Click chemistry-based enrichment and mapping of 5-carboxylcytosine (5caC) in DNA

Abstract

The application discloses a method for enriching and positioning 5-carboxyl cytosine (5caC) in DNA based on click chemistry, and belongs to the technical field of chemistry. The method comprises the following steps: firstly, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBT) are used to catalyze the dehydration condensation of 5caC and propargylamine to form 5-propargyl amino cytosine (5caC-PA); then, the 5caC-PA is further subjected to CuAAC click chemistry reaction with biotin-polyethylene glycol-azide (Biotin-PEG3-N3) under the catalysis of copper (II) -TBTA complex; finally, the biotin group is modified on the 5caC. Finally, the 5caC modified DNA fragment can be efficiently enriched through streptavidin affinity purification, and is used for subsequent high-throughput sequencing analysis, so that the modification spectrum of 5caC is obtained.

Description

Technical Field

[0001] This invention relates to the field of chemical technology, specifically to a method for enrichment and localization analysis of 5-carboxycytosine (5caC) in DNA based on click chemistry. Background Technology

[0002] The DNA sequence, composed of guanine (G), cytosine (C), adenine (A), and thymine (T), constitutes the genetic information of most life on Earth. Chemical modifications to DNA do not alter its basic sequence, but can regulate the expression of genetic information at new levels. 5-methylcytosine (5mC), the earliest discovered base, is the most common and widely studied modified base in vertebrate genomes. 5mC primarily occurs on CpG dinucleotides and is crucial for gene expression regulation in mammals. Produced by DNA methyltransferases (DNMTs), 5mC participates in regulating genomic imprinting, X chromosome inactivation, transposon silencing, and embryonic development, and is considered the fifth base. During development and differentiation, DNA methylation patterns recombine, either through replication-dependent passive dilution of 5mC or through active erasure of this epigenetic marker from DNA. In recent years, the mechanism of active DNA demethylation has been extensively studied. 5mC in DNA can be oxidized by the dioxygenase TET protein to 5-hydroxymethylcytosine (5hmC), 5-aldehyde cytosine (5fC), and 5-carboxycytosine (5caC). Subsequently, 5fC / 5caC is cleaved by thymine DNA glycosidase, and the resulting abase-free sites are repaired by base excision repair (BER). Therefore, oxidized derivatives of 5mC can serve as markers of active DNA demethylation. Furthermore, numerous indications suggest that, despite their extremely low concentrations, both 5fC and 5caC are involved in transcription and chromosomal localization, and are considered functional epigenetic modifications in mammals.

[0003] To investigate the function of 5mC and its oxidation derivatives, localization analysis is required, and the detection and analysis of cytosine modifications has always been a challenge. For the extremely low abundance of 5caC modification, current sequencing methods mainly fall into two categories: antibody-enrichment sequencing and chemical or enzymatic deamination sequencing. Antibody-enrichment methods are simple and rapid, suitable for sequencing low-abundance modifications. However, the specificity of 5caC antibodies limits the widespread application of this method. Traditional bisulfite sequencing (BS-seq) has long been the gold standard for 5mC sequencing. In BS-seq, cytosine is deamination to uracil after bisulfite treatment, but 5mC resists deamination, thus allowing sequencing to distinguish C and 5mC. Based on this, chemical modification-assisted bisulfite sequencing (CAB-seq) has been developed for the localization analysis of 5caC in DNA. In CAB-seq, 5caC reacts with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and the resulting product is resistant to deamination by bisulfite. Therefore, by comparing the results of BS-seq and CAB-seq, the location information of 5caC can be obtained. However, bisulfite conversion requires stringent chemical deamination conditions, resulting in up to 99% DNA degradation. Therefore, it has been proposed to use the cytosine deaminase APOBEC3A (A3A) for enzymatic deamination. A3A can efficiently deaminate other cytosines and their modifications (5mC, 5hmC, and 5fC) besides 5caC. 5caC, due to its large steric hindrance, is resistant to deamination by A3A, and based on this, 5caC in DNA can be distinguished (AMD-seq). In addition, some researchers have used pyridineboronic acid for localization analysis of 5caC (PS-seq, PS-c-seq). Pyridineboronic acid can convert 5fC and 5caC to dihydrouracil (DHU), while C, 5mC, and 5hmC do not react. Combined with hydroxylamine-labeled 5fC, the deamination of 5fC is inhibited, enabling localization analysis of 5caC. However, the above-mentioned chemical and enzymatic deamination-based methods cannot achieve the enrichment of 5caC. For 5caC with an abundance of 0.1–1 ppm, accurate localization analysis remains a significant challenge.

[0004] Therefore, this invention proposes a chemical labeling method for the enrichment and localization analysis of 5caC. This invention utilizes EDC to catalyze the dehydration condensation of 5caC with propargylamine, and then uses click chemistry to label 5caC with biotin. By utilizing the strong affinity of biotin for streptavidin, efficient enrichment of 5caC is achieved, which can then be used for subsequent sequencing analysis. Summary of the Invention

[0005] The purpose of this invention is to provide a method for enrichment and localization analysis of 5caC in DNA based on click chemistry, namely, a 5caC localization analysis method that does not rely on antibody enrichment, does not require bisulfite treatment, is highly selective, and is simple to operate.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A click chemistry-based method for the enrichment and localization analysis of 5caC in DNA, comprising the following steps:

[0008] (1) 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBT) are used to catalyze the dehydration condensation of 5caC in the DNA to be tested with propargylamine to form 5-propargylaminocytosine (5caC-PA).

[0009] (2) Recover 5caC-PA labeled DNA;

[0010] (3) The copper(II)-TBTA complex catalyzes a click chemical reaction between 5caC-PA and biotin-polyethylene glycol-azide (Biotin-PEG3-N3) to label 5caC-PA with biotin.

[0011] (4) Recover biotin-labeled DNA;

[0012] (5) Using streptavidin magnetic beads to affinity enrich biotin-labeled DNA containing 5caC modification;

[0013] (6) The magnetically captured DNA was used to construct a library and perform high-throughput sequencing analysis to draw a 5caC modification spectrum.

[0014] In step (1), the reaction conditions for DNA with propargylamine are as follows: the DNA to be tested is reacted at 40-60°C for 1.5-3 hours in a reaction system containing 10-50 mM of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 1-10 mM of 1-hydroxybenzotriazole (HOBT), 50-150 mM of 2-(N-morpholine)ethanesulfonic acid (MES), pH 4.0-5.5, and 5-20 mM of propargylamine.

[0015] In step (3), the biotin-labeled click chemistry reaction conditions of 5caC-PA are as follows: the DNA labeled with propargylamine is reacted at 30-45°C for 0.5-4 hours in a reaction system of 50-200 mM triethylamine acetic acid, pH 6.0-8.0, 3-6 M dimethyl sulfoxide, 0.2-2 mM ascorbic acid, 0.05-0.5 mM biotin-PEG3-N3, and 0.5-5 mM copper(II)-TBTA complex (Cu(II)-TBTA).

[0016] In step (5), the method for affinity enrichment of biotinylated DNA is as follows: streptavidin magnetic beads are used to bind biotinylated DNA, and the DNA bound to the streptavidin magnetic beads is separated by magnetic capture.

[0017] In step (6), the magnetically captured DNA to be tested is used to construct the experimental group DNA library; at the same time, the DNA recovered in step (4) is used as the control DNA to construct the control group DNA library. The experimental group and control group libraries are subjected to high-throughput sequencing to draw 5caC modification patterns.

[0018] The principle of the method of this invention is as follows: Figure 1 and Figure 2 First, 5caC and propargylamine were dehydrated and condensed using EDC and HOBT catalysis to form 5-propargylaminocytosine (5caC-PA). The latter then underwent a CuAAC click chemistry reaction with biotin-polyethylene glycol-azide (Biotin-PEG3-N3) under the catalysis of a copper(II)-TBTA complex, ultimately modifying 5caC with a biotin group. Finally, the 5caC-modified DNA fragments were efficiently enriched through streptavidin affinity purification and used for subsequent high-throughput sequencing analysis, thus obtaining the 5caC modification profile.

[0019] The method of this invention has high labeling efficiency and strong specificity, does not require bisulfite treatment, and can efficiently enrich 5caC for localization analysis of 5caC in DNA.

[0020] The advantages and beneficial effects of this invention are as follows:

[0021] 1. This invention does not require bisulfite treatment, the reaction conditions are mild, and large-scale DNA degradation is avoided.

[0022] 2. The two-step labeling reaction involved in the method of the present invention has a high conversion rate. The first step, the EDC catalytic condensation reaction, has an efficiency of 97.5%, and the second step, the click chemistry reaction, has a conversion rate of 99.6%, which is beneficial for the comprehensive analysis of 5caC.

[0023] 3. The chemical labeling method of the present invention has high specificity, only labeling 5caC and not reacting with normal cytosine or other cytosine modifications (such as 5mC, 5hmC and 5fC).

[0024] 4. The method of this invention can achieve efficient labeling of both single-stranded and double-stranded DNA, with a labeling efficiency of >97%.

[0025] 5. The method of this invention has high enrichment efficiency for 5caC, which is beneficial for the localization analysis of 5caC with extremely low abundance.

[0026] 6. The method of this invention can be widely applied to the localization analysis of 5caC modification in DNA, which helps to conduct more in-depth research on the biological functions of 5caC. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the method of the present invention. EDC and HOBT catalyze the dehydration condensation of 5caC in DNA with propargylamine to form 5-propargylaminocytosine (5caC-PA). The latter undergoes a CuAAC click chemistry reaction, ultimately modifying 5caC with a biotin group. Through streptavidin affinity purification, 5caC-modified DNA fragments can be enriched and used for subsequent high-throughput sequencing analysis, thereby obtaining the 5caC modification profile.

[0028] Figure 2 The reaction formula for biotin labeling of 5caC.

[0029] Figure 3 This is a high-resolution mass spectrum of 5-propargylcytosine (5caC-PA) from Example 1.

[0030] Figure 4 This is a high-resolution mass spectrum of biotin-labeled 5-caC (5caC-biotin) in Example 1.

[0031] Figure 5 The image shows the extracted ion spectra of 5 CaC before and after labeling with propargylamine, as detected by LC-MS / MS in Example 1.

[0032] Figure 6 The extracted ion spectra of the intermediate product 5caC-PA and the final product 5caC-biotin were obtained by LC-MS / MS detection in Example 1.

[0033] Figure 7 The labeling efficiency of DNA strands containing different pyrimidine modifications in Example 1.

[0034] Figure 8 The recovery rate of DNA strands containing different pyrimidine modifications in Example 1 after enrichment.

[0035] Figure 9 The enrichment factor is the DNA strand containing different pyrimidine modifications in Example 1.

[0036] Figure 10 This is a flowchart illustrating the localization analysis of 5caC in genomic DNA using the method of this invention.

[0037] Figure 11 This is the quality control result of 5caC enrichment and high-throughput sequencing of mouse embryonic stem cell (mESC) DNA using the method of the present invention in Example 2. Input_1 and Input_2 are the control library before enrichment, and IP_1 and IP_2 are the experimental library after enrichment. Raw reads: Number of raw sequencing sequences. Valid reads: Number of reads retained after filtering. Read validity rate: Percentage of filtered bases relative to the original bases. Match rate: Percentage of valid reads that match the reference sequence. Q20 ratio: Percentage of bases with a Phred value greater than 20 in the filtered data. Q30 ratio: Percentage of bases with a Phred value greater than 30 in the filtered data.

[0038] Figure 12 This document describes the enrichment and high-throughput sequencing of 5caC in mESC cell DNA using the method of the present invention in Example 2, and the statistical distribution of 5caC in gene functional regions. The results show that 5caC is mainly distributed in near-gene promoter regions (≤1kb), introns, and distant intergenic sequences.

[0039] Figure 13 This image shows the distribution of 5caC in the gene matrix and its upstream and downstream regions during high-throughput sequencing of mESC cell DNA using the method of this invention in Example 2. The top image represents the experimental group, and the bottom image represents the control group. The results indicate that 5caC is enriched in the gene initiation region.

[0040] Figure 14 The figure shows the distribution statistics of 5caC in the transcription start site (TSS) and its upstream and downstream regions in mESC cell DNA enrichment and high-throughput sequencing using the method of the present invention in Example 2. The results of the experimental group and the control group are shown in the figure. The results show that 5caC is mainly enriched in the TSS site and 150 bp downstream of it.

[0041] Figure 15 Example 2 shows the enrichment and high-throughput sequencing of 5caC in mESC cell DNA using the method of the present invention, and the distribution of 5caC in TSS and its upstream and downstream.

[0042] Figure 16This is Example 2, which describes the distribution of 5caC in the mouse genome by enriching and high-throughput sequencing of 5caC in mESC cell DNA using the method of the present invention. Detailed Implementation

[0043] In some embodiments, the present invention is based on a click chemistry-based method for the enrichment and localization analysis of 5caC in DNA, comprising the following steps:

[0044] (1) Prepare a high concentration of MES buffer: 750mM MES, pH 4.0~5.5.

[0045] (2) Prepare a high concentration of TEAA buffer: 1M triethylamine acetic acid, pH 6.0-8.0.

[0046] (3) Add EDC to DNA sample (10-100 μg) to a final concentration of 10-50 mM, HOBT to a final concentration of 1-10 mM, add high concentration MES buffer from step (1) to a final concentration of 50-150 mM, pH 4.0-5.5, add propargylamine (Bidepharmatech) to a final concentration of 5-20 mM, add water to 100 μL and react at 40-60 °C for 1.5-3 hours.

[0047] (4) Add ammonium acetate to the DNA obtained in step (3) to a final concentration of 20-100 mM, 5-10 μg of glycogen, 800-900 μL of ethanol, add water to a total volume of 1 mL, place at -60--80℃ for 8-16 hours, centrifuge at 12000-13000 rpm for 20-40 minutes at 4-10℃, discard the supernatant, and wash the precipitate twice with 80% ethanol.

[0048] (5) Add the high-concentration triethylamine acetate buffer from step (2) to the DNA recovered in step (4) to a final concentration of 50-200 mM, pH 6.0-8.0, 3-6 M dimethyl sulfoxide, 0.2-2 mM ascorbic acid, 0.05-0.5 mM biotin-PEG3-N3, and 0.5-5 mM copper(II)-TBTA complex (Cu(II)-TBTA, Aladdin), and react at 30-45°C for 0.5-4 hours.

[0049] (6) The DNA after the biotin-labeled reaction is recovered using the method described in step (4).

[0050] (7) Biotin-labeled DNA containing 5caC was enriched by affinity using streptavidin magnetic beads (PuriMag Biotech), and DNA bound to streptavidin magnetic beads was separated by magnetic capture.

[0051] (8) Construct an experimental DNA library from the magnetically captured DNA samples; simultaneously, construct a control DNA library from the DNA samples recovered in step (6) as control DNA. Perform high-throughput sequencing on the experimental and control libraries to plot 5caC modification patterns.

[0052] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

[0053] Unless otherwise specified, the techniques used in the examples (including ethanol precipitation to recover DNA, DNA enzymatic hydrolysis into nucleosides, and polymerase chain reaction, etc.) are conventional techniques well known to those skilled in the art. The techniques involved in the examples using commercially available kits (such as DNA extraction, streptavidin affinity separation, and DNA library preparation, etc.) all adopt the operating methods recommended by the reagent vendors.

[0054] Example 1. Evaluation of 5caC labeling and enrichment efficiency using artificially synthesized DNA strands

[0055] A. Identification of 5caC-labeled products using LC-HRMS

[0056] To identify the 5caC-labeled product, an 80-nt DNA strand modified with 5caC was synthesized. The 80-nt-caC sequence is: 5'-AGATGGTGATGGTAGTTATGAGGAAAGAGTGGAGTGTAGATATCA 5ca C GTCGTAACGTCATACAATAGGGTGTTGGGGGGAG-3' (SEQ ID NO. 1).

[0057] (1) The total reaction volume was 100 μL, containing 2 μL of 5 μM 80nt-caC, 10 mM propargylamine (Bidepharmatech), 20 mM EDC (MackLin), 2 mM HOBT (Aladdin), and 75 mM MES buffer, pH 5.0, and reacted at 37 °C for 4 hours. After the reaction, 38 μL of water, 70 μL of 7.5 M sodium acetate, 2 μL of 5 mg / mL glycogen (Sangon Biotech), and 840 μL of anhydrous ethanol were added. The mixture was incubated at -80 °C for 12 hours, centrifuged at 12,000 rpm for 30 minutes at 4 °C, the supernatant was discarded, the precipitate was washed twice with 80% ethanol, and the recovered DNA was dissolved in 20 μL of water.

[0058] (2) Take 10 μL of the DNA recovered in step A(1) and digest it into nucleosides. Identify the 5caC-labeled products using liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS). Figure 3 As shown, the precise molecular weight (309.1205) of the reaction product 5caC-propargylamine is consistent with the theoretical value (309.1193), indicating that the reaction occurred successfully.

[0059] (3) The remaining 10 μL of DNA from step A(1) was used for the following reaction. The total reaction volume was 40 μL, containing 10 μL of DNA, 100 mM TEAA, pH 7.0, 16 μL DMSO, 1 mM Biotin-PEG3-N3 (Aladdin), 0.5 mM ascorbic acid (Aladdin), and 0.5 mM Cu(II)-TBTA (Aladdin). The reaction was maintained at 37 °C for 2 hours. After the reaction was completed, 98 μL of water, 70 μL of 7.5 M sodium acetate, 2 μL of 5 mg / mL glycogen (Sangon Biotech), and 840 μL of anhydrous ethanol were added. The mixture was placed at -80 °C for 12 hours, centrifuged at 12,000 rpm for 30 minutes at 4 °C, the supernatant was discarded, the precipitate was washed twice with 80% ethanol, and the recovered DNA was dissolved in 20 μL of water.

[0060] (4) The DNA recovered in step A(3) was enzymatically digested into nucleosides, and the biotinylated 5caC-labeled product was identified using liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS). For example... Figure 4 As shown, the precise molecular weight (753.3382) of the reaction product 5caC-PA and biotin azide, 5caC-biotin, is consistent with the theoretical value (753.3348), indicating that the reaction occurred successfully.

[0061] B. Examining the 5caC labeling efficiency using LC-MS / MS

[0062] To investigate the labeling efficiency of 5caC, 80nt-caC from step A was selected for propargylamine labeling and click chemistry reaction. The reaction conditions were the same as in step A. The nucleosides after DNA digestion were analyzed by LC-MS / MS. Multiple reaction monitoring (MRM) was used in positive ion mode to detect the substrate 5caC (m / z 292.2>156.1), the intermediate 5caC-PA (m / z 309.2>193.1), and the final product 5caC-biotin (m / z 753.4>637.3). Figure 5-6 As shown, 2.5% remained after the 5CaC reaction, indicating that the conversion rate of the first-step EDC-catalyzed dehydration condensation reaction reached 97.5%. Figure 6As shown, the conversion rate of the second click chemical reaction is as high as 99.6%, indicating that the reaction can be used for efficient labeling of 5caC.

[0063] C. The labeling specificity of 5caC was examined using LC-MS / MS.

[0064] To evaluate the specificity of the labeling reaction, unmodified DNA strands with cytosine, 5mC, 5hmC, 5fC, and 5caC modifications were selected for the labeling reaction, and the reaction conditions were the same as in step A. The synthesized 304bp Oligo-C sequence is shown in SEQ ID NO.2; the synthesized 237bp Oligo-5mC sequence is shown in SEQ ID NO.3; the synthesized 382bp Oligo-5hmC sequence is shown in SEQ ID NO.4; the synthesized 393bp Oligo-5fC sequence is shown in SEQ ID NO.5; and the synthesized 258bp Oligo-5caC sequence is shown in SEQ ID NO.6.

[0065]

[0066]

[0067] The DNA after the reaction was digested into nucleosides, and then analyzed by LC-MS / MS. The conversion rate was calculated by detecting the contents of C, 5mC, 5hmC, 5fC, and 5caC before and after the reaction. The results showed that only the 5caC content decreased significantly after the labeling reaction, and the conversion rate reached 97%, while the contents of other modifications did not change significantly, indicating that the labeling reaction only occurred at 5caC (…). Figure 7 Furthermore, the effect of single-stranded or double-stranded DNA on the labeling reaction was investigated. The results showed that 5caC in both single-stranded and double-stranded DNA could be efficiently labeled. Figure 7 ).

[0068] D. Use quantitative polymerase chain reaction (qPCR) to investigate the enrichment efficiency of biotin-labeled 5caC.

[0069] The DNA strands modified with different pyrimidines in step C were used as substrates for biotin labeling reaction and enrichment analysis.

[0070] (1) Mix 200 ng Oligo-C, 10 ng Oligo-5mC, 10 ng Oligo-5hmC, 10 ng Oligo-5fC, and 10 ng Oligo-5caC, and perform a two-step labeling reaction according to the labeling method in step A of this embodiment. Divide the reacted DNA into two parts, one part as a control (volume adjusted to 40 μL), and the other part for streptomycin magnetic bead enrichment.

[0071] (2) Affinity enrichment: DNA was dissolved in 10 μL of water, and 10 μL of streptomycin magnetic beads (Premig) and 20 μL of binding buffer (100 mM Tris-HCl, pH 8.0, 200 mM NaCl) were added. After mixing well, the mixture was incubated at room temperature for 15 minutes. Magnetic adsorption occurred, and the supernatant was discarded. The mixture was washed three times with 1 mL of washing buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl). Finally, the magnetic beads were evenly dispersed in 40 μL of water.

[0072] (3) DNA before and after enrichment was analyzed by qPCR. The amplification system included:

[0073]

[0074] For DNA strands with different modifications, qPCR was performed using different primers. The primer sequences are as follows:

[0075]

[0076]

[0077] The amplification procedure is as follows:

[0078]

[0079] (4) Results Analysis. The relative content of different modified DNA strands was calculated based on the cycle number of qPCR. The results showed that the recovery rate of streptomycin magnetic beads combined with biotin-labeled 5caC modified DNA strands reached 80%, and the enrichment factor for Oligo-5caC strands was 180, while other modified and unmodified DNA strands showed almost no enrichment effect. Figure 8 and Figure 9 ).

[0080] In summary, the click chemistry-based labeling method of this invention can react with 5caC with high conversion rate and high selectivity, and can efficiently enrich and sequence 5caC in actual sample DNA using streptomycin magnetic beads.

[0081] Example 2. Localization analysis of 5caC in mouse embryonic stem cell genomic DNA

[0082] To demonstrate the application of the method of the present invention in actual samples, chemical labeling, enrichment and sequencing analysis of 5caC in mouse embryonic stem cell (mESC) genomic DNA were performed, specifically including the following steps (10):

[0083] A. Culture mESCs, harvest cells, and extract DNA using a tissue DNA extraction kit (Omega).

[0084] B. Use an ultrasonic homogenizer (Scientz) to segment genomic DNA into fragments with an average size of around 300 bp.

[0085] C. Then, using the method in Example 1, 5caC was first labeled with propargylamine, and then biotin was labeled onto 5caC via a CuAAC click chemical reaction. After enrichment with streptavidin magnetic beads, a library was constructed using a library construction kit (Yeason). A control library was constructed from biotin-labeled DNA that had not been enriched with streptomycin, and an experimental library was constructed from biotin-labeled DNA enriched with streptomycin. Finally, the control and experimental libraries were analyzed using Illumina high-throughput sequencing (Novogene) to obtain the sequencing data of 5caC. Figure 10 ).

[0086] High-throughput sequencing was performed on the control libraries (Input_1, Input_2) and experimental libraries (IP_1, IP_2). 24,757,062–36,163,912 raw reads were obtained from the four libraries, with a valid read rate greater than 88.7% and a matching rate greater than 89.7%, indicating good library quality. Figure 11 ).

[0087] Next, the distribution characteristics of 5caC in the mESC genome were analyzed. First, the distribution of 5caC in functional regions was analyzed. The results showed that approximately half of the 5caC was enriched in adjacent gene promoter regions (≤1kb, 47.5%), and also partially enriched in distal intergenic regions (Distal Intergenic, 18.04%) and other introns (Other Intron, 14.65%). Figure 12 Further analysis showed that 5caC was abundant at the 3' end of the gene, near the transcription start site (TSS), suggesting that 5caC may be involved in gene expression regulation. Figure 13-15 Finally, a distribution map of 5caC in the mESC genome was plotted. Figure 16 The results showed that 5caC was widely distributed in all 21 chromosomes of mice. The content of 5caC was lower in the Y chromosome (NC_000087.8), while the distribution was more even in other chromosomes.

[0088] In summary, the method of this invention can be used for the localization analysis of 5caC in extremely low amounts in the genome, and the biological functions of 5caC can be further studied using this method.

Claims

1. The application of a click chemistry-based enrichment and localization analysis method for 5-carboxycytosine (5caC) in DNA in the preparation of a kit for detecting 5-carboxycytosine (5caC), characterized in that, The method for enrichment and localization analysis of 5-carboxycytosine (5caC) in DNA based on click chemistry includes the following steps: (1) The 5caC in the DNA to be tested was dehydrated and condensed with propargylamine using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBT) to form 5-propargylaminocytosine (5caC-PA); the reaction was carried out at 37°C for 4 hours in a reaction system containing 20 mM EDC, 2 mM HOBT, 75 mM 2-(N-morpholine)ethanesulfonic acid (MES), pH 5.0 and 10 mM propargylamine. (2) Recovery of 5caC-PA-labeled DNA; (3) The 5caC-PA was biotin-labeled by a click chemical reaction catalyzed by the copper(II)-TBTA complex with biotin-PEG3-N3. The conditions for the click chemical reaction were as follows: the DNA labeled with propargylamine was reacted at 37°C for 2 hours in a reaction system of 100 mM triethylamineacetic acid, pH 7.0, 3-6 M dimethyl sulfoxide, 0.5 mM ascorbic acid, 1 mM Biotin-PEG3-N3, and 0.5 mM copper(II)-TBTA complex. (4) Recover biotin-labeled DNA; (5) Using streptavidin magnetic beads to bind biotinylated DNA, and using magnetic capture to separate DNA containing 5caC modified with streptavidin magnetic beads; (6) Construct an experimental DNA library from the magnetically captured DNA to be tested, and construct a control DNA library from the DNA recovered in step (4) as a control. Perform high-throughput sequencing on the two libraries and draw a 5caC modification spectrum by comparing the sequencing results of the experimental group and the control group.

2. The application according to claim 1, characterized in that, The DNA to be tested is either single-stranded DNA or double-stranded DNA.

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