM Cu / approximately 50 mM Bpy). This was vortexed for approximately 30 seconds. In an Eppendorf tube, approximately 4 μL of the complex was diluted with approximately 34 μL of water and mixed with approximately 10 mM ABNO in approximately 2 μL of acetonitrile. Approximately 5 μL of approximately 100 mM NaOH was added to this solution, followed by the addition of approximately 0.1 to approximately 2 μg of hmC DNA oligo in approximately 1 μL of water (or approximately 10 mM sodium hydroxide or approximately 10 mM Tris buffer pH approximately 8). Overall, the reaction mixture was assumed to be approximately 45 μL. If the oligonucleotide was diluted and used in quantities exceeding approximately 1 μL, the volume of water used to dilute the complex should be reduced accordingly. The reaction mixture was vortexed for approximately 5 seconds and then incubated at approximately 25°C for approximately 50–60 minutes without shaking. The reaction mixture was then immediately treated with approximately 7.5 μL of approximately 1 M malononitrile in acetonitrile. This was shaken at approximately 25°C for approximately 60 minutes and purified using a Zymo DNA clean filter according to the corresponding instructions.

[0270] Example 8

[0271] Approximately 1 μg of 5-hmC oligonucleotide (SEQ ID NO: 4, 5'-Phos-CACGTCCAGATCAAT(hmC)GACTATGAGCAGTACA) was treated with approximately 2.2 mM Cu(ClO4)2, approximately 4.4 mM Bpy, approximately 2.2 mM proline, approximately 0.44 mM ABNO, and approximately 10 mM NaOH for approximately 1 hour, and then reacted with approximately 150 mM malononitrile for approximately 60 minutes. The reaction product was purified using a Zymo DNA clean filter. The LC-MS results are shown in Figure 6.

[0272] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in application data sheets are incorporated herein by reference in their entirety. The embodiments of the above embodiments may be modified to provide further embodiments by adopting various patent, application and publication concepts as needed.

[0273] While this disclosure has been described with reference to several exemplary embodiments, it should be understood that many other modifications and embodiments that would fall within the spirit and scope of the principles of this disclosure can be devised by those skilled in the art. More specifically, reasonable variations and alterations are possible in the components and / or arrangements of the subject matter in combination configurations of the subject matter in the foregoing disclosure, drawings, and appended claims, without departing from the spirit of this disclosure. In addition to variations and alterations of components and / or arrangements, alternative uses will also be apparent to those skilled in the art.

Claims

1. A composition comprising one or more nucleic acid molecules, each nucleic acid molecule comprising (a) one or more 5-hydroxymethylcytosine bases, (b) a copper salt, (c) a chelating agent selected from the group consisting of bipyridine or phenanthroline, and (d) an N-oxide reagent, wherein the pH of the composition is in the range of about 7 to about 12, and the N-oxide reagent is selected from the group consisting of ABNO, AZADO, and Me-AZADO.

2. The composition according to claim 1, wherein the copper salt forms a complex with the chelating agent.

3. The composition according to claim 1, wherein the ratio of the copper salt to the chelating agent in the composition is in the range of about 1:3 to about 1:

1.

4. The composition according to claim 1, wherein the ratio of the copper salt to the chelating agent in the composition is about 1:2.

5.

5. The composition according to claim 1, wherein the ratio of the copper salt to the chelating agent in the composition is about 1:2.

2.

6. The composition according to claim 1, wherein the ratio of the copper salt to the chelating agent in the composition is about 1:

2.

7. The composition according to claim 1, wherein the chelating agent is 2,2'-bipyridine or a derivative thereof.

8. The composition according to claim 1, wherein the bipyridine is selected from the group consisting of 4,4'-dimethyl-2,2'-bipyridine, 5,5'-dimethyl-2,2'-bipyridine, 4,4'-diethyl-2,2'-bipyridine, 5,5'-diethyl-2,2'-bipyridine, 4,4'-dimethoxy-2,2'-bipyridine, and 5,5'-dimethoxy-2,2'-bipyridine.

9. The copper salt is Cu(ClO ４ ) ２ , CdSO ４ Cu (ACN) ４ Triflate and Cu(OAc) ２ A composition according to claim 1, selected from the group consisting of the following.

10. The composition according to claim 1, wherein the N-oxide reagent is AZADO.

11. The composition according to claim 1, wherein the N-oxide reagent is Me-AZADO.

12. The composition according to claim 1, wherein the N-oxide reagent comprises ABNO.

13. The composition according to claim 1, wherein the ratio of the copper salt to the N-oxide in the composition is in the range of about 1:0.5 to about 1:0.

1.

14. The composition according to claim 1, wherein the ratio of the copper salt to the N-oxide in the composition is about 1:about 0.

2.

15. The composition according to claim 1, further comprising a solvent.

16. The composition according to claim 15, wherein the solvent is acetonitrile.

17. The composition according to claim 1, wherein the composition comprises a base selected from the group consisting of NaOH, KOH, and LiOH.

18. The composition according to claim 1, wherein the composition comprises TRIS, TAPSO, TEA, EPPS, tricine, Gly-Gly, Bicine, TABS, AMPSO, CHES, CAPS, AMP, and CAPS.

19. The composition according to claim 1, wherein one or more nucleic acid molecules are single-stranded.

20. The composition according to claim 1, wherein one or more nucleic acid molecules are double-stranded.

21. The composition according to claim 1, wherein the pH of the composition is in the range of about 8 to about 12.

22. A composition comprising one or more nucleic acid molecules, each nucleic acid molecule comprising (a) one or more 5-formylcytosine bases, (b) a copper salt, (c) a chelating agent selected from one of bipyridine or phenanthroline, and (d) formula (I): 【Chemistry 1】 (In the formula, R is an electron-withdrawing group selected from cyano, nitro, C １ -C ６ alkyl, carboxylic acid ester, unsubstituted carboxamide, C １ -C ６ alkyl-monosubstituted carboxamide, C １ -C ６ alkyl-disubstituted carboxamide, a substituted carbonyl moiety, and a substituted sulfonyl moiety, wherein the substitution is C １ -C ６ a linear or branched alkyl group, C ４ -C ６ selected from cycloalkyl group, phenyl, 5- or 6-membered heteroaryl, and 5- or 6-membered heteroaryl fused with a benzene ring), and has a compound The composition wherein the pH of the composition is in the range of approximately 7 to approximately 12.

23. R stands for cyano, nitro, C １ -C ６ The composition according to claim 22, wherein the alkyl is a carboxylic acid ester.

24. R is cyano or C １ -C ６ The composition according to claim 22, wherein it is alkyl.

25. The composition according to claim 22, wherein the compound of formula (I) is malononitrile.

26. The composition according to claim 22, wherein the chelating agent is phenanthroline.

27. The composition according to claim 22, wherein the chelating agent is bipyridine.

28. The composition according to claim 22, wherein the copper salt forms a complex with the chelating agent.

29. The composition according to claim 22, wherein the ratio of the copper salt to the chelating agent in the composition is in the range of about 1:3 to about 1:1.

75.

30. The composition according to claim 22, wherein the ratio of the copper salt to the chelating agent in the composition is about 1:2.

75.

31. The composition according to claim 22, wherein the ratio of the copper salt to the chelating agent in the composition is about 1:2.

5.

32. The composition according to claim 22, wherein the ratio of the copper salt to the chelating agent in the composition is about 1:2.

25.

33. The composition according to claim 22, wherein the chelating agent is 2,2'-bipyridine or a derivative thereof.

34. The composition according to claim 22, wherein the derivative of 2,2'-bipyridine is selected from the group consisting of 4,4'-dimethyl-2,2'-bipyridine, 5,5'-dimethyl-2,2'-bipyridine, 4,4'-diethyl-2,2'-bipyridine, 5,5'-diethyl-2,2'-bipyridine, 4,4'-dimethoxy-2,2'-bipyridine, and 5,5'-dimethoxy-2,2'-bipyridine.

35. The copper salt is Cu(ClO ４ ) ２ , CdSO ４ Cu (ACN) ４ Triflate and Cu(OAc) ２ The composition according to claim 22, selected from the group consisting of the following.

36. The copper salt is (Cu(ClO) ４ ) ２ The composition according to claim 22, wherein the chelating agent is 2,2'-bipyridine.

37. The composition according to claim 22, wherein one or more nucleic acid molecules are single-stranded.

38. The composition according to claim 22, wherein one or more nucleic acid molecules are double-stranded.

39. The composition according to claim 22, wherein the pH is in the range of about 8 to about 12.

40. The composition according to claim 22, wherein the composition further comprises an N-oxide reagent.

41. A method for preparing one or more nucleic acid molecules, each having one or more 5-formylcytosine bases, comprising: (a) obtaining a sample containing one or more nucleic acid molecules, each having one or more 5-hydroxymethylcytosine bases; and (b) contacting the obtained sample with a first composition containing a chelating agent selected from one of copper salts, bipyridine, or phenanthroline, and an N-oxide reagent, wherein the N-oxide reagent is selected from the group consisting of ABNO, AZADO, and Me-AZADO.

42. The method according to claim 41, wherein the first composition further comprises at least one base or buffer.

43. The method according to claim 41, wherein the reaction is carried out at a pH in the range of approximately 7 to approximately 12.

5.

44. The method according to claim 41, wherein the reaction is carried out at a pH in the range of about 8 to about 12.

45. The method according to claim 41, wherein the reaction is carried out at a temperature in the range of about 20°C to about 35°C.

46. The method according to claim 45, wherein the temperature is in the range of about 20°C to about 30°C.

47. The method according to claim 45, wherein the temperature is in the range of about 25°C to about 30°C.

48. The method according to claim 41, wherein the reaction is carried out over a period of time ranging from approximately 30 minutes to approximately 90 minutes.

49. The method according to claim 48, wherein the period is approximately 60 minutes.

50. The method according to claim 41, wherein the ratio of the copper salt to the chelating agent in the first composition is about 1:2.

75.

51. The method according to claim 41, wherein the ratio of the copper salt to the chelating agent in the first composition is about 1:2.

5.

52. The method according to claim 41, wherein the ratio of the copper salt to the chelating agent in the first composition is about 1:2.

25.

53. The method according to claim 41, wherein the chelating agent is 2,2'-bipyridine or a derivative thereof.

54. The method according to claim 41, wherein the bipyridine is selected from the group consisting of 4,4'-dimethyl-2,2'-bipyridine, 5,5'-dimethyl-2,2'-bipyridine, 4,4'-diethyl-2,2'-bipyridine, 5,5'-diethyl-2,2'-bipyridine, 4,4'-dimethoxy-2,2'-bipyridine, and 5,5'-dimethoxy-2,2'-bipyridine.

55. The copper salt is Cu(ClO ４ ) ２ , CdSO ４ Cu (ACN) ４ Triflate and Cu(OAc) ２ The method according to claim 41, selected from the group consisting of the following.

56. The method according to claim 41, wherein the N-oxide is ABNO.

57. The copper salt is Cu(ACN) ４ Triflate or Cu(OAc) ２ The method according to claim 41, wherein the chelating agent is 2,2'-bipyridine and the N-oxide is ABNO.

58. The method according to claim 41, further comprising monitoring the reaction by liquid chromatography and / or mass spectrometry.

59. The method according to claim 41, further comprising carrying out at least one additional downstream reaction after the reaction of the obtained sample with the first composition.

60. The method according to claim 59, wherein the at least one additional downstream reaction converts the 5-formylcytosine base in the one or more nucleic acid molecules into adducts of 5-formylcytosine, thereby producing one or more nucleic acid molecules having one or more adducts of 5-formylcytosine.

61. The method according to claim 60, wherein the adduct of 5-formylcytosine is a malononitrile adduct of 5-formylcytosine.

62. The method according to claim 60, further comprising contacting one or more nucleic acid molecules having one or more adducts of 5-formylcytosine with a polymerase to obtain one or more amplified nucleic acid molecules, wherein each of the one or more amplified nucleic acid molecules has a thymine base at a position corresponding to the position of the adduct of 5-formylcytosine in each of the one or more nucleic acid molecules having one or more adducts of 5-formylcytosine.

63. The method according to claim 62, further comprising sequencing one or more amplified nucleic acid molecules.

64. The method according to claim 63, wherein the sequencing includes next-generation sequencing.

65. A method for preparing one or more nucleic acid molecules, each having one or more adducts of 5-formylcytosine, comprising: (a) obtaining a sample containing one or more nucleic acid molecules, each having one or more 5-formylcytosine bases; and (b) chelating the obtained sample with a chelating agent selected from one of copper salts, bipyridine, or phenanthroline, and a compound having formula (I): 【Chemistry 2】 (In the formula, R stands for cyano, nitro, C １ -C ６ Alkyl, carboxylic acid ester, unsubstituted carboxamide, C １ -C ６ Alkyl monosubstituted carboxamide, C １ -C ６ The electron-withdrawing group is selected from alkyl disubstituted carboxamides, substituted carbonyl moieties, and substituted sulfonyl moieties, wherein the substitution is C １ -C ６ Linear or branched alkyl groups, C ４ -C ６ A method comprising reacting with a composition comprising a cycloalkyl group, a phenyl group, a five- or six-membered heteroaryl group, and a five- or six-membered heteroaryl group formed by the condensation of a benzene ring.

66. R is cyano or C １ -C ６ The method according to claim 65, wherein the alkyl group is alkyl.

67. The method according to claim 65, wherein the compound of formula (I) is malononitrile.

68. The method according to claim 65, wherein the reaction is carried out at a pH in the range of approximately 7 to approximately 12.

5.

69. The method according to claim 65, wherein the reaction is carried out at a pH in the range of about 8 to about 12.

70. The method according to claim 65, wherein the reaction is carried out at a temperature in the range of about 20°C to about 35°C.

71. The method according to claim 70, wherein the temperature is in the range of about 20°C to about 30°C.

72. The method according to claim 70, wherein the temperature is in the range of about 25°C to about 30°C.

73. The method according to claim 65, wherein the reaction is carried out over a period of time ranging from approximately 30 minutes to approximately 90 minutes.

74. The method according to claim 73, wherein the aforementioned period is approximately 60 minutes.

75. The method according to claim 65, wherein the chelating agent is phenanthroline.

76. The method according to claim 65, wherein the chelating agent is bipyridine.

77. The method according to claim 65, wherein the ratio of the copper salt to the chelating agent in the first composition is about 1:2.

75.

78. The method according to claim 65, wherein the ratio of the copper salt to the chelating agent in the first composition is about 1:2.

5.

79. The method according to claim 65, wherein the ratio of the copper salt to the chelating agent in the first composition is about 1:2.

25.

80. The method according to claim 65, wherein the chelating agent is 2,2'-bipyridine or a derivative thereof.

81. The method according to claim 65, wherein the bipyridine is selected from the group consisting of 4,4'-dimethyl-2,2'-bipyridine, 5,5'-dimethyl-2,2'-bipyridine, 4,4'-diethyl-2,2'-bipyridine, 5,5'-diethyl-2,2'-bipyridine, 4,4'-dimethoxy-2,2'-bipyridine, and 5,5'-dimethoxy-2,2'-bipyridine.

82. The copper salt is Cu(ClO ４ ) ２ , CdSO ４ Cu (ACN) ４ Triflate and Cu(OAc) ２ The method according to claim 65, selected from the group consisting of the following.

83. The method according to claim 65, further comprising monitoring the reaction by liquid chromatography and / or mass spectrometry.

84. The method according to claim 65, further comprising contacting one or more nucleic acid molecules having one or more adducts of 5-formylcytosine with a polymerase to obtain one or more amplified nucleic acid molecules, wherein each of the one or more amplified nucleic acid molecules has a thymine base at a position corresponding to the position of the adduct of 5-formylcytosine in each of the one or more nucleic acid molecules having one or more adducts of 5-formylcytosine.

85. The method according to claim 84, further comprising sequencing one or more amplified nucleic acid molecules.

86. The method of claim 85, wherein the sequencing includes next-generation sequencing.

87. The method according to claim 65, wherein the obtained sample is prepared by (i) obtaining a solution containing one or more nucleic acid molecules each having one or more 5-hydroxymethylcytosine bases, and (ii) oxidizing the 5-hydroxymethylcytosine bases of the one or more nucleic acid molecules in the solution to obtain the sample containing one or more nucleic acid molecules each having one or more 5-formylcytosine bases.

88. The method according to claim 87, wherein the oxidation comprises exposing the solution to a formulation comprising a chelating agent selected from one of a copper salt, bipyridine, or phenanthroline, and an N-oxide.

89. A method for synthesizing one or more nucleic acid molecules, each comprising one or more adducts of 5-formylcytosine, the method comprising: (a) obtaining a sample comprising one or more nucleic acid molecules each comprising one or more 5-hydroxymethylcytosine bases; (b) contacting the obtained sample with a first composition at a first temperature and for a first period to obtain a mixture comprising one or more nucleic acid molecules each comprising one or more 5-formylcytosine bases, wherein the first composition comprises a chelating agent selected from one of copper salt, bipyridine or phenanthroline, and an N-oxide reagent; and (c) contacting the obtained mixture at a second temperature for a second period, wherein the second composition comprises a compound having formula (I): 【Transformation 3】 (In the formula, R stands for cyano, nitro, C １ -C ６ Alkyl, carboxylic acid ester, unsubstituted carboxamide, C １ -C ６ Alkyl monosubstituted carboxamide, C １ -C ６ The electron-withdrawing group is selected from alkyl disubstituted carboxamides, substituted carbonyl moieties, and substituted sulfonyl moieties, wherein the substitution is C １ -C ６ Linear or branched alkyl groups, C ４ -C ６ The process involves contacting a mixture containing (selected from cycloalkyl groups, phenyl groups, five- or six-membered heteroaryl groups, and five- or six-membered heteroaryl groups with a fused benzene ring), A method for obtaining one or more nucleic acid molecules, each containing one or more adducts of 5-formylcytosine.

90. The method according to claim 89, wherein the obtained mixture is brought into contact with the second composition without first purifying the mixture.

91. The method according to claim 89, wherein the obtained sample is brought into contact with the first composition at a pH in the range of about 8 to about 12.

92. The method according to claim 89, wherein the obtained sample is brought into contact with the second composition at a pH in the range of about 8 to about 12.

93. The method according to claim 89, wherein the first temperature is in the range of about 20°C to about 35°C.

94. The method according to claim 89, wherein the second temperature is in the range of about 20°C to about 35°C.

95. The method according to claim 89, wherein the first and second temperatures are substantially the same.

96. The method according to claim 89, wherein the first duration is approximately 30 minutes to approximately 90 minutes, and the second duration is approximately 30 minutes to approximately 90 minutes.

97. The copper salt is Cu(ACN) ４ Triflate or Cu(OAc) ２ The method according to claim 89, wherein the chelating agent is 2,2'-bipyridine.

98. The Cu(ACN) present in the first composition ４ Triflate or the Cu(OAc) ２ The method according to claim 97, wherein the ratio of the amount of to the amount of 2,2'-bipyridine is in the range of about 1:1 to about 1:

3.

99. The method according to claim 89, wherein the compound having formula (I) is malononitrile.

100. The method according to claim 89, wherein the bipyridine is selected from the group consisting of 4,4'-dimethyl-2,2'-bipyridine, 5,5'-dimethyl-2,2'-bipyridine, 4,4'-diethyl-2,2'-bipyridine, 5,5'-diethyl-2,2'-bipyridine, 4,4'-dimethoxy-2,2'-bipyridine, and 5,5'-dimethoxy-2,2'-bipyridine.

101. The method according to claim 89, wherein one or more nucleic acid molecules in the obtained sample are single-stranded nucleic acid molecules.

102. The method according to claim 89, wherein one or more nucleic acid molecules in the obtained sample are double-stranded nucleic acid molecules.

103. The method according to claim 89, wherein one or more nucleic acid molecules in the acquired sample are ligated to one or more adapters.

104. The method according to claim 89, wherein one or more nucleic acid molecules in the acquired sample contain one or more barcodes.

105. The method according to claim 104, wherein one or more barcodes are unique molecular identifiers.

106. The method according to claim 89, wherein the obtained sample is prepared by (i) obtaining a solution containing one or more nucleic acid molecules each having one or more 5-methylcytosine bases, and (ii) oxidizing the 5-methylcytosine bases of the one or more nucleic acid molecules in the solution to obtain the sample containing one or more nucleic acid molecules each having one or more 5-hydroxymethylcytosine bases.

107. The method according to claim 89, further comprising contacting one or more nucleic acid molecules having one or more adducts of 5-formylcytosine with a polymerase to obtain one or more amplified nucleic acid molecules, wherein each of the one or more amplified nucleic acid molecules has a thymine base at a position corresponding to the position of the adduct of 5-formylcytosine in each of the one or more nucleic acid molecules having one or more adducts of 5-formylcytosine.

108. The method according to claim 107, wherein the adduct of 5-formylcytosine is a malononitrile adduct of 5-formylcytosine.

109. The method according to claim 107, further comprising ligating one or more adapters to the one or more nucleic acid molecules having one or more adducts of 5-formylcytosine before contacting the one or more nucleic acid molecules having one or more adducts of 5-formylcytosine with the polymerase.

110. The method according to claim 107, wherein the polymerase is DNA polymerase.

111. The method according to claim 110, wherein the DNA polymerase is a uracil-resistant polymerase.

112. The method according to claim 107, further comprising sequencing one or more amplified nucleic acid molecules.

113. The method according to claim 112, wherein the sequencing includes next-generation sequencing.

114. The method according to claim 89, wherein the sample obtained is obtained from a tumor.

115. The method according to claim 89, wherein the obtained sample is obtained from a specimen suspected of having a tumor.

116. A kit comprising (i) a first container containing a copper salt and a chelating agent selected from bipyridine or phenanthroline, and (ii) a second container containing a polymerase.

117. Adductor forming reagent having formula (I) 【Chemistry 4】 (In the formula, R stands for cyano, nitro, C １ -C ６ Alkyl, carboxylic acid ester, unsubstituted carboxamide, C １ -C ６ Alkyl monosubstituted carboxamide, C １ -C ６ The electron-withdrawing group is selected from alkyl disubstituted carboxamides, substituted carbonyl moieties, and substituted sulfonyl moieties, wherein the substitution is C １ -C ６ Linear or branched alkyl groups, C ４ -C ６ The kit according to claim 116, further comprising a third container containing (selected from cycloalkyl groups, phenyl groups, five- or six-membered heteroaryl groups, and five- or six-membered heteroaryl groups formed by condensation of a benzene ring).

118. The kit according to claim 116, wherein the ratio of the amount of chelating agent to the amount of copper salt in the first container is in the range of about 2:

1.

119. The kit according to claim 1, wherein the polymerase is a heat-stable polymerase.

120. The kit according to claim 116, further comprising at least one buffer solution or strong base.

121. A method for detecting one or more epigenetic changes in a target nucleic acid molecule, comprising: (a) obtaining a sample comprising one or more nucleic acid molecules each having one or more 5-hydroxymethylcytosine bases; (b) contacting the obtained sample with a first composition at a first temperature and for a first period of time to obtain a mixture comprising one or more nucleic acid molecules each having one or more 5-formylcytosine bases, wherein the first composition comprises a chelating agent selected from one of copper salts, bipyridine, or phenanthroline, and an N-oxide; and (c) contacting the obtained mixture with a second composition at a second temperature and for a second period of time, wherein the second composition comprises a compound having formula (I): 【Transformation 5】 (In the formula, R stands for cyano, nitro, C １ -C ６ Alkyl, carboxylic acid ester, unsubstituted carboxamide, C １ -C ６ Alkyl monosubstituted carboxamide, C １ -C ６ The electron-withdrawing group is selected from alkyl disubstituted carboxamides, substituted carbonyl moieties, and substituted sulfonyl moieties, wherein the substitution is C １ -C ６ Linear or branched alkyl groups, C ４ -C ６ (Selected from cycloalkyl groups, phenyl groups, 5-membered or 6-membered heteroaryl groups, and 5-membered or 6-membered heteroaryl groups with a fused benzene ring), A method comprising: (d) obtaining one or more nucleic acid molecules each containing one or more adducts of 5-formylcytosine; (e) contacting the one or more nucleic acid molecules having one or more adducts of 5-formylcytosine with a polymerase to obtain one or more amplified nucleic acid molecules, wherein the one or more amplified nucleic acid molecules have a thymine base at a position corresponding to the position of the adduct of 5-formylcytosine in each of the one or more nucleic acid molecules having one or more adducts of 5-formylcytosine; and (f) sequencing the one or more amplified nucleic acid molecules.

122. The method according to claim 121, wherein the adduct of 5-formylcytosine is a malononitrile adduct of 5-formylcytosine.

123. The method according to claim 121, further comprising ligating one or more adapters to the one or more nucleic acid molecules having one or more adducts of 5-formylcytosine before contacting the one or more nucleic acid molecules having one or more adducts of 5-formylcytosine with the polymerase.

124. The method according to claim 121, wherein the polymerase is DNA polymerase.

125. The method according to claim 121, wherein the DNA polymerase is a uracil-resistant polymerase.

126. The method according to claim 121, wherein the sequencing includes next-generation sequencing.

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