CN115125292A - Single-base resolution positioning analysis method for inosine modification in RNA assisted by endonuclease - Google Patents

Abstract

The invention discloses an endonuclease-assisted single-base resolution positioning method for inosine modification in RNA, belonging to the technical field of biology. According to the invention, 3 '-deoxy-A is used for blocking the end of RNA 3' OH, then hEndoV protein is cut at inosine to generate new RNA 3 'OH, the new 3' OH is subjected to polyuridylation and library construction, and sequencing is carried out to obtain inosine modified site information. The method has the advantages of direct principle, simple and convenient operation, no chemical reaction in the whole process, no degradation of RNA, no need of complex bioinformatics algorithm, and capability of directly obtaining single base resolution positioning information modified by inosine in RNA.

Description

An endonuclease-assisted single-base-resolution mapping analysis of inosine modifications in RNA method

technical field

The invention belongs to the field of biotechnology, and in particular relates to an endonuclease-assisted single-base resolution positioning method for inosine modification in RNA.

Background technique

RNA is composed of four typical bases including A, U, G, and C. In addition to the nucleotides of the four standard bases, more than 150 epigenetic modifications with important functions have been found on RNA. Inosine (Ino) is one of the most important post-transcriptional modifications on mammalian RNA and is widely present on mRNA, tRNA, lncRNA, miRNA and siRNA. Ino is produced by the adenosine deaminase (ADAR) family of proteins acting on RNA catalyzes the aminohydrolytic deamination of adenosine C6, a transition also known as A-to-InoRNA editing. Three ADAR proteins have been identified in mammals, ADAR1, ADAR2 with enzymatic activity and ADAR3 without enzymatic activity.

A-to-Ino editing has important biological functions. Since Ino is recognized as G by cellular machinery due to pairing with C during reverse transcription, A-to-Ino editing can alter gene information post-transcriptionally. A-to-Ino editing on mRNA may result in amino acid changes, creation or deletion of splice sites and stop codons, resulting in post-translational protein changes that favor protein diversity. Ino on tRNA is located at position 34 of the anticodon loop, and Ino at this position allows multiple codons to be recognized by wobble pairing, enhancing the decoding ability of tRNA. miRNAs and siRNAs can modulate target genes in a highly specific manner, and A-to-Ino editing regulates the maturation, structure, and function of miRNAs and siRNAs.

Aberrant A-to-Ino editing levels have been shown to be associated with a variety of human diseases, including various cancers, neurological and neurodegenerative diseases, psychiatric disorders, and autoimmune diseases. Aberrant editing levels at A-to-Ino editing sites on some transcripts have been shown to be associated with disease mechanisms. For example, A-to-Ino editing of serotonin 2C receptor pre-mRNA occurs at 5 conserved sites (designated A to E) in exon 5, the level of editing at the A site in the brains of suicide victims of depression E-site editing levels were significantly increased in patients with major depressive disorder. However, the significance of most A-to-Ino editing sites is still unclear, and accurate identification of the location of A-to-Ino editing in the whole transcriptome is the basis for in-depth understanding of the biological function and significance of each site.

The current single-base mapping method for transcriptome-wide A-to-Ino editing is mainly RNA-seq. Ino is paired with a C during reverse transcription so that it is read as a G during sequencing, so A-to-Ino editing sites can be identified based on the A-to-G transition. Although the principle is simple, in the actual analysis process, RNA-seq will generate a large number of false positive sites due to interference factors such as alignment errors, single nucleotide polymorphisms, and somatic mutations. Although bioinformatic approaches such as statistical modeling, filtering and/or alignment, and integration of other genomic information have been developed to reduce false positives, RNA-seq remains less accurate in identifying A-to-Ino editing sites. One of the chemical methods for the identification of A-to-Ino editing sites on a transcriptome scale is "ICE-seq", N1 ^- cyanoethylinosine produced by the reaction of Ino with acrylonitrile hinders cDNA extension during reverse transcription, resulting in truncation The cDNA was screened during library construction due to the short fragments, so the "erasure" signal generated in sequencing can be used to identify A-to-Ino editing sites, but this method cannot identify 100% edited sites . Another method involving chemical reactions is Ino-specific sequencing. The property of nuclease T1 is to cleave RNA at G or Ino, and G forms a stable adduct with glyoxal in the presence of borate to resist nuclease T1. cleavage, while Ino is cleaved by nuclease T1 due to its inability to form stable adducts. This method begins by chemically oxidizing and biotinylating the 3' end of the RNA, followed by glyoxal and borate treatment to protect the G on the RNA, followed by biotin-streptomycin affinity to immobilize the RNA to the streptomycin function On the magnetic beads, nuclease T1 cleaved RNA at Ino, and then the cleaved RNA was subjected to library construction and sequencing analysis of A-to-Ino editing sites. The disadvantage of this method is that the procedure is complicated, and if 100% protection of G cannot be achieved, G can also be cleaved by nuclease T1, resulting in false positive sites. A common disadvantage of chemical methods is that severe chemical reactions can cause severe RNA degradation, so mild enzymatic methods should be developed for single-base resolution mapping analysis of A-to-Ino editing.

hEndoV (human endonuclease V), as an endonuclease of Ino on RNA, can specifically recognize and cleave Ino on RNA, and generate a 3'OH and a second phosphodiester bond at its downstream 3' end. 5' Phosphate cut. According to this feature, we propose the invention of an endonuclease-assisted method for single-base resolution mapping of Ino in RNA.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide an endonuclease-assisted single-base resolution localization analysis method for inosine modification in RNA, that is, a method that can be applied to the field of RNA modification sequencing, No chemical reagent processing, no complex bioinformatics algorithm, high sensitivity, high selectivity, easy to operate Ino single base resolution localization analysis method.

The object of the present invention is achieved through the following technical solutions:

An endonuclease-assisted single-base resolution localization analysis method for inosine modification in RNA, comprising the following steps:

(1) Fragmenting the RNA of the cell or tissue sample;

(2) 3'OH end blocking of fragmented RNA: use polyA polymerase to catalyze 3'deoxy-ATP to add 3'deoxy-A to the 3'OH end of fragmented RNA to block the 3'OH end of RNA end, followed by purification of RNA;

(3) Use hEndoV protein to cleave above-mentioned 3'OH end-blocked RNA to generate new RNA 3'OH at inosine;

(4) 3' polyuridylation of the cleaved RNA: using polyU polymerase to catalyze UTP to add a U tail to the 3' OH end of the RNA to carry out 3' polyuridylation;

(5) using 3' polyuridylated RNA as a template, using reverse transcription primers with polyadenosine and 3' linkers to carry out reverse transcription reaction to obtain cDNA;

(6) using a primer with a 5' linker to add a 5' linker to the cDNA through a DNA polymerase extension reaction to obtain DNA for amplification;

(7) PCR amplification is carried out with the above-mentioned DNA as a template, and the amplified product is sequenced;

(8) Inosine-modified single-base recognition: in the sequencing results, the second base G from 3' to 5' after removing the double-end linker is an inosine-modified base.

The method of the present invention obtains the single-base resolution positioning information of Ino through the specific cleavage of Ino by hEndoV protein and sequencing.

The above-mentioned hEndoV protein cleavage reaction: NEBuffer 4 (NEB company, product number B7004) was used as the reaction buffer, the buffer system of the buffer reaction solution was Tris-CH ₃ COOH, the concentration of Mg ²⁺ was 10mM, and a certain amount of hEndoV protein was used. (specifically determined according to the activity of the actual protein), the reaction conditions are preferably 37°C for 30min.

The above hEndoV protein cleavage reaction refers to the specific cleavage reaction of Ino on RNA by hEndoV protein.

The advantages and beneficial effects of the present invention are as follows:

1) The principle of the present invention is direct, the method is simple, and no complicated pretreatment is required.

2) The present invention does not require chemical reaction to process RNA, and will not cause severe degradation of RNA.

3) In the present invention, the hEndoV protein cleavage reaction can be completed in only 30 minutes, which greatly shortens the overall experimental time.

4) The cleavage reaction involved in the present invention has high specificity (only Ino cleavage, not A) and high efficiency (complete cleavage), which is beneficial to localization analysis.

5) The RNA library construction method involved in the present invention does not require single-strand ligation reaction, has high library construction efficiency, and is beneficial to sequencing and site analysis.

6) The present invention does not involve complex bioinformatics algorithms, which is beneficial to the identification of A-to-Ino editing sites.

Description of drawings

FIG. 1 is a schematic diagram of the single-base resolution localization analysis method of inosine modification in endonuclease-assisted RNA of the present invention. The 3'OH terminus of the RNA was first blocked by 3'-deoxy-A, and then a new 3'OH was generated at the 3' second phosphodiester bond of Ino by hEndoV protein cleavage, and the newly generated 3'OH was subsequently blocked by 3'-OH 'Polyuridylation, enabling single-base resolution mapping analysis of Ino via library construction and sequencing.

Fig. 2 is a PAGE gel image of the specific cleavage of synthetic RNA containing Ino modified by hEndoV protein in the present invention. (A) Schematic diagram of hEndoV cleaving RNA with Ino modifications; (B) Using synthetic RNA strands containing Ino modifications (Ino-RNA-1 and Ino-RNA-2, sequences are shown in Table 2) as substrates to evaluate hEndoV protein Cleavage effect, (C) The cleavage specificity of hEndoV protein was evaluated by using RNA strand without Ino modification (A-RNA-1, see Table 2 for sequence) as substrate.

FIG. 3 is a diagram showing the sequencing effect of the single-base resolution localization analysis of inosine modification on an artificially synthesized 45nt Ino-RNA-1 oligonucleotide chain in the present invention.

Detailed ways

In some embodiments, the single-base resolution localization analysis method of inosine modification in endonuclease-assisted RNA of the present invention specifically comprises the following steps:

(1) Fragmentation: Take 5 μg of mRNA extracted from biological sample tissues or cells, and use NEBNext RNA Mg ⁺⁺ Fragmentation Module (NEB, Cat. No. E6150) to fragment the mRNA with a length of about 1000 nt.

(2) 3'OH end blocking of the above RNA: add 5U polyA polymerase (NEB, Cat. No. M0276), 1×polyA reaction buffer (delivered with the enzyme), 1mM 3'deoxy-ATP (Sigma-Aldrich) to the above mRNA , Cat. No. C9137) and 20U RNase inhibitor, after 30 min of reaction at 37°C, RNA was purified (10 μL elution) with RNA Clean & Concentrator Kits (ZYMORESEARCH, Cat. No. R1013).

(3) hEndoV cleavage of the above RNA: add a certain amount of hEndoV protein, 1×NEBuffer 4 buffer (NEB, Cat. No. B7004) and 20U RNase inhibitor to the above 10 μL RNA, the final volume is 20 μL, and the cleavage reaction is carried out at 37°C 30min.

Among them, the expression process of hEndoV protein is as follows. The full-length coding sequence of hEndoV (see Table 1) is inserted into pET-28a(+) plasmid to obtain hEndoV-pET-28a(+) recombinant plasmid, which is transformed into E. coli BL21(DE3); induction The conditions for protein expression were IPTG concentration of 1 mM, induction temperature of 25 °C, and induction time of 10 h; the protein was purified by BeyoGold His-tag Purification Resin (reduction-resistant chelation type) (Biyuntian Biotechnology, Cat. No. P2210). The evaluation process of hEndoV protein activity is as follows, containing 1 μM RNA (Ino-RNA-1, Ino-RNA-2 and A-RNA-1, sequences are shown in Table 2), a certain amount of hEndoV, 20U RNase inhibitor and 1×NEBuffer 4 buffer (NEB, Cat. No. B7004) in a total of 10 μL of reaction system was incubated at 37°C for 30 min, then 1 μL of 10× loading buffer (Takara, Cat. No. 9157) was added to the system to stop the reaction, and the reaction results were identified by 20% PAGE (Figure 2).

(4) 3' polyuridylation of the above RNA: add 1U polyU polymerase (NEB, Cat. No. M0337), 1×NEBuffer 2 (delivered with the enzyme), 100μM UTP and 20U RNase inhibitor to the above 20μL reaction system , the final volume is 40 μL. RNA was purified (20 μL elution) using RNA Clean & Concentrator Kits (ZYMORESEARCH, Cat. No. R1013) after the system was incubated at 37°C for 8 min.

(5) The above RNA was reverse transcribed to synthesize cDNA: 30 μL of reverse transcription system was composed of 20 μL of the above RNA, 200 UM-MuLV reverse transcriptase (Sangong Bio, Cat. No. B500517), 1× RT reaction buffer (delivered with the enzyme), 0.33 μM 3'adaptor-A ₂₀ NC primer (see Table 2 for the sequence), 0.5mM dNTP and 20U RNase inhibitor, this system was reacted at 42°C for 90min.

(6) Removal of RNA and purification of cDNA: To the reverse transcription reaction (30 μL), add 1 μL of Exonuclease I (NEB, Cat. No. M0293), 5 μL of Exonuclease I Reaction Buffer (delivered with the enzyme) and 14 μL of H ₂ O , react at 37°C for 30min to remove excess reverse transcription primer; then add 10μL NaOH (1M) and 10μL EDTA (0.5M) to the exonuclease I reaction system (50μL), and react at 65°C for 15min to remove RNA in the system, cDNA was recovered (10 μL elution) using Oligo Clean & Concentrator Kits (ZYMORESEARCH, Cat. No. D4060).

(7) Carry out the extension reaction of the above cDNA and add 5' adapter: The reaction system (30 μL) consists of 10 μL of the above cDNA, 5U BsuDNA polymerase (NEB, Cat. No. M0330), 1×NEBuffer 2 (delivered with the enzyme), 0.33 μM 5' The adaptor-N ₆ primer (see Table 2 for the sequence) and 1 mM dNTP were used to react in this system for 1 h at 37°C.

(8) DNA purification: add 1 μL exonuclease I (NEB, Cat. No. M0293), 5 μL exonuclease I reaction buffer (delivered with the enzyme) and 14 μL H ₂ O to the previous system (30 μL), at 37°C The excess 5'adaptor-N ₆ primer was removed after the reaction for 30 min, and DNA was recovered (15 μL elution) using Oligo Clean & Concentrator Kits (ZYMO RESEARCH, Cat. No. D4060).

(9) PCR amplification of the above DNA: The PCR system (50 μL) consists of 15 μL of the above DNA, 2× amplification buffer (Yisheng Bio, Cat. No. 10136ES01), 0.5 μM P5 and P7 primers (see Table 2 for the sequence). Amplification cycle time program: ① 98°C denaturation for 1 min; ② 98°C denaturation for 10 sec; ③ 60°C annealing for 25 sec; ④ 72°C extension for 50 sec;

(10) The amplified product obtained is sequenced.

Table 1 hEndoV full-length coding sequence

Table 2 Oligonucleotide chains involved in the specific embodiment

The following examples are used to further illustrate the present invention, but should not be construed as a limitation of the present invention. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present invention should be equivalent. The replacement modes are all included in the protection scope of the present invention.

Unless otherwise specified, the technical means used in the examples including nucleic acid extraction, kit purification, reverse transcription reaction, and polymerase chain reaction are conventional means well known to those skilled in the art.

Example 1: Standard Synthetic RNA Analysis

(1) Add 100ng of artificially synthesized Ino-RNA-1 chain (see Table 3 for the sequence), 5U polyA polymerase, 1mM 3'deoxy-ATP, 1×polyA reaction buffer and 20U RNase inhibitor to a 10μL reaction system, 37 After 30 min of reaction at °C, the RNA was purified with a kit.

(2) The above RNA is subjected to hEndoV protein cleavage reaction. A certain amount of hEndoV protein, 1×NEBuffer 4 and 20U RNase inhibitor were added to the above RNA to make the final volume 20 μL, and the cleavage reaction was carried out at 37°C for 30min.

(3) The above RNA is subjected to a polyuridylation reaction. 1U polyU polymerase, 100 μM UTP, 1×NEBuffer 2 and 20U RNase inhibitor were added to the above 20 μL reaction system to a final volume of 40 μL. After this system was carried out at 37°C for 8 min, the kit was used to purify the RNA.

(4) The above RNA is subjected to reverse transcription reaction. The reverse transcription system (30 μL) includes 10 μL of the above RNA, 0.33 μM 3’adaptor-A ₂₀ GC primer (see Table 3 for the sequence), 0.5 mM dNTP, 200 U reverse transcriptase, 1× reverse transcription buffer, 20 U RNaseinhibitor, this system is in The reaction was carried out at 42 °C for 90 min. Since the standard synthetic RNA analysis system using Ino-RNA-1 is simple, the step of recovering cDNA here is simplified as adding 1 μL of ribonuclease A to the reverse transcription system at the end of the reaction, and reacting at 37°C for 30 minutes to remove RNA, and then pass through the kit. cDNA was recovered.

(5) The above cDNA is subjected to a 5' adapter addition reaction. The 5' adapter addition reaction system (30 μL) included 10 μL of the above cDNA, 5U Bsu DNA polymerase, 0.33 μM 5’ adapter primer (see Table 3 for the sequence), 1 mM dNTP, and 1×NEBuffer 2. The system was reacted at 37 °C for 1 h.

(6) Take the above-mentioned reaction DNA and carry out polymerase chain amplification reaction. The 25 μL PCR reaction system consisted of 2 μL of the above DNA, 12.5 μL of 2× amplification buffer, 10 μM of P5 and P7 primers (see Table 3 for sequences), 1.25 μL of each, and 8 μL of H ₂ O. Amplification cycle time program: ① 98°C denaturation for 1 min; ② 98°C denaturation for 10 sec; ③ 60°C annealing for 25 sec; ④ 72°C extension for 50 sec; The sample can be directly sequenced by Sanger. The sequencing result is shown in Figure 3. The second base from 3' to 5' in the sequencing diagram is G (marked by a box), and this site is the muscle in the original Ino-RNA-1. glycoside modification.

The oligonucleotide chains involved in Table 3 Example 1

sequence listing

<110> Wuhan University

<120> An endonuclease-assisted single-base resolution mapping method for inosine modification in RNA

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 846

<212> DNA

<213> Homo sapiens

<400> 1

atggccctgg aggcggcggg agggccgccg gaggaaacgc tgtcactgtg gaaacgggag 60

caagctcggc tgaaggccca cgtcgtagac cgggacaccg aggcgtggca gcgagacccc 120

gccttctcgg gtctgcagag ggtcgggggc gttgacgtgt ccttcgtgaa aggggacagt 180

gtccgcgctt gtgcttccct ggtggtgctc agcttccctg agctcgaggt ggtgtatgag 240

gagagccgca tggtcagcct cacagccccc tacgtgtcgg gcttcctggc cttccgagag 300

gtgcccttct tgctggagct ggtgcagcag ctgcgggaga aggagccggg cctcatgccc 360

caggtccttc ttgtggatgg aaacggggta ctccaccacc gaggctttgg ggtggcctgc 420

caccttggcg tccttacaga cctgccgtgt gttggggtgg ccaagaaact tctgcaggtg 480

gatgggctgg agaacaacgc cctgcacaag gagaagatcc gactcctgca gactcgagga 540

gactcattcc ctctgctggg agactctggg actgtcctgg gaatggccct gaggagccac 600

gaccgcagca ccaggcccct ctacatctcc gtgggccaca ggatgagcct ggaggccgct 660

gtgcgcctga cttgctgctg ctgcaggttc cggatcccag agcccgtgcg ccaggctgac 720

atctgctccc gagagcacat ccgcaagtcg ctgggactcc ccgggccacc cacaccgagg 780

agcccgaagg cgcagaggcc agtggcatgc cccaaaggag actccggaga gtcctcagca 840

ctttgt 846

Claims (5)

1. a single base resolution positioning analysis method of inosine modification in an endonuclease-assisted RNA, is characterized in that: comprise the following steps: (1) Fragmenting the RNA of the cell or tissue sample; (2) 3'OH end blocking of the fragmented RNA; (3) using hEndoV protein to cleave above-mentioned 3'OH end-blocked RNA; (4) 3' polyuridylation of the cleaved RNA; (5) using 3' polyuridylated RNA as a template, using reverse transcription primers with polyadenosine and 3' linkers to carry out reverse transcription reaction to obtain cDNA; (6) using a primer with a 5' linker to add a 5' linker to the cDNA through a DNA polymerase extension reaction to obtain DNA for amplification; (7) PCR amplification is carried out with the above-mentioned DNA as a template, and the amplified product is sequenced; (8) Inosine-modified single-base recognition: in the sequencing results, the second base G from 3' to 5' after removing the double-end linker is an inosine-modified base. 2. the single base resolution localization analysis method of inosine modification in endonuclease-assisted RNA according to claim 1, is characterized in that: step (2) is: use polyA polymerase to catalyze 3 'deoxy-ATP to add 3'deoxy-A to the 3'OH terminus of fragmented RNA. 3. the single base resolution localization analysis method of inosine modification in endonuclease-assisted RNA according to claim 1, is characterized in that: in step (3), use the reaction buffer of hEndoV protein to cut RNA with Tris -CH ₃ COOH is a buffer system, and the concentration of Mg ²⁺ in the buffer system is 10 mM. 4 . The single-base resolution localization analysis method for inosine modification in endonuclease-assisted RNA according to claim 3 , wherein the reaction conditions for cutting the RNA are 37° C. for 30 minutes. 5 . 5. the single base resolution localization analysis method of inosine modification in endonuclease-assisted RNA according to claim 1, is characterized in that: step (4) is: use polyU polymerase catalyzed UTP to add U tail to RNA 3' OH terminus, 3' polyuridylation.

Images