Detection of epigenetic modifications

Abstract

The present invention provides a method comprising the steps of: a) providing a nucleic acid comprising 5fC, 5hmC or 5caC; b) providing a reactant comprising two reactive groups, wherein a first reactive group is capable of reacting with a formyl group, a hydroxymethyl group, or a carboxyl group and a second reactive group is a nucleophilic group; c) reacting the first reactive group with the formyl group, hydroxymethyl group, or carboxyl group, thereby obtaining a modified 5fC, 5hmC or 5caC; d) reacting the second reactive group with the C6 position of the modified 5fC, 5hmC or 5caC, thereby obtaining a bicyclic or tricyclic molecule comprising a 5,6-dihydrocytosine entity; and e) deaminating the 5,6-dihydrocytosine entity to a 5,6-dihydro-uracil entity.

Description

[Technical Field]

[0001] The present invention relates to the field of mapping epigenetic modifications of DNA and RNA, which is becoming increasingly important because these modifications play a role in several biological processes and diseases, including development, aging and last but not least, cancer. [Background technology]

[0002] The most relevant for identifying epigenetic modifications is their detection by sequencing-based methods. The distinction between cytosine (C) and 5-methylcytosine (5mC) has been demonstrated by direct sequencing of DNA (without pre-amplification) via nanopores (e.g., PacificBioscience SingleMolecule Real-Time (SMRT) sequencing technology), which allows for the differential kinetics of nucleotide incorporation by polymerases towards C and mC.

[0003] Another technique is to use methods to convert C or mC to T(U) equivalents and subsequent amplification, allowing identification when comparing untreated and converted sample DNA (for review, see L. Zhao et al., Protein Cell 2020, 11, 792-808).

[0004] The most important methods are bisulfite sequencing (conversion of C to U by bisulfite treatment), NEB's EM-seq method (oxidation of mC by TET2 enzyme and glucosylation by β-glucosyltransferase (blocking the enzymatic deaminase reaction) and conversion of C to U by APOBEC deaminase), TAPS method (TET-assisted pyridine borane sequencing) which applies TET enzyme oxidation of mC and subsequent reduction of the oxidized mC species with pyridine borane to give dihydrocytosine nucleosides that are easily deaminated to give dihydrouracils (T equivalents), and the CLEVER method (C. Zhu et al., Cell Stem Cell 2017, 20, 720-731), which is based on the oxidation of mC by TET enzyme and the subsequent reaction of 5-formyl-C with malononitrile to give adducts that primarily act as T equivalents in subsequent PCR amplification. As an alternative to the enzymatic oxidation of mC by TET, the enzymatic oxidation can also be carried out by chemical means, for example using potassium perruthenate (KRuO4).

[0005] Additionally, a partial modification of the above method can be used to distinguish between 5-hydroxymethyl-dC (5hmC), 5-formyl (5fC), or 5-carboxyl-C (5caC) modifications.

[0006] Recently, WO 2022 / 096751 also disclosed a method for generating dihydrothymine (DHT) or dihydrouracil (DHU) residues from nucleosides or polynucleotides containing 5-methylcytosine (5mC) or 5-carboxyl-cytosine (5caC) by using a radical initiator that can be used with a nucleophilic compound.

[0007] However, all of the disclosed methods have several drawbacks. Bisulfite sequencing involves harsh conditions that degrade most of the input DNA, allowing only indirect detection of methylated cytosines. Similarly, enzymatic methyl sequencing by enzymatic deamination (EM-Seq) is inefficient because most unmethylated cytosines must be converted. TAPS requires toxic reagents, such as pyridine borane, for reduction, and dihydrouridine, which is unstable under mildly acidic conditions, cannot be easily converted back to the more stable uridine derivative. In the CLEVER method, enzymatic incorporation of nucleotides by polymerase when the condensation product of 5fC and malononitrile serves as a substrate is not highly accurate, and incorporation of dA is favored but not exclusive, resulting in a conversion yield far below 100% for the condensation reaction of 5fC and malononitrile (see also F. Galardi et al., Biomolecules 2020, 10, 1677). The method of WO 2022 / 096751 is not completely specific for caC and also converts unmodified C to some extent. Another drawback is that dihydrouridine derivatives cannot be easily converted back to more stable uridine derivatives. Summary of the Invention

[0008] The present invention is based on the basic idea that introduction of a nucleophile at 5fC, 5hmC or caC and subsequent intramolecular addition of a 5-substituted cytosine at C-6 allows deamination at C-4 of a cytosine derivative.

[0009] In a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a) providing a nucleic acid comprising 5fC, 5hmC or 5caC; b) providing a reactant comprising two reactive groups, wherein a first reactive group is capable of reacting with a formyl group, a hydroxymethyl group, or a carboxyl group, and a second reactive group is a nucleophilic group; c) reacting the first reactive group with a formyl group, a hydroxymethyl group, or a carboxyl group, thereby obtaining modified 5fC, 5hmC, or 5caC; d) reacting a second reactive group with the C6 position of the modified 5fC, 5hmC, or 5caC, thereby obtaining a bicyclic or tricyclic molecule containing a 5,6-dihydrocytosine entity; and e) deaminating 5,6-dihydrocytosine entities to 5,6-dihydro-uracil entities The present invention relates to a method, comprising:

[0010] The cyclic structure newly formed in step d) is a 5- to 7-membered ring.

[0011] In one embodiment, the method further comprises step f), i.e., reversing the ring formation of step d), thereby obtaining a 5-substituted uracil.

[0012] Since naturally occurring DNA methylation is 5mC, the method may also include a step of converting 5mC to 5fC, 5hmC, or 5caC prior to step a), thus facilitating the analysis of the methylation status of naturally occurring DNA.

[0013] In certain embodiments, the nucleic acid contains at least one 5fC residue. In this case, the first reactive group may be a CH acid group, an amine, or a phosphorus ylide. In another specific embodiment, the nucleic acid contains at least one 5hmC residue. The first reactive group may then constitute a glycosyltransferase substrate. In yet another embodiment, the nucleic acid contains a 5caC residue. The first reactive group may then be an amine.

[0014] In all cases, the second reactive group may be a nucleophilic group selected from the group consisting of a thiol or a sulfinate.

[0015] All of the methods disclosed above may further comprise a subsequent step of amplifying the nucleic acid, which may preferably be carried out by PCR amplification. Similarly, all of the methods disclosed above may further comprise a subsequent step of sequencing the nucleic acid, with or without a pre-amplification step, which is preferably PCR amplification. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 provides a scheme for the detection of methylated sequences according to the present invention. [Figure 2] FIG. 2 shows a reaction scheme for the modification of 5fC by Knoevenagel condensation using reactants bearing a CH acidic group. [Figure 3] FIG. 3 shows a reaction scheme for the modification of 5fC by aldol condensation reaction with a CH-acidic reagent containing a sulfinate moiety. [Figure 4] FIG. 4 shows the reaction scheme for the modification of 5fC by the Wittig reaction. [Figure 5] FIG. 5 shows a specific example of the Wittig reaction according to FIG. 4 using a sulfinate-modified Wittig reagent. [Figure 6] FIG. 6 shows the reaction scheme for the modification of 5fC by reductive amination. [Figure 7] FIG. 7 shows a specific example of a reductive amination, as in FIG. 6, in which 5fC is reacted with 2-aminomethylsulfinate. [Figure 8] FIG. 8 shows a reaction scheme for the modification of hmC in which the 2-position of the glucose moiety is replaced with UDP-glucose, and a subsequent β-glucosyltransferase reaction is carried out using nucleophilically substituted UDP-glucose with a reagent that reacts with nucleobases. [Figure 9] FIG. 9 shows a reaction scheme similar to Example 8 in which 2-thioglucose-UDP is used. [Figure 10] FIG. 10 shows an example of modification of the carboxyl group of caC. DETAILED DESCRIPTION OF THE INVENTION

[0017] Abbreviation C-cytosine or cytidine T-thymine or thymidine U-uracil or uridine DHU-Dihydrouracil or Dihydrouridine 5mC-5-methylcytosine or 5-methylcytidine 5hmC-5-hydroxymethylcytosine or 5-hydroxymethylcytidine 5fC-5-formylcytosine or 5-formylcytidine 5caC-5-carboxyl cytosine or 5-carboxyl cytidine dC-2'-deoxycytidine dU-2'-deoxyuridine TET-10-11 translocation dioxygenase TAPS-TET assisted pyridine-borane sequencing CAPS - Chemically Assisted Pyridine-Borane Sequencing

[0018] definition Nucleophilic groups are functional groups that are attracted to electron-deficient or positively charged centers and donate electrons, especially functional groups that donate electron pairs to electrophiles or electrophilic centers to form covalent bonds.Nucleophilic groups are, for example, alcohols, alcoholates, thiols, thiolates, amines, sulfinates, or carbanions.The term deamination herein refers to the substitution reaction of exocyclic amino groups with hydroxyl groups, particularly the substitution of the amino group at C-4 of cytosine nucleobase to convert it to uracil nucleobase.C-4 deamination is enhanced in 5,6-dihydrocytosine to obtain 5,6-dihydrouracil derivatives, for example, as applied after adding bisulfite to the C5-C6 double bond of cytosine.

[0019] The term CH acidic group is used herein for a carbon atom in a compound that has at least one electron-withdrawing group and is therefore more easily deprotonated by base.

[0020] Electron-withdrawing groups are, for example, carbonyl-containing groups, nitro groups, cyano groups, sulfoxide or sulfone groups.

[0021] As used herein, the term Wittig reagent or phosphorus ylide refers to a neutral dipolar molecule containing a formally negatively charged carbon atom (carbanion) directly bonded to a formally positively charged phosphorus atom. In phosphorus ylides (phosphonium ylides), two adjacent carbon atoms and the phosphorus atom are linked by both covalent and ionic bonds. Thus, phosphorus ylides are a subclass of 1,2-dipolar compounds and zwitterions. In the Wittig reaction, triphenylphosphonium ylides react with aldehydes or ketones to obtain alkene bonds. In the Horner-Wadsworth-Emmons reaction, stabilized phosphonate carbanions are used as reagents.

[0022] As used herein, a glycosyltransferase substrate refers to a reagent containing an activating group, such as a UDP moiety, and a sugar residue, such as glucose or a substituted glucose. Glycosyltransferase catalyzes the transfer of a sugar moiety from an activated donor sugar (glycosyltransferase substrate) to a sugar or non-sugar acceptor. The non-sugar acceptor can be, for example, 5-hydroxymethyl-dC. T4 phage β-glucosyltransferase (e.g., from New England Biolabs) specifically transfers the glucose moiety of uridine diphosphoglucose to 5-hydroxymethylcytosine (5-hmC) residues in double-stranded DNA, generating β-glucosyl-5-hydroxymethylcytosine.

[0023] Methods for DNA methylation status analysis by modification and subsequent sequencing The general scheme for detecting methylated C residues (mC) is shown schematically in Figure 1. Without any specific processing, the epigenetic dC modifications 5-methyl-dC (mC), 5-hydroxymethyl-dC (hmC), 5-formyl-dC (fC), and 5-carboxyl-dC (caC) are read as unmodified dC when amplified and / or sequenced. However, oxidation of mC by enzymatic and / or chemical means results in the oxidized mC modifications hmC, fC, and caC (general: xC).

[0024] The oxidation or conversion of 5mC to 5fC can be achieved by using TET enzymes (Ten-Eleven Transfer (TET) Methylcytosine Dioxygenase). The same class of enzymes is suitable for converting 5mC to 5hmC. 5hmC can be further oxidized to 5fC enzymatically by laccase enzymes or chemically using either KRuO or Cu(II) / TEMPO. Further details are disclosed in Pfeifer et al., Epigenetics & Chromatin 2013, 6:10, pp. 1-9.

[0025] Subsequently, 5fC, 5hmC, or caC is reacted with a reagent of the invention containing two reactive groups, where the first reactive group forms a covalent bond with hmC, fC, or caC, thereby introducing the second reactive group containing a nucleophile. The intramolecular addition of the nucleophile to the C5-C6 double bond of hmC, fC, or caC initiates deamination at the C4 position, generating a modified dU derivative that is read as T in subsequent amplification or sequencing steps, thereby enabling detection of the epigenetic modification.

[0026] Such sequencing according to the present invention allows for the generation of data on the methylation status of the DNA of interest. Also described herein is a method for analyzing the methylation status of naturally occurring DNA, comprising: a) converting 5mC to 5fC, 5hmC or 5caC; b) providing a reactant comprising two reactive groups, wherein a first reactive group is capable of reacting with a formyl group, a hydroxymethyl group, or a carboxyl group, and a second reactive group is a nucleophilic group; c) reacting the first reactive group with a formyl, hydroxymethyl, or carboxyl group, thereby obtaining modified 5fC, 5hmC, or 5caC; d) reacting a second reactive group with the C6 position of the modified 5fC, 5hmC, or 5caC, thereby obtaining a bicyclic or tricyclic molecule containing a 5,6-dihydrocytosine entity; and e) deaminating 5,6-dihydrocytosine entities to 5,6-dihydro-uracil entities The present invention provides a method comprising:

[0027] In some embodiments, the sample is derived from a subject or patient. In some embodiments, the sample may include solid tissue or fragments of a solid tumor derived from a subject or patient, for example, by biopsy. The sample may also include bodily fluids that may contain nucleic acids (e.g., urine, sputum, serum, blood or blood fractions, 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 samples). In other embodiments, the sample is a culture sample, e.g., a tissue culture containing cells and fluids from which nucleic acids can be isolated. In some embodiments, the nucleic acid of interest in the sample is derived from an infectious agent, such as a virus, bacterium, protozoan, or fungus. The present invention includes manipulating isolated nucleic acids isolated or extracted from a sample. Nucleic acid extraction methods are well known in the art (see Sambrook et al., and references thereto; Molecular Cloning: A Laboratory Manual, and references thereto; 1989, 2nd Ed., Cold Spring Harbor Laboratory Press: New York, NY). A variety of kits for extracting nucleic acids (DNA or RNA) from biological samples are commercially available, such as KAPA Express Extract (Roche Sequencing Solutions, Pleasanton, Calif.), and other similar products from BD Biosciences Clontech (Palo Alto, Calif.), Epicentre Technologies (Madison, Wis.); Gentra Systems (Minneapolis, Minn.); and Qiagen (Valencia, Calif.), Ambion (Austin, Tex.); BioRad Laboratories (Hercules, Calif.).

[0028] The present invention involves detecting epigenetic modifications, specifically epigenetic cytosine modifications (including, but not limited to, cytosine methylation) in nucleic acids. Nucleic acid sequences that undergo conditional epigenetic modifications are target sequences analyzed by the methods disclosed herein. The same nucleic acid sequence may or may not have an epigenetic modification characterized by cytosine methylation at position 5 (5mC or 5hmC). In some embodiments, a set or panel of target nucleic acids is probed for the presence of methylation. For example, as shown in Patai et al., "Comprehensive DNA Methylation Analysis Reveals a Common Ten-Gene Methylation Signature in Colorectal Adenomas and Carcinomas," PLOS ONE 10(8):e0133836 (2015), and Onwuka et al., "A panel of DNA methylation signature from peripheral blood may predict colorectal cancer susceptibility," BMC Cancer 20,692 (2020), methylation of biomarkers in a panel of methylation biomarkers indicates the presence of colorectal cancer in a patient. Thus, testing of any known or future panel of methylation biomarkers for prognostic or diagnostic purposes is contemplated by the methods disclosed herein. In some embodiments, the entire genome of an organism is probed for the presence of methylation. The methods of the present invention include detecting methylation at all sites throughout an organism's genome to diagnose a disease or condition, or a predisposition to a disease or condition, using sequence analysis and artificial intelligence tools, for example, as described in Shull et al., "Sequencing the cancer methylome," Methods Mol Biol. 1238:627-635 (2015).

[0029] In some embodiments, the method for detecting epigenetic modifications comprises sequencing.The nucleic acid processed as described herein is subjected to sequencing, preferably massively parallel single molecule sequencing.Analyzing individual molecules by massively parallel sequencing typically requires different levels of barcoding for sample identification and error correction.Using molecular barcodes as 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, marking the molecule and its descendants (for example, the original molecule and its amplicon generated by PCR). Unique molecular barcodes (UIDs) have multiple uses, including counting the number of original target molecules in a sample and error correction (Newman et al., "An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage," Nature Medicine doi:10.1038 / nm.3519 (2014)).

[0030] Nanopore sequencing is a unique, scalable technology that enables the direct, real-time analysis of long DNA or RNA fragments. It works by monitoring changes in electrical current as nucleic acids pass through a protein nanopore. The resulting signal is then decoded to obtain specific DNA or RNA sequences. Nanopore-based sequencing technology uses a semiconductor-based electronic detection system to detect the unique electrical signals generated by various molecules as they pass through the nanopore. This technology offers a high-throughput, cost-effective sequencing solution. At the heart of this technology is a biological nanopore—a protein pore embedded in a membrane. The technology's brain lies in the electronic circuitry and unique chemistry of semiconductor integrated circuits. Chip-embedded electronic sensor technology enables automated membrane assembly and nanopore insertion, while allowing active control of individual sensors on the circuit. Pairing various sequencing chemistries with nanopore and electronic sensor technology enables high-throughput, high-precision sequencing with faster data transfer times.

[0031] In some embodiments, unique molecular barcodes (UIDs) are used for error correction in sequencing. All descendants of a single target molecule are labeled with the same barcode, forming a barcoded family. Sequence variations that are not shared by all members of the barcoded family are discarded as artifacts. Because barcodes represent the entire family of a single molecule in the original sample, they can also be used for positional deduplication and target quantification (Newman et al., "Integrated digital error suppression for improved detection of circulating tumor DNA," Nature Biotechnology 34:547 (2016)).

[0032] In some embodiments, the method includes forming a library containing nucleic acids from the sample. The library consists of a plurality of nucleic acids that are ready for sequencing or another type of detection method, such as PCR. The library may be stored and used multiple times for further processing, such as amplification or sequencing of the nucleic acids in the library. In some embodiments, the library is the input nucleic acid whose methylation is detected by the methods described herein. In other embodiments, the library is formed from nucleic acids that have been subjected to a methylation detection reaction described herein.

[0033] In some embodiments, the nucleic acid processed for detecting epigenetic modifications by the methods described herein is sequenced. Any of several sequencing techniques or sequencing assays can be utilized. As used herein, the term "next-generation sequencing (NGS)" refers to a sequencing method that allows for massively parallel sequencing of clonally amplified molecules and single nucleic acid molecules.

[0034] Non-limiting examples of sequence assays suitable for use in the methods disclosed herein include nanopore sequencing (U.S. Patent Application Publication Nos. 2013 / 0244340, 2013 / 0264207, 2014 / 0134616, 2015 / 0119259, 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)), and mass spectrometry sequencing, such as matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS; Fu et al., Nature 1999, 10:103-104 (1999)). Biotech., 16:381-384 (1998)), sequencing by hybridization (Drmanac et al., Nature Biotech., 16:54-58 (1998)), and NGS methods such as, but not limited to, sequencing by synthesis (e.g., HiSeq™, MiSeq™, or Genome Analyzer, each available from Illumina), sequencing by ligation (e.g., SOLiD™, Life Technologies), ion semiconductor sequencing (e.g., Ion Torrent™, Life Technologies), and SMRT™ sequencing (e.g., Pacific Biosciences).

[0035] Commercially available sequencing technologies include the sequencing-by-hybridization platform from Affymetrix Inc. (Sunnyvale, Calif.), the sequencing-by-synthesis platforms from Illumina / Solexa (San Diego, Calif.) and Helicos Biosciences (Cambridge, Mass.), and the sequencing-by-ligation platform from Applied Biosystems (Foster City, Calif.). Other sequencing technologies include, but are not limited to, Ion Torrent technology (ThermoFisher Scientific), and nanopore sequencing (Genia Technology, Roche Sequencing Solutions, Santa Clara, Calif.), Oxford Nanopore Technologies (Oxford, UK), and sequencing by amplification (Stratos Genomics, Roche Sequencing Solutions).

[0036] In some embodiments, the sequencing step includes sequence alignment. In some embodiments, alignment is used to determine a consensus sequence from multiple sequences, for example, multiple sequences with the same unique molecular ID (UID). The molecular ID is a barcode that can be added to each molecule prior to sequencing or, if included, prior to the amplification step. In some embodiments, the UID is present in the 5' portion of the RT primer. Similarly, the UID can be present at the 5' end of the last barcode subunit that is added to the compound barcode. In other embodiments, the UID is present in an adapter and is added to one or both ends of the target nucleic acid by ligation.

[0037] In some embodiments, a consensus sequence is determined from multiple sequences that all share the same UID. Sequences with the same UID are presumed to be derived from the same original molecule by amplification. In other embodiments, UIDs are used to eliminate artifacts, i.e., variations present in the progeny of a single molecule (characterized by a specific UID). Such artifacts resulting from PCR or sequencing errors can be eliminated using UIDs.

[0038] In some embodiments, the number of each sequence in a sample can be quantified by quantifying the relative number of sequences with each UID in a population with the same multiplex sample ID (MID). Because each UID represents a single molecule in the original sample, counting the distinct UIDs associated with each sequence variant can determine the fraction of each sequence variant in the original sample in which all molecules share the same MID. One skilled in the art can determine the number of sequence reads required to determine a consensus sequence. In some embodiments, the relevant number is the reads per UID ("sequence depth") required for accurate quantification. In some embodiments, the desired depth is 5-50 reads per UID.

[0039] 5fC modification Modification of 5fC according to the present invention can be achieved when the first reactive group of the reagent according to the present invention is a CH acid group, an amine or a phosphorus ylide.

[0040] When the first reactive group is a C-H acid group, the reaction can be carried out as a Knoevenagel condensation, as shown schematically in Figure 2. A C-H-acidic reagent containing a nucleophilic group is added to fC under Knoevenagel conditions, followed by intramolecular addition of the nucleophile to the C5-C6 double bond, which allows deamination at C4 to obtain the "T equivalent." The yield of the intramolecular addition of the nucleophile to the C5-C6 double bond can be increased by photoisomerization of the double bond and / or by applying radical initiation or catalysis. If desired, reversal of the ring closure can be achieved under alkaline conditions.

[0041] Alternatively, an aldol condensation reaction of 5fC with a C-H-acidic reagent containing a sulfinate moiety (e.g., 1-(methylsulfonyl)methanesulfinate), followed by the addition of the sulfinate moiety to the C-6 position of the substituted cytosine, followed by deamination and desulfurization (conversion of 5fC to T) can be performed (Figure 3). The desulfurization step under alkaline conditions may not be necessary. Instead of a sulfinate moiety, a thiol moiety can be applied as a nucleophile, and the addition reaction to the C5-C6 double bond can be achieved by applying thiol-ene chemistry using a radical initiator or catalyst. The yield can be increased by photoisomerization of the newly formed double bond.

[0042] Modification of 5fC according to the present invention can also be carried out by the Wittig reaction (Figure 4). In this case, the reagent according to the present invention is a phosphorus ylide containing a nucleophilic group. The reagent is added to 5fC under Wittig conditions, followed by subsequent intramolecular addition of the nucleophile to the C5-C6 double bond, thereby allowing deamination at the C4 position to obtain a "T equivalent."

[0043] Reaction details are shown in Figure 5 for a specific example. In this case, 5fC is reacted with a sulfinate-modified Wittig reagent. This is followed by the addition of a sulfinic acid moiety to the C-6 position of the substituted cytosine, followed by deamination and desulfurization (conversion of 5fC to T). The desulfurization step under alkaline conditions may not always be necessary.

[0044] Alternatively, a thiol moiety may be used in place of the sulfinate moiety by applying thiol-ene chemistry using a radical initiator or catalyst. The yield of the intramolecular addition of a nucleophile to the C5-C6 double bond may be increased by photoisomerization of the newly formed double bond and / or by applying radical initiation or catalyst. If desired, reversal of the ring closure may be achieved under alkaline conditions. Alternatively, the reaction may be carried out using Horner-Wadsworth-Emmons reaction conditions, specifically using a sulfinate group as the nucleophile.

[0045] Yet another alternative is the modification of 5fC by reductive amination, as shown in Figure 6. In this case, a reagent of the present invention containing an amine and an additional nucleophilic group is added to 5fC by reductive amination. This is followed by intramolecular addition of a nucleophile to the C5-C6 double bond, which allows deamination at the C4 position to obtain a "T equivalent." If necessary, reversal of ring closure can be achieved under alkaline conditions. The application of radical initiation or a catalyst can increase yields.

[0046] A more detailed example for performing a reductive amination reaction is shown in Figure 7. 5fC is reacted with 2-aminomethylsulfinate, followed by the addition of a sulfinate moiety at the C-6 position of the substituted cytosine. This is followed by a deamination and desulfurization step to convert 5fC to T. Again, the desulfurization step under alkaline conditions may not be necessary. Alternatively, the sulfinic acid moiety can be replaced with a thiol moiety using thiol-ene chemistry for the addition reaction to the double bond C5-C6, optionally involving the use of a radical initiator or catalyst.

[0047] hmC Modification: Figure 8 shows a schematic reaction scheme for the modification of hmC. In this case, the reagent used by the present invention is UDP-glucose modified with a nucleophilic group at the 2-position. This is added to hmC via a β-glucosyltransferase enzyme. Subsequent intramolecular addition of a nucleophile to the C5-C6 double bond then occurs, allowing deamination at the C4 position to obtain a "T equivalent." As mentioned above, the yield of the intramolecular addition of a nucleophile to the C5-C6 double bond can be increased by applying radical initiation or a catalyst, and, if necessary, reversal of ring closure can be achieved under alkaline conditions.

[0048] An alternative example is shown in Figure 9. In this case, 5hmC is reacted with 2-thioglucose-UDP and β-glucosyltransferase. Thiol-ene chemistry is then applied, optionally using a radical initiator or catalyst, to add a thiol moiety to the C6 position of the substituted cytosine, followed by a deamination reaction, converting 5hmC to T. Alternatively, 2-sulfinato-glucose-UDP can also be applied as a substrate for β-glucosyltransferase.

[0049] caC modification: In this embodiment, shown in Figure 10, the method of the present invention uses a reagent containing an amine and an additional nucleophilic group, which is added after EDC / NHS activation of the carboxyl group of caC. This allows caC to form an amide bond, followed by intramolecular addition of a nucleophile to the C5-C6 double bond, thereby allowing deamination at the C4 position to obtain a "T equivalent." Again, the application of a radical initiator or catalyst can increase the yield of the intramolecular addition of the nucleophile to the C5-C6 double bond, and under alkaline conditions, the reverse cyclization reaction can be achieved.

Claims

1. a) providing a nucleic acid comprising 5fC, 5hmC or 5caC; b) providing a reactant comprising two reactive groups, wherein a first reactive group is capable of reacting with a formyl group, a hydroxymethyl group, or a carboxyl group, and a second reactive group is a nucleophilic group; c) reacting the first reactive group with a formyl group, a hydroxymethyl group, or a carboxyl group, thereby obtaining modified 5fC, 5hmC, or 5caC; d) reacting the second reactive group with the C6 position of the modified 5fC, 5hmC, or 5caC, thereby obtaining a bicyclic or tricyclic molecule containing a 5,6-dihydrocytosine entity; and e) deaminating said 5,6-dihydrocytosine entities to 5,6-dihydro-uracil entities.

2. 10. The method of claim 1, further comprising the step of reversing the ring formation of step d), thereby obtaining a 5-substituted uracil.

3. 3. The method of claim 1 or 2, further comprising, before step a), converting 5mC to 5fC, 5hmC or 5caC.

4. The method of any one of claims 1 to 3, wherein the nucleic acid comprises 5fC.

5. The method of claim 4, wherein the first reactive group is a C—H acidic group, an amine, or a phosphorus ylide.

6. The method of any one of claims 1 to 3, wherein the nucleic acid comprises 5hmC.

7. 7. The method of claim 6, wherein the first reactive group is a glycosyltransferase substrate.

8. The method of any one of claims 1 to 3, wherein the nucleic acid comprises 5caC.

9. The method of claim 8 , wherein the first reactive group is an amine.

10. The method of any one of claims 1 to 9, wherein the second reactive group is a nucleophilic group selected from the group consisting of a thiol or a sulfinate.

11. The method of any one of claims 1 to 10, further comprising the subsequent step of amplifying the nucleic acid.

12. The method of any one of claims 1 to 11, further comprising the subsequent step of sequencing the nucleic acid.

13. A composition comprising nucleic acid processed according to the method of any one of claims 1 to 10.

14. A kit for carrying out the method according to any one of claims 1 to 12.

Images