A single-base resolution localization analysis method for inosine modification in RNA assisted by endonuclease

Abstract

The present invention discloses a single-base resolution positioning method for inosine modification in RNA assisted by an endonuclease, and belongs to the field of biotechnology. The present invention first uses 3'-deoxy-A to block the 3'OH end of RNA, and then the hEndoV protein cuts at the inosine to produce a new RNA 3'OH, and the new 3'OH is polyuridylated and library constructed, and sequencing is used to obtain the site information of inosine modification. The present invention has a direct principle and is easy to operate. There is no chemical reaction throughout the process to prevent RNA from degradation. No complex bioinformatics algorithm is required, and single-base resolution positioning information of inosine modification in RNA can be directly obtained.

Description

Technical Field

[0001] 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 Art

[0002] 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 in mRNA, tRNA, lncRNA, miRNA, and siRNA. Ino is produced by the hydrolysis and deamination of the amino group at the C6 position of adenosine catalyzed by the adenosine deaminase (ADAR) family proteins acting on RNA. This conversion is also called A-to-InoRNA editing. Currently, three ADAR proteins have been identified in mammals, ADAR1 and ADAR2 with enzymatic activity and ADAR3 without enzymatic activity.

[0003] A-to-Ino editing has important biological functions. Since Ino is recognized as G by the cellular machinery during reverse transcription due to pairing with C, A-to-Ino editing can change genetic information after transcription. A-to-Ino editing on mRNA may lead to amino acid changes, the generation or loss of splice sites and stop codons, resulting in changes in post-translational proteins, which is beneficial to protein diversity. Ino on tRNA is located at position 34 of the anticodon loop. Ino at this position allows recognition of multiple codons through wobble pairing, enhancing the decoding ability of tRNA. miRNA and siRNA can regulate target genes in a highly specific manner. A-to-Ino editing regulates the maturation, structure and function of miRNA and siRNA.

[0004] Abnormal A-to-Ino editing levels have been shown to be associated with a variety of human diseases, including various cancers, neurological and neurodegenerative diseases, mental disorders, and autoimmune diseases. Abnormal editing levels of A-to-Ino editing sites on some transcripts have been shown to be associated with the pathogenesis of the disease. For example, A-to-Ino editing of serotonin 2C receptor pre-mRNA occurs at five conserved sites in exon 5 (named A to E), the editing level of site A is increased in the brains of suicide victims of depression, and the editing level of site E is 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 entire transcriptome is the basis for in-depth understanding of the biological function and significance of each site.

[0005] At present, the main single-base localization method for transcriptome-wide A-to-Ino editing is RNA-seq. Ino pairs with C during the reverse transcription process so that it is read as G during sequencing. Therefore, the A-to-Ino editing site can be identified based on the A-to-G conversion. Although the principle is simple, in the actual analysis process, RNA-seq will produce a large number of false positive sites due to interference factors such as alignment errors, single nucleotide polymorphisms, somatic mutations, etc. Although bioinformatics methods such as statistical modeling, filtering and / or alignment, and integration of other genomic information have been developed to reduce false positives, the accuracy of RNA-seq in identifying A-to-Ino editing sites is still poor. One of the chemical methods for identifying A-to-Ino editing sites within the transcriptome is "ICE-seq", in which N produced by the reaction of Ino with acrylonitrile 1 -Cyanoethyl inosine hinders cDNA extension during reverse transcription, and the resulting truncated cDNA is screened out during library construction because the fragment is too short. The "erased" signal generated during 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 characteristic of nuclease T1 is to cut RNA at G or Ino. In the presence of borate, G forms a stable adduct with glyoxal to resist the cutting of nuclease T1, while Ino is cut by nuclease T1 because it cannot form a stable adduct. This method first oxidizes and biotinylates the 3' end of RNA through chemical reactions, then treats glyoxal and borate to protect the G on RNA, then fixes the RNA on streptavidin functional magnetic beads through biotin-streptavidin affinity, and nuclease T1 cuts RNA at Ino. The cut RNA is then used for 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 cut by nuclease T1, resulting in false positive sites. The common disadvantage of chemical methods is that the violent chemical reaction will cause severe degradation of RNA, so mild enzymatic reaction methods should be developed for single-base resolution positioning analysis of A-to-Ino editing.

[0006] hEndoV (human endonuclease V) is an endonuclease for Ino on RNA, which can specifically recognize and cleave Ino on RNA, and produce a 3'OH and 5' phosphate incision at the second phosphodiester bond at its downstream 3' end. Based on this characteristic, we propose an invention of an endonuclease-assisted single-base resolution localization method for Ino in RNA. Summary of the invention

[0007] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide an endonuclease-assisted single-base resolution positioning analysis method for inosine modification in RNA, that is, an Ino single-base resolution positioning analysis method that can be applied to the field of RNA modification sequencing, does not require chemical reagent treatment, does not require complex bioinformatics algorithms, and has high sensitivity, high selectivity, and is easy to operate.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A single-base resolution localization analysis method for inosine modification in RNA assisted by an endonuclease comprises the following steps:

[0010] (1) fragmenting RNA from cell or tissue samples;

[0011] (2) Blocking the 3'OH termini of the fragmented RNA: using polyA polymerase to catalyze 3'deoxy-ATP to add 3'deoxy-A to the 3'OH termini of the fragmented RNA to block the 3'OH termini of the RNA, and then purifying the RNA;

[0012] (3) using hEndoV protein to cleave the above 3'OH-terminated RNA to generate a new RNA 3'OH at the inosine position;

[0013] (4) 3' polyuridylation of the cut RNA: using polyU polymerase to catalyze the addition of UTP to the 3'OH end of RNA for 3' polyuridylation;

[0014] (5) using 3' polyuridine-modified RNA as a template, and using a reverse transcription primer with polyadenylation and a 3' linker to perform a reverse transcription reaction to obtain cDNA;

[0015] (6) using a primer with a 5' adapter to add a 5' adapter to the cDNA through a DNA polymerase extension reaction to obtain a DNA for amplification;

[0016] (7) performing PCR amplification using the above DNA as a template, and sequencing the amplified product;

[0017] (8) Identification of inosine-modified single bases: In the sequencing results, the second base G from 3' to 5' after removing the double-end adapter is the inosine-modified base.

[0018] The method of the invention obtains the single-base resolution positioning information of Ino by sequencing through the specific cleavage of Ino by hEndoV protein.

[0019] The hEndoV protein cleavage reaction was performed by using NEBuffer 4 (NEB, catalog number B7004) as the reaction buffer. The buffer system of the buffer reaction solution was Tris-CH3COOH, Mg 2+ The concentration is 10 mM, a certain amount of hEndoV protein is used (determined according to the actual activity of the protein), and the reaction conditions are preferably 37° C. for 30 minutes.

[0020] The hEndoV protein cleavage reaction mentioned above refers to the specific cleavage reaction of the hEndoV protein on Ino on RNA.

[0021] The advantages and beneficial effects of the present invention are as follows:

[0022] 1) The principle of the present invention is direct, the method is simple, and no complicated pre-treatment is required.

[0023] 2) The present invention does not require chemical reaction to treat RNA and will not cause severe degradation of RNA.

[0024] 3) The hEndoV protein cleavage reaction in the present invention can be completed in only 30 minutes, which greatly shortens the overall experimental time.

[0025] 4) The cleavage reaction involved in the present invention is highly specific (only Ino is cleaved, not A) and efficient (complete cleavage), which is conducive to positioning analysis.

[0026] 5) The RNA library construction method of the present invention does not require single-stranded ligation reaction, has high library construction efficiency, and is conducive to sequencing and site analysis.

[0027] 6) The present invention does not involve complex bioinformatics algorithms and is conducive to the identification of A-to-Ino editing sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The schematic diagram of the single-base resolution localization analysis method of inosine modification in RNA assisted by the endonuclease of the present invention. The 3'OH end of RNA is first blocked by 3'-deoxy-A, and then a new 3'OH is generated at the second phosphodiester bond of 3' of Ino by cleavage of hEndoV protein, and the newly generated 3'OH is subsequently 3' polyuridylated, and the single-base resolution localization analysis of Ino is achieved through library construction and sequencing.

[0029] Figure 2The PAGE gel images of the specific cleavage of the artificially synthesized RNA containing Ino modification by the hEndoV protein of the present invention. (A) Schematic diagram of hEndoV cleavage of RNA with Ino modification; (B) The cleavage effect of the hEndoV protein was evaluated using the synthetic RNA chain containing Ino modification (Ino-RNA-1 and Ino-RNA-2, the sequence is shown in Table 2) as a substrate; (C) The cleavage specificity of the hEndoV protein was evaluated using the RNA chain without Ino modification (A-RNA-1, the sequence is shown in Table 2) as a substrate.

[0030] Figure 3 This is a sequencing effect diagram of the single-base resolution positioning analysis of inosine modification in the artificially synthesized 45nt Ino-RNA-1 oligonucleotide chain in the present invention. DETAILED DESCRIPTION

[0031] In some embodiments, the endonuclease-assisted single-base resolution localization analysis method for inosine modification in RNA of the present invention specifically comprises the following steps:

[0032] (1) Fragmentation: Take 5 μg of mRNA extracted from biological sample tissues or cells and use NEBNext RNA Mg ++ The mRNA was fragmented into fragments of about 1000 nt in length using the fragmentation module (NEB, Cat. No. E6150).

[0033] (2) Blocking the 3'OH end of the above RNA: Add 5U polyA polymerase (NEB, Catalog No. M0276), 1× polyA reaction buffer (provided with the enzyme), 1mM 3'deoxy-ATP (Sigma-Aldrich, Catalog No. C9137) and 20U RNase inhibitor to the above mRNA. After reacting at 37°C for 30min, purify the RNA using RNA Clean & Concentrator Kits (ZYMORESEARCH, Catalog No. R1013) (elution with 10μL).

[0034] (3) hEndoV cleavage of the above RNA: a certain amount of hEndoV protein, 1×NEBuffer 4 buffer (NEB, catalog number B7004) and 20 U RNase inhibitor were added to the above 10 μL RNA to a final volume of 20 μL. The cleavage reaction was carried out at 37°C for 30 min.

[0035] The hEndoV protein expression process is as follows: the full-length coding sequence of hEndoV (see Table 1) is inserted into the pET-28a(+) plasmid to obtain the hEndoV-pET-28a(+) recombinant plasmid, which is transformed into Escherichia coli BL21(DE3); the conditions for inducing protein expression are IPTG concentration of 1 mM, induction temperature of 25°C, and induction time of 10 h; the protein is purified using BeyoGold His-tag Purification Resin (reduction-resistant chelating type) (Beyotime Biotechnology, product number P2210). The hEndoV protein activity evaluation process is as follows: a total of 10 μL reaction system 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, 20 U RNase inhibitor and 1×NEBuffer 4 buffer (NEB, Product No. B7004) was incubated at 37°C for 30 min, and then 1 μL 10×loading buffer (Takara, Product No. 9157) was added to the system to terminate the reaction. The reaction results were identified by 20% PAGE ( Figure 2 ).

[0036] (4) 3' polyuridylation of the above RNA: 1U polyU polymerase (NEB, Catalog No. M0337), 1×NEBuffer 2 buffer (provided with the enzyme), 100μM UTP and 20U RNase inhibitor were added to the above 20μL reaction system, and the final volume was 40μL. After this system was carried out at 37°C for 8 minutes, RNA was purified using RNA Clean & Concentrator Kits (ZYMORESEARCH, Catalog No. R1013) (20μL elution).

[0037] (5) Reverse transcription of the above RNA to synthesize cDNA: 30 μL of reverse transcription system consists of 20 μL of the above RNA, 200 μM-MuLV reverse transcriptase (Sangon Biotechnology, catalog number B500517), 1× RT reaction buffer (provided with the enzyme), 0.33 μM 3'adaptor-A 20 The system consisted of NC primer (sequence shown in Table 2), 0.5 mM dNTP and 20 U RNase inhibitor, and the reaction was carried out at 42°C for 90 min.

[0038] (6) Removal of RNA and purification of cDNA: 1 μL of nuclease I (NEB, catalog number M0293), 5 μL of nuclease I reaction buffer (provided with the enzyme), and 14 μL of H2O were added to the reverse transcription reaction (30 μL), and the reaction was carried out at 37°C for 30 min to remove excess reverse transcription primer; then, 10 μL of NaOH (1 M) and 10 μL of EDTA (0.5 M) were added to the exonuclease I reaction system (50 μL), and the reaction was carried out at 65°C for 15 min to remove RNA in the system, and cDNA was recovered using Oligo Clean & Concentrator Kits (ZYMORESEARCH, catalog number D4060) (10 μL elution).

[0039] (7) The above cDNA was extended to add a 5' linker: the reaction system (30 μL) consisted of 10 μL of the above cDNA, 5 U Bsu DNA polymerase (NEB, catalog number M0330), 1× NEBuffer 2 buffer (distributed with the enzyme), 0.33 μM 5'adaptor-N6 primer (sequence see Table 2), and 1 mM dNTP. The system was reacted at 37°C for 1 h.

[0040] (8) DNA purification: 1 μL of exonuclease I (NEB, Catalog No. M0293), 5 μL of exonuclease I reaction buffer (provided with the enzyme), and 14 μL of H2O were added to the previous system (30 μL). The reaction was incubated at 37°C for 30 min to remove excess 5'adaptor-N6 primer. The DNA was recovered using Oligo Clean & Concentrator Kits (ZYMO RESEARCH, Catalog No. D4060) (elution in 15 μL).

[0041] (9) PCR amplification of the above DNA: The PCR system (50 μL) consists of 15 μL of the above DNA, 2× amplification buffer (Yisheng Biotechnology, catalog number 10136ES01), and 0.5 μM P5 and P7 primers (sequences are shown in Table 2). 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; ②-④ steps are repeated 30 times; 72°C extension for 10 min, and stored at 4°C.

[0042] (10) The obtained amplified product is sequenced.

[0043] Table 1 hEndoV full-length coding sequence

[0044]

[0045] Table 2 Oligonucleotide chains involved in specific embodiments

[0046]

[0047] The following examples are used to further illustrate the present invention, but should not be construed as limiting 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 replacement methods and are included in the protection scope of the present invention.

[0048] 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.

[0049] Example 1: Standard synthetic RNA analysis

[0050] (1) Add 100 ng of artificially synthesized Ino-RNA-1 chain (sequence shown in Table 3), 5 U polyA polymerase, 1 mM 3'deoxy-ATP, 1× polyA reaction buffer and 20 U RNase inhibitor to a 10 μL reaction system, react at 37°C for 30 min, and then purify the RNA using a kit.

[0051] (2) hEndoV protein cleavage reaction of the above RNA: A certain amount of hEndoV protein, 1×NEBuffer 4 and 20 U 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 30 min.

[0052] (3) The above RNA was subjected to polyuridylation reaction. 1U polyU polymerase, 100μM UTP, 1×NEBuffer 2 and 20U RNase inhibitor were added to the above 20μL reaction system to a final volume of 40μL. This system was carried out at 37°C for 8 minutes, and the RNA was purified using a kit.

[0053] (4) The above RNA was subjected to reverse transcription reaction. The reverse transcription system (30 μL) included 10 μL of the above RNA, 0.33 μM 3'adaptor-A 20 GC primer (sequence in Table 3), 0.5mM dNTP, 200U reverse transcriptase, 1× reverse transcription buffer, 20U RNaseinhibitor, this system was reacted at 42°C for 90min. Since the standard synthetic RNA analysis system using Ino-RNA-1 is simple, the steps for recovering cDNA here are simplified to adding 1μL ribonuclease A to the reverse transcription system at the end of the reaction, reacting at 37°C for 30min to remove RNA, and recovering cDNA using the kit.

[0054] (5) The cDNA was subjected to a 5' adapter addition reaction. The 5' adapter addition reaction system (30 μL) included 10 μL of the cDNA, 5 U of Bsu DNA polymerase, 0.33 μM of 5'adaptor primer (sequence shown in Table 3), 1 mM dNTP, and 1× NEBuffer 2. The system was reacted at 37° C. for 1 h.

[0055] (6) Take the above reaction DNA and perform polymerase chain amplification reaction. The 25μL PCR reaction system consists of 2μL of the above DNA, 12.5μL 2× amplification buffer, 10μM P5, P7 primers (sequences see Table 3) 1.25μL each and 8μL H2O. Amplification cycle time program: ① 98℃ denaturation for 1min; ② 98℃ denaturation for 10sec; ③ 60℃ annealing for 25sec; ④ 72℃ extension for 50sec; ②-④ steps repeated 30 times; 72℃ extension for 10min, and stored at 4℃. The sample can be directly sequenced by Sanger sequencing, and the sequencing results are shown in Figure 3 , in the sequencing map, the second base from 3' to 5' after removing the adapter is G (marked by a box), and this site is the inosine modification in the initial Ino-RNA-1.

[0056] Table 3 Oligonucleotide chains involved in Example 1

[0057] Sequence Listing <110> Wuhan University <120> A single-base resolution localization analysis method for inosine modification in RNA assisted by endonuclease <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 846 <212> DNA <213> Homo sapiens <400> 1 atggccctgg aggcggcggg agggccgccg gaggaaacgc tgtcactgtg gaaacggggag 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

1. A single-base resolution localization analysis method for inosine modification in RNA assisted by an endonuclease, characterized in that: The following steps are involved: (1) Fragmenting RNA from cell or tissue samples; (2) Blocking the 3'OH end of the fragmented RNA: using polyA polymerase to catalyze 3'deoxy-ATP to add 3'deoxy-A to the 3'OH end of the fragmented RNA; (3) Using hEndoV protein to cleave the above 3'OH-terminated RNA; the reaction buffer for using hEndoV protein to cleave RNA is Tris-CH3COOH as the buffer system, and the Mg content in the buffer system is 2+ The concentration is 10mM; (4) 3' polyuridylation of the cut RNA: using polyU polymerase to catalyze UTP to add a U tail to the 3'OH end of RNA for 3' polyuridylation; (5) Using 3' polyuridine-modified RNA as a template, reverse transcription is performed using a reverse transcription primer with polyadenylation and a 3' linker to obtain cDNA; (6) using a primer with a 5' adapter to add a 5' adapter to the cDNA through a DNA polymerase extension reaction to obtain DNA for amplification; (7) performing PCR amplification using the above DNA as a template, and sequencing the amplified product; (8) Identification of inosine-modified single bases: The second base from 3' to 5' after removing the double-end adapter in the sequencing result is the inosine-modified base.

2. The method for single-base resolution localization analysis of inosine modification in RNA assisted by endonuclease according to claim 1, characterized in that: In step (3), the reaction conditions for RNA cleavage are 37°C for 30 minutes.

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