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, hydroxymethyl or carboxyl group and a second reactive group is a nucleophilic group; c) reacting the first reactive group with a formyl, hydroxymethyl or carboxy 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) subjecting the 5, 6-dihydrocytosine entity to deamination to form a 5, 6-dihydrouracil entity.

Description

Technical Field

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

[0002] Most relevant for identifying epigenetic modifications is detection by sequencing-based methods. The distinction between cytosine (C) and 5-methylcytosine (5mC) is described by direct sequencing of DNA through nanopores (without preamplification) (e.g., by Pacific Bioscience single molecule real-time (SMRT) sequencing technology), thereby reading out the different kinetics of nucleotide incorporation relative to C versus mC by the polymerase.

[0003] Other techniques include using methods that convert C or mC to T(U) equivalents followed by amplification to allow identification when comparing untreated and converted sample DNA (see review: 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 (mC oxidation by TET2 enzyme and glycosylation by β-glucosyltransferase (to block the enzymatic deaminase reaction) and C conversion to U by APOBEC deaminase); the TAPS method (TET-assisted pyridine borane sequencing), which applies TET enzyme to oxidize mC and subsequent reduction of the oxidized mC species by pyridine borane to obtain dihydrocytidine nucleosides that are easily deaminated to give dihydrouracil (T equivalent); and the CLEVER method, which is based on the oxidation of mC by TET enzyme and the subsequent reaction of 5-formyl-C with malononitrile to obtain an adduct that is mainly used as a T equivalent in subsequent PCR amplification (C. Zhu et al., Cell Stem Cell 2017, 20, 720-731). Instead of enzymatic oxidation of mC by TET enzymes, it can also be oxidized by chemical means using, for example, potassium perruthenate (KRuO4).

[0005] There are also methods for distinguishing between modifications of 5-hydroxymethyl-dC (5hmC), 5-formyl (5fC) or 5-carboxyl-C (5caC) by partially modifying the above method.

[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-carboxy-cytosine (5caC) by using a free radical initiator (which can be used together with a nucleophilic compound).

[0007] However, all disclosed methods have some disadvantages. Bisulfite sequencing contains harsh conditions that cause most input DNA to degrade and can only detect methylated cytosine indirectly. Similarly, enzymatic methyl sequencing (EM-Seq) via enzymatic deamination is inefficient because most unmethylated cytosines must be converted. TAPS requires toxic reagents required for reduction, such as pyridine borane, and dihydrouridine is unstable under slightly acidic conditions and is not easily converted back to more stable uridine derivatives. For the CLEVER method, the conversion rate of the condensation reaction of 5fC with malononitrile is much lower than 100%, because when the condensation product of 5fC with malononitrile is used as a substrate, the enzymatic incorporation of nucleotides by the polymerase is not highly accurate, and the incorporation of dA is only favorable, not unique (see also: F.Galardi et al., Biomolecules 2020, 10, 1677). The method of WO2022 / 096751 is not entirely for caC, but also converts unmodified C to a certain extent. Another disadvantage 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 the introduction of a nucleophile at 5fC, 5hmC or caC and subsequent intramolecular addition at C-6 of a 5-substituted cytosine enables deamination at C-4 of a cytosine derivative.

[0009] Therefore, in a first aspect, the present invention relates to a method comprising the following steps:

[0010] a) Providing a nucleic acid containing 5fC, 5hmC or 5caC

[0011] b) providing a reactant comprising two reactive groups, wherein the first reactive group is capable of reacting with a formyl, hydroxymethyl or carboxyl group and the second reactive group is a nucleophilic group,

[0012] c) reacting the first reactive group with a formyl group, a hydroxymethyl group or a carboxyl group, thereby obtaining a modified 5fC, 5hmC or 5caC

[0013] 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

[0014] e) deaminating the 5,6-dihydrocytosine entity to a 5,6-dihydrouracil entity.

[0015] The newly formed cyclic structure in step d) is a 5- to 7-membered ring.

[0016] In one embodiment, the method further comprises step f) of reversing the cyclization of step d) to obtain a 5-substituted uracil.

[0017] Since naturally occurring DNA methylation is 5mC, the method may further include a step of converting 5mC to 5fC, 5hmC or 5caC before step a). Therefore, the present invention facilitates the analysis of the methylation status of naturally occurring DNA.

[0018] In one embodiment, the nucleic acid comprises at least one 5fC residue. In this case, the first reactive group may be a CH acidic group, an amine or a phosphorus ylide. In another embodiment, the nucleic acid comprises at least one 5hmC residue. The first reactive group may then constitute a glycosyltransferase substrate. In another embodiment, the nucleic acid comprises a 5caC residue. The first reactive group may then be an amine.

[0019] In all cases, the second reactive group may be a nucleophilic group selected from the group consisting of thiols or sulfinates.

[0020] All methods disclosed above may further include a subsequent step of amplifying the nucleic acid, which is preferably performed by PCR amplification. Similarly, all methods disclosed above may further include a subsequent step of sequencing the nucleic acid with or without a prior amplification step, which is preferably PCR amplification. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A scheme for detecting methylated sequences according to the present invention is provided.

[0022] Figure 2 Reaction scheme showing the modification of 5fC by Knoevenagel condensation using reactants with CH acidic groups

[0023] Figure 3 Reaction scheme showing the modification of 5fC by aldol condensation reaction with a CH-acidic reagent containing a sulfinate moiety

[0024] Figure 4 A reaction scheme for modifying 5fC by the Wittig reaction is shown.

[0025] Figure 5 Show according to Figure 4 A specific example of a Wittig reaction using a sulfinate-modified Wittig reagent.

[0026] Figure 6 Reaction scheme showing modification of 5fC by reductive amination

[0027] Figure 7 Shown as Figure 6 A specific example of a reductive amination is shown, in which 5fC is reacted with 2-aminomethanesulfinate

[0028] Figure 8 A reaction scheme is shown for modifying hmC using UDP-glucose substituted with a nucleophile at the 2-position of the glucose moiety as a reagent and for the subsequent β-glucosyltransferase reaction.

[0029] Fig. 9 A similar reaction scheme to Example 8 is shown, where 2-thio-glucose-UDP is used.

[0030] Fig.10 Examples of modifications of the carboxyl group of caC are shown. DETAILED DESCRIPTION

[0031] abbreviation

[0032] C – Cytosine or Cytidine

[0033] T – thymine or thymidine

[0034] U – uracil or uridine

[0035] DHU – dihydrouracil or dihydrouridine

[0036] 5mC – 5-methylcytosine or 5-methylcytidine

[0037] 5hmC – 5-hydroxymethylcytosine or 5-hydroxymethylcytidine

[0038] 5fC – 5-formylcytosine or 5-formylcytidine

[0039] 5caC – 5-carboxycytosine or 5-carboxycytidine

[0040] dC – 2'-deoxycytidine

[0041] dU – 2'-deoxyuridine

[0042] TET – 10-11 translocation dioxygenase

[0043] TAPS–TET-assisted pyridine-borane sequencing

[0044] CAPS – Chemically Assisted Pyridine-Borane Sequencing

[0045] definition

[0046] Nucleophilic groups are functional groups that attract electron-deficient or positively charged centers and provide electrons, particularly provide electron pairs to electrophilic reagents 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 reaction in which an exocyclic amino group is replaced by a hydroxyl group, particularly the replacement of the amino group at the C-4 position of a cytosine nucleobase to be converted into a uracil nucleobase. The deamination at the C-4 position in 5,6-dihydrocytosine is enhanced to obtain 5,6-dihydrouracil derivatives, such as applied after bisulfite addition to the C5-C6 double bond of cytosine.

[0047] The term CH acidic group is used herein to denote a carbon atom in a compound which carries at least one electron withdrawing group and is therefore more easily deprotonated by a base. Electron withdrawing groups are, for example, carbonyl-containing groups, nitro groups, cyano groups, sulfoxide or sulfone groups.

[0048] As used herein, the term Wittig reagent or phosphorus ylide refers to a neutral dipole molecule containing a formally negatively charged carbon atom (carbanion) directly attached to a formally positively charged phosphorus atom. In phosphorus ylide (phosphonium ylide), two adjacent carbon atoms and phosphorus atoms are connected by both covalent bonds and ionic bonds. Therefore, phosphorus ylide is a 1,2-dipole compound and a subclass of zwitterions. In the Wittig reaction, triphenylphosphonium ylide reacts with an aldehyde or ketone to obtain an olefinic bond. In the Horner-Wadsworth-Emmons reaction, a stable phosphonic acid carbanion is used as a reagent.

[0049] As used herein, a glycosyltransferase substrate refers to a reagent comprising an activating group (e.g., a UDP moiety) and a sugar residue (e.g., glucose or substituted glucose). Glycosyltransferases catalyze the transfer of a sugar moiety from an activated donor sugar (glycosyltransferase substrate) to a sugar or non-sugar acceptor. The non-sugar acceptor may be, for example, 5-hydroxymethyl-dC. T4 bacteriophage β-glucosyltransferase (e.g., from New England Biolabs) specifically transfers the glucose moiety of uridine diphosphate glucose to 5-hydroxymethylcytosine (5-hmC) residues in double-stranded DNA to obtain β-glucosyl-5-hydroxymethylcytosine.

[0050] Method for analyzing DNA methylation status by modification and subsequent sequencing

[0051] Figure 1Schematic showing a general scheme of how to detect methylated C residues mC. Without any special treatment, 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 oxidized mC modifications hmC, fC, and caC (collectively referred to as xC).

[0052] The oxidation or conversion of 5mC to 5fC can be achieved by using TET enzymes (10-11 translocation (TET) methylcytosine dioxygenase). Similar enzymes are suitable for converting 5mC to 5hmC. 5hmC can be further enzymatically oxidized to 5fC by laccase, or chemically oxidized using KRuO4 or Cu(II) / TEMPO. Further details are disclosed in the following literature: Pfeifer et al., Epigenetics & Chromatin 2013, 6:10, pages 1-9.

[0053] Subsequently, 5fC, 5hmC or caC reacts with a reagent of the present invention comprising two reactive groups, the first reactive group forms a covalent bond with hmC, fC or caC, and thus introduces a second reactive group comprising a nucleophile. By intramolecular addition of a nucleophile to the C5-C6 double bond of hmC, fC or caC, deamination begins at the C4 position to obtain a modified dU derivative, which is read as T in the subsequent amplification or sequencing step, thereby enabling the detection of epigenetic modifications.

[0054] Such sequencing according to the present invention allows the generation of data on the methylation state of the target DNA. Therefore, the present invention also provides a method for analyzing the methylation state of naturally occurring DNA, the method comprising the following steps:

[0055] a) Convert 5mC to 5fC, 5hmC or 5caC

[0056] b) providing a reactant comprising two reactive groups, wherein the first reactive group is capable of reacting with a formyl, hydroxymethyl or carboxyl group and the second reactive group is a nucleophilic group,

[0057] c) reacting the first reactive group with a formyl group, a hydroxymethyl group or a carboxyl group, thereby obtaining a modified 5fC, 5hmC or 5caC,

[0058] 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

[0059] e) deaminating the 5,6-dihydrocytosine entity to a 5,6-dihydrouracil entity.

[0060] In some embodiments, the sample is obtained from a subject or patient. In some embodiments, the sample may include, for example, a fragment of a solid tissue or solid tumor obtained from the subject or patient by biopsy. The sample may also include a body fluid (e.g., urine, sputum, serum, blood or blood components (i.e., plasma), lymph, saliva, sputum, sweat, tears, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cystic fluid, bile, gastric juice, intestinal fluid or fecal sample) that may contain nucleic acid. In other embodiments, the sample is a culture sample, for example, a tissue culture containing cells and liquids from which nucleic acid can be isolated. In some embodiments, the target nucleic acid in the sample is from an infectious agent, such as a virus, bacteria, protozoa or fungus. The present invention relates to an isolated nucleic acid separated or extracted from a sample. The method for nucleic acid extraction is well known in the art (see, Sambrook et al., "Molecular Cloning: A Laboratory Manual", 1989, 2nd edition, Cold Spring Harbor Laboratory Press: New York, NY). 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, Pleasanton, 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.), etc.

[0061] The present invention relates to detecting epigenetic modifications (specifically, epigenetic cytosine modifications in nucleic acids (including but not limited to cytosine methylation)). 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 methylation of cytosine at position 5 (5mC or 5hmC). In some embodiments, a set or group of target nucleic acids is detected for the presence or absence of methylation. For example, methylation of a biomarker in a set of methylation biomarkers indicates that the patient has colorectal cancer, as shown in the following documents: 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). Therefore, it is contemplated that any known or future sets of methylation biomarkers are tested for prognostic or diagnostic purposes using 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 using sequence analysis and artificial intelligence tools such as those described in the following literature to detect methylation in all sites of the entire genome of an organism to diagnose a disease or condition or a predisposition to a disease or condition: Shull et al., "Sequencing the cancer methylome", Methods Mol Biol. 1238: 627-635 (2015).

[0062] In some embodiments, the method for detecting epigenetic modification includes sequencing. The nucleic acid treated as described herein is sequenced; preferably, large-scale parallel single molecule sequencing. Analysis of single molecules by large-scale parallel sequencing usually requires a separate level of barcoding for sample identification and error correction. The use of molecular barcodes, such as described in U.S. Patents 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 mark the molecule and its progeny (e.g., the original molecule and its amplicon generated by PCR). Unique molecular barcodes (UIDs) have multiple uses, including calculating the number and error correction of the original target molecule in the sample (Newman, A. et al., "An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage", Nature Medicine doi: 10.1038 / nm.3519 (2014)).

[0063] Nanopore sequencing is a unique, scalable technology that enables 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 decoded to provide a specific DNA or RNA sequence. Nanopore-based sequencing technology uses semiconductor-based electronic detection systems to detect the unique electrical signals of different molecules as they pass through a nanopore. The technology can provide a high-throughput, cost-effective sequencing solution. The core of the technology is a biological nanopore, a protein pore embedded in a membrane, while the "brains" of the technology lie in the electronics and proprietary chemistry of the semiconductor integrated circuit. The electronic sensor technology embedded in the chip enables automated membrane assembly and nanopore insertion, while allowing active control of individual sensors on the circuit. Different sequencing chemistries can be used in conjunction with nanopore and electronic sensor technology to achieve high-throughput, high-accuracy sequencing and faster data acquisition time.

[0064] In certain embodiments, unique molecular barcodes (UIDs) are used for sequencing error correction. The entire progeny of a single target molecule are labeled with the same barcode and form a barcode family. The variation in the sequence not shared by all members of the barcoded family is discarded as an artifact. Barcodes can also be used for positional deduplication and target quantification, because the entire family represents a single molecule in the original sample (Newman, A. et al., "Integrated digital error suppression for improved detection of circulating tumor DNA", Nature Biotechnology 34: 547 (2016)).

[0065] In some embodiments, the method involves forming a library comprising nucleic acids from a sample. The library consists of a plurality of nucleic acids ready for sequencing or another type of detection method (e.g., PCR). The library can 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 an input nucleic acid, wherein methylation is detected by the methods described herein. In other embodiments, the library is formed by nucleic acids that have undergone methylation detection reactions described herein.

[0066] In some embodiments, nucleic acids processed according to the methods described herein for detecting epigenetic modifications are sequenced. Any of a variety of sequencing techniques or sequencing analyses can be used. As used herein, the term "Next Generation Sequencing (NGS)" refers to a sequencing method that allows for large-scale parallel sequencing of molecules and single nucleic acid molecules that are cloned and amplified.

[0067] Non-limiting examples of sequence determination suitable for use with the methods disclosed herein include nanopore sequencing (U.S. Patent Publication Nos. US2013 / 0244340, US2013 / 0264207, US2014 / 0134616, US2015 / 0119259, and US2015 / 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)), hybridization sequencing (Drmanac et al., Nature Biotech., 16:381-384 (1998)), and sequencing by PCR (Drmanac et al., Nature Biotech., 16:382-384 (1998)). Biotech., 16:54-58 (1998)) and NGS methods, including but not limited to sequencing by synthesis (e.g., HiSeq TM 、MiSeq TM or GenomeAnalyzer, both available from Illumina), sequencing by ligation (e.g., SOLiD TM , Life Technologies), ion semiconductor sequencing (e.g., Ion Torrent TM , Life Technologies) and Sequencing (eg, Pacific Biosciences).

[0068] Commercially available sequencing technologies include the hybridization sequencing platform of Affymetrix Inc. (Sunnyvale, Calif.), the sequencing-by-synthesis platform of Illumina / Solexa (San Diego, Calif.) and Helicos Biosciences (Cambridge, Mass.), and the sequencing-by-ligation platform of 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, CA), Oxford Nanopore Technologies (Oxford, UK), and sequencing by expansion (Stratos Genomics, Roche Sequencing Solutions).

[0069] In some embodiments, the sequencing step involves sequence alignment. In some embodiments, alignment is used to determine a consensus sequence from multiple sequences (e.g., multiple sequences with the same unique molecule ID (UID)). The molecule ID is a barcode that can be added to each molecule prior to sequencing, or if an amplification step is included, it is added to each molecule prior to the amplification step. In some embodiments, the UID is present in the 5' portion of the RT primer. Similarly, the UID can appear at the 5' end of the last barcode subunit to be added to the composite 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.

[0070] In some embodiments, the consensus sequence is determined from multiple sequences that all have the same UID. It is inferred that the sequences with the same UID are derived from the same original molecule by amplification. In other embodiments, the UID is used to eliminate artifacts, that is, variations (characterized by a specific UID) present in single molecule offspring. Such artifacts derived from PCR errors or sequencing errors can be eliminated using UIDs.

[0071] In some embodiments, the number of each sequence in the sample can be quantified by quantifying the relative number of sequences of each UID in a population with the same multiple sample ID (MID). Each UID represents a single molecule in the original sample, and counting the different UIDs associated with each sequence variation can determine the proportion of each sequence variation in the original sample, where all molecules share the same MID. Those skilled in the art will be able to determine the number of sequence reads necessary for determining a consensus sequence. In some embodiments, for accurate quantitative results, each UID ("sequence depth") needs to read a related number. In some embodiments, the desired depth is 5-50 reads per UID.

[0072] Modification of 5fC

[0073] The modification of 5fC according to the invention can be achieved if the first reactive group of the reagent according to the invention is a CH acidic group, an amine or a phosphorus ylide.

[0074] If the first reactive group is a CH acidic group, the reaction can be performed as a Knoevenagel condensation, such as Figure 2 Schematically depicted in . A CH-acidic reagent containing a nucleophilic group is added to fC via Knoevenagel conditions, and then an intramolecular addition of a nucleophilic reagent to the C5-C6 double bond occurs, thereby deaminating C4 to obtain a "T-equivalent". The yield of the intramolecular addition of a nucleophilic reagent to the C5-C6 double bond can be increased by photoisomerization of the double bond and / or by the use of free radical initiation or catalysts. If desired, reversal of the cyclization can be achieved under alkaline conditions.

[0075] Alternatively, 5fC is subjected to an aldol condensation reaction with a CH-acidic reagent containing a sulfinate moiety (e.g., 1-(methylsulfonyl)methanesulfinate), followed by addition of the sulfinate moiety at the C-6 position of the substituted cytosine, and then deamination and desulfination (5fC to T conversion) can be performed ( Figure 3 ). Even the desulfinylation step under alkaline conditions may not be required. Instead of the sulfinate moiety, the thiol moiety can also be used as a nucleophile and the addition reaction of the C5-C6 double bond can be achieved by thiol-ene chemistry using free radical initiators or catalysts. The yield can be improved by photoisomerization of the newly formed double bond.

[0076] The modification of 5fC according to the present invention can also be accomplished by the Wittig reaction ( Figure 4 ). In this case, the reagent according to the invention is a phosphorus ylide comprising a nucleophilic group. This reagent is added to 5fC via Wittig conditions, and then a subsequent intramolecular addition of a nucleophile to the C5-C6 double bond occurs, thereby deaminating the C4 position, so as to obtain a "T-equivalent".

[0077] Figure 5 In the specific example, reaction details are shown. In this case, 5fC is reacted with the Wittig reagent modified with sulfinate. Subsequently, sulfinate moiety is added at the C-6 position of the substituted cytosine, and then deamination and desulfinylation (5fC to T conversion) are carried out. The desulfinylation step under alkaline conditions is not necessarily required. Alternatively, instead of the sulfinate moiety, the thiol moiety can also be used by applying thiol-ene chemistry, using free radical initiators or catalysts. The yield of the nucleophilic agent intramolecular addition to the C5-C6 double bond can be improved by the photoisomerization of the newly formed double bond and / or by applying free radical initiation or catalysts. If necessary, the reversal of cyclization can be achieved under alkaline conditions. Alternatively, the reaction can be carried out using Honer-Wadsworth-Emmons reaction conditions, particularly using sulfinate groups as nucleophilic agents.

[0078] Yet another alternative is to modify 5fC by reductive amination, such as Figure 6 As shown. In this case, the reagent of the invention comprising an amine and an additional nucleophilic group is added to 5fC via reductive amination. Subsequently, the nucleophile is intramolecularly added to the C5-C6 double bond, which deaminates the C4 position to obtain a "T-equivalent". If desired, the reversal of the cyclization can be achieved under alkaline conditions. The yield can be improved by using free radical initiation or catalysts.

[0079] A more detailed example for carrying out the reductive amination reaction is Figure 7 As shown. 5fC is reacted with 2-aminomethylsulfinate, and then the sulfinate moiety is added to the C-6 position of the substituted cytosine. This is followed by a deamination and desulfinylation step, which converts 5fC to T. Likewise, a desulfinylation step under alkaline conditions may not be required. Likewise, the sulfinate moiety can be replaced by a thiol moiety using thiol-ene chemistry for addition reaction to the double bond C5-C6, which optionally includes the use of a free radical initiator or catalyst.

[0080] Modification of hmC:

[0081] Figure 8 The reaction scheme for hmC modification is schematically shown. In this case, the reagent used according to the invention is UDP-glucose, which is modified with a nucleophilic group at the 2 position. It is added to hmC via β-glucosyltransferase. Then, subsequent intramolecular addition of the nucleophile to the C5-C6 double bond occurs, thereby deaminating the C4 position to obtain a "T-equivalent". As mentioned above, if necessary, the yield of intramolecular addition of the nucleophile to the C5-C6 double bond can be increased by applying free radical initiation or catalysts, and reversal of the cyclization can be achieved under alkaline conditions.

[0082] An alternative example is shown in Fig. 9 In this case, 5hmC is reacted with 2-thioglucose-UDP and β-glucosyltransferase. Subsequently, applying thiol-ene chemistry and optionally using a free radical initiator or catalyst, a thiol moiety is added at the C6 position of the substituted cytosine, followed by a deamination reaction, resulting in the conversion of 5hmC to T. Alternatively, 2-sulfinyl-glucose-UDP can also be used as a substrate for β-glucosyltransferase.

[0083] Modification of caC:

[0084] for Fig.10 In the embodiment shown, the method of the present invention uses a reagent comprising an amine and an additional nucleophilic group. It is added after the carboxyl group of caC is activated by EDC / NHS. This leads to the formation of an amide bond of caC, and the subsequent intramolecular addition of the nucleophilic reagent to the C5-C6 double bond, thereby deaminating the C4 position to obtain a "T-equivalent". Similarly, the yield of the intramolecular addition of the nucleophilic reagent to the C5-C6 double bond can be increased by applying free radical initiation or catalysts, and the reversal of the cyclization can be achieved under alkaline conditions.

Claims

1. A method comprising: a) providing a nucleic acid comprising 5fC, 5hmC or 5caC, b) providing a reactant comprising two reactive groups, wherein the first reactive group is capable of reacting with a formyl, hydroxymethyl or carboxyl group and the 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 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-dihydrouracil entity.

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

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

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

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

6. The method of claims 1 to 3, wherein the nucleic acid comprises 5hmC. The method of claim 6 , wherein the first reactive group is a glycosyltransferase substrate.

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

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

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

13. A composition comprising nucleic acid treated according to the method of claims 1 to 10.

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

Images