CN116806266A

CN116806266A - Method for detection of methylated base level in nucleic acids

Info

Publication number: CN116806266A
Application number: CN202280013794.8A
Authority: CN
Inventors: F·贝格曼; S·S·常; P·克里萨利; A·德比尔; D·海因德尔; O·卡克斯胡尔; D·L·彭克勒; J-A·彭克勒; M·拉尼克; M·塔英
Original assignee: F Hoffmann La Roche AG; Kapa Biosystems Inc
Current assignee: F Hoffmann La Roche AG; Kapa Biosystems Inc
Priority date: 2021-02-09
Filing date: 2022-02-08
Publication date: 2023-09-26

Abstract

The present invention includes improved methods and compositions for detecting methylation in nucleic acids. In particular, the present disclosure relates to a method of converting 5-hydroxymethylcytosine (5 hmC) and/or 5-methylcytosine (5 mC) to thymine (T). Furthermore, the present disclosure also relates to methods of detecting 5hmC and/or 5mC in a sample.

Description

Method for detection of methylated base level in nucleic acids

Technical Field

The present invention relates to the field of nucleic acid-based diagnostics. More particularly, the invention relates to a method for detecting epigenetic modifications in a nucleic acid, wherein the epigenetic modifications may have biological and clinical significance.

Background

Epigenetic modifications, and in particular DNA methylation, play a role in developmental and pathological processes. Detecting methylation includes detecting modified cytosine bases (methylcytosine and hydroxymethylcytosine (5 mC and 5 hmC)) in nucleic acids. Until recently, gold standards for detecting methylation involved treating DNA with bisulfite. This treatment will convert unmethylated cytosine (C) to uracil (U), while methylated cytosine (5 mC and 5 hmC) will remain intact. The C to U change will then be detected by, for example, nucleic acid sequencing. Unfortunately, bisulphite treatment results in the degradation of most sample DNA. Alternative, less stringent methods for detecting methylated cytosines include enzymatic treatment with 10-11 translocator (TET) dioxygenase and detection of any of the oxidation products. One particular method, known as TAPS (TET assisted pyridine-borane sequencing), involves oxidizing methylated cytosines in nucleic acids with TET and cocatalysts (e.g., fe (II) ions and alpha-ketoglutarate), and treating the oxidized products with borane derivatives 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. Other methods also utilize TET, but provide an alternative method of borane reduction.

In one example, the oxidation product may react with malononitrile to form an adduct, which is also read as T during sequencing, see Zhu C., et al, (2017) Single-Cell 5-Formylcytosine Landscapes ofMammalian Early Embryos and ESCs at Single-Base Resolution, cell Stem Cell,20:720-731.e5. Malononitrile reacts only with 5-formyl cytosine (5 fC). Another method of detecting 5fC is to use Wittig reagent in an organic solvent followed by irradiation with ultraviolet light. The reaction products were detected using fluorescent recognition techniques as described in International patent publication No. WO 2020155742.

Unfortunately, newer methods face obstacles before they can be widely adopted by clinical laboratories: the chemical reactions in the TAPS and malononitrile processes either require high temperatures (70 ℃) or require days to complete. There is a need for a rapid, convenient methylation detection method that can be deployed in a clinical laboratory.

Unfortunately, the oxidation processes currently used, such as TET in the presence of Fe (II) ions and alpha-ketoglutarate, produce a mixture of 5-carboxycytosine (5 caC) and 5-formylcytosine (5 fC). In order to be able to accurately perform the base level detection of cytosine methylation, an enzymatic method is required that selectively oxidizes methylated cytosine and preferentially or exclusively forms 5fC for use in downstream detection procedures.

Disclosure of Invention

In some embodiments, the invention is a method of detecting a 5-formyl cytosine (5 fC) nucleotide in a nucleic acid, the method comprising: (i) By combining a sample containing a nucleic acid comprising 5fC with a sample comprising a nucleic acid of formula R ₁ —CH ₂ -contacting a composition of CN compounds to form a reaction mixture, the compounds being capable of reacting with 5fC in the nucleic acid to form adducts according to the following reaction scheme:

wherein R is ₁ Is an electron withdrawing group selected from the group consisting of substituted or unsubstituted cyano, nitro, formyl, carbonyl compounds, wherein the substitution is selected from the group consisting of C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, C1-C30 linear or branched alkynyl, cycloalkyl, aryl, or heteroaryl; (ii) Incubating the reaction mixture for less than 3 hours, wherein at least 90% of the 5fC has formed an adduct; (iii) Sequencing nucleic acids from the reaction mixture to obtain a test sequence, wherein the adduct is read as thymine (T) during sequencing; and (iii) comparing the test sequence to a reference sequence, wherein a transition from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of 5fC in the nucleic acid. In some embodiments, R1 is a cyano group (CN). In some embodiments, a compound of formula R ₁ —CH ₂ The composition of the-CN compound contains an organic acid moiety. In some embodiments, the organic acid has the formula R-COOH and R is selected from the group consisting of C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, and C1-C30 linear or branched alkynyl. In some embodiments, the organic acid is acetic acid. In some embodiments, a compound of formula R ₁ —CH ₂ The combination of-CN compounds is present in a non-aqueous solvent. In some embodiments, the nonaqueous solvent has the formula R-OH, wherein R is selected from the group consisting of C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, C1-C30 linear or branched alkynyl, cycloalkyl, aryl, or heteroaryl. In some embodiments, the nonaqueous solvent is 10% to 100%, e.g., 90%-100% methanol or ethanol. In some embodiments, the reaction mixture further comprises formula R _x NH _y A compound wherein x and y are 0, 1, 2 or 3 such that x+y = 3 and each R is independently selected from C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, C1-C30 linear or branched alkynyl, cycloalkyl, aryl or heteroaryl. In some embodiments, R _x NH _y Is triethanolamine. In some embodiments, the reaction mixture is incubated for 1 hour. In some embodiments, prior to sequencing in step (iii), the nucleic acid is amplified, for example with a B family polymerase, effectively incorporating adenine (a) nucleotides as opposed to adducts. In some embodiments, the sequencing in step (iii) is performed by a sequencing-by-synthesis (SBS) method, for example using nanopores.

In some embodiments, the nucleic acid comprising 5fC is obtained by contacting a nucleic acid comprising methylated cytosine with a composition comprising 10-11 translocation (TET) dioxygenase and 5 to 100 μm Fe (II) ions at a pH of 7 to 8. In some embodiments, the composition comprises a 10-11 translocation (TET) dioxygenase and 5 to 10 μm of Fe (II) ions at pH 8. In some embodiments, the composition comprises a 10-11 translocation (TET) dioxygenase and 80 to 100 μm of Fe (II) ion at pH 7. In some embodiments, the Fe (II) ion is produced by contacting the sample with a metal selected from FeSO ₄ 、(NH ₄ ) ₂ Fe(SO ₄ ) ₂ 、FeSO ₄ 7H ₂ O、(NH ₄ ) ₂ Fe(SO ₄ ) ₂ 6H ₂ O and FeCl ₂ Is produced by contacting the compounds of (a) with one another. In some embodiments, the composition further comprises one or more of ascorbic acid, alpha-ketoglutarate, and a reducing agent. In some embodiments, the nucleic acid comprising 5fC is obtained by contacting a nucleic acid comprising methylated cytosine with a composition comprising a Cu (II) compound and 2, 6-tetramethylpiperidin-1-yloxy (TEMPO). In some embodiments, the nucleic acid comprising 5fC is prepared by contacting a nucleic acid comprising methylated cytosine with a nucleic acid selected from potassium ruthenate (K ₂ RuO ₄ ) And potassium homoruthenate (KRUO) ₄ ) Is obtained by contacting potassium ruthenium salt.

In some embodiments, the invention is a method of detecting methylation in a nucleic acid A method of cytosine (C) nucleotides, the method comprising: (i) By combining a sample containing a nucleic acid comprising 5-methylcytosine (5 mC) and/or 5-hydroxymethylcytosine (5 hmC) with a nucleic acid comprising a 10-11 metathesis (TET) dioxygenase capable of converting 5mC and 5hmC in the nucleic acid to 5-formylcytosine (5 fC) and a nucleic acid of formula R ₁ —CH ₂ -contacting a composition of CN compounds to form a reaction mixture, the compounds being capable of reacting with 5fC in a nucleic acid to form an adduct according to the following reaction scheme:

wherein R is ₁ Is an electron withdrawing group selected from the group consisting of substituted or unsubstituted cyano, nitro, formyl, carbonyl compounds, wherein the substitution is selected from the group consisting of C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, C1-C30 linear or branched alkynyl, cycloalkyl, aryl, or heteroaryl; (ii) Incubating the reaction mixture for less than 3 hours, wherein at least 90% of the 5fC has formed an adduct; (iii) Sequencing nucleic acids from the reaction mixture to obtain a test sequence, wherein the adduct is read as thymine (T) during sequencing; and (iii) comparing the test sequence to a reference sequence, wherein a transition from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence is indicative of the presence of a methylated cytosine in the nucleic acid. In some embodiments, R1 is cyano group (CN) and the composition added in step (i) contains a nonaqueous solvent having the formula R-OH, wherein R is selected from C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, C1-C30 linear or branched alkynyl, cycloalkyl, aryl or heteroaryl, and the concentration of solvent in the reaction mixture is at least 90%. In some embodiments, R1 is cyano group (CN) and the composition added in step (i) contains ethanol or methanol in a concentration of at least 90% in the reaction mixture, and further comprises triethanolamine.

In some embodiments, the invention is a method of detecting methylated cytosine nucleotides in a nucleic acid, the method comprising: (i) Ligating an adapter to a nucleic acid comprising 5-methylcytosine (5 mC) and/or 5-hydroxymethylcytosine (5 hmC), wherein the adapter comprises an amplification primer binding site; (ii) Forming a reaction mixture by contacting a sample containing adapter-ligated nucleic acids with a 10-11 translocation (TET) dioxygenase capable of converting 5mC and 5hmC in the nucleic acids to 5-formylcytosine (5 fC);

(iii) Allowing the reaction mixture to react with a compound of formula R ₁ —CH ₂ -CN compound, which compound is capable of reacting with 5fC in a nucleic acid to form an adduct according to the following reaction scheme:

wherein R is ₁ Is an electron withdrawing group selected from the group consisting of substituted or unsubstituted cyano, nitro, formyl, carbonyl compounds, wherein the substitution is selected from the group consisting of C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, C1-C30 linear or branched alkynyl, cycloalkyl, aryl, or heteroaryl; (iv) Incubating the reaction mixture for less than 3 hours, wherein at least 90% of the 5fC has formed an adduct; (v) Amplifying the ligated nucleic acids using a DNA polymerase and a primer capable of binding to the primer binding site, wherein the DNA polymerase reads the adduct as thymine (T) during amplification; (vi) Sequencing the amplified nucleic acid to obtain a test sequence; (vii) Comparing the test sequence to a reference sequence, wherein a transition from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of a methylated cytosine in the nucleic acid. In some embodiments, R1 is cyano group (CN) and the composition added in step (i) contains a nonaqueous solvent having the formula R-OH, wherein R is selected from C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, C1-C30 linear or branched alkynyl, cycloalkyl, aryl or heteroaryl, and the concentration of solvent in the reaction mixture is at least 90%. In some embodiments, R1 is cyano group (CN) and the composition added in step (i) contains ethanol or methanol in a concentration of at least 90% in the reaction mixture, and further comprises triethanolamine.

In some embodiments, the invention is a kit for detecting 5-formyl cytosine (5 fC) in a nucleic acid in less than 3 hours, the kit comprising an ethanol solution of malononitrile. In some embodiments, the kit further comprises one or more of the following: nucleic acid sequencing reagents, nucleic acid amplification reagents, nucleic acid purification reagents, solutions of acetic acid, solutions of triethanolamine and instructions for reacting 5fC in a nucleic acid with malononitrile in the presence of an organic acid and an alkylamine.

In some embodiments, the invention is a kit for detecting methylated cytosine nucleotides in a nucleic acid in less than 3 hours, the kit comprising 10-11 translocation (TET) dioxygenase, an ethanol solution of malononitrile, and further comprising reagents for nucleic acid purification, amplification, and sequencing.

In some embodiments, the invention is a method of detecting 5-formyl cytosine (5 fC) and 5-carboxyl cytosine (5 caC) nucleotides in a nucleic acid, the method comprising: (i) Forming a reaction mixture by contacting a sample containing a nucleic acid comprising 5fC and/or 5 cat with a composition comprising a borane derivative; (ii) Incubating the reaction mixture for less than 3 hours, wherein at least 90% of the 5fC and 5caC have been reduced to Dihydropyrimidine (DHU);

(iii) Sequencing nucleic acids from the reaction mixture to obtain a test sequence, wherein DHU is read as thymine (T) during sequencing; and (iii) comparing the test sequence to a reference sequence, wherein a transition from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of 5fC or 5caC in the nucleic acid. In some embodiments, the borane derivative is picoline borane. In some embodiments, the reaction mixture contains an organic acid moiety. In some embodiments, the organic acid has the formula R-COOH and R is selected from the group consisting of C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, and C1-C30 linear or branched alkynyl. In some embodiments, the organic acid is acetic acid. In some embodiments, the borane derivative is present in a non-aqueous solvent. In some embodiments, the nonaqueous solvent has the formula R-OH, wherein R is selected from the group consisting of C1-C30 linear or branched alkyl, C1-C30 linear or branched alkylAlkenyl, C1-C30 straight or branched chain alkynyl, cycloalkyl, aryl or heteroaryl. In some embodiments, the nonaqueous solvent is methanol or ethanol. In some embodiments, the reaction mixture is incubated for 1 hour. In some embodiments, prior to sequencing in step (iii), the nucleic acid is amplified, for example with a B family polymerase, effectively incorporating adenine (a) nucleotides as opposed to DHU. In some embodiments, the sequencing in step (iii) is performed by a sequencing-by-synthesis (SBS) method, for example using nanopores. In some embodiments, the nucleic acid comprising 5fC and/or 5caC is obtained by contacting a nucleic acid comprising methylated cytosine with a composition comprising 10-11 translocation (TET) dioxygenase and 5 to 100 μm Fe (II) ions at a pH of 7 to 8. In some embodiments, the composition comprises a 10-11 translocation (TET) dioxygenase and 5 to 10 μm of Fe (II) ions at pH 8. In some embodiments, the composition comprises a 10-11 translocation (TET) dioxygenase and 80 to 100 μm of Fe (II) ion at pH 7. In some embodiments, the Fe (II) ion is produced by contacting the sample with a metal selected from FeSO ₄ 、(NH ₄ ) ₂ Fe(SO ₄ ) ₂ 、FeSO ₄ 7H ₂ O、(NH ₄ ) ₂ Fe(SO ₄ ) ₂ 6H ₂ O and FeCl ₂ Is produced by contacting the compounds of (a) with one another. In some embodiments, the composition further comprises one or more of ascorbic acid, alpha-ketoglutarate, and a reducing agent. In some embodiments, the nucleic acid comprising 5fC is obtained by contacting a nucleic acid comprising methylated cytosine with a composition comprising a Cu (II) compound and 2, 6-tetramethylpiperidin-1-yloxy (TEMPO). In some embodiments, the nucleic acid comprising 5fC is prepared by contacting a nucleic acid comprising methylated cytosine with a nucleic acid selected from potassium ruthenate (K ₂ RuO ₄ ) And potassium homoruthenate (KRUO) ₄ ) Is obtained by contacting potassium ruthenium salt.

In some embodiments, the invention is a method of detecting methylated cytosine (C) nucleotides in a nucleic acid, the method comprising: (i) Forming a reaction mixture by contacting a sample containing a nucleic acid comprising 5-methylcytosine (5 mC) and/or 5-hydroxymethylcytosine (5 hmC) with a composition comprising a 10-11 metathesis (TET) dioxygenase capable of converting 5mC and 5hmC in the nucleic acid to 5-formylcytosine (5 fC) and 5-carboxycytosine (5 caC) and a borane derivative in a non-aqueous solvent;

(ii) Incubating the reaction mixture for less than 3 hours, wherein at least 90% of the 5fC and 5caC have been reduced to Dihydropyrimidine (DHU); (iii) Sequencing nucleic acids from the reaction mixture to obtain a test sequence, wherein the DHU is read as thymine (T) during sequencing; and (iv) comparing the test sequence to a reference sequence, wherein a transition from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence is indicative of the presence of a methylated cytosine in the nucleic acid. In some embodiments, the borane derivative is selected from the group consisting of pyridinium borane, 2-methylpyridinium borane (pic-BH 3), borane, sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride, and the nonaqueous solvent is selected from the group consisting of methanol and methanol, and the reaction mixture further comprises acetic acid.

In some embodiments, the invention is a method of detecting methylated cytosine nucleotides in a nucleic acid, the method comprising: (i) Ligating an adapter to a nucleic acid comprising 5-methylcytosine (5 mC) and/or 5-hydroxymethylcytosine (5 hmC), wherein the adapter comprises an amplification primer binding site; (ii) Forming a reaction mixture by contacting a sample containing adapter-ligated nucleic acids with a 10-11 translocation (TET) dioxygenase capable of converting 5mC and 5hmC in the nucleic acids to 5-formyl cytosine (5 fC) and 5-carboxyl cytosine (5 caC); (iii) Contacting the reaction mixture with a borane derivative in a non-aqueous solvent; (iv) Incubating the reaction mixture for less than 3 hours, wherein at least 90% of the 5fC and 5caC have been reduced to Dihydropyrimidine (DHU); (v) Amplifying the ligated nucleic acids using a DNA polymerase and a primer capable of binding to the primer binding site, wherein the DNA polymerase reads DHU as thymine (T) during amplification; (vi) Sequencing the amplified nucleic acid to obtain a test sequence; (vii) Comparing the test sequence to a reference sequence, wherein a transition from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of a methylated cytosine in the nucleic acid. In some embodiments, the borane derivative is selected from the group consisting of pyridinium borane, 2-methylpyridinium borane (pic-BH 3), borane, sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride, and the nonaqueous solvent is selected from the group consisting of methanol and methanol, and the reaction mixture further comprises acetic acid.

In some embodiments, the invention is a kit for detecting 5-formyl cytosine (5 fC) and 5-carboxyl cytosine (5 caC) in a nucleic acid in less than 3 hours, the kit comprising a borane derivative in an ethanol solution. In some embodiments, the borane derivative is selected from the group consisting of pyridinium borane, 2-picolinate borane (pic-BH 3), borane, sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride. In some embodiments, the kit further comprises one or more of the following: nucleic acid sequencing reagents, nucleic acid amplification reagents, nucleic acid purification reagents, solutions of acetic acid and instructions for reacting 5fC and 5caC in a nucleic acid with a borane compound in the presence of an organic acid.

In some embodiments, the invention is a kit for detecting methylated cytosine nucleotides in a nucleic acid in less than 3 hours, the kit comprising a 10-11 translocation (TET) dioxygenase, an ethanol solution of a borane derivative, and further comprising reagents for nucleic acid purification, amplification, and sequencing.

In some embodiments, the invention is a single tube method of detecting a 5-formyl cytosine (5 fC) nucleotide in a nucleic acid, the method comprising: (i) By contacting a sample containing a nucleic acid comprising 5fC with a 10-11 translocation (TET) dioxygenase and a nucleic acid of formula R ₁ —CH ₂ -contacting a composition of CN compounds to form a reaction mixture, the compounds being capable of reacting with 5fC in the nucleic acid to form adducts according to the following reaction scheme:

wherein R is ₁ Is an electron withdrawing group selected from the group consisting of substituted or unsubstituted cyano, nitro, formyl, carbonyl compounds, wherein the substitution is selected from the group consisting of C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, C1-C30 linear or branched alkynyl, cycloalkyl, arylA group or heteroaryl; (ii) incubating the reaction mixture to form an adduct;

(iii) Sequencing nucleic acids from the reaction mixture to obtain a test sequence, wherein the adduct is read as thymine (T) during sequencing; and (iii) comparing the test sequence to a reference sequence, wherein a transition from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of 5fC in the nucleic acid. In some embodiments, formula R ₁ —CH ₂ The CN compound is malononitrile.

In some embodiments, the invention is a method of detecting tissue of origin of a nucleic acid in a sample, the method comprising detecting the presence and location of methylated cytosines in the nucleic acid by the methods disclosed herein, comparing the methylation pattern to known methylation patterns of several tissues; and identifying the tissue of origin of the nucleic acid in the sample.

In some embodiments, the invention is a method of detecting organ transplant rejection in a transplant recipient comprising obtaining a blood sample containing cell free nucleic acid from the transplant recipient; detecting the presence and location of methylated cytosines in the cell free nucleic acid by the methods disclosed herein; comparing the methylation pattern with known methylation patterns of several organs; if cell free nucleic acid having a methylation pattern specific for the transplanted organ is detected in the sample, then transplant rejection is detected. In some embodiments, the invention is a method of monitoring graft rejection by: periodically, circulating cell free DNA is sampled and the presence and location of methylated cytosines is detected according to the disclosure herein, and changes in the level of cell free DNA having a specific methylation pattern of the transplanted organ are measured, wherein an increase in such level of cell free DNA is indicative of organ transplant rejection.

In some embodiments, the invention is a method of screening for the presence of a cancerous tumor in a patient, the method comprising obtaining a blood sample from the patient containing cell free nucleic acids; detecting the presence and location of methylated cytosines in the cell free nucleic acid by the methods disclosed herein; the methylation pattern is compared to known methylation patterns of tumor and non-tumor tissue, and if a tumor-specific methylation pattern is detected, the presence of a tumor is detected. In some embodiments, the invention is a method of monitoring tumor volume in a patient, the method comprising periodically sampling circulating cell free DNA and detecting the presence and location of methylated cytosines by the methods disclosed herein, measuring changes in the level of cell free DNA having a tumor-specific methylation pattern, wherein an increase in such cell free DNA level is indicative of tumor growth and a decrease in such cell free DNA level is indicative of tumor shrinkage. In some embodiments, the invention is a method of monitoring the effectiveness of a treatment for cancer in a patient by a method comprising the steps of: periodically, circulating cell free DNA is sampled and the presence and location of methylated cytosines is detected by the methods disclosed herein, and changes in the level of cell free DNA having a tumor-specific methylation pattern are measured, wherein an increase in such level of cell free DNA is indicative of the treatment being ineffective and a decrease in such level of cell free DNA is indicative of the effectiveness of the treatment.

In some embodiments, the invention is a method of diagnosing or treating Minimal Residual Disease (MRD) in a cancer patient, the method comprising obtaining a blood sample from the patient comprising cell free nucleic acid, detecting the presence and location of methylated cytosines in the nucleic acid by the methods disclosed herein, comparing the methylation pattern to known methylation patterns of several tissues; and identifying the tissue of origin of the nucleic acid in the sample.

In some embodiments, the invention is a method of diagnosing an autoimmune disease in a patient, the method comprising obtaining a blood sample from the patient comprising cell free nucleic acid, detecting the presence and location of methylated cytosines in the nucleic acid by the methods disclosed herein, comparing the methylation pattern to a known methylation pattern of tissue damaged by the immune disease; if such methylation patterns are found, an immune disease is diagnosed.

In some embodiments, the invention is a method of detecting the presence and location of methylated cytosines as disclosed herein, further comprising chemically blocking 5-hydroxymethylcytosine (5 hmC) in the nucleic acid from reacting with TET prior to contacting the reaction mixture with TET dioxygenase. In some embodiments, 5hmC is blocked by contacting the reaction mixture with a glycosyltransferase and a glucose moiety. In some embodiments, the reaction mixture is contacted with β -glucosyltransferase and UDP glucose.

In some embodiments, the invention is a method of forming 5-formyl cytosine (5 fC) in a nucleic acid comprising contacting a reaction mixture comprising a nucleic acid comprising at least one 5-hydroxymethyl cytosine (5 hmC) with a laccase. In some embodiments, the laccase is isolated from a species selected from the group consisting of cellulites graminea (hexagona tenuis), phoenix mushroom (Pleurotis sajor caju), oyster mushroom (Pleutoris ostreatus), carbo-trichum multiforme (Xylariapolymorpha), trametes hirsuta (Trametes hirsuta), coriolus versicolor (Trametes versicolor), and Coprinus spp. In some embodiments, the laccase is isolated from a strain selected from the group consisting of Pleurotus sajor-caju MTCC-141, pleurotus ostreatus MTCC-1801, xylobacter multiforme MTCC-1100 and Brevibacterium MTCC-1171. In some embodiments, the reaction mixture further comprises cofactors, such as 2, 6-tetramethylpiperidine-1-oxyl (TEMPO), acetosyringone, syringaldehyde, p-coumaric acid 2,2' -azine-bis (3-ethylbenzothiazoline-6-sulfonate (ABTS), violuric acid (VLA), N-acetyl-N-phenylhydroxylamine (NHA), N-Hydroxybenzotriazole (HBT), and N-Hydroxyphthalimide (HPI).

In some embodiments, the invention is a method of detecting 5-hydroxymethylcytosine (5 hmC) in a nucleic acid, comprising the steps of: contacting a sample comprising a nucleic acid comprising 5hmC with a laccase under conditions suitable for oxidizing 5hmC to 5-formyl cytosine (5 fC); allowing the sample to react with a sample containing R ₁ —CH ₂ -a composition of CN compounds capable of reacting with 5fC in a nucleic acid under conditions suitable for forming the 5fC adduct to form an adduct according to the following reaction scheme:

wherein R is ₁ Is an electron withdrawing group selected from the group consisting of cyano, nitro, C1-C6 alkyl carboxylate, unsubstituted carboxamide, C1-C6 alkyl mono-and C1-C6 alkyl di-substituted carboxamide, substituted carbonyl moiety, substituted sulfonyl moiety, wherein the substitution is selected from the group consisting of C1-C6 straight or branched chain alkyl, C4-C6 cycloalkyl, phenyl, 5-or 6-membered heteroaryl, and benzocyclized 5-or 6-membered heteroaryl; sequencing nucleic acids from the reaction mixture to obtain a test sequence, wherein the adduct is read as thymine (T) during sequencing; and comparing the test sequence to a reference sequence, wherein a transition from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of 5hmC in the nucleic acid. In some embodiments, the alkyl substitution may include heteroatoms such as O or N, e.g., -CH ₂ -CH ₂ -O-CH ₃ . In some embodiments, formula R ₁ —CH ₂ The CN compound is malononitrile. Alternatively, the same transformation can be performed with Wittig reagent. Then, R1-CH 2-CN is replaced with ph3p=c (R2) CN, wherein R2 is one of hydrogen, cyano, halogen, alkyl, and alkyl containing O, N, halogen, P, S, or Si. This is disclosed in WO 2020/155742.

The reaction is as follows:

the above disclosed R1-CH 2-CN and Wittig reagents are hereinafter defined as "fC converting reagents" because they are capable of converting cytosine to thymine equivalents when used as substrates for a polymerase.

In addition, the reaction product of R1-CH 2-CN and the Wittig reagent with 5-formyl-cytosine (5 fC) is hereinafter defined as the "5fC adduct" or "adduct" which acts as a thymine equivalent when used as a polymerase substrate.

In some embodiments, the reaction mixture further contains one or more of the following: organic acid, nonaqueous solvent and formula R _x NH _y A compound wherein x and y are 0, 1, 2 or 3 such that x+y=3 and each R is independently selected from C1-C6 linear or branched alkyl, C6-C10-aryl or 5-or 6-membered heteroaryl groups which may contain heteroatoms such as O and N. In some embodiments, the organic acid is acetic acid, the non-aqueous solvent is ethanol or methanol, and formula R _x NH _y The compound is triethanolamine or piperidine. In other embodiments, the compound is a buffer, such as ammonium acetate or Tris. In some embodiments, the nonaqueous solvent is present in the reaction mixture at a concentration of 10% to 100%, for example 90% to 100%. In some embodiments, the reaction mixture is incubated for 1 hour. In some embodiments, prior to sequencing, the nucleic acid is amplified with a B family polymerase, effectively incorporating adenine (a) nucleotides as opposed to thymine equivalents.

In some embodiments, the invention is a method of detecting methylated cytosine (C) in a nucleic acid, the method comprising: contacting a sample containing nucleic acid comprising 5-methylcytosine (5 mC) and/or 5-hydroxymethylcytosine (5 hmC) with a 10-11 translocation (TET) dioxygenase capable of converting 5mC to 5hmC and with a laccase capable of converting 5hmC to 5-formylcytosine (5 fC); allowing the sample to react with R ₁ —CH ₂ -CN compound, which compound is capable of reacting with 5fC in a nucleic acid to form an adduct according to the following reaction scheme:

wherein R is ₁ Is an electron withdrawing group selected from the group consisting of cyano, nitro, C1-C6 alkyl carboxylate, unsubstituted carboxamide, C1-C6 alkyl mono-and C1-C6 alkyl di-substituted carboxamide, substituted carbonyl moiety, substituted sulfonyl moiety, wherein the substitution is selected from the group consisting of C1-C6 straight or branched chain alkyl, C4-C6 cycloalkyl, phenyl, 5-or 6-membered heteroaryl, and benzocyclized 5-or 6-membered heteroaryl; sequencing nucleic acids from the sample to obtain a test sequence, wherein the adduct is read as thymine (T) during sequencing; and comparing the test sequence with a reference sequence, wherein the sequence is selected from the group consisting ofThe transition of cytosine (C) in the reference sequence to thymine (T) in the corresponding position in the test sequence indicates the presence of methylated cytosine in the nucleic acid. In some embodiments, formula R ₁ —CH ₂ The CN compound is malononitrile. In some embodiments, the reaction mixture further contains one or more of the following: organic acid, nonaqueous solvent and formula R _x NH _y A compound wherein x and y are 0, 1, 2 or 3 such that x+y=3 and each R is independently selected from C1-C6 linear or branched alkyl, C6-C10-aryl or 5-or 6-membered heteroaryl groups which may contain heteroatoms such as O and N. In some embodiments, the organic acid is acetic acid, the non-aqueous solvent is ethanol or methanol, and formula R _x NH _y The compound is triethanolamine or piperidine. In some embodiments, the nonaqueous solvent is present in the reaction mixture at a concentration of 10% to 100%, for example 90% to 100%. In some embodiments, the reaction mixture is incubated for 1 hour. In some embodiments, prior to sequencing, the nucleic acid is amplified with a B family polymerase, effectively incorporating adenine (a) nucleotides as opposed to adducts. In some embodiments, the TET and laccase are active in the same reaction mixture. In other embodiments, the TET and laccase are inactive in the same reaction mixture and are added continuously to the sample.

In some embodiments, the invention is a method of detecting 5-hydroxymethylcytosine (5 hmC) in a nucleic acid, comprising the steps of: contacting a sample comprising nucleic acid comprising 5hmC with a Wittig reagent under conditions suitable for oxidizing 5hmC to 5-formyl cytosine (5 fC); irradiating the sample with ultraviolet light to form a product; sequencing nucleic acids from the reaction mixture to obtain a test sequence, wherein the product is read as thymine (T) during sequencing; comparing the test sequence to a reference sequence, wherein a transition from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of 5hmC in the nucleic acid. In some embodiments, the 5fC conversion reagent is of formula Ph ₃ P=c (R2) CN compound. In some embodiments, the compound is Ph ₃ P＝C(CN) ₂ (R2＝-CN)。

In some embodiments, the invention is a method of forming 5-formylcytosine (5 fC) in a nucleic acid comprising contacting a reaction mixture comprising a nucleic acid comprising at least one 5-methylcytosine (5 mC) and/or 5-hydroxymethylcytosine (5 hmC) with an enzyme selected from the group consisting of xylene monooxygenase, toluene methyl monooxygenase (EC 1.14.15.26), P450 monooxygenase (EC 1.14.14.1), alcohol dehydrogenase, alcohol oxidase, galactose oxidase, chloroperoxidase, and peroxidase.

In some embodiments, the invention is a kit for detecting methylated cytosines in a nucleic acid comprising a laccase. In some embodiments, the laccase is isolated from a species selected from the group consisting of cellulites graminea (hexagona tenuis), phoenix mushroom (Pleurotis sajor caju), oyster mushroom (Pleutoris ostreatus), carbo-trichum multiforme (Xylariapolymorpha), trametes hirsuta (Trametes hirsuta), coriolus versicolor (Trametes versicolor), and Coprinus spp. In some embodiments, the laccase is isolated from a strain selected from the group consisting of Pleurotus sajor-caju MTCC-141, pleurotus ostreatus MTCC-1801, xylobacter multiforme MTCC-1100 and Brevibacterium MTCC-1171. In some embodiments, the kit further comprises a laccase cofactor selected from the group consisting of 2, 6-tetramethylpiperidine-1-oxyl (TEMPO), acetosyringone, syringaldehyde, p-coumaric acid 2,2' -azine-bis (3-ethylbenzothiazoline-6-sulfonate (ABTS), violet uric acid (VLA), N-acetyl-N-phenylhydroxylamine (NHA), N-Hydroxybenzotriazole (HBT), and N-Hydroxyphthalimide (HPI).

In some embodiments, the invention is a kit for detecting methylated cytosines in a nucleic acid comprising an enzyme capable of converting 5mC to 5hmC and/or 5fC selected from the group consisting of xylene monooxygenase, toluene methyl monooxygenase (EC 1.14.15.26), and P450 monooxygenase (EC 1.14.14.1). In some embodiments, the invention is a kit for detecting methylated cytosines in a nucleic acid comprising an enzyme capable of converting 5hmC to 5fC selected from the group consisting of alcohol dehydrogenase, alcohol oxidase, galactose oxidase, chloroperoxidase, and peroxidase.

In some embodiments, the invention is a method of detecting a hydroxymethylated cytosine (5 hmC) nucleotide in a nucleic acid, the method comprising: (i) Ligating an adapter to a nucleic acid comprising 5-hydroxymethylcytosine (5 hmC), wherein the adapter comprises an amplification primer binding site; (ii) Forming a reaction mixture by contacting a sample containing an adaptor-ligated nucleic acid with a laccase capable of converting 5hmC in the nucleic acid to 5-formylcytosine (5 fC); (iii) Contacting the reaction mixture with malononitrile to form a 5fC adduct; (iv) Amplifying the nucleic acid from step (iii) using a DNA polymerase and a primer capable of binding to the primer binding site, wherein the DNA polymerase reads the 5fC adduct as thymine (T) during amplification; (v) Sequencing the amplified nucleic acid to obtain a test sequence; (vi) Comparing the test sequence to a reference sequence, wherein a transition from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of hydroxymethylated cytosine in the nucleic acid.

In some embodiments, the invention is a method of detecting methylated cytosine (5 mC) nucleotides in a nucleic acid, the method comprising: (i) Ligating an adapter to a nucleic acid comprising 5-methylcytosine (5 mC), wherein the adapter comprises an amplification primer binding site; (ii) Forming a reaction mixture by contacting a sample containing an adaptor-ligated nucleic acid with a TET enzyme capable of converting 5mC in the nucleic acid to 5hmC and a laccase capable of converting 5hmC in the nucleic acid to 5-formyl cytosine (5 fC); (iii) Contacting the reaction mixture with malononitrile to form a 5fC adduct; (iv) Amplifying the nucleic acid from step (iii) using a DNA polymerase and a primer capable of binding to the primer binding site, wherein the DNA polymerase reads the 5fC adduct as thymine (T) during amplification; (v) Sequencing the amplified nucleic acid to obtain a test sequence; (vi) Comparing the test sequence to a reference sequence, wherein a transition from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of a methylated cytosine in the nucleic acid.

In some embodiments, the invention is a method of detecting tissue of origin of a nucleic acid in a sample, the method comprising detecting the presence and location of methylated cytosines in the nucleic acid by a method as disclosed herein, comparing methylation patterns to known methylation patterns of several tissues; and identifying the tissue of origin of the nucleic acid in the sample.

In some embodiments, the invention is a method of detecting organ transplant rejection in a transplant recipient comprising obtaining a blood sample containing cell free nucleic acid from the transplant recipient; detecting the presence and location of methylated cytosines in the cell free nucleic acid by the methods described herein, comparing the methylation pattern to known methylation patterns of several organs; if cell free nucleic acid having a methylation pattern specific for the transplanted organ is detected in the sample, then transplant rejection is detected.

In some embodiments, the invention is a method of monitoring graft rejection by: periodically, circulating cell free DNA is sampled and the presence and location of methylated cytosines is detected according to the methods described herein, and changes in the level of cell free DNA having a specific methylation pattern of the transplanted organ are measured, wherein an increase in such level of cell free DNA is indicative of organ transplant rejection.

In some embodiments, the invention is a method of screening for the presence of a cancerous tumor in a patient, the method comprising obtaining a blood sample from the patient containing cell free nucleic acids; detecting the presence and location of methylated cytosines in the cell free nucleic acid by the methods described herein; the methylation pattern is compared to known methylation patterns of tumor and non-tumor tissue, and if a tumor-specific methylation pattern is detected, the presence of a tumor is detected.

In some embodiments, the invention is a method of monitoring tumor volume in a patient, the method comprising periodically sampling circulating cell free DNA and detecting the presence and location of methylated cytosines according to the methods described herein, measuring changes in the level of cell free DNA having a tumor-specific methylation pattern, wherein an increase in such cell free DNA level is indicative of tumor growth and a decrease in such cell free DNA level is indicative of tumor shrinkage.

In some embodiments, the invention is a method of monitoring the effectiveness of a treatment for cancer in a patient by a method comprising the steps of: periodically, circulating cell free DNA is sampled and the presence and location of methylated cytosines is detected according to the methods described herein, and changes in the level of cell free DNA having a tumor-specific methylation pattern are measured, wherein an increase in such level of cell free DNA is indicative of the treatment being ineffective and a decrease in such level of cell free DNA is indicative of the effectiveness of the treatment.

In some embodiments, the invention is a method of diagnosing or treating Minimal Residual Disease (MRD) in a cancer patient, the method comprising obtaining a blood sample from the patient comprising cell free nucleic acid, detecting the presence and location of methylated cytosines in the nucleic acid by the methods described herein, comparing the methylation pattern to known methylation patterns of several tissues; and identifying the tissue of origin of the nucleic acid in the sample.

In some embodiments, the invention is a method of diagnosing an autoimmune disease in a patient, the method comprising obtaining a blood sample from the patient comprising cell free nucleic acid, detecting the presence and location of methylated cytosines in the nucleic acid by the methods described herein, comparing the methylation pattern to a known methylation pattern of tissue damaged by the immune disease; if such methylation patterns are found, an immune disease is diagnosed.

In some embodiments, the invention is a method of distinguishing 5-hydroxymethylcytosine (5 hmC) from 5-methylcytosine (5 mC) in a nucleic acid in a sample, the method comprising: dividing the sample into two aliquots; in a first aliquot, a nucleic acid comprising 5mC and 5hmC is reacted with 10-11 translocation (TET) dioxygenase to convert 5hmC and 5mC to 5-formylcytosine(5 fC) contacting under conditions; in a second aliquot, contacting a nucleic acid comprising 5mC and 5hmC with a laccase under conditions that convert 5hmC to 5-formylcytosine (5 fC); separately associating two aliquots with formula R ₁ —CH ₂ -CN compound, which compound is capable of reacting with 5fC in a nucleic acid to form an adduct according to the following reaction scheme:

Wherein R is ₁ Is an electron withdrawing group selected from the group consisting of cyano, nitro, C1-C6 alkyl carboxylate, unsubstituted carboxamide, C1-C6 alkyl mono-and C1-C6 alkyl di-substituted carboxamide, substituted carbonyl moiety, substituted sulfonyl moiety, wherein the substitution is selected from the group consisting of C1-C6 straight or branched chain alkyl, C4-C6 cycloalkyl, phenyl, 5-or 6-membered heteroaryl, and benzocyclized 5-or 6-membered heteroaryl; sequencing the nucleic acids from the two aliquots to obtain a first test sequence and a second test sequence, respectively, wherein the adduct is read as thymine (T) during sequencing; and comparing the first test sequence to a reference sequence, wherein a transition from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of 5hmC and 5mC in the nucleic acid; comparing the second test sequence to a reference sequence, wherein a transition from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of 5hmC in the nucleic acid; the first test sequence is compared to the second test sequence, wherein only such cytosine of the first test sequence is 5mC, which is not detected as hydroxymethylated cytosine (5 hmC) in the second test sequence. Alternatively, the two aliquots may be separately contacted with the Wittig reagent.

Drawings

Figure 1 shows DHU conversion using ethanol as co-solvent for picoline-borane in methylation detection assays (TAPS).

Figure 2 shows DHU conversion using methanol and acetic acid as co-solvents for picoline-borane in methylation detection assays (TAPS).

Figure 3 shows the formation of malononitrile adducts using malononitrile in sodium acetate buffer in methylation detection assays.

FIG. 4 shows the formation of malononitrile adducts using malononitrile in ethanol-TRIS buffer in methylation detection assays.

Figure 5 shows the formation of malononitrile adducts using malononitrile in ethanol-triethylamine buffer in methylation detection assays.

FIG. 6 shows the conversion of CpG sites in a single tube methylation assay using TET and malononitrile

FIG. 7 shows that laccase oxidizes 5hmC in oligonucleotides to 5fC in the presence of TEMPO.

FIG. 8 shows that under the same conditions, 5mC in the oligonucleotide was not oxidized by laccase.

Fig. 9A and 9B show liquid chromatography-mass spectrometry (LC-MS) data on how various amine buffer catalysts were tuned to TET activities of 5hmC and 5fC. In particular, FIG. 9A shows the effect of 2-amino-5-methoxybenzoic acid, and FIG. 9B shows the effect of 2- (aminomethyl) imidazole dihydrochloride on TET oxidation of 5mC to 5hmC/5 fC.

FIG. 10 shows a table depicting the amounts of 5fC and 5caC produced via oxidation of 5mC with TEMPO at two different temperatures, 25℃and 37 ℃.

FIG. 11A shows LC-MS data for the effect of malononitrile on the conversion of 5fC to 5fC-M adducts under various buffer conditions. The upper plot of fig. 11A shows a buffer condition of 40 ℃ for 1 hour, the middle plot of fig. 11A shows a buffer condition of 60 ℃ for 1 hour, and the lower plot of fig. 11A shows a buffer condition of 95 ℃ for 10 minutes. Fig. 11B shows data showing the effect of pre-denaturation with NaOH on malononitrile activity.

FIG. 12 shows LC-MS data showing oxidation of 5hmC in Cu 2, 6-tetramethylpiperidine-1-oxyl (CuTEMPO) in a short 22 hour period. The upper plot of fig. 12 shows oxidation of 5hmC in CuTEMPO, and the lower plot of fig. 12 shows derivatization of the product from the upper plot with DMEAH.

FIG. 13A shows the conversion of 5fC-M adducts to thymine (T), which is mediated by the activity of the polymerase. FIG. 13B shows the composition of an optimized buffer for polymerase ("DOE_1"). FIG. 13C shows the conversion of the 5fC-M adduct to T using standard buffer ("buffer A") and optimized buffer ("DOE_1").

Detailed Description

Abbreviations (abbreviations)

Some abbreviations used throughout the present disclosure are listed below.

C-cytosine

T-thymine

U-uracil

DHU-Dihydrouracil

5 mC-5-methylcytosine

5 hmC-5-hydroxymethylcytosine

5 ghmC-5-glucosyl-hydroxymethylcytosine

5 fC-5-formyl cytosine

5 caC-5-carboxycytosine

TET-10-11 translocation dioxygenase

TAPS-TET assisted picoline-borane sequencing

CAPS-chemically assisted picoline-borane sequencing

oxBS or oxBS-Seq-oxidative bisulfite sequencing

5-methylcytosine and 5-hydroxymethylcytosine (5 mC and 5 hmC) are important epigenetic biomarkers and have many clinical applications in the fields of oncology, prenatal detection, etc. Until recently, detection of methylated base levels was accomplished by reacting unmethylated cytosine with bisulfite, followed by PCR, array hybridization, or sequencing. Unmethylated cytosine (C) will be read as thymine (T) after reaction with bisulfite, and methylated cytosine (5 mC and 5 hmC) will be read as C. Unfortunately, bisulphite treatment results in degradation of most of the sample nucleic acid, making it unsuitable for applications requiring high sensitivity. For example, this method is not suitable for the latest applications for analyzing cell-free nucleic acids such as cell-free DNA.

More recently, less stringent methods for detecting methylated cytosines have been disclosed. The latest approach involves modifying methylated cytosines, rather than unmethylated cytosines, liu, y, as is the case for Bisulfite treatment, et al (2019) bisufite-free direct detection of5-methylcytosine and-hydroxymethylcytosine at base resolution. Nat biotechnol.37,424-429. Methyl cytosine (5 mC) is stepwise oxidized via 5-hydroxymethyl cytosine (5 hmC) to formyl cytosine (5 fC) and carboxyl cytosine (5 caC) using 10-11 metathesis dioxygenase (TET) in the presence of Fe (II) ions and α -ketoglutarate.

Liu et al further describe the use of borane derivatives such as pyridinium borane, picolinium borane, and the like, to reduce 5fC (and 5 caC) to Dihydrouracil (DHU). Then, in subsequent amplification and sequencing, DHU is read as T by uracil-resistant nucleic acid polymerase. As a result, methylated C is read as T, while unmethylated C remains unchanged. This TET and picoline-borane based approach, known as tap (TET assisted picoline-borane sequencing), does not lead to DNA degradation as bisulfite treatment does, and allows direct detection of the signal, rather than subtracting the background to obtain the signal. Both advantages will allow higher alignment rates, potentially lower sequencing depths, and restore higher molecular diversity from the sample.

Another technique known as CAPS (chemically assisted picoline borane sequencing) involves the use of potassium homoruthenate (KRUO ₄ ) 5hmC is selectively converted to 5fC. KRUO ₄ The use as a chemical alternative to TET is known from the technique known as oxidative bisulfite sequencing or oxBS-seq, see Booth M.J. et al (2012) Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine atsingle base resolution, science 5 month 12 days 934-937. The 5fC obtained by potassium perruthenate conversion is an advantageous target for further processing by e.g. borane treatment or any other downstream method.

Another sequencing technique is an alternative method of reducing 5fC with borane. The method involves forming a 5fC adduct identified as T. The adduct is formed using malononitrile, see Zhu c, et al, (2017) Single-Cell 5-Formylcytosine Landscapes ofMammalian Early Embryos andESCs atSingle-BaseResolution, cell Stem Cell,20:720-731.e5.

The TAPS, CAPS and malononitrile methods of Zhu et al are preferred over the bisulfite method because they avoid harsh chemical treatments and the resulting loss of sample nucleic acid. However, the newer methods have the disadvantage that they take a long time to complete or require high temperatures: the borane reaction of TAPS is carried out at 70℃for 3 hours or at 37℃for 16 hours (see Liu et al, nature Biotech.37, pages 424-429 (2019)) or 1 to 2 days to form malononitrile adducts (see U.S. Pat. No. 10,519,184 and application publication No. US 20200165661). The present disclosure includes improved and more practical methods for detecting cytosine methylation in a nucleic acid.

Various aspects of the invention are described in further detail below.

In some embodiments, the invention is a method of detecting epigenetic modifications, particularly cytosine methylation, in a nucleic acid. The most advanced method for detecting methylated cytosines in nucleic acids comprises the following key steps: 1) Oxidation of methylated cytosine; 2) Reducing the oxidation product to a form that can be read as thymine (T) during sequencing; 3) Sequencing the nucleic acid; 4) Comparing the treated sequence to the untreated sequence, wherein a change in sequence read from cytosine (T) to thymine (T) indicates the presence of methylated cytosine. The present invention includes several useful improvements to the general scheme described above.

In some embodiments, the invention is a method comprising the modified step 1) oxidizing methylated cytosines and steps 2) through 4) according to the most advanced technique. In some embodiments, the invention is a process comprising the modified step 2) of reducing the oxidation product, and steps 1), 3) and 4) according to the most advanced technique. In some embodiments, the invention is a method comprising the steps of modified step 1) oxidizing methylated cytosine, modified step 2) reducing the oxidation product, and steps 3) and 4) according to the prior art.

The present invention relates to a method of manipulating nucleic acids of a sample. In some embodiments, the sample is obtained from a subject or patient. In some embodiments, the sample may comprise fragments of solid tissue or solid tumors obtained from the subject or patient, for example, by biopsy. The sample may also include a bodily fluid (e.g., urine, sputum, serum, blood or blood component (i.e., plasma), lymph, saliva, sputum, sweat, tears, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cyst fluid, bile, gastric fluid, intestinal fluid, or fecal sample) that may contain nucleic acids. In other embodiments, the sample is a culture sample, e.g., a tissue culture containing cells and fluid from which nucleic acids can be isolated. In some embodiments, the nucleic acid of interest in the sample is from an infectious agent, such as a virus, bacterium, protozoan, or fungus.

The present invention relates to manipulating isolated nucleic acids isolated or extracted from a sample. Nucleic acid extraction methods are known in the art. See J.Sambrook et al, "Molecular Cloning: ALaboratory Manual",1989, 2 nd edition, cold Spring Harbor Laboratory Press: new York, n.y. A variety of kits are commercially available for extracting nucleic acids (DNA or RNA) from biological samples (e.g., KAPA expression extracts (Roche Sequencing Solutions, plasanton, cal.) and other similar products from BD Biosciences Clontech (Palo Alto, cal.), epicentre Technologies (Madison, wisc.), gentra Systems (Minneapolis, minn.) and Qiagen (Valencia, cal.), ambion (Austin, tex.), bioRad Laboratories (Hercules, cal.), and the like.

In some embodiments, the nucleic acids are extracted, separated by size and optionally concentrated by accelerated electrophoresis, as described, for example, in WO2019092269 and WO 2020074742.

The present invention relates to detecting epigenetic modifications in nucleic acids. The nucleic acid sequence subjected to conditional epigenetic modification is a target sequence analyzed by the methods disclosed herein. The same nucleic acid sequence may or may not have an epigenetic modification (5 mC or 5 hmC) characterized by methylation of a cytosine at position 5. In some embodiments, a set or group of target nucleic acids is probed for the presence of methylation. For example, methylation of a biomarker in a panel of methylation biomarkers can be indicative of colorectal Cancer in a patient as described in Patai AV, et al (2015) Comprehensive DNA Methylation Analysis Reveals a Common Ten-Gene Methylation Signature in Colorectal Adenomas and Carcinomas. PLOS ONE 10 (8): e0133836 and Onwuka, J.U., et al (2020) A panel of DNA methylation signature from peripheral blood may predict colorectal Cancer persistence. BMC Cancer 20,692. Thus, it is contemplated to test any known or future sets of methylation biomarkers for prognostic or diagnostic purposes using the methods disclosed herein.

In some embodiments, the entire genome of the organism is probed for the presence of methylation. The methods of the invention include the use of sequence analysis and artificial intelligence tools such as described in the following documents to detect methylation in all loci throughout the genome of an organism to diagnose a disease or disorder or predisposition to a disease or disorder: shull AY, et al, (2015) Sequencing the cancer methyl methods Mol biol.1238:627-5.

In some embodiments, it is desirable to separately detect or distinguish between 5mC and 5hmC in the sample. In this example, 5hmC is blocked from oxidation and does not convert to a compound read as T during sequencing. The blocking process utilized reactive hydroxyl groups present at 5hmC but not at 5 mC. In some embodiments, the blocking group added to 5hmC is a sugar moiety. In some embodiments, the sugar moiety is a modified or unmodified glucose moiety and forms 5-glucosyl-hydroxymethylcytosine (5 ghmC). According to the known protocols for 5fC and 5hmC, 5ghmC does not undergo adduct formation or reduction with borane derivatives. In some embodiments, the addition of blocking groups is catalyzed by glycosyltransferases, such as glucosyltransferase. In some embodiments, 5hmC in the nucleic acid is reacted with modified glucose in the presence of β -glycosyltransferase. In some embodiments, the modified glucose is UDP-glucose and the catalyst is phage t4β -glucosyltransferase (T4 BGT).

In some embodiments, the method includes the step of oxidizing the methylated cytosine for downstream detection. In some embodiments, the method comprises the step of converting 5-methylcytosine (5 mC) and/or 5-hydroxymethylcytosine (5 hmC) to 5-formylcytosine (5 fC) or 5-carboxycytosine (5 caC) or a mixture of 5fC and 5 caC. In some embodiments, the invention includes the step of contacting the sample or reaction mixture with a 10-11 metathesis (TET) dioxygenase, as described, for example, in U.S. patent No. 9,115,386 or U.S. application publication No. US 20200370114. In some embodiments, the TET enzyme is selected from TET1, TET2, TET3, and related protein CXXC4. In some embodiments, TET is selected from mouse TET1, TET2, or TET3 (mTET 1, mTET2, or mTET 3); human TET1, TET2, or TET3 (hTET 1, hTET2, or hTET 3); naegleria (Naegleria) TET (NgTET); coprinus cinereus (Coprinopsis cinerea) (CcTET) or any other analogue or equivalent thereof having similar or equivalent enzymatic activity.

In some embodiments, the invention utilizes a step of converting 5mC and/or 5hmC in the sample to mainly or exclusively 5 fC. In some embodiments, the invention utilizes a step of converting 5mC and 5hmC in the sample to mainly or exclusively 5 caC. In some embodiments, the invention utilizes a step of converting 5mC and 5hmC in a sample to a mixture of 5fC and 5 caC.

In some embodiments, the invention includes the step of converting 5mC and 5hmC in the sample to predominantly or exclusively 5fC by contacting the sample with TET in the presence of Fe (II) ions. In some embodiments, a suitable source or Fe (II) ion is selected from the group consisting of example FeSO ₄ 、(NH ₄ ) ₂ Fe(SO ₄ ) ₂ 、FeSO ₄ 7H ₂ O、(NH ₄ ) ₂ Fe(SO ₄ ) ₂ 6H ₂ O、FeCl ₂ And the like. In some embodiments, the invention includes the step of converting 5mC and 5hmC in the sample to 5fC by contacting the sample with TET and 5 to 100 μm Fe (II) ions at a pH of 7 to 8. In some embodiments, the invention utilizes a step of converting 5mC and 5hmC in a sample to 5fC by incubation with TET and 5 to 10 μm Fe (II) ions at pH 8. In some embodiments, the invention utilizes a step of converting 5mC and 5hmC in a sample to 5fC by incubation with TET and 80 to 100 μm Fe (II) ions at pH 7.

In some embodiments, the invention includes the step of converting 5mC and 5hmC in the sample to mainly or exclusively 5fC by contacting the sample with TET and Fe (II) ions in the presence of ascorbic acid, alpha-ketoglutarate, and a reducing agent.

In some embodiments, the present invention utilizes a step of converting 5hmC in a sample to predominantly or exclusively 5fC by contacting the sample with a Cu (II) compound and 2, 6-tetramethylpiperidin-1-yloxy (TEMPO).

In some embodiments, the invention includes an improved step of detecting methylated cytosines in a nucleic acid by detecting 5-formyl cytosine (5 fC) nucleotides in the nucleic acid, wherein 5fC is formed by one of the methods described herein above. The method involves contacting a sample containing a nucleic acid comprising 5fC with a nucleic acid of formula R contained in a modified solvent composition ₁ —CH ₂ -improved composition contact of CN compound capable of reacting with 5fC in the nucleic acid to form an adduct according to the following reaction scheme:

wherein R is ₁ Is an electron withdrawing group selected from the group consisting of substituted or unsubstituted cyano, nitro, formyl, carbonyl compounds, wherein the substitution is selected from the group consisting of C1-C30 straight or branched alkyl, C1-C30 straight or branched alkenyl, C1-C30 straight or branched alkynyl, cycloalkyl, aryl, or heteroaryl. The reactants of the above reactions are described, for example, in U.S. patent No. 10,519,184 to Yi et al and application publication No. US 20200165661. For example, R1 is a cyano group (CN) and the reactant is malononitrile.

The present invention provides an improved reaction mixture composition that improves the Yi process by allowing the reaction to proceed for less than 3 hours, wherein at least 90% of the 5fC has formed an adduct. In some embodiments, the reaction is performed for only 1 hour, wherein at least 90% of the 5fC forms adducts. In contrast, yi reaction takes no less than 20 hours and up to 48 hours (see US20200165661, examples).

In some embodiments, an improvement over the prior art involves performing 5fC with formula R in a solution comprising an organic acid moiety ₁ —CH ₂ -reaction between CN compounds. The organic acid has the formula R-COOH, and R is selected from C1-C30 straight or branched chainAlkyl, C1-C30 straight or branched alkenyl, C1-C30 straight or branched alkynyl, cycloalkyl, aryl or heteroaryl. In some embodiments, the reaction occurs in the presence of acetic acid. In some embodiments, the concentration of the organic acid in the reaction is between 1% and 30%, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%.

In some embodiments, an improvement over the prior art involves performing 5fC with formula R in a non-aqueous solvent ₁ —CH ₂ -reaction between compounds of CN. The nonaqueous solvent has the formula R-OH, wherein R is selected from the group consisting of C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, C1-C30 linear or branched alkynyl, cycloalkyl, aryl, or heteroaryl. In some embodiments, the reaction occurs in methanol or ethanol. In some embodiments, the reaction occurs in 10% -100% methanol or ethanol. In some embodiments, the reaction occurs in 90% or more methanol or ethanol.

In some embodiments, an improvement over the prior art relates to the use of a compound of formula R _x NH _y Performing 5fC with formula R in solution of the compound ₁ —CH ₂ -a reaction between CN compounds, wherein x and y are 0, 1, 2 or 3, such that x+y = 3, and each R is independently selected from C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, C1-C30 linear or branched alkynyl, cycloalkyl, aryl or heteroaryl. In some embodiments, formula R _x NH _y The compound is a primary, secondary or tertiary amine having an aliphatic or aromatic group. In some embodiments, the reaction occurs in the presence of triethanolamine.

In some embodiments, an improvement over the prior art involves performing 5fC with formula R simultaneously with TET oxidation ₁ —CH ₂ Reaction between CN compounds to achieve a simplified single tube workflow. In some embodiments, TET and malononitrile are added simultaneously, and oxidation to 5fC and 5 fC-malononitrile adducts occurs in the same tube.

In some embodiments, the invention includes an improved step of detecting methylated cytosines in a nucleic acid by forming and detecting 5-carboxycytosine (5 caC) and 5-formylcytosine (5 fC), wherein 5fC, 5caC, or a mixture of 5fC and 5caC is formed by one of the methods described above. The method involves contacting a sample containing a nucleic acid comprising 5fC and/or 5 cat with a modified composition comprising a borane derivative in a modified solvent composition, the borane derivative being capable of reacting with 5 cat and reacting with 5fC in the nucleic acid with less efficiency to form Dihydrouracil (DHU). Examples of borane derivatives include 2-picoline borane (picoline-borane), pyridine borane, t-butylamine borane, ethylenediamine borane, and dimethylamine borane, as described by Song and Liu in WO2019136413 (TET-Assisted Picoline borane Sequencing or TAPS).

The present invention provides an improved composition of a borane-containing reaction mixture that improves the TAPS process by allowing the reaction to proceed at 35 ℃ for less than one hour, wherein substantially all 5caC is converted to DHU. In some embodiments, the reaction is performed for only 1/2 hour, with almost all 5caC being converted. In contrast, the TAPS borane reaction described by Liu et al requires no less than 3 hours at 70℃or 16 hours at 37℃ (see WO2019136413, examples: borane reduction).

In some embodiments, the improvement over the prior art involves conducting the reaction between 5fC or 5caC and the borane derivative in a solution comprising an organic acid moiety. The organic acid has the formula R-COOH, and R is selected from the group consisting of C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, C1-C30 linear or branched alkynyl, cycloalkyl, aryl, or heteroaryl. In some embodiments, the reaction occurs in the presence of acetic acid.

In some embodiments, the improvement over the prior art involves conducting the reaction between 5fC or 5caC and the borane derivative in a non-aqueous solvent. The nonaqueous solvent has the formula R-OH, wherein R is selected from the group consisting of C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, C1-C30 linear or branched alkynyl, cycloalkyl, aryl, or heteroaryl. In some embodiments, the reaction occurs in methanol or ethanol. In some embodiments, the reaction occurs in 90% or more methanol or ethanol.

After the formation of the adduct (in the case of malononitrile treatment) or of the DHU (in the case of borane treatment), the nucleic acid with the adduct or with the DHU is sequenced. In some embodiments, sequencing is performed by a next generation massively parallel sequencing process. Sequencing results in a test sequence, wherein the adduct or DHU is read as thymine (T), i.e., the sequencing polymerase is able to accommodate the adduct or DHU in the replicated strand and incorporate adenine (a) as opposed to the adduct or DHU. The method further comprises the step of comparing the test sequence to a reference sequence, wherein a change from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of a methylated cytosine in the test nucleic acid.

In some embodiments, nucleic acids in the sample are amplified prior to sequencing. In some embodiments, amplification utilizes a B family polymerase to efficiently incorporate adenine (a) nucleotides as opposed to malononitrile adducts or DHU. In this embodiment, the sequencing may be performed with any polymerase suitable for the sequencing process, as the adduct or DHU has been recognized as T by the amplifying polymerase.

In some embodiments, nucleic acids in a sample are ligated to adaptors, wherein the adaptors comprise elements useful for amplification and sequencing. The adapter comprises at least one of: bar codes, primer binding sites, and ligation sites.

In some embodiments, the invention is an improved method of detecting methylated cytosine nucleotides in a nucleic acid, the method comprising: (i) Ligating an adapter to the nucleic acid in the sample, wherein the adapter comprises an amplification primer binding site; (ii) Forming a reaction mixture by contacting a sample containing an adaptor-ligated nucleic acid with a TET capable of converting 5mC in the nucleic acid to 5-formylcytosine (5 fC); (iii) Bringing the reaction mixture into contact with a reaction product of formula R ₁ —CH ₂ -CN compound, which compound is capable of reacting with 5fC in a nucleic acid to form an adduct according to the following reaction scheme:

wherein R is ₁ Is an electron withdrawing group selected from the group consisting of substituted or unsubstituted cyano, nitro, formyl, carbonyl compounds, wherein the substitution is selected from the group consisting of C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, C1-C30 linear or branched alkynyl, cycloalkyl, aryl, or heteroaryl (e.g., malononitrile); (iv) Incubating the reaction mixture for less than 3 hours, wherein at least 90% of the 5fC has formed an adduct; (v) Amplifying the ligated nucleic acids using a DNA polymerase and a primer capable of binding to the primer binding site, wherein the DNA polymerase reads the adduct as thymine (T) during amplification; (vi) Sequencing the amplified nucleic acid to obtain a test sequence; (vii) Comparing the test sequence to a reference sequence, wherein a change from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of 5mC in the nucleic acid. In some embodiments, in step (iii), formula R ₁ —CH ₂ the-CN compound is present in a non-aqueous solvent such as ethanol or methanol. In some embodiments, in step (iii), formula R ₁ —CH ₂ The CN compound is present in a solution comprising an organic acid such as acetic acid. In some embodiments, in step (iii), formula R ₁ —CH ₂ The CN compound is present in a solution comprising an amine such as triethanolamine.

In some embodiments, the invention is an improved method of detecting methylated cytosine nucleotides in a nucleic acid, the method comprising: (i) Ligating an adapter to the nucleic acid in the sample, wherein the adapter comprises an amplification primer binding site; (ii) Forming a reaction mixture by contacting a sample containing adapter-ligated nucleic acids with a TET capable of converting methylated cytosines in the nucleic acids to 5-carboxycytosine (5 caC) or a mixture of 5-formylcytosine (5 fC) and 5 caC; (iii) Contacting the reaction mixture with a borane derivative capable of reacting with 5fC and 5caC in a nucleic acid to form DHU; (iv) Incubating the reaction mixture for no more than about 1 hour, wherein at least 90% of the 5fC and 5caC have formed DHU; (v) Amplifying the ligated nucleic acids using a DNA polymerase and a primer capable of binding to the primer binding site, wherein the DNA polymerase reads DHU as thymine (T) during amplification; (vi) Sequencing the amplified nucleic acid to obtain a test sequence; (vii) Comparing the test sequence to a reference sequence, wherein a change from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of 5mC in the nucleic acid. In some embodiments, in steps (iii) and (iv), the borane derivative is present in a non-aqueous solvent, such as ethanol or methanol. In some embodiments, in steps (iii) and (iv), the borane derivative is present in a solution comprising an organic acid, such as acetic acid.

In some embodiments, the invention is a single tube method of detecting methylation in a nucleic acid. In some embodiments, the method comprises: (i) Ligating an adapter to the nucleic acid in the sample, wherein the adapter comprises an amplification primer binding site; (ii) Forming a reaction mixture by contacting a sample containing an adaptor-ligated nucleic acid with a TET capable of converting 5mC in the nucleic acid to 5-formyl cytosine (5 fC) or a mixture of 5-carboxy cytosine (5 caC) and 5 fC; (iii) Allowing the same reaction mixture to react with the formula R ₁ —CH ₂ -CN compound, which compound is capable of reacting with 5fC in a nucleic acid to form an adduct according to the following reaction scheme:

wherein R is ₁ Is an electron withdrawing group selected from the group consisting of substituted or unsubstituted cyano, nitro, formyl, carbonyl compounds, wherein the substitution is selected from the group consisting of C1-C30 linear or branched alkyl, C1-C30 linear or branched alkenyl, C1-C30 linear or branched alkynyl, cycloalkyl, aryl, or heteroaryl (e.g., malononitrile); (iii) Incubating the reaction mixture to allow 5fC to form an adduct; (iv) Amplifying the ligated nucleic acids using a DNA polymerase and a primer capable of binding to the primer binding site, wherein the DNA polymerase reads the adduct as thymine (T) during amplification; (v) Sequencing the amplified nucleic acid to obtain a test A sequence; (vi) Comparing the test sequence to a reference sequence, wherein a change from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of 5mC in the nucleic acid. In some embodiments, in step (ii), the conditions are optimized to improve the performance of the TET. In some embodiments, in steps (ii) and (iii), the reaction mixture comprises a non-aqueous solvent, such as ethanol or methanol. In some embodiments, in steps (ii) and (iii), the reaction mixture comprises an organic acid such as acetic acid. In some embodiments, in steps (ii) and (iii), the reaction mixture comprises an organic amine such as triethanolamine.

In some embodiments, the invention includes a step of amplifying the nucleic acid. In some embodiments, the amplification occurs prior to the sequencing step. In some embodiments, the amplification occurs after the step of forming an adduct of 5fC and malononitrile. In some embodiments, the amplification occurs after the step of reducing the oxidized methylated cytosine with the borane derivative. In some embodiments, the amplification occurs prior to the target enrichment step. Amplification utilizes an upstream primer and a downstream primer. In some embodiments, both primers are target-specific primers, i.e., primers comprising sequences complementary to the target sequence of the methylation biomarker. In some embodiments, one or both primers are not universal primers. In some embodiments, the universal primer binding site is present in an adapter that is ligated to a target sequenced as described herein. In some embodiments, the universal primer binding site is present in the 5' region (tail) of the target-specific primer. Thus, after one or more rounds of primer extension with the tailed target-specific primer, the universal primer can be used for subsequent rounds of amplification. In some embodiments, a universal primer is paired with another universal primer (having the same or a different sequence). In other embodiments, the universal primer pairs with a target-specific primer.

In some embodiments, the invention relates to nucleic acid polymerases. Nucleic acid polymerases for amplification and sequencing are known and commercially available from a variety of sources. In some embodiments, the invention relates to replicating a chain comprising a 5fC adduct formed as described herein. Such replication needs to accommodate 5fC plusPolymerase of the compound. In some embodiments, the polymerase is a B family polymerase. In some embodiments, the polymerase is capable of replicating a strand comprising a 5fC adduct by recognizing the adduct as T (i.e., incorporating a opposite to the adduct). The polymerases capable of containing the 5fC adducts described herein include DNA polymerases known to contain uracil (U) in a DNA strand. The polymerase may be a naturally occurring or engineered polymerase. In some embodiments, the polymerase is isolated from a hyperthermophilic archaebacterium, such as a genus of Pyrococcus (e.g., pyrococcus furiosus) or a genus of Thermus (e.g., thermus aquaticus (Thermus aquaticus)). In some embodiments, the polymerase is isolated from a species of archaea mesophilic bacteria, such as methanosarcina (metanosarccina) (e.g., methanosarcina acetate (Methanosarcina acetivorans)). Examples of engineered uracil-resistant polymerases include KAPA HiFi uracil+dna polymerase (Roche Sequencing Solutions, plasanton, cal.), takara Terra (Takara Bio USA, mountain View, cal.) and Taq DNA polymerase (New England Biolabs, waltham, mass.) was hot started.

In some embodiments, the DNA polymerase is a type a DNA polymerase (DNA-dependent DNA polymerase). Some DNA polymerases have limited terminal transferase activity (Taq polymerase adds a separate dA to the 3' end of the copy strand). Other DNA polymerases do not have detectable terminal transferase activity. In such embodiments, a separate terminal transferase is used to add non-templated nucleotides to the 3' end of the copy strand.

In some embodiments, the DNA polymerase is a hot start polymerase or a similar condition activated polymerase. For the amplification step, a thermostable DNA polymerase is used, for example, the polymerase is Taq or Taq-derived polymerase (e.g., KAPA 2G polymerase from KAPA Biosystems, wilmington, mass.).

In some embodiments, the invention utilizes adaptors added to one or both ends of the nucleic acid or nucleic acid strand. Adapters of various shapes and functions are known in the art (see, e.g., PCT/EP2019/05515, US8822150 and US8455193 submitted at 28, 2, 2019). In some embodiments, the function of the adapter is to introduce the desired element into the nucleic acid. The adapter-carrying element comprises at least one of a nucleic acid barcode, a primer binding site, or a ligation enabling site.

The adaptors may be double stranded, partially single stranded or single stranded. In some embodiments, a Y-shaped, hairpin or stem loop adaptor is used, wherein the double stranded portion of the adaptor is ligated to the double stranded nucleic acid formed as described herein.

In some embodiments, the adapter molecule is an artificial sequence synthesized in vitro. In other embodiments, the adapter molecule is a naturally occurring sequence synthesized in vitro. In other embodiments, the adaptor molecule is an isolated naturally occurring molecule or an isolated non-naturally occurring molecule.

Double-stranded or partially double-stranded adaptor oligonucleotides may have overhangs or blunt ends. In some embodiments, double-stranded DNA may comprise blunt ends to which blunt end ligation may be applied to ligate blunt end adaptors. In other embodiments, blunt-ended DNA undergoes a tailing, where a single a nucleotide is added to the blunt end to match an adapter designed with a single T nucleotide extending from the blunt end to facilitate ligation between the DNA and the adapter. Commercially available kits for performing adaptor ligation include the aveno ctDNA library preparation kit, or KAPA HyperPrep and HyperPlus kits (roche sequencing solutions company, plaston, california). In some embodiments, the adaptor-ligated (ligated) DNA may be separated from excess adaptor and unligated DNA.

In some embodiments, the invention includes the use of bar codes. In some embodiments, the method of detecting an epigenetic modification comprises sequencing. Sequencing a nucleic acid treated as described herein; preferably, large-scale parallel single molecule sequencing. Analysis of individual molecules by large-scale parallel sequencing typically requires a separate level of barcoding for sample identification and error correction. The use of molecular barcodes, for example, is described in U.S. patent nos. 7,393,665, 8,168,385, 8,481,292, 8,685,678 and 8,722,368. A unique molecular barcode is added to each molecule to be sequenced to label the molecule and its progeny (e.g., the original molecule and its amplicon generated by PCR). Unique molecular barcodes (UIDs) have a variety of uses, including counting the number and error correction of original target molecules in a sample (Newman, a., et al, (2014) An ultrasensitive methodfor quantitating circulating tumor DNA with broad patient coverage, nature Medicine doi: 10.1038/nm.3519).

In some embodiments, a unique molecular barcode (UID) is used for sequencing error correction. The entire progeny of a single target molecule is labeled with the same barcode and forms a family of barcodes. Variations in sequences that are not shared by all members of the barcoded family are discarded as artifacts. Bar codes can also be used for positional deduplication (positional deduplication) and target quantification, as the entire family represents a single molecule in the original sample (Newman, a., et al, (2016) Integrated digital error suppression for improved detection of circulating tumor DNA, nature Biotechnology 34:547).

In some embodiments of the invention, the adaptors ligated to one or both ends of the barcoded target nucleic acids comprise one or more barcodes for sequencing. The barcode may be a UID or multiple sample ID (MID or SID) for identifying the sample source in the mixed (multiple) sample. The bar code may also be a combination of UID and MID. In some embodiments, a single bar code is used as both the UID and MID. In some embodiments, each bar code includes a predefined sequence. In other embodiments, the bar code includes a random sequence. In some embodiments of the invention, the length of the barcode is between about 4-20 bases, thereby adding 96 to 384 different adaptors to the human genome sample, each adaptor having a different identical barcode pair. In some embodiments, the number of UIDs in the reaction may exceed the number of molecules to be labeled. One of ordinary skill will recognize that the number of barcodes depends on the complexity of the sample (i.e., the expected number of unique target molecules), and will be able to create an appropriate number of barcodes for each experiment.

In some embodiments, the method involves forming a library comprising nucleic acids from the sample. The library is composed of a plurality of nucleic acids that are ready for sequencing or another type of detection method (e.g., PCR). The library may be stored and used multiple times for further processing, such as amplification or sequencing of nucleic acids in the library. In some embodiments, the library is an input nucleic acid, wherein methylation is detected by the methods described herein. In other embodiments, the library is formed from nucleic acids that have undergone the methylation detection reactions described herein.

In some embodiments, nucleic acids treated according to the methods described herein for detection of epigenetic modifications are sequenced. Any of a variety of sequencing techniques or sequencing assays may be utilized. As used herein, the term "new generation sequencing" (NGS) refers to a sequencing method that allows for large-scale parallel sequencing of clonally amplified molecules and single nucleic acid molecules.

Non-limiting examples of sequencing suitable for use with the Methods disclosed herein include nanopore sequencing (U.S. patent publication nos. 2013/024340, 2013/0264207, 2014/0134516, 2015/0110859, and 2015/0337366), sanger sequencing, capillary array sequencing, thermal cycle sequencing (Sears et al, biotechniques,13:626-633 (1992)), solid phase sequencing (Zimmerman et al, methods mol. Cell biol.,3:39-42 (1992)), sequencing using mass spectrometry such as matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; fu et al, nature biotech, 16:381-384 (1998)), sequencing by hybridization (draac et al, nature biotech, 16:54-58 (1998)), and NGS Methods including, but not limited to sequencing by synthesis (e.g., hiSeq) ^TM 、MiSeq ^TM Or Genome Analyzer, all available from Illumina), by ligation sequencing (e.g., SOLiD ^TM Life Technologies), ion semiconductor sequencing (e.g., ion Torrent ^TM Life Technologies) and/or a combination thereofSequencing (e.g., pacific Biosciences).

Commercially available sequencing technologies include the Affymetrix limited (Sentrel, calif.), the sequencing-by-hybridization platform, the Illumina/Solexa (san Diego, calif.), and Helicos Biosciences (Cannabis, massachusetts) sequencing-by-synthesis platform, the Applied Biosystems (Foster City, calif.), the sequencing-by-ligation platform. Other sequencing techniques include, but are not limited to, ion Torrent technology (ThermoFisher Scientific) and nanopore sequencing (Genia Technology from Roche Sequencing Solutions, santa Clara, cal.) and OxfordNanopore Technologies (Oxford, UK).

In some embodiments, the sequencing step involves sequence analysis. In some embodiments, the consensus sequence is determined from multiple sequences (e.g., multiple sequences having the same unique molecular ID (UID)) using alignment. The molecular ID is a barcode, which may be added to each molecule prior to sequencing, or if an amplification step is included, to each molecule prior to the amplification step. In some embodiments, the UID is present in the 5' portion of the RT primer. Likewise, the UID may appear 5' to the last barcode subunit to be added to the composite barcode. In other embodiments, the UID is present in the adapter and added to one or both ends of the target nucleic acid by ligation.

In some embodiments, the consensus sequence is determined from a plurality of sequences all having the same UID. Sequences with the same UID are presumed to originate from the same original molecule by amplification. In other embodiments, the UID is used to eliminate artifacts, i.e., variations (characterized by a particular UID) that exist in the offspring of a single molecule. Such artifacts, which originate from PCR errors or sequencing errors, can be eliminated using UID.

In some embodiments, the number of each sequence in a sample may be quantified by quantifying the relative number of sequences for each UID in a population having the same multiple sample IDs (MIDs). Each UID represents a single molecule in the original sample, and counting the different UIDs associated with each sequence variant can determine the proportion of each sequence variant in the original sample, where all molecules share the same MID. The person skilled in the art will be able to determine the number of sequence reads necessary to determine the consensus sequence. In some embodiments, each UID ("sequence depth") needs to read the relevant number for accurate quantitative results. In some embodiments, the desired depth is 5-50 reads per UID.

In some embodiments, the invention is a kit comprising components and means for performing the improved methods of detecting DNA methylation described herein. In some embodiments, the kit includes components for detecting cytosine methylation in a nucleic acid by detecting a product of in vitro oxidized 5-methylcytosine (5 mC) or 5-hydroxymethylcytosine (5 hmC). In some embodiments, the product is 5-formyl cytosine (5 fC) or 5-carboxy cytosine (5 caC). In other embodiments, the kit further comprises components for performing in vitro oxidation of 5-methylcytosine (5 mC) or 5-hydroxymethylcytosine (5 hmC) to 5-formylcytosine (5 fC) or 5-carboxycytosine (5 caC).

In some embodiments, the kit includes a borane derivative and a non-aqueous solvent. The borane derivative is selected from the group consisting of pyridinium borane, 2-methylpyridinium borane (pic-BH 3), borane, sodium borohydride, sodium cyanoborohydride and sodium triacetoxyborohydride, and the non-aqueous solvent is selected from the group consisting of ethanol and methanol. In other embodiments, the kit does not include a non-aqueous solvent, but rather includes a borane derivative and instructions for using a non-aqueous solvent (such as ethanol or methanol) with the borane derivative in the methods of detecting DNA methylation described herein. In some embodiments, the kit further comprises an organic acid. In some embodiments, the kit includes instructions for using an organic acid (such as acetic acid) in a method of detecting DNA methylation including a borane derivative in a non-aqueous solvent as described herein. In some embodiments, the kit further comprises a buffer such as MES or TRIS.

In some embodiments, the kit comprises malononitrile and a non-aqueous solvent. The nonaqueous solvent is selected from ethanol and methanol. In other embodiments, the kit does not include a non-aqueous solvent, but rather includes instructions for using a non-aqueous solvent (such as ethanol or methanol) in the method of detecting DNA methylation with malononitrile as described herein. In some embodiments, the kit further comprises an organic acid and a primary, secondary or tertiary amine. The organic acid may be acetic acid and the amine may be triethanolamine. In other embodiments, the kit includes instructions for using organic acids and amines (such as acetic acid and triethanolamine) in the method of detecting DNA methylation with malononitrile as described herein. In some embodiments, the kit further comprises a buffer such as MES or TRIS.

In some embodiments, the kit further comprises a TET enzyme for oxidizing 5-methylcytosine (5 mC) or 5-hydroxymethylcytosine (5 hmC) to 5-carboxycytosine (5 caC) in vitro. In some embodiments, TET is selected from mouse TET1, TET2, or TET3 (mTET 1, mTET2, or mTET 3); human TET1, TET2, or TET3 (hTET 1, hTET2, or hTET 3); nardostachys TET (NgTET); coprinus cinereus (CcTET) or any other analogue or equivalent thereof having similar or equivalent enzymatic activity. In some embodiments, the TET is a nakai genus TET-like oxygenase (NgTET 1). In some embodiments, TET is a wild-type protein. In other embodiments, TET is a mutein. In some embodiments, the kit further comprises one or more cofactors selected from the group consisting of alpha-ketoglutarate and Fe (II) ion sources.

In some embodiments, as an alternative to TET, the kit includes a chemical oxidizing agent, such as potassium homoruthenate (KRuO ₄ ) Or potassium ruthenate (K) ₂ RuO ₄ )。

In some embodiments, the kit further comprises reagents for chemically blocking 5hmC from reactions involving 5 mC. In some embodiments, the kit includes a glucose compound and a glycosyltransferase capable of transferring a glucose moiety to a 5-hydroxy moiety of 5 hmC. In some embodiments, the kit comprises beta-glucosyltransferase (BGT) and UDP-glucose. In some embodiments, the BGT is a T4 BGT.

In some embodiments, the method further comprises assessing the status of the subject (e.g., patient) based on the methylation status of one or more genetic loci in the genome of the patient. In some embodiments, the method comprises determining genomic location in a patient sample and optionally determining the amount of methylated cytosines (5 mC and/or 5 hmC) in the genome. In some embodiments, methylation of genetic loci known as disease biomarkers is assessed. The method further comprises diagnosing a disease or disorder in the patient or selecting or altering a treatment based on the presence or amount of methylation in nucleic acid isolated from the patient.

There are a variety of methods for identifying disease or disorder specific methylation loci, the methylation of which can be assessed using the methods disclosed herein, see, e.g., US20200385813"ystems and methods for estimating cell source fractions using methylation information"; US20200239965"Source of origin deconvolution based on methylation fragments in cell-free DNA samples"; US20190287652"Anomalous fragment detection and classification" (methylation markers indicate disease states); US20190316209"Multi-assay prediction model for cancer detection"; US20190390257A1"Tissue-specific methylation marker"; WO2011/070441"Categorization of DNA samples"; WO2011/101728"Identification of source of DNA samples"; WO2020/188561"Methods and systems for detecting methylation changes in DNA samples).

In some embodiments, the invention includes methods of detecting tissue-specific DNA methylation patterns using the methylation detection methods disclosed herein. In one aspect of this embodiment, the method may further comprise identifying the tissue of origin of the methylated DNA present in the sample. In some embodiments, the method further comprises identifying the tissue of origin of the cell free DNA isolated from the blood. In another aspect of this embodiment, the invention includes using methylation patterns of cell free DNA to detect organ failure or organ damage, including organ transplant rejection in a transplant recipient. The invention includes detecting circulating cell free DNA having an organ specific methylation pattern, wherein the presence of such cell free DNA is indicative of organ transplant rejection. In some embodiments, the invention includes monitoring transplant rejection by periodically sampling circulating cell free DNA and measuring changes in the level of cell free DNA with an organ specific methylation pattern, wherein an increase in such level of cell free DNA is indicative of organ transplant rejection.

In some embodiments, the invention includes a method of diagnosing or screening for the presence of a cancerous tumor in a patient or subject. In some embodiments, the invention includes using methylation detection methods disclosed herein to detect tumors using methylation patterns of cell free DNA. In some embodiments, the invention includes detecting a tumor derived from a particular tissue or organ by detecting circulating cell free DNA having a tissue or organ specific methylation pattern using the methylation detection methods disclosed herein, wherein the presence of such cell free DNA is indicative of the presence of a tumor derived from the tissue or organ. In some embodiments, the invention includes monitoring tumor growth or reduction by periodically sampling circulating cell free DNA and measuring changes in the level of cell free DNA with a tumor specific methylation pattern, wherein an increase in such level of cell free DNA is indicative of tumor growth and a decrease in such level of cell free DNA is indicative of tumor reduction.

In some embodiments, the invention includes a method of monitoring the effectiveness of a cancer treatment in a patient or subject. In some embodiments, the invention includes detecting tumor dynamics associated with treatment using methylation patterns of cell free DNA detected using the methylation detection methods disclosed herein. In some embodiments, the invention includes detecting therapeutic effects on tumors derived from a particular tissue or organ by periodically sampling circulating cell free DNA and measuring changes in the levels of free DNA having a tissue or organ specific methylation pattern, wherein an increase in such levels of cell free DNA is indicative of tumor growth and treatment inefficiency, while a decrease in such levels of cell free DNA is indicative of tumor shrinkage and treatment effectiveness, and a stabilization in such levels of cell free DNA is indicative of disease stabilization and treatment effectiveness.

In some embodiments, the invention includes a method of diagnosing or treating Minimal Residual Disease (MRD) in a cancer patient after treatment. MRD is defined by the national cancer institute as the very few cancer cells that remain in the body during or after treatment when the patient is free of signs or symptoms of disease. In some embodiments, the invention includes a method of detecting MRD using a methylation pattern of cell free DNA detected using a methylation detection method disclosed herein. In some embodiments, the invention includes detecting MRD from a tumor derived from a particular tissue or organ by detecting circulating cell free DNA having a tissue or organ specific methylation pattern, wherein the presence of such cell free DNA indicates the presence of MRD from the tumor.

In some embodiments, the invention includes a method of diagnosing or screening for the presence or status of an autoimmune disease in a patient or subject. In some embodiments, the invention includes detecting a tumor using a methylation pattern of cell free DNA detected using a methylation detection method disclosed herein. In some embodiments, the invention includes detecting an autoimmune disease characterized by damage to a particular tissue or organ by detecting circulating cell free DNA having a tissue or organ specific methylation pattern, wherein the presence of such cell free DNA is indicative of organ damage caused by the autoimmune disease and the presence of the autoimmune disease. In some embodiments, the invention includes monitoring onset or remission of autoimmune disease by periodically sampling circulating cell free DNA and measuring changes in free DNA levels having a tissue or organ specific methylation pattern, wherein an increase in such cell free DNA levels is indicative of increased onset of organ damage and autoimmune disease, and a decrease in such cell free DNA levels is indicative of decreased remission of organ damage and autoimmune disease.

More recently, less stringent methods for detecting methylated cytosines have been disclosed. Rao et al (U.S. Pat. No. 9,115,386) have found that the 10-11 translocation dioxygenase (TET) family converts 5mC to 5hmC in vitro. Liu et al disclose a process for the stepwise oxidation of methylcytosine (5 mC) to formylcytosine (5 fC) and carboxycytosine (5 caC) via 5-hydroxymethylcytosine (5 hmC) using TET in the presence of Fe (II) ions and alpha-ketoglutarate. Liu, Y., et al (2019) Bisulfofield-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine atbase resolution. Nat. Biotechnol.37,424-429.

Liu et al further describe the use of borane derivatives such as pyridinium borane, picolinium borane, and the like, to reduce 5fC (and 5 caC) to Dihydrouracil (DHU). Then, in subsequent amplification and sequencing, DHU is read as T by uracil-resistant nucleic acid polymerase. As a result, methylated C is read as T, while unmethylated C remains unchanged. This TET and pyridine-borane based approach, known as tap (TET assisted pyridine-borane sequencing), does not lead to DNA degradation like bisulfite treatment and allows direct detection of the signal, rather than subtracting the background to obtain the signal. Both advantages will allow higher alignment rates, potentially lower sequencing depths, and restore higher molecular diversity from the sample.

Another technique known as CAPS (chemically assisted pyridine borane sequencing) involves the use of potassium homoruthenate (KRUO ₄ ) 5hmC is selectively converted to 5fC. KRUO ₄ The use as a chemical alternative to TET is known from the technique known as oxidative bisulfite sequencing or oxBS-seq, see Booth M.J. et al (2012) Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine atsingle base resolution, science 5 month 12 days 934-937. The 5fC obtained by potassium perruthenate conversion is an advantageous target for further processing by e.g. borane treatment or any other downstream method.

All of the above methods rely at least in part on the oxidation of 5hmC and 5mC to 5fC with TET family enzymes. Another known oxidation technique involves the conversion of 5hmC to 5fC mainly or exclusively with Cu (II) compounds and 2, 6-tetramethylpiperidin-1-yloxy (TEMPO).

Disclosed herein are methods and compositions for enzymatic oxidation of 5hmC to 5fC only or predominantly. The oxidation is catalyzed by laccase, an enzyme that has been previously known to catalyze the oxidation of phenol-containing and non-phenol compounds under certain conditions. The inventors have found that, surprisingly, the enzyme functions in a nucleic acid environment to convert a 5-hydroxy group of 5-hmC to a 5-formyl group.

Various aspects of the invention are described in further detail below.

In some embodiments, the invention is a method of detecting epigenetic modifications, particularly cytosine methylation, in a nucleic acid. The most advanced method for detecting methylated cytosines in nucleic acids comprises the following key steps: 1) Oxidation of methylated cytosine; 2) Converting the oxidation product into a form that can be read as thymine (T) during sequencing; 3) Sequencing the nucleic acid; 4) Comparing the treated sequence to the untreated sequence, wherein a change in sequence read from cytosine (C) to thymine (T) indicates the presence of methylated cytosine. The invention includes a novel method for performing step 1) of oxidative methylation of cytosines. After the oxidation step, steps 2) to 4) are carried out according to the most advanced technique.

In some embodiments, it is desirable to separately detect or distinguish between 5mC and 5hmC in the sample. In some embodiments, it is desirable to detect only 5hmC in a sample by treating the sample with laccase as described herein. In other embodiments, it is desirable to detect only 5mC.

In one embodiment, two procedures are run in parallel on two aliquots of the sample. In one of the parallel experiments, 5hmC was blocked while only 5mC was detected by converting the TET and malononitrile procedure described in U.S. provisional application serial No. 63/147,307, filed 2/9/2021, to T equivalent, for example. The blocking of 5hmC takes advantage of reactive hydroxyl groups present on 5hmC but not on 5mC. In some embodiments, the blocking group added to 5hmC is a sugar moiety. In some embodiments, the sugar moiety is a modified or unmodified glucose moiety and forms 5-glucosyl-hydroxymethylcytosine (5 ghmC). In some embodiments, the addition of blocking groups is catalyzed by glycosyltransferases, such as glucosyltransferase. In some embodiments, 5hmC in the nucleic acid is reacted with modified glucose in the presence of β -glycosyltransferase. In some embodiments, the modified glucose is UDP-glucose and the catalyst is phage t4β -glucosyltransferase (T4 BGT).

In one embodiment, two procedures are run in parallel on two aliquots of the sample. In one of the parallel experiments, 5hmC was converted to 5fC using laccase as described herein, and 5fC was detected as T via the malononitrile process. 5mC did not react and was detected as C. In a second parallel experiment, both 5hmC and 5mC were detected as T without distinction, for example, by the TET and malononitrile procedures described in U.S. provisional application Ser. No. 63/147,307 filed on 9, 2, 2021. The first of the two parallel programs showed 5hmC, while the second of the two parallel programs showed 5hmC plus 5mC.

In some embodiments, the invention is a method of distinguishing 5-hydroxymethylcytosine (5 hmC) from 5-methylcytosine (5 mC) in a nucleic acid in a sample, the method comprising: (i) dividing the sample into two aliquots; (ii) Contacting in a first aliquot a nucleic acid comprising 5mC and 5hmC with a 10-11 translocation (TET) dioxygenase under conditions of 5hmC and 5mC conversion to 5-formylcytosine (5 fC); (iii) Contacting in a second aliquot a nucleic acid comprising 5mC and 5hmC with a laccase under conditions whereby 5hmC is converted to 5-formyl cytosine (5 fC); (iv) Separately associating two aliquots with formula R ₁ —CH ₂ -CN moiety, which moiety is capable of reacting with 5fC in a nucleic acid to form an adduct according to the following reaction scheme:

wherein R is ₁ Is an electron withdrawing group selected from the group consisting of cyano, nitro, C1-C6 alkyl carboxylate, unsubstituted carboxamide, C1-C6 alkyl mono-and C1-C6 alkyl di-substituted carboxamide, substituted carbonyl moiety, substituted sulfonyl moiety, wherein the substitution is selected from the group consisting of C1-C6 straight or branched chain alkyl, C4-C6 cycloalkyl, phenyl, 5-or 6-membered heteroaryl, and benzocyclized 5-or 6-membered heteroaryl; (v) Sequencing the nucleic acids from the two aliquots to obtain a first test sequence and a second test sequence, respectively, wherein the adduct is read as thymine (T) during sequencing; (vi) Comparing the first test sequence to a reference sequence, wherein a transition from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of 5hmC and 5mC in the nucleic acid; (vii) The second test sequence is compared to the reference sequence,wherein a transition from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of 5hmC in the nucleic acid; and (viii) comparing the first test sequence to the second test sequence, wherein only such cytosine of the first test sequence is 5mC that is not detected as hydroxymethylated cytosine (5 hmC) in the second test sequence. Alternatively, the two aliquots may be separately contacted with the Wittig reagent.

In some embodiments, it is desirable to detect 5mC and 5hmC indiscriminately. In this example, 5mC is reacted with TET, e.g., as described in us patent No. 9,115,386, to produce 5hmC, which will be oxidized by laccase, as described herein.

In some embodiments, the method includes the step of oxidizing the methylated cytosine for downstream detection. In some embodiments, the method includes the step of converting 5-hydroxymethylcytosine (5 hmC) to 5-formylcytosine (5 fC) with a laccase. In some embodiments, oxidation occurs in the presence of a cofactor. In some embodiments, the cofactor is 2, 6-tetramethylpiperidin-1-oxy (TEMPO). In some embodiments, oxidation occurs at a low pH, e.g., pH < 6. Laccase catalyzes the oxidation of phenolic compounds (including lignin) by reducing oxygen to water; the presence of the mediator also allows for the oxidation of non-phenolic compounds such as benzyl alcohol according to the following scheme:

(see Catalysis Communications 2020,135,105887).

In one embodiment, a laccase type oxidoreductase (EC number 1.10.3.2) is used. In some embodiments, the laccase is from a fungal source. In some embodiments, the fungal source is selected from the group consisting of a species of the genus scilla, phoenix mushroom, oyster mushroom, carbon horn fungus, trametes, coriolus versicolor, and coprinus. In some embodiments, the fungal source is selected from the group consisting of a phoenix dactyloides, a phoenix mushroom MTCC-141, a oyster mushroom MTCC-1801, a carbon black fungus MTCC-1100, a trametes MTCC-1171, a coprinus species, or any other analog or equivalent thereof having similar or equivalent enzymatic activity, such as alcohol dehydrogenase, alcohol oxidase, galactose oxidase, chloroperoxidase, and peroxidase. In some embodiments, 5mC is converted to 5hmC using a toluylmethyl-monooxygenase (EC 1.14.15.26) and a P450 monooxygenase (EC 1.14.14.1).

In some embodiments, the sample is contacted with laccase in the presence of a cofactor. In some embodiments, the cofactor is 2, 6-tetramethylpiperidin-1-oxy (TEMPO). In some embodiments, the cofactor is selected from the group consisting of: laccase natural cofactors selected from acetosyringone, syringaldehyde, p-coumaric acid and laccase synthetic cofactors selected from 2,2' -azino-bis (3-ethylbenzothiazoline-6-sulfonate (ABTS), N-hydroxy mediators such as violuric acid (VLA), N-acetyl-N-phenylhydroxylamine (NHA), N-Hydroxybenzotriazole (HBT), N-Hydroxyphthalimide (HPI), see: two decades of laccases: advancing sustainability in the chemical industry: M.D.Cannatelli, A.J.Ragauskas, chem.Rec.2017,17 (1), 122-140.

In some embodiments, the method includes a preliminary step of converting 5mC to 5hmC with an enzyme, such as TET, prior to reacting the 5hmC with laccase. In some embodiments, the TET and laccase are present in the same convenient "one-pot" reaction. In some embodiments, the TET, laccase, and malononitrile are present in the same convenient "one-pot" reaction.

In some embodiments, the invention further comprises a downstream step of detecting a 5-formyl cytosine (5 fC) nucleotide in a nucleic acid, wherein the 5fC is formed by a method described above. In some embodiments, the downstream step involves contacting a sample containing a nucleic acid comprising 5fC with formula R contained in a solvent composition ₁ —CH ₂ -improved composition contact of CN compound capable of reacting with 5fC in the nucleic acid to form an adduct according to the following reaction scheme:

wherein R is ₁ Is selected from cyano, nitro, C1-C6 alkyl carboxylic acidEsters, unsubstituted carboxamides, C1-C6 alkyl monosubstituted and C1-C6 alkyl disubstituted carboxamides, substituted carbonyl moieties, electron withdrawing groups of substituted sulfonyl moieties wherein the substitution is selected from the group consisting of C1-C6 straight or branched chain alkyl, C4-C6 cycloalkyl, phenyl, 5-or 6-membered heteroaryl and benzocyclized 5-or 6-membered heteroaryl. The reactants of the above reactions are described, for example, in U.S. patent No. 10,519,184 to Yi et al and application publication No. US 20200165661. For example, R1 is a cyano group (CN) and the reactant is malononitrile. Alternatively, 1, 3-indandione compounds may be used instead of R1-CH ₂ CN as a 5fC converting reagent (see B.Xia et al, nature Methods 2015,12 (11), 1047-1050). Still alternatively, 5fC may be reacted with Wittig reagent.

In other embodiments, the downstream step is designed to contact a sample containing a nucleic acid comprising 5fC with a Wittig reagent in an organic solvent and then irradiated with ultraviolet light. The reaction products were detected using fluorescent recognition techniques as described in WO 2020155742.

In some embodiments, formula R ₁ —CH ₂ The CN compound is provided in a reaction mixture capable of allowing the reaction to proceed for less than 3 hours, wherein at least 90% of the 5fC has formed adducts as described in filing U.S. provisional application serial No. 63/147,307 at month 9 of 2021. In some embodiments, the reaction is performed for only 1 hour, wherein at least 90% of the 5fC forms adducts. In some embodiments, the reaction mixture comprises an organic acid moiety. The organic acid has the formula R-COOH and R is selected from the group consisting of C1-C30 linear or branched alkyl, C2-C30 linear or branched alkenyl, C2-C30 linear or branched alkynyl (which may contain heteroatoms such as O and N), aryl, or heteroaryl. In some embodiments, the reaction occurs in the presence of acetic acid. In some embodiments, the concentration of the organic acid in the reaction is between 1% and 30%, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%.

In some embodiments, the reaction mixture comprises a nonaqueous solvent. The nonaqueous solvent has the formula R-OH, wherein R is selected from C1-C3 straight or branched chain alkyl groups and may contain heteroatoms such as O and N. In some embodiments, the reaction occurs in methanol or ethanol. In some embodiments, the reaction occurs in 10% -100% methanol or ethanol. In some embodiments, the reaction occurs in 90% or more methanol or ethanol.

In some embodiments, the reaction mixture comprises formula R _x NH _y A compound wherein x and y are 0, 1, 2 or 3 such that x+y = 3 and each R is independently selected from C1-C6 linear or branched alkyl (optionally containing heteroatoms such as O and N), C6-C10-aryl, or 5-or 6-membered heteroaryl. In some embodiments, formula R _x NH _y The compound is a primary, secondary or tertiary amine having an aliphatic or aromatic group. In some embodiments, rx may form a 5-or 6-membered cyclic heteroalkyl group with N, such as piperidine. In some embodiments, the reaction occurs in the presence of triethanolamine.

After the adduct is formed, the nucleic acid having the adduct is sequenced. In some embodiments, sequencing is performed by a next generation massively parallel sequencing process. Sequencing results in a test sequence in which the adduct is read as thymine (T), i.e., the sequencing polymerase is able to accommodate the adduct in the replicated strand and incorporate adenine (a) as opposed to the adduct. The method further comprises the step of comparing the test sequence to a reference sequence, wherein a change from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence is indicative of the presence of methylated and/or hydroxymethylated cytosine in the test nucleic acid.

In some embodiments, nucleic acids in the sample are amplified prior to sequencing. In some embodiments, amplification utilizes a B family polymerase to efficiently incorporate adenine (a) nucleotides as opposed to malononitrile adducts. In this embodiment, the sequencing may be performed with any polymerase suitable for the sequencing process, as the adduct has been identified as T by the amplifying polymerase.

In some embodiments, the invention is an improved method of detecting methylated and/or hydroxymethylated cytosine nucleotides in a nucleic acid, the method comprising: (i) Ligating an adapter to the nucleic acid in the sample, wherein the adapter comprises an amplification primer binding site; (ii) Forming a reaction mixture by contacting a sample containing an adaptor-ligated nucleic acid with a laccase capable of converting 5hmC in the nucleic acid to 5 fC; (iii) Bringing the reaction mixture into contact with a reaction product of formula R ₁ —CH ₂ -CN compound, which compound is capable of reacting with 5fC in a nucleic acid to form an adduct according to the following reaction scheme:

Wherein R is ₁ Is an electron withdrawing group selected from the group consisting of cyano, nitro, C1-C6 alkyl carboxylate, unsubstituted carboxamide, C1-C6 alkyl mono-and C1-C6 alkyl di-substituted carboxamide, substituted carbonyl moiety, substituted sulfonyl moiety, wherein the substitution is selected from the group consisting of C1-C6 straight or branched chain alkyl, C4-C6 cycloalkyl, phenyl, 5-or 6-membered heteroaryl, and benzocyclized 5-or 6-membered heteroaryl; (iv) Incubating the reaction mixture for less than 3 hours, wherein at least 90% of the 5fC has formed an adduct; (v) Amplifying the ligated nucleic acids using a DNA polymerase and a primer capable of binding to the primer binding site, wherein the DNA polymerase reads the adduct as thymine (T) during amplification; (vi) Sequencing the amplified nucleic acid to obtain a test sequence; (vii) Comparing the test sequence to a reference sequence, wherein a change from cytosine (C) in the reference sequence to thymine (T) in a corresponding position in the test sequence indicates the presence of 5mC and/or 5hmC in the nucleic acid. In some embodiments, in step (iii), formula R ₁ —CH ₂ the-CN compound is present in a non-aqueous solvent such as ethanol or methanol. In some embodiments, in step (ii), the reaction mixture is further contacted with a cofactor of a laccase, such as 2, 6-tetramethylpiperidin-1-oxy (TEMPO). In some embodiments, prior to step (ii), the sample is contacted with TET to convert 5mC in the nucleic acid to 5hmC for reaction with laccase in step (ii). In some embodiments, in step (iii), formula R ₁ —CH ₂ The CN compound is present in a solution comprising an organic acid such as acetic acid. In some embodiments, in step (iii), formula R ₁ —CH ₂ The CN compound is present in a solution comprising an amine such as triethanolamine or piperidine. Alternatively, a Wittig reagent may be used in step (iii).

In some embodiments, the invention relates to nucleic acid polymerases. Nucleic acid polymerases for amplification and sequencing are known and commercially available from a variety of sources. In some embodiments, the invention relates to replicating a chain comprising a 5fC adduct formed as described herein. Such replication requires a polymerase that accommodates the 5fC adduct. In some embodiments, the polymerase is a B family polymerase. In some implementationsIn an embodiment, the polymerase is able to replicate a strand comprising the 5fC adduct by recognizing the adduct as T (i.e., incorporating a as opposed to the adduct). The polymerases capable of containing the 5fC adducts described herein include DNA polymerases known to contain uracil (U) in a DNA strand. The polymerase may be a naturally occurring or engineered polymerase. In some embodiments, the polymerase is isolated from a hyperthermophilic archaebacterium, such as a genus of Pyrococcus (e.g., pyrococcus furiosus) or a genus of Thermus (e.g., thermus aquaticus (Thermus aquaticus)). In some embodiments, the polymerase is isolated from a species of archaea mesophilic bacteria, such as methanosarcina (metanosarccina) (e.g., methanosarcina acetate (Methanosarcina acetivorans)). Examples of engineered uracil-resistant polymerases include KAPA HiFi uracil+dna polymerase (Roche Sequencing Solutions, plasanton, cal.), takara Terra (Takara Bio USA, mountain View, cal.) and Taq DNA polymerase (New England Biolabs, waltham, mass.) was hot started.

In some embodiments, the consensus sequence is determined from a plurality of sequences all having the same UID. It is assumed that sequences with the same UID are derived from the same original molecule by amplification. In other embodiments, the UID is used to eliminate artifacts, i.e., variations (characterized by a particular UID) that exist in the offspring of a single molecule. Such artifacts, which originate from PCR errors or sequencing errors, can be eliminated using UID.

In some embodiments, the invention is a kit comprising components and means for performing the improved methods of detecting DNA methylation described herein. In some embodiments, the kit includes reagents for detecting cytosine methylation in a nucleic acid by performing in vitro oxidation of 5-hydroxymethylcytosine (5 hmC) to 5-formylcytosine (5 fC). In some embodiments, the kit further comprises reagents for detecting 5-formyl cytosine (5 fC) in the nucleic acid.

In some embodiments, the kit further comprises a laccase. In some embodiments, the laccase is from a fungal source. In some embodiments, the fungal source is selected from the group consisting of a species of the genus scilla, phoenix mushroom, oyster mushroom, carbon horn fungus, trametes, coriolus versicolor, and coprinus. In some embodiments, the fungal source is selected from the group consisting of Botrytis cinerea, pleurotus sajor-co, MTCC-141, pleurotus ostreatus, MTCC-1801, xylobacter multiforme, xylobacter makino, MTCC-1171, coprinus species, or any other analog or equivalent thereof having similar or equivalent enzymatic activity, such as F.

In some embodiments, the kit further comprises a cofactor for laccase oxidation. In some embodiments, the cofactor is selected from the group consisting of 2, 6-tetramethylpiperidine-1-oxy (TEMPO), acetosyringone, syringaldehyde, p-coumaric acid 2,2' -azine-bis (3-ethylbenzothiazoline-6-sulfonate (ABTS), violuric acid (VLA), N-acetyl-N-phenylhydroxylamine (NHA), N-Hydroxybenzotriazole (HBT), and N-Hydroxyphthalimide (HPI).

In some embodiments, the kit further comprises a 10-11 translocation dioxygenase (TET). In some embodiments, TET is selected from mouse TET1, TET2, or TET3 (mTET 1, mTET2, or mTET 3); human TET1, TET2, or TET3 (hTET 1, hTET2, or hTET 3); naegleria (Naegleria) TET (NgTET); coprinus cinereus (Coprinopsis cinerea) (CcTET) or any other analogue or equivalent thereof having similar or equivalent enzymatic activity.

In some embodiments, the kit further comprises malononitrile. In some embodiments, malononitrile is present in a non-aqueous solvent. The nonaqueous solvent is selected from ethanol and methanol. In other embodiments, the kit does not include a non-aqueous solvent, but rather includes instructions for using a non-aqueous solvent (such as ethanol or methanol) in the method of detecting DNA methylation with malononitrile as described herein. In some embodiments, the kit further comprises an organic acid and a primary, secondary or tertiary amine. The organic acid may be acetic acid and the amine may be triethanolamine. In other embodiments, the kit includes instructions for using an organic acid and an amine (such as acetic acid and triethanolamine or piperidine) in a method for detecting DNA methylation with malononitrile as described herein. In some embodiments, the kit further comprises a buffer such as MES or TRIS.

In some embodiments, the kit further comprises reagents for distinguishing 5mC from 5hmC in the nucleic acid by protecting 5hmC while the 5mC is chemically reacted. In some embodiments, the kit includes a glucose compound and a glycosyltransferase capable of transferring a glucose moiety to a 5-hydroxy moiety of 5hmC to form 5-glycosylhydroxymethylcytosine (5 ghmC). In some embodiments, the kit comprises beta-glucosyltransferase (BGT) and UDP-glucose. In some embodiments, the BGT is a T4 BGT.

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Selective Enzymic Oxidation of Aromatic Methyl Groups to Aldehydes

Some embodiments of the present disclosure relate to a method for detecting 5-hydroxymethylcytosine (5 hmC) in a target nucleic acid from a sample, wherein the method comprises the steps of: (a) Contacting the target nucleic acid with laccase or copper (II) perchlorate and 2, 6-tetramethylpiperidin-1-oxy (Cu (II)/TEMPO), wherein the laccase or Cu (II)/TEMPO converts 5hmC to 5-formylcytosine (5 fC), thereby producing a nucleic acid comprising one or more 5-formylcytosine (5 fC); (b) Contacting the nucleic acid comprising one or more 5fC of step (a) with malononitrile, wherein the malononitrile converts 5fC to a 5fC-M adduct, thereby producing a nucleic acid comprising one or more 5fC-M adducts; (c) Contacting the nucleic acid comprising one or more 5fC-M adducts of step (b) with a polymerase, wherein the polymerase converts the 5fC-M adducts to thymine (T), thereby producing a nucleic acid comprising one or more T; and (d) sequencing the nucleic acid comprising one or more T of step (c), wherein if T is detected in the nucleic acid comprising one or more T of step (c) at a location where 5hmC was originally present in the target nucleic acid, then 5hmC has been detected in the target nucleic acid. In related embodiments, the target nucleic acid is contacted with laccase in step (a), and wherein step (a) occurs in less than 22 hours, or step (a) occurs in less than 5 hours, or step (a) occurs in less than 4 hours, or step (a) occurs in less than 3 hours, or step (a) occurs in 3 hours. In related embodiments, step (a) occurs at about 25 ℃, or step (a) occurs at about 37 ℃, or step (a) occurs at 37 ℃. In another embodiment, the target nucleic acid is contacted with Cu (II)/TEMPO in step (a), and wherein step (a) occurs in less than 24 hours, or step (a) occurs in 22 hours. In another embodiment, step (b) occurs at about 60 ℃. In a related embodiment, step (b) occurs at 60 ℃ and/or step (b) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer. In one embodiment, the buffer comprises 25mM Tris. In another embodiment, the pH of the buffer is around 8. In another embodiment, a nucleic acid comprising one or more 5fC is contacted with malononitrile for 1.5 hours. In another embodiment, the method further comprises an additional step between step (a) and step (b), wherein the additional step between step (a) and step (b) comprises contacting the nucleic acid comprising one or more 5fC with NaOH. In a related embodiment, the nucleic acid comprising one or more 5fC is contacted with malononitrile in step (b) for less than 1 hour. In another embodiment, the nucleic acid comprising one or more 5fC is contacted with malononitrile in step (b) for about 1 hour. In another embodiment, the nucleic acid comprising one or more 5fC is contacted with malononitrile in step (b) for less than 30 minutes. In another embodiment, the nucleic acid comprising one or more 5fC is contacted with malononitrile in step (b) for about 30 minutes. In another embodiment, step (c) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer. In another embodiment, the buffer comprises the following components: (i) 5% dimethyl sulfoxide (DMSO); (ii) 0.85M betaine; (iii) 70mM tetramethyl ammonium chloride (TMAC); (iv) 2.1mM dATP; (v) 2.25mM MgCl2; and (vi) 15mM ammonium sulfate.

Another embodiment of the present disclosure relates to a method for detecting 5-methylcytosine (5 mC) in a target nucleic acid from a sample, wherein the method comprises the steps of: (a) Contacting the target nucleic acid with a 10-11 translocation (TET), wherein the TET converts 5mC to 5-hydroxymethylcytosine (5 hmC), thereby producing a nucleic acid comprising one or more 5 hmcs; (b) Contacting the nucleic acid comprising one or more 5 hmcs of step (a) with laccase or copper (II) perchlorate and 2, 6-tetramethylpiperidin-1-oxy (Cu (II)/TEMPO), wherein the laccase or Cu (II)/TEMPO converts 5 hmcs to 5-formylcytosine (5 fC), thereby producing a nucleic acid comprising one or more 5-formylcytosine (5 fC); (c) Contacting the nucleic acid comprising one or more 5fC of step (b) with malononitrile, wherein the malononitrile converts 5fC to a 5 fC-malononitrile adduct (55 fC-M adduct) thereby producing a nucleic acid comprising one or more 5fC-M adducts; (d) Contacting the nucleic acid comprising one or more 5fC-M adducts of step (c) with a polymerase, wherein the polymerase converts the 5fC-M adducts to thymine (T), thereby producing a nucleic acid comprising one or more T; and (e) sequencing the nucleic acid comprising one or more T of step (d), wherein if T is detected in the nucleic acid comprising one or more T of step (d) at a location where 5hmC was originally present in the target nucleic acid, then 5hmC has been detected in the target nucleic acid. In another embodiment, step (a) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer comprising an amine catalyst. In another embodiment, the amine catalyst is 2-amino-5-methoxybenzoic acid. In another embodiment, the buffer comprises sodium phosphate and has a pH of about 5.2. In another embodiment, the amine catalyst is 2- (aminomethyl) imidazole dihydrochloride. In another embodiment, the buffer comprises Tris and has a pH of about 8. In another embodiment, the target nucleic acid is contacted with laccase in step (b). In another embodiment, step (b) occurs in less than 22 hours. In another embodiment, step (b) occurs in less than 5 hours. In another embodiment, step (b) occurs in less than 4 hours. In another embodiment, step (b) occurs in less than 3 hours. In another embodiment, step (b) occurs within 3 hours. In another embodiment, step (b) occurs at about 25 ℃. In another embodiment, step (b) occurs at 25 ℃. In another embodiment, step (b) occurs at about 37 ℃. In another embodiment, step (b) occurs at 37 ℃. In another embodiment, step (a) and step (b) are combined into a single step. In another embodiment, the combining of step (a) and step (b) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer, and wherein the buffer comprises both TET and laccase. In another embodiment, the target nucleic acid is contacted with a TET, wherein the TET converts 5mC to 5hmC, thereby producing a nucleic acid comprising one or more 5 hmcs; and wherein the laccase converts 5hmC to 5fC, thereby producing a nucleic acid comprising one or more 5 fcs. In another embodiment, the target nucleic acid is contacted with Cu (II)/TEMPO in step (b). In another embodiment, step (b) occurs in less than 24 hours. In another embodiment, step (b) occurs within 22 hours. In another embodiment, step (c) occurs at about 60 ℃. In another embodiment, step (c) occurs at 60 ℃. In another embodiment, step (c) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer. In another embodiment, the buffer comprises 25mM Tris. In another embodiment, the pH of the buffer is around 8. In another embodiment, a nucleic acid comprising one or more 5fC is contacted with malononitrile for 1.5 hours. In another embodiment, the method further comprises an additional step between step (b) and step (c), wherein the additional step between step (b) and step (c) comprises contacting the nucleic acid comprising one or more 5fC with NaOH. In another embodiment, the nucleic acid comprising one or more 5fC is contacted with malononitrile in step (c) for less than 1 hour. In another embodiment, the nucleic acid comprising one or more 5fC is contacted with malononitrile in step (c) for about 1 hour. In another embodiment, the nucleic acid comprising one or more 5fC is contacted with malononitrile in step (c) for less than 30 minutes. In another embodiment, the nucleic acid comprising one or more 5fC is contacted with malononitrile in step (c) for about 30 minutes. In another embodiment, step (d) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer. In another embodiment, the buffer comprises the following components: (i) 5% dimethyl sulfoxide (DMSO); (ii) 0.85M betaine; (iii) 70mM tetramethyl ammonium chloride (TMAC); (iv) 2.1mM dATP; (v) 2.25mM MgCl2; and (vi) 15mM ammonium sulfate.

Another embodiment of the present disclosure relates to a method for converting 5-hydroxymethylcytosine (5 hmC) to thymine (T) in a target nucleic acid from a sample, wherein the method comprises the steps of: (a) Contacting the target nucleic acid with laccase or copper (II) perchlorate and 2, 6-tetramethylpiperidin-1-oxy (Cu (II)/TEMPO), wherein the laccase or Cu (II)/TEMPO converts 5hmC to 5-formylcytosine (5 fC), thereby producing a nucleic acid comprising one or more 5-formylcytosine (5 fC); (b) Contacting the nucleic acid comprising one or more 5fC of step (a) with malononitrile, wherein the malononitrile converts 5fC to a 5fC-M adduct, thereby producing a nucleic acid comprising one or more 5fC-M adducts; and (c) contacting the nucleic acid comprising one or more 5fC-M adducts of step (b) with a polymerase, wherein the polymerase converts the 5fC-M adducts to thymine (T), thereby producing a nucleic acid comprising one or more T; and wherein 5hmC in the target nucleic acid has been converted to T. In another embodiment, the target nucleic acid is contacted with laccase in step (a). In another embodiment, step (a) occurs in less than 22 hours. In another embodiment, step (a) occurs in less than 5 hours. In another embodiment, step (a) occurs in less than 4 hours. In another embodiment, step (a) occurs in less than 3 hours. In another embodiment, step(s) occurs within 3 hours. In another embodiment, step (a) occurs at about 25 ℃. In another embodiment, step (a) occurs at 25 ℃. In another embodiment, step (a) occurs at about 37 ℃. In another embodiment, step (a) occurs at 37 ℃. In another embodiment, the target nucleic acid is contacted with Cu (II)/TEMPO in step (a). In another embodiment, step (a) occurs in less than 24 hours. In another embodiment, step(s) occurs within 22 hours. In another embodiment, step (b) occurs at about 60 ℃. In another embodiment, step (b) occurs at 60 ℃. In another embodiment, step (b) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer. In another embodiment, the buffer comprises 25mM Tris. In another embodiment, the pH of the buffer is around 8. In another embodiment, a nucleic acid comprising one or more 5fC is contacted with malononitrile for 1.5 hours. In another embodiment, the method further comprises an additional step between step (a) and step (b), wherein the additional step between step (a) and step (b) comprises contacting the nucleic acid comprising one or more 5fC with NaOH. In another embodiment, the nucleic acid comprising one or more 5fC is contacted with malononitrile in step (b) for less than 1 hour. In another embodiment, the nucleic acid comprising one or more 5fC is contacted with malononitrile in step (b) for about 1 hour. In another embodiment, the nucleic acid comprising one or more 5fC is contacted with malononitrile in step (b) for less than 30 minutes. In another embodiment, the nucleic acid comprising one or more 5fC is contacted with malononitrile in step (b) for about 30 minutes. In another embodiment, step (c) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer. In another embodiment, the buffer comprises the following components: (i) 5% dimethyl sulfoxide (DMSO); (ii) 0.85M betaine; (iii) 70mM tetramethyl ammonium chloride (TMAC); (iv) 2.1mM dATP; (v) 2.25mM MgCl2; and (vi) 15mM ammonium sulfate.

Another embodiment of the present disclosure relates to a method for converting 5-methylcytosine (5 mC) in a target nucleic acid from a sample to thymine (T), wherein the method comprises the steps of: (a) Contacting the target nucleic acid with a 10-11 translocation (TET), wherein the TET converts 5mC to 5-hydroxymethylcytosine (5 hmC), thereby producing a nucleic acid comprising one or more 5 hmcs; (b) Contacting the nucleic acid comprising one or more 5 hmcs of step (a) with laccase or copper (II) perchlorate and 2, 6-tetramethylpiperidin-1-oxy (Cu (II)/TEMPO), wherein the laccase or Cu (II)/TEMPO converts 5 hmcs to 5-formylcytosine (5 fC), thereby producing a nucleic acid comprising one or more 5-formylcytosine (5 fC); (c) Contacting the nucleic acid comprising one or more 5fC of step (b) with malononitrile, wherein the malononitrile converts 5fC to a 5 fC-malononitrile adduct (5 fC-M adduct), thereby producing a nucleic acid comprising one or more 5fC-M adducts; (d) Contacting the nucleic acid comprising one or more 5fC-M adducts of step (c) with a polymerase, wherein the polymerase converts the 5fC-M adducts to thymine (T), thereby producing a nucleic acid comprising one or more T; and wherein 5mC in the target nucleic acid has been converted to T. In another embodiment, step (a) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer comprising an amine catalyst. In another embodiment, the amine catalyst is 2-amino-5-methoxybenzoic acid. In another embodiment, the buffer comprises sodium phosphate and has a pH of about 5.2. In another embodiment, the amine catalyst is 2- (aminomethyl) imidazole dihydrochloride. In another embodiment, the buffer comprises Tris and has a pH of about 8. In another embodiment, the target nucleic acid is contacted with laccase in step (b). In another embodiment, step (b) occurs in less than 22 hours. In another embodiment, step (b) occurs in less than 5 hours. In another embodiment, step (b) occurs in less than 4 hours. In another embodiment, step (b) occurs in less than 3 hours. In another embodiment, step (b) occurs within 3 hours. In another embodiment, step (b) occurs at about 25 ℃. In another embodiment, step (b) occurs at 25 ℃. In another embodiment, step (b) occurs at about 37 ℃. In another embodiment, step (b) occurs at 37 ℃. In another embodiment, step (a) and step (b) are combined into a single step. In another embodiment, the combining of step (a) and step (b) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer, and wherein the buffer comprises both TET and laccase. In another embodiment, the target nucleic acid is contacted with a TET, wherein the TET converts 5mC to 5hmC, thereby producing a nucleic acid comprising one or more 5 hmcs; and wherein the laccase converts 5hmC to 5fC, thereby producing a nucleic acid comprising one or more 5 fcs. In another embodiment, the target nucleic acid is contacted with Cu (II)/TEMPO in step (b). In another embodiment, step (b) occurs in less than 24 hours. In another embodiment, step (b) occurs within 22 hours. In another embodiment, step (c) occurs at about 60 ℃. In another embodiment, step (c) occurs at 60 ℃. In another embodiment, step (c) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer. In another embodiment, the buffer comprises 25mM Tris. In another embodiment, the pH of the buffer is around 8. In another embodiment, a nucleic acid comprising one or more 5fC is contacted with malononitrile for 1.5 hours. In another embodiment, the method further comprises an additional step between step (b) and step (c), wherein the additional step between step (b) and step (c) comprises contacting the nucleic acid comprising one or more 5fC with NaOH. In another embodiment, the nucleic acid comprising one or more 5fC is contacted with malononitrile in step (c) for less than 1 hour. In another embodiment, the nucleic acid comprising one or more 5fC is contacted with malononitrile in step (c) for about 1 hour. In another embodiment, the nucleic acid comprising one or more 5fC is contacted with malononitrile in step (c) for less than 30 minutes. In another embodiment, the nucleic acid comprising one or more 5fC is contacted with malononitrile in step (c) for about 30 minutes. In another embodiment, step (d) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer. In another embodiment, the buffer comprises the following components: (i) 5% dimethyl sulfoxide (DMSO); (ii) 0.85M betaine; (iii) 70mM tetramethyl ammonium chloride (TMAC); (iv) 2.1mM dATP; (v) 2.25mM MgCl2; and (vi) 15mM ammonium sulfate.

Examples

Example 1. Ethanol was used as a co-solvent for picoline-borane in methylation detection assays (TAPS).

In this example, a synthetic oligonucleotide with a caC nucleotide is reduced by DHU under novel conditions. 2-methylpyridine borane was dissolved in absolute ethanol or methanol (1 mg/5uL,1.87 mM). The synthetic oligonucleotide TAPS-caC SEQ ID NO. 1 (2.7 nmol,5' -Phos-CACGTCCAGATCAAT (caC) GACTATGAGCAGTACA) was dissolved in 35uL sodium acetate (3M, pH 4.3) and mixed with 25uL picoline borane solution to a final concentration of 790mM. The resulting cloudy solution was placed on a hot mixer and shaken at 35C for 3 hours. The solution was diluted with DI water (300 uL) and purified by HPLC (C18, eluent: CH3CN/0.1M TEAA; gradient: 2% -15% CH3CN/0.1M TEAA over 35 minutes) to give 1.33nmol (49%) of TAPS-DHU as identified by mass spectrometry (M/z 9869.43, calculated 9869.0). DHU product formation was monitored by taking aliquots (2 uL) of the reaction solution over 30, 60, 180 minutes and analyzing by LCMS. The results are shown in figure 1, with surface DHU conversion completed within one hour.

Example 2. Methanol and acetic acid were used as co-solvents for picoline-borane in methylation detection assays (TAPS).

In this example, the oligonucleotide of example 1 (SEQ ID NO: 1) was used under the conditions described in example 1, except that picoline-borane was present in a methanol/acetic acid solution. The reaction contained 1.39nmol of TAPS ca oligonucleotide, 250mM methyl borane (10:1 v: v acetic acid solution) and 200mM MES pH 6. We were able to reduce the effective borane concentration to 25mM and the reaction time was further reduced to 1 hour. The reaction products were detected as described in example 1. The results are shown in FIG. 2.

Example 3 malononitrile in sodium acetate buffer was used in methylation detection assays.

In this example we show an improved process for the formation of adducts of malononitrile on 5fC oligonucleotides at 35C over 5 hours using 1M sodium acetate as buffer. The reaction was performed as follows: 1nmol (4 uL) of 5fC containing the oligonucleotide (TAPS-fC: SEQ ID NO:15' -Phos-CACGTCCAGATCAAT (fC) GACTATGAGCAGTACA)) was reacted with a solution of malononitrile (100 mM,50 uL) in sodium acetate (1M, pH 8.4). LCMS analysis of samples at different incubation times showed that more than 90% of TAPS-fC was consumed after 5 hours at 35C. The results are shown in FIG. 3. The mass of TAPS-fC was 9894, and the mass of the reaction product was 9942.

Example 4 malononitrile in ethanol-TRIS buffer was used in methylation detection assays.

In this example we show an improved process for the formation of adducts of malononitrile on 5fC oligonucleotides in 4 hours at 35C using ethanol/TRIS as buffer. The reaction was performed as follows: 1nmol (4 uL) of TAPS-fC oligonucleotide SEQ ID NO 1 was reacted with malononitrile solution (200 mM,25 uL) in ethanol and TRIS (pH 8, 20mM,26 uL). LCMS analysis of the samples at different incubation times showed that after 4 hours at 37C, most of the 5fC was converted to product. The results are shown in FIG. 4. The mass of TAPS-fC was 9894, and the mass of the reaction product was 9942.

Example 5 malononitrile in ethanol-triethylamine buffer was used in methylation detection assays.

In this example we show an improved process for the formation of adducts of malononitrile on 5fC oligonucleotides in one hour at 35C using ethanol/triethylamine as buffer. The reaction was performed as follows: 1nmol (4 uL) of TAPS-5fC oligomer (SEQ ID NO: 1) was reacted with malononitrile solution (100 mM,46 uL) in ethanol and 1uL of triethylamine. LCMS analysis was performed on samples at different incubation times. After 1 hour of incubation at 35C, more than 90% of the oligomers reacted. The results are shown in FIG. 5. The mass of TAPS-fC was 9894, and the mass of the reaction product was 9942.

Example 6. Single tube methylation detection assay using TET and malononitrile.

In this example, we demonstrate that TET is active in malononitrile and can achieve a single tube methylation detection reaction. Lambda DNA (unmethylated and methylated) was sheared and ligated to adaptors to prepare a sequencing library. 10ng of unmethylated and methylated library was used as input DNA. 10nM, 100nM or 500nM mTET2 (NEB) or NgTET was incubated with DNA at a final concentration of 150mM malononitrile. The reaction was incubated at 37C for 20 hours. One quarter of the sample was added directly to the PCR and 14 cycles were performed using kappa HiFi polymerase. The library was sequenced and the conversion at CpG sites is shown in figure 6. The conversion of 0.9% compared to 0.1% without the TET control indicates that TET is capable of functioning in the presence of malononitrile and that oxidation and adduct formation can occur in a single tube.

Example 7 oxidation of 5hmC with laccase.

In this example, a synthetic oligonucleotide with 5hmC nucleotides is subjected to oxidation using laccase. The 5hmC synthetic oligonucleotide (SEQ ID NO: 2) has the sequence 5'-ATT ATT TAT TTA TThmC GTA TTA TTT ATT ATT-3'.150 μl reaction mixture contains 50nmol of oligonucleotide, 1.6mg (10 μmol) 2, 6-tetramethylpiperidin-1-yloxy (TEMPO), 2mg laccase from Coriolus versicolor (Sigma, catalog number 38429), 50mM phosphate buffer pH 5.2. The reaction was allowed to proceed at room temperature for 5 hours. The first control reaction contained all of the above reagents (including TEMPO) except laccase. The second control reaction contained all of the above reagents (including laccase and TEMPO), except that laccase and TEMPO were added in a 1:1000 dilution. The third control reaction contained all of the above reagents except that oligonucleotide SEQ. ID. NO:2 contained 5mC instead of 5hmC:5'-ATT ATT TAT TTA TTmC GTA TTA TTT ATT ATT-3'.

The reaction mixture was analyzed by liquid chromatography-mass spectrometry (LCMS). The molecular weight is as follows:

5hmC–9176Da

5mC–9160Da

5fC–9174Da

FIG. 7 shows the reaction generated 5fC peak at 9174 Da. Another peak was observed at 9229Da, corresponding to the artifact of imine formation of 5-formyl-dC with n-butylamine from LC-MS eluate (fc+55 da=9229 Da). To some extent, additional oxidation (+14 Da) occurs at the hydroxyl group of the ribose.

FIG. 8 shows that there is no reaction with 5mC (5 mC peak is unchanged at 9160 Da). Oxidation (+14 Da) occurs to some extent at the hydroxyl group of ribose. The control reaction without enzyme and diluted enzyme indicated no change in the 5hmC starting material (data not shown).

Example 8 (predictive). The 5mC is oxidized to 5hmC before reacting the 5hmC with laccase.

To convert 5mC to 5hmC, the reaction mixture contains 3ug TET protein and 2 μg of oligonucleotide substrate in 50mM HEPES pH 8, 50mM NaCl, 2mM ascorbic acid, 1mM 2-ketoglutarate, 100 μM ferrous ammonium sulfate (Fe2+) and 1mM DTT, and incubated for 3 hours at 37℃as described in Tahliani et al, (2009) Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1, science 324 (5929): 930-935. The nucleic acids obtained are purified, for example, by SPRI. An aliquot of the reaction mixture was incubated with laccase as described in example 7.

Although the invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various modifications can be made within the scope of the invention. Accordingly, the scope of the invention should not be limited by the examples described herein, but by the claims presented below.

Example 9: TET activity was adjusted to 5hmC/5fC using an amine buffered catalyst.

A study was conducted to determine if the amine buffered catalyst would modulate TET activity to oxidize 5mC to 5hmC/5fC. To investigate this, 1. Mu.g of double stranded DNA containing 5mC was oxidized with 3.2. Mu.M NgTET at 37℃for 1 hour in the presence or absence of a catalyst under certain buffer conditions. In one experiment, the catalyst 2-amino-5-methoxybenzoic acid (AMBA) was added to modulate TET activity in a buffer containing sodium phosphate and having a pH of 5.2 (the results are depicted in fig. 9A). The upper diagram of fig. 9A shows a TET without any AMBA, the middle diagram of fig. 9A shows a TET with 5mM AMBA, and the lower diagram of fig. 9A shows a TET with 10mM AMBA. FIG. 9A shows that AMBA effectively promoted TET-mediated oxidation of 5mC to 5hmC/5fC in a dose-dependent manner without excessive accumulation of unwanted 5caC product. In another experiment, the catalyst 2- (aminomethyl) imidazole dihydrochloride (AMI) was added to modulate TET activity in Tris-containing buffer at pH 8 (the results are depicted in fig. 9B). The upper diagram of fig. 9B shows a TET without any AMI, the middle diagram of fig. 9B shows a TET with 5mM AMI, and the lower diagram of fig. 9B shows a TET with 10mM AMI. FIG. 9B shows that AMI effectively promotes TET-mediated oxidation of 5mC to 5hmC/5fC in a dose-dependent manner without excessive accumulation of unwanted 5caC product. Thus, these studies indicate that the amine catalysts AMBA and AMI in the buffer favour oxidation of 5mC to 5hmC/5fC in the presence of TET.

Taken together, these studies indicate that amine catalysts, such as 2-amino-5-methoxybenzoic acid (AMBA) and 2- (aminomethyl) imidazole dihydrochloride (AMI), may be useful to promote TET-mediated 5mC oxidation, thereby facilitating the production of 5hmC and 5fC species while preventing complete oxidation to 5caC species. Thus, amine catalysts such as 2-amino-5-methoxybenzoic acid (AMBA) and 2- (aminomethyl) imidazole dihydrochloride (AMI) may be ultimately useful and used in a workflow to detect 5mC and 5hmC species by ultimately facilitating the production of thymine.

Example 10: 5fC material was detected and accumulated by oxidation of 5hmC with laccase as early as 3 hours.

Studies have shown that laccase converts 5hmC to 5fC after 22 hours. A study was performed to determine if 5fC would be detected earlier/faster within 22 hours. To this end, laccase (2 mg laccase (from Coriolus versicolor (Sigma catalog number 38429)) was tested for its ability to oxidize 5hmC from a 50nM 5 hmC-containing oligonucleotide substrate in the presence of 1.6mg (10. Mu.M) of 2, 6-tetramethylpiperidin-1-oxyl (TEMPO) and 150. Mu.l of 50mM phosphate buffer (pH 5.2) at two different temperatures (25℃and 37 ℃).

These studies showed that laccase can efficiently convert 5hmC to 5fC in as early as 3 hours at 25 ℃ and 37 ℃ without unwanted accumulation/conversion of 5caC (as shown in fig. 10). Thus, these studies indicate that laccase can be used as an important enzyme for detecting methylated species (e.g., 5 hmC) by facilitating conversion to 5fC, which can then be subsequently converted to thymine.

Example 11: the activity of malononitrile to convert 5fC to 5fC-M adducts was optimized.

Here studies were performed to optimize the activity of malononitrile to convert 5fC to 5fC-M adducts.

In particular, studies were performed here to determine if elevated temperatures would accelerate the malononitrile-mediated reaction of 5fC to 5fC-M adducts. Early studies in the art have demonstrated that malononitrile can be used to convert 5fC to 5fC-M adducts, however, these studies were performed in 10mM Tris at 37℃for 20 hours (see U.S. Pat. No. 10,519,184 and U.S. Pat. publication No. US 2020/0165661). To evaluate the effect of elevated temperature, 33. Mu.M of 5fC was added to 100mM malononitrile in 25mM Tris HCl at different temperatures and incubation times (40℃1 hour, 60℃1 hour and 95℃10 minutes). The results are shown in fig. 11A. FIG. 11A shows LC-MS data for the effect of malononitrile on the conversion of 5fC to 5fC-M adducts under various buffer conditions. The upper plot of fig. 11A shows a buffer condition of 40 ℃ for 1 hour, the middle plot of fig. 11A shows a buffer condition of 60 ℃ for 1 hour, and the lower plot of fig. 11A shows a buffer condition of 95 ℃ for 10 minutes. FIG. 11A shows the significant accumulation of 5fC-M adducts after 1 hour at elevated temperature (60 ℃). Figure 11A also shows significant accumulation of 5fC-M adducts (but the presence of degradation products) after only 10 minutes at elevated temperature (95 ℃). Thus, FIG. 11A shows that malononitrile activity, which converts 5fC to a 5fC-M adduct, is optimized at elevated temperatures (60 ℃) which represents a significant improvement over the prior art.

In another study, the effect of NaOH (pre-denaturing double stranded nucleic acid) on malononitrile activity to convert 5fC to 5fC-M adducts was evaluated. For this, two different synthetic 5fC oligonucleotides (CGA and CGC) were used as substrates in the presence of copper (II) perchlorate and malononitrile and NaOH in 2, 6-tetramethylpiperidin-1-oxy (TEMPO) (Cu (II)/TEMPO) over 30 minutes. Malononitrile activity was evaluated to convert 5fC to a 5fC-M adduct, and the results are depicted in fig. 11B. Figure 11B shows significant malononitrile activity in the presence of NaOH in as little as 30 minutes. These studies indicate that NaOH pre-denaturation enhances malononitrile activity.

In addition, malononitrile activity can be optimized with a 25mM Tris buffer at pH 8 at 60℃for a shortened single strand DNA incubation time (1.5 hours). It is believed that an increased/increased amount of Tris (e.g., 25mM Tris) and at an increased temperature (e.g., 50C-60C) will result in an accelerated reaction and improved malononitrile activity efficiency (data not shown). In addition, the addition of copper (II) perchlorate and 2, 6-tetramethylpiperidin-1-oxyl (TEMPO) (Cu (II)/TEMPO) components also enhanced the reaction efficiency (data not shown).

Taken together, these studies indicate that malononitrile activity can be optimized by: (i) increasing the incubation temperature (to 60 ℃), (II) shortening the incubation time of single stranded DNA (to 1.5 hours) by using a high Tris buffer (e.g., 25mM Tris) at pH 8, (iii) using a pre-denaturation step with NaOH (which can shorten the incubation of double stranded DNA), and (iv) adding copper (II) perchlorate and 2, 6-tetramethylpiperidin-1-oxy (Cu (II)/TEMPO) components (which can enhance the reaction efficiency). Thus, the ability to detect methylated species in an oligonucleotide sample can be improved/enhanced by optimizing malononitrile activity in a variety of ways.

Example 12: optimization of copper (II) perchlorate and 2, 6-tetramethylpiperidin-1-oxyl (Cu (II)/TEMPO).

A study was performed to optimize the copper (II) perchlorate and 2, 6-tetramethylpiperidin-1-oxyl (Cu (II)/TEMPO) reaction conditions to evaluate its effect on CuTEMPO mediated oxidation of 5hmC to 5 fC. Early studies in the art showed that the Cu (II)/TEMPO reaction occurred at room temperature for up to 48 hours (see, e.g., matsushita et al, "DNA-friendly Cu (II)/TEMPO-ctalyzed 5-hydroxyymethylythosone-specific oxidation," chem. Commun.53:5756-5759 (2017)). These studies were performed to assess whether the Cu (II)/TEMPO reaction would be shortened to 22 hours. To evaluate this, the following reagents were combined: 49 μl of H ₂ O, 10. Mu.l Cu (ClO) ₄ ) ₂ (100 mM), 15. Mu.l of Bipyr (100 mM), 10. Mu.l of TEMPO (100 mM), 10. Mu.l of NaOH (50 mM) and 6. Mu.l of hMC (169. Mu.M) were mixed at 500rpm for 22 hours at 25℃in the presence or absence of dimethylaminoethyl hydrazine (DMAEH), which reacts only to 5 fC. The results are depicted in fig. 12. The upper plot of FIG. 12 shows Cu (II)/TEMPO oxidation of 5mC to 5hmC/5fC, and the lower plot of FIG. 12 shows the derivatization of the product from the upper plot with DMAEH.

Thus, these data show that copper (II) perchlorate and 2, 6-tetramethylpiperidin-1-oxyl (Cu (II)/TEMPO) reactions can be shortened to 22 hours, which represents an improvement over the prior art.

Example 13: buffer conditions for optimizing polymerase activity for conversion of 5fC-M adducts to thymine (T)

The polymerase mediates the step of converting the 5fC-M adduct to thymine (T), as depicted in fig. 13A. A study was performed to evaluate the effect of buffer on conversion of 5fC-M adducts to T. To this end, the standard buffer ("buffer a") was compared with the optimized buffer ("doe_1"). The composition of the optimized buffer doe_1 is shown in fig. 13B. Oligonucleotides containing purified 5fC-M adducts in the context of "CGA" or "CGC" were amplified with polymerase in standard buffer ("buffer a") or optimized buffer ("doe_1"). FIG. 13C shows data showing the conversion of 5fC-M adducts to T using standard buffer ("buffer A") and optimized buffer ("DOE_1"). In particular, FIG. 13C shows that optimizing buffer ("DOE_1") increases/enhances the conversion of 5fC-M adducts to T as compared to standard buffer. Indeed, as shown in fig. 13C, for "CGA", the conversion with optimized buffer (94.12) was significantly greater than the conversion with standard buffer (76.94), and for "CGC", the conversion with optimized buffer (86.28) was significantly higher than the conversion with standard buffer (69.46). Thus, these studies showed that polymerase activity was enhanced in the presence of the optimized buffer compared to the standard buffer.

Taken together, these studies indicate that optimizing the buffer enhances the polymerase activity for converting the 5fC-M adduct to thymine (T). This means that improved buffers for improved increased polymerase activity can be used to improve the method of detecting methylated species in an oligonucleotide sample.

Claims

1. A method for detecting 5-hydroxymethylcytosine (5 hmC) in a target nucleic acid from a sample, wherein the method comprises the steps of:

(a) Contacting the target nucleic acid with laccase or copper (II) perchlorate and 2, 6-tetramethylpiperidine-1-oxyl (Cu (II)/TEMPO), wherein the laccase or Cu (II)/TEMPO converts 5hmC to 5-formylcytosine (5 fC), thereby producing a nucleic acid comprising one or more 5-formylcytosine (5 fC);

(b) Contacting the nucleic acid comprising one or more 5fC of step (a) with malononitrile, wherein the malononitrile converts 5fC to a 5fC-M adduct, thereby producing a nucleic acid comprising one or more 5fC-M adducts;

(c) Contacting the nucleic acid comprising one or more 5fC-M adducts of step (b) with a polymerase, wherein the polymerase converts the 5fC-M adducts to thymine (T), thereby producing a nucleic acid comprising one or more T; and

(d) Sequencing the nucleic acid comprising one or more T of step (c), wherein if T is detected in the nucleic acid comprising one or more T of step (c) at a location where 5hmC was originally present in the target nucleic acid, then 5hmC has been detected in the target nucleic acid.

2. The method of claim 1, wherein the target nucleic acid is contacted with laccase or Cu (II)/TEMPO in step (a).

3. The method of claim 1, further comprising an additional step between step (a) and step (b), wherein the additional step between step (a) and step (b) comprises contacting the nucleic acid comprising one or more 5fC with NaOH.

4. A method for detecting 5-methylcytosine (5 mC) in a target nucleic acid from a sample, wherein the method comprises the steps of:

(a) Contacting the target nucleic acid with a 10-11 translocation (TET), wherein the TET converts 5mC to 5-hydroxymethylcytosine (5 hmC), thereby producing a nucleic acid comprising one or more 5 hmcs;

(b) Contacting the nucleic acid comprising one or more 5 hmcs of step (a) with laccase or copper (II) perchlorate and 2, 6-tetramethylpiperidin-1-oxy (Cu (II)/TEMPO), wherein the laccase or Cu (II)/TEMPO converts 5 hmcs to 5-formylcytosine (5 fC), thereby producing a nucleic acid comprising one or more 5-formylcytosine (5 fC);

(c) Contacting the nucleic acid comprising one or more 5fC of step (b) with malononitrile, wherein the malononitrile converts 5fC to a 5 fC-malononitrile adduct (55 fC-M adduct) thereby producing a nucleic acid comprising one or more 5fC-M adducts;

(d) Contacting the nucleic acid comprising one or more 5fC-M adducts of step (c) with a polymerase, wherein the polymerase converts the 5fC-M adducts to thymine (T), thereby producing a nucleic acid comprising one or more T; and

(e) Sequencing the nucleic acid comprising one or more T of step (d), wherein if T is detected in the nucleic acid comprising one or more T of step (d) at a location where 5hmC was originally present in the target nucleic acid, then 5hmC has been detected in the target nucleic acid.

5. The process of claim 4, wherein step (a) occurs in a reaction mixture comprising a buffer containing an amine catalyst, preferably 2-amino-5-methoxybenzoic acid or 2- (aminomethyl) imidazole dihydrochloride.

6. The method of claim 4, wherein the target nucleic acid is contacted with laccase in step (b).

7. The method of claim 6, wherein step (a) and step (b) are combined in a single step, and wherein the reaction mixture comprises a buffer, and wherein the buffer comprises both TET and laccase.

8. The method of claim 4, wherein the target nucleic acid is contacted with Cu (II)/TEMPO in step (b).

9. The method of claim 4, further comprising an additional step between step (b) and step (c), wherein the additional step between step (b) and step (c) comprises contacting the nucleic acid comprising one or more 5fC with NaOH.

10. A method for converting 5-hydroxymethylcytosine (5 hmC) to thymine (T) in a target nucleic acid from a sample, wherein the method comprises the steps of:

(b) Contacting the nucleic acid comprising one or more 5fC of step (a) with malononitrile, wherein the malononitrile converts 5fC to a 5fC-M adduct, thereby producing a nucleic acid comprising one or more 5fC-M adducts; and

(c) Contacting the nucleic acid comprising one or more 5fC-M adducts of step (b) with a polymerase, wherein the polymerase converts the 5fC-M adducts to thymine (T), thereby producing a nucleic acid comprising one or more T; and wherein the 5hmC in the target nucleic acid has been converted to T.

11. The method of claim 10, wherein the target nucleic acid is contacted with laccase or Cu (II)/TEMPO in step (a).

12. The method of claim 1 β, further comprising an additional step between step (a) and step (b), wherein the additional step between step (a) and step (b) comprises contacting the nucleic acid comprising one or more 5fC with NaOH.

13. A method for converting 5-methylcytosine (5 mC) in a target nucleic acid from a sample to thymine (T), wherein the method comprises the steps of:

(c) Contacting the nucleic acid comprising one or more 5fC of step (b) with malononitrile, wherein the malononitrile converts 5fC to a 5 fC-malononitrile adduct (5 fC-M adduct) thereby producing a nucleic acid comprising one or more 5fC-M adducts;

(d) Contacting the nucleic acid comprising one or more 5fC-M adducts of step (c) with a polymerase, wherein the polymerase converts the 5fC-M adducts to thymine (T), thereby producing a nucleic acid comprising one or more T; and wherein the 5mC in the target nucleic acid has been converted to T.

14. The process according to claim 13, wherein step (a) occurs in a reaction mixture, wherein the reaction mixture comprises a buffer comprising an amine catalyst, preferably 2-amino-5-methoxybenzoic acid or 2- (aminomethyl) imidazole dihydrochloride.

15. The method of claim 13, wherein the target nucleic acid is contacted with Cu (II)/TEMPO in step (b).