Methods and kits for deaminase-assisted single base resolution mapping of n6-methyladenine in dna

Abstract

The application provides an analysis method and a kit for deaminase-assisted single-base resolution positioning of N6-methyladenine in DNA, the method comprising: performing a deamination reaction on DNA by using adenine deaminase ABE8e protein, and then performing high-temperature inactivation treatment to inactivate the adenine deaminase ABE8e protein, to obtain a deaminated DNA sample; performing a PCR reaction on the deaminated DNA sample to obtain an amplification product; and sequencing the amplification product to obtain site information of N6-methyladenine. 6 The application uses ABE8e to effectively deaminate normal adenine in DNA and not deaminate N6-methyladenine, and then performs a polymerase chain reaction amplification to obtain the site information of N6-methyladenine by sequencing. The method is simple to operate and can directly obtain single-base resolution positioning of N6-methyladenine in DNA.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an analytical method and kit for single-base resolution localization of N6-methyladenine in DNA with the assistance of deaminase. Background Technology

[0002] 6mA is a naturally occurring DNA modification that plays a crucial role in the restriction modification system in *E. coli*, resisting cleavage by restriction endonucleases and protecting DNA integrity. 6mA in *E. coli* also plays an important role in DNA mismatch repair and gene regulation. 6mA is a widespread epigenetic modification that plays a key role in various biological processes. Recent studies have found 6mA in different eukaryotic genomes, including *Chlamydomonas reinhardtii*, *C. elegans*, *Drosophila melanogaster*, fungi, vertebrates, mammals, and plants. In *Chlamydomonas reinhardtii*, 6mA is present in more than 14,000 genes and exhibits a periodic distribution pattern near transcription start sites, suggesting that 6mA may play an important role in nucleosome localization and gene activation. In *C. elegans* and *Drosophila melanogaster*, 6mA modifications have been found in certain transposons, and these modifications may affect intergenerational inheritance. In fungi, 6mA modifications are mainly concentrated around transcription start sites and are symmetrically present in ApT dinucleotides. While 6mA modification levels are relatively high in some invertebrate genomes, they are generally low in vertebrate and mammalian genomes. Although 6mA modification is well-known as a DNA modification in prokaryotes, its presence in eukaryotes has only recently been confirmed. In the human genome, 6mA content is approximately 0.051%. 6mA modification is also considered closely related to embryonic development and tumorigenesis. It is crucial for stress, neuropsychiatric disorders, and mESC function. In mouse mESCs, 6mA is abundant in the LINE1 transposon on the X chromosome, reaching levels as high as 25-30 ppm. In glioblastoma cancer, 6mA has been found to be a repressive marker, closely associated with the repressive histone marker H3K9me3 in genes related to neurogenesis and neuronal development. In adult mice undergoing fear extinction training, 6mA has also shown an association with stress responses, including chronic restraint stress in the mouse brain and stress adaptation in the mitochondria of *C. elegans*. These studies all suggest that 6mA modification plays a potential regulatory role in gene activation, indicating that 6mA could serve as an additional epigenetic marker. However, the status of 6mA as an epigenetic marker in mammals remains controversial. Isotope labeling and mass spectrometry analysis results indicate that 6mA in the genome primarily originates from 6mA nucleotides in RNA, which are processed via nucleotide repair pathways and mistakenly incorporated into DNA by DNA polymerase. These studies oppose considering 6mA modification as a heritable epigenetic marker in the mammalian genome. Nevertheless, some researchers have found that 6mA modification is mainly present in mammalian mitochondrial DNA (mtDNA) and regulates mitochondrial transcription, replication, and mitochondrial activity.In mtDNA, the level of 6mA modification can be 1300 times higher than that in nuclear DNA, especially under certain stress conditions. The presence of 6mA modification can also significantly reduce the transcriptional activity of the mitochondrial transcription complex in vitro, thus leading to reduced RNA transcription in human mitochondria. This suggests that 6mA modification is an epigenetic marker in mammalian mtDNA.

[0003] To reveal the function of 6mA modifications, high-accuracy methods for locating 6mA modifications in DNA are needed. The ability to map methylation sites in the human genome has revolutionized our understanding of many cellular processes and diseases, so the exploration of 6mA function is inseparable from 6mA localization analysis. Since adenine and 6mA can form complementary base pairs with thymine, direct high-throughput sequencing using traditional methods is challenging. In recent years, several strategies have been developed to locate 6mA modifications in DNA; however, these methods all have different drawbacks. For example, (1) the method of using immunoprecipitation combined with high-throughput sequencing to locate 6mA has been used for the localization analysis of 6mA modifications in the genomes of various species, but this method has low resolution. There is also a DpnI-assisted 6mA modification localization method, which uses DpnI to cut the 6mA site in double-stranded DNA and then uses the cutting site to locate the 6mA position. This method is limited by the recognition site of Dpn I. If 6mA exists in other sequences, it will cause the loss of 6mA site information, so it cannot completely locate the 6mA site in the genome. (2) Another method is to use antibody-crosslinked exonuclease-assisted sequencing to locate 6mA. This method combines immunoprecipitation enrichment, photocrosslinking and exonuclease digestion to locate 6mA. The experimental steps are cumbersome and depend on the specificity of the antibody. SMRT technology has also been used to detect the 6mA modification site in mESC and plant genomes. However, recent studies have shown that this method usually produces false positive results and overestimates the content of 6mA modification in the genome.

[0004] Therefore, how to establish a single-base resolution 6mA localization sequencing method that can be applied to the field of DNA modification sequencing, with simple operation steps and high deamination efficiency, has become an urgent technical problem to be solved. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an analytical method for single-base resolution localization of N6-methyladenine in DNA using deaminase-assisted methods. The method is simple to operate, and the deamination reaction involved has a high deamination efficiency (99%), which is beneficial for localization analysis.

[0006] In a first aspect of the present invention, a deaminase-assisted analytical method for single-base resolution localization of N6-methyladenine in DNA is provided, the method comprising: DNA was deaminated using adenine deaminase ABE8e protein, followed by high-temperature inactivation to inactivate the adenine deaminase ABE8e protein, thus obtaining a deaminated DNA sample. The deaminated DNA sample was subjected to a PCR reaction to obtain the amplification product; The amplified product was sequenced to obtain... N 6 Site information for methyladenine.

[0007] Furthermore, the deamination reaction is carried out at a temperature of 36–38°C and for a time of 0.5–2 hours.

[0008] Furthermore, in the deamination reaction, the reaction system components include adenine deaminase ABE8e protein, DNA, and reaction buffer.

[0009] Furthermore, the reaction buffer comprises Tris-HCl with a final concentration of 45–55 mM, pH 6–8, and DTT with a concentration of 5–15 mM.

[0010] Furthermore, the conditions for the high-temperature inactivation treatment include: incubation in a water bath at 92–98°C for 5–20 minutes.

[0011] In a second aspect of the invention, the application of adenine deaminase ABE8e protein in an analytical method for single-base resolution localization of N6-methyladenine in DNA is provided.

[0012] In a third aspect of the invention, a kit is provided for single-base resolution localization of N6-methyladenine in DNA, the kit comprising: adenine deaminase ABE8e protein.

[0013] Furthermore, the kit also includes a reaction buffer; the reaction buffer comprises Tris-HCl at a final concentration of 45–55 mM, pH 6–8, and DTT at 5–15 mM.

[0014] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: The present invention provides a method for single-base resolution localization of N6-methyladenine in DNA using deaminase-assisted methods, comprising: deaminating DNA with adenine deaminase ABE8e protein, followed by high-temperature inactivation treatment to inactivate the adenine deaminase ABE8e protein, thereby obtaining a deaminized DNA sample; performing a PCR reaction on the deaminized DNA sample to obtain an amplification product; and sequencing the amplification product to obtain... N 6 -Methyladenine site information. This invention uses adenine deaminase to deaminate DNA. Because unmethylated adenine is deaminated to form hypoxanthine while 6mA is not deaminated, 6mA and adenine can be effectively distinguished during sequencing, thereby enabling the localization analysis of 6mA in the genome. This has the following advantages: (1) The method of the present invention is simple to operate and does not require complicated sample pretreatment. After deamination, polymerase chain amplification reaction can be performed directly.

[0015] (2) The present invention does not require the use of reagents to oxidize or derivatize reagents for labeling, which greatly shortens the experimental time.

[0016] (3) The deamination reaction involved in this invention has a high deamination efficiency (99%), which is beneficial for localization analysis. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 In this invention, ABE8e is effective against adenine and N 6 Schematic diagram of the deamination of methyladenine; Figure 2 This invention utilizes the ABE8e protein to target DNA... N 6 α-methyladenine is used for localization; Figure 3 The results of LC-MS / MS detection of the 60-nucleotide long DNA strand containing A, which was artificially synthesized in this invention, before and after deamination treatment; Figure 4 The results of LC-MS / MS detection of the 60-nucleotide long DNA strand containing 6 mA artificially synthesized in this invention before and after deamination. Figure 5This is a comparison of the sequencing results of the artificially synthesized 60-nucleotide DNA strand containing A and 6mA before and after deamination in this invention. Figure 6 A flowchart illustrating a deaminase-assisted method for single-base resolution localization of N6-methyladenine in DNA, provided in an embodiment of the present invention; Figure 7 The results show the statistical results of the deamination rate of the 314-nucleotide-length DNA strand artificially synthesized in this invention, which contains 16 different A sequences, after deamination treatment. Detailed Implementation

[0019] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0020] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0021] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0022] The technical solution provided by the embodiments of the present invention is to solve the above-mentioned technical problems, and the general idea is as follows: According to a typical embodiment of the present invention, a deaminase-assisted analytical method for single-base resolution localization of N6-methyladenine in DNA is provided, such as... Figure 6 As shown, the method includes: S1. DNA was deaminated using adenine deaminase ABE8e protein, followed by high-temperature inactivation to inactivate the adenine deaminase ABE8e protein, thus obtaining a deaminated DNA sample. In step S1 The deamination reaction is carried out at a temperature of 36–38°C for 0.5–2 hours. Temperatures that are too low or too high, or reaction times that are too short or too long, are detrimental to the deamination reaction and thus reduce its sensitivity.

[0023] In the deamination reaction, the reaction system comprises adenine deaminase ABE8e protein, DNA, and a reaction buffer. The reaction buffer consists of Tris-HCl at a final concentration of 45–55 mM, pH 6–8, and DTT at 5–15 mM. The choice of these components ensures the activity of the deaminase and the efficiency of the deamination reaction.

[0024] The conditions for the high-temperature inactivation treatment include incubation in a water bath at 92–98°C for 5–20 minutes. These reaction conditions are beneficial for the inactivation of adenine deaminase ABE8e protein, thereby facilitating the subsequent PCR amplification process.

[0025] In specific implementations, the concentration of ABE8e is determined based on the actual activity of the protein.

[0026] S2. Perform a PCR reaction on the deaminated DNA sample to obtain the amplification product; S3. Sequencing the amplified product to obtain... N 6 Site information for methyladenine.

[0027] This application uses adenine deaminase to deaminate DNA. Since unmethylated adenine is deaminated to form hypoxanthine while 6mA is not deaminated, 6mA and adenine can be effectively distinguished during sequencing, thereby enabling the localization analysis of 6mA in the genome.

[0028] According to another typical embodiment of the present invention, an application of adenine deaminase ABE8e protein in the analysis method for single-base resolution localization of N6-methyladenine in DNA is provided.

[0029] According to another typical embodiment of the present invention, a kit for single-base resolution localization of N6-methyladenine in DNA is provided, the kit comprising: adenine deaminase ABE8e protein.

[0030] The kit also includes a reaction buffer; the reaction buffer comprises Tris-HCl at a final concentration of 45–55 mM, pH 6–8, and DTT at 5–15 mM.

[0031] The effects of this application will be described in detail below with reference to embodiments and experimental data. Unless otherwise specified, the technical means used in the embodiments, including nucleic acid extraction, enzymatic digestion, and polymerase chain reaction, are conventional methods well known to those skilled in the art.

[0032] Example 1 1. A kit for single-base resolution localization of N6-methyladenine in DNA, the kit comprising: Adenine deaminase ABE8e protein.

[0033] Reaction buffer: consisting of 50 mM Tris-HCl, pH 5.0, 10 mM EDTA, and 2 mM MgCl2.

[0034] 2. A deaminase-assisted analytical method for single-base resolution localization of N6-methyladenine in DNA, the method comprising: (1) Use an ultrasonic disruptor to fragment the DNA; denature the fragmented DNA, incubate it in a 95°C water bath for 10 minutes, and then immediately transfer it to an ice box for quenching. (2) Take 10 ng of the DNA sample, add ABE8e protein to a final concentration of 2.8 μM, 1 μL of Tris-HCl reaction buffer, and add deionized water to a reaction volume of 50 μL. At 37°C... o React in a small heated water bath at C for 1–4 hours; after the reaction, incubate the sample at 95°C. o Incubate at C for 10 minutes.

[0035] (3) Subsequently, polymerase chain reaction (PCR) was performed to amplify the DNA, followed by sequencing. Specifically: 10 μL of the above-mentioned DNA was used for PCR amplification. Reaction system: 2 μL of 10× amplification buffer, 1 μL each of 10 μmol / L forward and reverse primers, 10 ng of template DNA, Q5 DNA polymerase final concentration of 10 U, and deionized water was added to a final volume of 20 μL. The annealing temperature for the amplification reaction was selected according to different regions; the amplification cycle time program was as follows: ① 95℃ denaturation for 5 min; ② 95℃ denaturation for 30 sec; ③ 50-68℃ annealing for 30 sec; ④ 72℃ extension for 30 sec; steps ②-④ were repeated 25 times; extension at 72℃ for 10 min, and then stored at 4℃. The samples can be directly sequenced using Sanger sequencing.

[0036] The DNA sample was a synthetically produced 60-nucleotide oligonucleotide DNA containing 6 mA. The LC-MS / MS detection results of the synthetically produced 60-nucleotide oligonucleotide DNA strand before and after deamination treatment are as follows: Figure 4 As shown, it indicates Escherichia coli genomic DNA, human mitochondrial DNA, and other DNA containing N6-methyladenine.

[0037] Example 2 1. A kit for single-base resolution localization of N6-methyladenine in DNA, the kit comprising: Adenine deaminase ABE8e protein.

[0038] Reaction buffer: consisting of Tris-HCl to a final concentration of 55 mM, pH 5.5, 12 mM EDTA, and 3 mM MgCl2.

[0039] 2. A deaminase-assisted analytical method for single-base resolution localization of N6-methyladenine in DNA, the method comprising: (1) Use an ultrasonic disruptor to fragment the DNA; denature the fragmented DNA, incubate it in a 95°C water bath for 10 minutes, and then immediately transfer it to an ice box for quenching. (2) Take 10 ng of the DNA sample, add ABE8e protein to a final concentration of 2.8 μM, 1 μL of Tris-HCl reaction buffer, and add deionized water to a reaction volume of 50 μL. At 37°C... o The reaction was carried out in a small heated water bath at C for 1 hour; after the reaction, the sample was heated at 95°C. o Incubate at C for 10 minutes.

[0040] (3) Subsequently, polymerase chain reaction (PCR) was performed to amplify the DNA, followed by sequencing. Specifically: 10 μL of the above-mentioned DNA was used for PCR amplification. Reaction system: 2 μL of 10× amplification buffer, 1 μL each of 10 μmol / L forward and reverse primers, 10 ng of template DNA, Q5 DNA polymerase final concentration of 10 U, and deionized water was added to a final volume of 20 μL. The annealing temperature for the amplification reaction was selected according to different regions; the amplification cycle time program was as follows: ① 95℃ denaturation for 5 min; ② 95℃ denaturation for 30 sec; ③ 50-68℃ annealing for 30 sec; ④ 72℃ extension for 30 sec; steps ②-④ were repeated 25 times; extension at 72℃ for 10 min, and then stored at 4℃. The samples can be directly sequenced using Sanger sequencing.

[0041] Example 3 1. A kit for single-base resolution localization of N6-methyladenine in DNA, the kit comprising: Adenine deaminase ABE8e protein.

[0042] Reaction buffer: consisting of Tris-HCl to a final concentration of 45 mM, pH 4.5, 8 mM EDTA, and 1 mM MgCl2.

[0043] 2. A deaminase-assisted analytical method for single-base resolution localization of N6-methyladenine in DNA, the method comprising: (1) Use an ultrasonic disruptor to fragment the DNA; denature the fragmented DNA, incubate it in a 95°C water bath for 10 minutes, and then immediately transfer it to an ice box for quenching. (2) Take 10 ng of the DNA sample, add ABE8e protein to a final concentration of 2.8 μM, 1 μL of Tris-HCl reaction buffer, and add deionized water to a reaction volume of 50 μL. At 37°C... o The reaction was carried out in a small heated water bath at C for 1 hour; after the reaction, the sample was heated at 95°C. o Incubate at C for 10 minutes.

[0044] (3) Subsequently, polymerase chain reaction (PCR) was performed to amplify the DNA, followed by sequencing. Specifically: 10 μL of the above-mentioned DNA was used for PCR amplification. Reaction system: 2 μL of 10× amplification buffer, 1 μL each of 10 μmol / L forward and reverse primers, 10 ng of template DNA, Q5 DNA polymerase final concentration of 10 U, and deionized water was added to a final volume of 20 μL. The annealing temperature for the amplification reaction was selected according to different regions; the amplification cycle time program was as follows: ① 95℃ denaturation for 5 min; ② 95℃ denaturation for 30 sec; ③ 50-68℃ annealing for 30 sec; ④ 72℃ extension for 30 sec; steps ②-④ were repeated 25 times; extension at 72℃ for 10 min, and then stored at 4℃. The samples can be directly sequenced using Sanger sequencing.

[0045] Comparative Example 1 In this comparative example, the DNA sample is a synthetically produced 60-nucleotide oligonucleotide DNA containing A, with the sequence shown in SEQ ID NO.2; all other steps are the same as in Example 1.

[0046] The LC-MS / MS detection results of the artificially synthesized 60-nucleotide oligonucleotide DNA strand containing A before and after deamination treatment are as follows: Figure 3 As shown.

[0047] Experiment Example 1, Performance Measurement 1. A comparison of sequencing results before and after deamination of the artificially synthesized DNA strand containing A in Comparative Example 1 and the 60mA oligonucleotide in Example 1 is shown in the figure. Figure 5 As shown; Depend on Figure 5 It is known that during the sequencing process, 6mA and adenine can be effectively distinguished, thereby enabling the localization analysis of 6mA in the genome.

[0048] 2. In this invention, after deamination of a 314-nucleotide-length DNA strand containing 16 different A sequences synthesized artificially, the deamination rate was statistically analyzed. The results of the deamination rate analysis are as follows: Figure 7 As shown.

[0049] Depend on Figure 7 It is known that the deamination reaction involved in this invention has a high deamination efficiency (99%), which is beneficial for localization analysis.

[0050] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0051] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0052] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. An analytical method for deaminase-assisted single base resolution mapping of N6-methyladenine in DNA for non-disease diagnostic and therapeutic purposes, characterized by, The method includes: DNA was deaminated using adenine deaminase ABE8e protein, followed by high-temperature inactivation to inactivate the adenine deaminase ABE8e protein, thus obtaining a deaminated DNA sample. The deaminated DNA sample was subjected to a PCR reaction to obtain the amplification product; The amplification products were sequenced to obtain N 6 - site information of methyladenine.

2. The analytical method according to claim 1, characterized in that, The deamination reaction is carried out at a temperature of 36–38°C for 0.5–2 h.

3. The analytical method according to claim 1, characterized in that, In the deamination reaction, the reaction system components include adenine deaminase ABE8e protein, DNA, and reaction buffer.

4. The analytical method according to claim 1, characterized in that, The conditions for the high-temperature inactivation treatment include incubation in a water bath at 92–98°C for 5–20 minutes.

5. Application of an adenine deaminase ABE8e protein in a single-base resolution analysis method for N6-methyladenine localization in DNA for non-disease diagnostic and therapeutic purposes.

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