CN111434778A - A kind of RNA molecule m6A modification level detection method and application - Google Patents

Abstract

The present invention relates to a method for detecting the level of modification of RNA m6A in a biological or medical sample. The method is characterized in that the method is based on the characteristic that YTHDF2 protein is combined with RNA molecule m6A to interfere reverse transcription reaction of reverse transcriptase on the RNA molecule, and then the specific RNA molecule modified at a specific site m6A is detected by a PCR amplification technology. The method can be used for detection and analysis of m6A modified RNA molecules in biological or medical samples.

Description

A kind of RNA molecule m6A modification level detection method and application

Technical Field The present invention belongs to the field of biomedicine, and specifically relates to a method for detecting m6A-modified RNA molecules, which can be used for the detection and analysis of m6A-modified RNA molecules and the detection of biological and clinical specimens.

Background of the Invention RNA base modifications are ubiquitous in RNA molecules. Recent studies have shown that different base modifications in RNA molecules have important biological functions, and the dynamic modification of bases in RNA molecules is related to the occurrence and development of various diseases (Batista PJ. The RNA Modification N(6)-methyladenosine and Its Implications in Human Disease. Genomics Proteomics Bioinformatics. 2017, 15(3): 154-163). Therefore, more and more attention has been paid to the study of base modification of RNA molecules. At present, the most researched and relatively clear is the m6A modification of RNA molecules. However, the current analysis methods for the m6A modification of RNA molecules are mainly based on mA antibody-based RNA methylation (m6A) co-immunoprecipitation high-throughput sequencing (meRIP-seq) method to obtain data, and then use bioinformatics methods to analyze and obtain data. Information on m6A modification of RNA molecules (Dominissini D, et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature. 2012, 485(7397): 201-206; Chen K, et al. High-resolution N6-methyladenosine (m6A) map using photo-crosslinking-assisted m(6)A sequencing. Angew Chem Int Ed Engl. 2015, 54(5):1587-1590). Although a series of different methods have been derived based on the above methods, these methods are more suitable for analyzing the overall modification status of RNA molecules. At present, RNA molecular detection and analysis methods for specific site modification are still in development. Golovina et al. established a melting curve change detection method (Golovina AY, et al. Method for site-specific detection of m6A nucleoside presence in RNA based on high-resolutionmelting (HRM) analysis. Nucleic Acids Res. 2014, 42(4): e27 ), Hong et al. established an interference reverse transcription detection method based on the involvement of 4SedTTP and FTO (fat mass and obesity associated protein) (Hong T, et al. Precise Antibody-Independent m6A Identification via4SedTTPInvolved and FTO-Assisted Strategy at Single-Nucleotide Resolution . JAm Chem Soc, 2018, 140:5886-5889). These methods can detect m6A-modified RNAs at specific sites. These methods have their own characteristics, but they also have shortcomings.

Detection and analysis of changes in the level of m6A modification at specific sites of specific RNA molecules For the biological function study of m6A modified RNAs, and for detection and analysis of specific sites in specific RNA molecules in disease-related clinical samples, m6A modification is found in both disease diagnosis and treatment marker molecules. of great significance. Due to the high similarity between m6A and A, and in most cases, the proportion of m6A modification in RNA molecules is low, which makes it impossible for conventional detection and analysis methods to monitor and analyze m6A modification at specific sites in a simple, rapid, and large-scale manner. specific RNA molecules. Recent studies have found that a variety of protein molecules are involved in the m6A modification of RNA molecules, including modification enzymes, removal enzymes and related binding protein molecules. Among them, YTHDF2 (YTH domain-containing family protein 2) has a natural and specific function of binding m6A (Zhu T, et al. Crystalstructure of the YTH domain of YTHDF2 reveals mechanism for recognition of N6-methyladenosine. Cell Res. 2014, 24(12) : 1493-1496; Li F, et al. Structure of the YTH domain of human YTHDF2 in complex with an m(6)A mononucleotide reveals anaromatic cage for m(6)A recognition. Cell Res. 2014, 24(12): 1490 -1492). The characteristic of YTHDF2 binding to m6A provides conditions for the establishment of a method for detecting m6A-modified RNA molecules.

SUMMARY OF THE INVENTION The purpose of the present invention is to establish a simple method for detecting m6A-modified RNA molecules, to realize the detection of m6A-modified RNA molecules at specific sites, and to be suitable for the detection and analysis of biological samples and clinical samples. The detection method of the present invention is characterized in that the YTHDF2 protein is added to the RNA reverse transcription reaction system by utilizing the characteristic of YTHDF2 protein binding to the m6A methylation modification site in the RNA molecule, and the YTHDF2 protein molecule binds RNA with m6A modification The site, which in turn hinders the extension of the reverse transcription primer of the RNA reverse transcription reaction, obtains the reverse transcription product of a short fragment of cDNA that terminates before the m6A modification site. By designing a pair of PCR reaction primers spanning the m6A modification site, and a pair of PCR amplification primers on the 3' side of the m6A modification site of RNA molecules, the reverse transcription products were amplified with two pairs of primers, respectively. As a result, the detection and analysis of m6A modified RNA molecules is realized.

The m6A modified RNA molecule detection method includes two main steps: (1) reverse transcription reaction. One set of normal reverse transcription reactions, and one set of reverse transcription reactions with the addition of YTHDF2 protein bound to RNA molecules. (2) PCR reaction. PCR reactions were carried out using PCR primers spanning the m6A modification site and PCR amplification primers on the 3' side of the m6A modification site of the RNA molecule, respectively. According to the different ways of detecting amplified products by PCR reaction, ordinary PCR reaction can be used, or real-time quantitative PCR reaction can be used by adding fluorescent dyes or molecular beacons. Depending on the purpose of detecting RNA molecules, PCR detection can also be performed only by using the reverse transcription reaction product in which YTHDF2 protein is added and combined with RNA molecules.

In the method of the present invention, the YTHDF2 protein used can be isolated and purified from human cell lines and tissues, or it can be a fusion protein expressed in prokaryotic and eukaryotic expression systems. In this example, the recombinant human YTHDF2 protein with His tag isolated and purified in E. coli was used. In another specific reaction, the amount of YTHDF2 protein used depends on the amount of RNA used in the reverse transcription reaction and the degree of m6A modification in the RNA, which has achieved the blocking effect that meets the detection requirements. In a commonly used reverse transcription system, the amount of YTHDF2 protein can be several micrograms to several hundred micrograms.

In the reverse transcription reaction, the amount of reverse transcriptase and the amount of dNTPs have an impact on the efficiency of the reverse transcription reaction. Under normal circumstances, within a certain range, the increase in the amount of reverse transcriptase used and the increase in the concentration of dNTPs are beneficial to the reverse transcription reaction. The method of the present invention utilizes the property of YTHDF2 to bind the m6A modification site in the RNA molecule to block the reverse transcription reaction. In order to achieve the blocking effect required by the detection, the dosage of reverse transcriptase can be the recommended dosage of the commonly used reverse transcription kit, or it can be reduced to one-eighth or less of the recommended dosage. The concentration of dNTPs can be the amount recommended for use with reverse transcription kits, or it can be reduced to less than 1 μmol/L. It is also determined by the amount of RNA in the reverse transcription reaction and the degree of m6A modification of the RNA molecule, and the blocking effect that meets the detection requirements has been achieved.

The method for RNA m6A methylation detection provided by the present invention comprises measuring one or more RNAs in a sample. In the actual detection process, any one of the RNA molecules can be detected, and the m6A methylation of different RNA molecules can also be detected to determine the changes in the m6A methylation level of RNA molecules in the sample.

The RNA m6A detection method of the present invention is suitable for detection of body fluid samples, including urine, saliva, blood, blood derivatives, etc., as well as detection of cell and tissue samples. The preparation of RNA in different samples was carried out using conventional RNA preparation methods.

For the convenience of detection and use, it can be assembled into a kit according to this method, and the kit is composed of conventional reverse transcription reagents, PCR or real-time quantitative PCR reagents, and YTHDF2 protein.

The present invention is illustrated by the following examples, which are intended to be illustrative only and not to limit the present invention in any way. The specific experimental methods not mentioned in the examples are usually carried out according to conventional experimental methods.

The specific implementation method of the present invention will be described with reference to the accompanying drawings.

Description of drawings

Figure 1: Schematic diagram of the detection method of m6A-modified RNA molecules

Figure 2: Detection of synthetic m6A-modified RNA molecules

Figure 3: Detection of m6A-modified 18S rRNA and 28S rRNA

Example

Example 1

Detection of synthetic m6A-modified RNA molecules

1. Synthesize m6A modified RNA molecules, reverse transcription primers and PCR reaction primers

The synthesized RNA fragment contains m6A modification at position 40.

m6A modified RNA molecule (MeA): 5'-UCGGCCCACGGCCCUGGCGGAGCGCUGAGAAGACGGUCGA(m6A)CUUGACUAUCUAGAGGAAG.

RNA molecule without m6A modification (MeU): 5'-UCGGCCCACGGCCCUGGCGGAGCGCUGAGAAGACGGUCGACUUGACUAUCUAGAGGAAG.

Reverse transcription primer (MeA-U-RT primer): 5'-CTTCCTCTAGATAGTCAAGTTC.

PCR Forward primer: 5'-TCGGCCCACGGCCCTGGCGG.

PCR reverse primer (Reversed primer): 5'-CTTCCTCTAGATAGTCAAGTTC.

2. Reverse Transcription Reaction

The reverse transcription reaction was performed with the GoScript ^™ Reverse Transcriptase Reaction Kit. Take the mixture of MeA and MeU as the reverse transcription template. 20% and 10%. Take 100ng of RNA to be detected as reverse transcription templates, add 1 μL (0.5 μg) of reverse transcription primers (MeA-U-RT ptimer) respectively, make up to 5 μL with DEPC water, place in a metal bath at 70 °C for 5 min, and then place on ice. Place for 5 min; add 4 μg YTHDF2 protein (recombinant His-YTHDF2 fusion protein expressed in E. coli used in this experiment), incubate at 37° C. for 20 min, and centrifuge at 3000 r/min for 20 s. Add 4 μl 5×RT buffer, 1 μL dNTP (0.1 mM), 2.4 μl Mgcl ₂ (25 mM), 0.5 μl (20 U) RNase inhibitor, 0.5 μl (100 U) GoScriptTM reverse transcriptase to each tube, Make up to 20 μl of RNase water. In a metal bath at 25 °C for 5 min, 42 °C for 60 min, and 70 °C for 15 min, the obtained cDNA was stored at -20 °C or directly used in PCR reaction.

3. PCR reaction and agarose gel electrophoresis analysis

Real-time quantitative PCR reaction system components and dosage: 1 μL cDNA template, 10 μL 2×TaqPCR starMix, 0.5 μL (20 mM) PCR forward primer, 0.5 μL (20 mM) PCR reverse primer, add water to make up to 20 μL. The reaction conditions were: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30s, 60°C for 30s, 72°C for 30s, and amplification for 30 cycles. The PCR reaction products were subjected to agarose gel electrophoresis to analyze the results.

The test results are shown in Figure 2, which shows the results of three repeated tests. In this experiment, 4 μg of YTHDF2 protein was used to prevent reverse transcription of m6A-modified synthetic RNA fragments.

Embodiment 2

Detection of RNA molecules with m6A-modified 28S rRNA

1. Design of primers for reverse transcription reaction and PCR reaction

In order to detect the m6A modification at the 4190th position of 28S rRNA, synthetic reverse transcription reaction primers and PCR reaction primers were designed.

28S rRNA reverse transcription primer (28S-RT): 5'-AATCAAGATCAAGCGAGCTT. This primer is also the downstream primer of the PCR amplification reaction.

28S rRNA forward primer 1 (28S-Fu): 5'-GGCGCGACCCGCTCCGGGGA. This primer is located on the 5' side of the m6A modification site.

28S rRNA forward primer 2 (28S-Fd): 5'-CGCAGGTGTCCTAAGGCGAG. This primer is located on the 3' side of the m6A modification site.

2. Reverse transcription reaction, PCR reaction and agarose gel electrophoresis conditions are the same as those in Example 1

比较Rx/R0比值。2 μg of YTHDF2 protein was used in this experiment. The detection results are shown in Figure 3, and the detection of m6A-modified RNA is realized. The results are calculated as follows: ΔCt0=Ct0(Fu)-Ct0(Fd), ΔCtx=Ctx(Fu)-Ctx(Fd),

Compare the Rx/R0 ratio.

Claims (8)

1. An assay for detecting a modified RNA molecule of m6A, comprising: based on the total RNA in purified cells, tissues and body fluids, m6A binding protein YTHDF2 is added in the reverse transcription reaction of the total RNA, so that the total RNA is bound with m6A in RNA molecules to interfere the progress of the reverse transcription reaction, a cDNA fragment which is terminated before a m6A modification site is obtained, a pair of primers spanning the m6A modification site and a pair of primers at one side of the m6A modification site are used for respectively carrying out PCR reaction on the cDNA obtained by reverse transcription, and the Ct values of the two groups of PCR reactions are compared to achieve the aim of detecting the m6A modified RNA.

2. The detection method according to claim 1, wherein: comprises two main steps of (1) reverse transcription reaction; (2) and (3) carrying out PCR reaction.

3. The m 6A-binding protein YTHDF2 used in reverse transcription reaction according to claim 1, wherein: YTHDF2 protein is purified YTHDF2 protein, or recombinant YTHDF2 protein or YTHDF2 fusion protein, and the dosage of YTHDF2 protein in reaction is 1-100 micrograms.

4. The reverse transcription reaction according to claim 1, wherein: the dosage of the reverse transcriptase is from the conventional dosage of the reverse transcription reaction to one eighth of the conventional dosage; the amount of dNTPs used is from the conventional amount for a reverse transcription reaction to one percent of the conventional amount.

5. The primers for PCR reaction as set forth in claim 1, wherein: wherein, the upstream primer and the downstream primer in one pair of primers are respectively positioned at two sides of the modification site of the RNA molecule m6A, and the upstream primer and the downstream primer in the other pair of primers are respectively positioned at one side of the 3' end of the modification site of the RNA molecule m 6A.

6. The PCR reaction of claim 1, wherein: and adding fluorescent dye or molecular beacon into a reaction system of the PCR reaction to perform real-time quantitative PCR reaction.

7. Use of the detection method of any one of claims 1 to 6 in a m6A modified RNA molecule detection assay.

8. The method for detecting an RNA molecule of any of claims 1 to 6, wherein the sample comprises a cell, urine, saliva, blood derivative or tissue sample.

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