Use of composition in detecting n6-methyladenosine modification in mRNA bound with plant protein

Abstract

A use of a composition in detecting N6-methyladenosine modification in mRNA bound with a plant protein. The composition is composed of ethanol, sodium acetate, and glycogen, and, in the composition, the ratio of ethanol to sodium acetate to glycogen is 500 ul of ethanol to 3 mol of sodium acetate to 0.1 g of glycogen.

Description

Application of the composition in detecting N6-adenylate methylation modification in mRNA bound to plant proteins This application claims priority to a Chinese patent application filed with the Patent Office of China on December 27, 2023, with application number 202311826572.0 and invention name “Application of a composition for detecting N6-adenylate methylation modification in mRNA bound to plant proteins”, the entire contents of which are incorporated by reference into this application. Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of a composition in detecting N6-adenosine methylation modification in protein-bound mRNA. Background Art

[0002] RNA immunoprecipitation (RIP), a key technique in epigenetics in recent years, is primarily used to identify target protein-RNA interactions. RIP primarily focuses on a class of RNA-binding proteins (RBPs), identifying RNAs such as mRNAs and long noncoding RNAs through immunoprecipitation. RNA-protein interactions can regulate the functions of mRNAs and noncoding RNAs. Studying and analyzing chemical modifications near target protein-bound RNAs will contribute to our understanding of RNA biological functions.

[0003] N6-adenosine methylation modification (modification to obtain N6-methyladenosine, m 6 A, CAS number 1867-73-8) is the most abundant modification in eukaryotic mRNA and the first endogenous modification detected in mRNA. 6 A plays an important role in regulating gene expression and mRNA stability. 6 A affects almost all aspects of RNA metabolism, however, in plants 6 The mechanism by which A regulates plant development remains unclear. 6 A is essential for embryonic development, which is conserved in mammals and plants. 6 Loss of function of the A methyltransferase complex (including METTL3, METTL14, and WTAP) leads to embryonic lethality. 6 A methyltransferase (including MTA, MTB, FIP37 and VIR) genes lead to embryonic lethality, with embryonic development arrested at the globular stage. Restorative mutations in MTA or FIP37 further confirmed the m 6 The function of A in the plant embryonic development stage.

[0004] The existing methods for detecting N6-adenylic acid methylation near target protein-bound RNA in vivo have been mainly reported in animal experiments, but this technology has not been reported in plants. The RIP method used in animal experiments enriches less RNA modifications in plant experiments. The purpose of this study is to develop and optimize a quantitative detection method for m6-adenylic acid methylation near plant protein-bound RNA. 6 A. Abundance method. Summary of the Invention

[0005] A technical problem to be solved by the present invention is how to detect the abundance of protein-bound N6-adenylate methylation modification.

[0006] In order to solve the above technical problems, the present invention first provides an application of a composition in detecting N6-adenylic acid methylation modification in protein-bound mRNA, wherein the composition is composed of ethanol, sodium acetate and glycogen, and the ratio of ethanol, sodium acetate and glycogen in the composition is 500ul:3mol sodium acetate:0.1g glycogen.

[0007] The present invention also provides the above composition.

[0008] The present invention also provides a method for detecting the abundance of N6-adenylic acid methylation modification in protein-bound mRNA (i.e., a method for detecting the abundance of N6-adenylic acid methylation modification near the target protein-bound RNA in vivo), specifically a method for detecting the abundance of protein-bound N6-adenylic acid methylation modification by RIP (RNA immunoprecipitation) combined with LC-MS (liquid chromatography-mass spectrometry) or MS (liquid chromatography), wherein the method for detecting the abundance of protein-bound N6-adenylic acid methylation modification by RIP (RNA immunoprecipitation) combined with LC-MS (liquid chromatography-mass spectrometry) comprises the following steps:

[0009] S1. Extract plant mRNA;

[0010] S2. fragmenting the mRNA with metal ions to obtain a fragmented mRNA solution, and then adding the above-mentioned composition to promote mRNA precipitation to obtain purified fragmented mRNA;

[0011] S3. expressing the target protein of the plant in vitro;

[0012] S4, incubating the target protein described in S3 with the fragmented mRNA purified in S2 in vitro to obtain a protein-RNA complex;

[0013] S5, eluting and separating the protein-RNA complex to obtain a target mRNA solution;

[0014] S6. Adding the above-mentioned composition to the target mRNA solution to promote mRNA precipitation, thereby obtaining purified target mRNA;

[0015] S7, preparing the purified target mRNA in S6 into mononucleotides, further dephosphorylating, and then purifying the mononucleotides to obtain a sample for use in an apparatus;

[0016] S8, perform liquid chromatography-mass spectrometry detection, and use external standard method to detect N6-methyladenosine (m 6 A) and adenine ribonucleoside (rA) content are quantified, and the abundance of protein-bound N6-adenylic acid methylation modification of the biological sample is obtained by dividing the N6-methyladenosine content by the adenine ribonucleoside content;

[0017] The N6-methyladenosine content is quantified based on the chromatographic peak corresponding to the ion peak at m / z 282.1-150.1 at a collision energy of 16V;

[0018] The adenine ribonucleoside content is quantified based on the chromatographic peak corresponding to the ion peak with m / z of 268.1-136.1 at a collision energy of 10V.

[0019] The present invention also provides a method for detecting the abundance of protein-bound N6-adenylate methylation modification by combining RIP (RNA immunoprecipitation) with MS (liquid chromatography), comprising the following steps:

[0020] S1. Extract plant mRNA;

[0021] S2. fragmenting the mRNA with metal ions to obtain a fragmented mRNA solution, and then adding the above-mentioned composition to promote mRNA precipitation to obtain purified fragmented mRNA;

[0022] S3. expressing the target protein of the plant in vitro;

[0023] S4, incubating the target protein described in S3 with the fragmented mRNA purified in S2 in vitro to obtain a protein-RNA complex;

[0024] S5, eluting and separating the protein-RNA complex to obtain a target mRNA solution;

[0025] S6. Adding the above-mentioned composition to the target mRNA solution to promote mRNA precipitation, thereby obtaining purified target mRNA;

[0026] S7, preparing the purified target mRNA in S6 into mononucleotides, further dephosphorylating, and then purifying the mononucleotides to obtain a sample for use in an apparatus;

[0027] S8', perform liquid chromatography detection, and use external standard method to detect N6-methyladenosine (m 6The content of N6-methyladenosine (A) and adenine ribonucleoside (rA) was quantified, and the abundance of protein-bound N6-adenylic acid methylation modification of the biological sample was obtained by dividing the N6-methyladenosine content by the adenine ribonucleoside content; the mobile phase used in the liquid chromatography was composed of phase A and phase B, wherein the phase A was a 0.1% by volume formic acid aqueous solution (the solvent was water and the solute was formic acid), and the phase B was a 0.1% by volume formic acid acetonitrile solution (the solvent was acetonitrile and the solute was formic acid); the elution procedure used in the liquid chromatography was As follows: from 0 min to 8 min, the volume ratio of phase A to the mobile phase linearly decreases from 100% to 99%; from more than 8 min to 10 min, the volume ratio of phase A to the mobile phase linearly decreases from less than 99% to 94%; from more than 10 min to 15 min, the volume ratio of phase A to the mobile phase linearly decreases from less than 94% to 0%; from more than 15 min to 20 min, the volume fraction of phase A to the mobile phase linearly increases from 0% to 100%; the chromatographic column used in the liquid chromatography is a Thermo Scientific Hypersil GOLD aQ reverse phase column with a column length of 100 mm, an inner diameter of 2.1 mm, and a filler particle size of 1.9 μm; the column temperature of the chromatographic column is 25° C., and the flow rate of the mobile phase is 0.3 ml / min; the retention time of the chromatographic peak of the N6-methyladenosine acid is 10.5±1 min; and the retention time of the chromatographic peak of the adenine ribonucleotide is 5.5±1 min.

[0028] In the above method, the addition of the above composition to promote mRNA precipitation in S2 or S6 is performed by adding the composition of claim 1 to the fragmented mRNA solution or the target mRNA solution to obtain a reaction system, wherein the ethanol content in the reaction system is 2.5 L, the sodium acetate content is 0.3 M, and the glycogen content is 100 μg ml -1 .

[0029] In the above method, the above composition is added in S2 or S6 to promote mRNA precipitation, and the precipitation is performed at -80°C.

[0030] In the above method, the purified fragmented mRNA and the purified target mRNA must be dissolved in enzyme-free sterile water to prepare a solution before use.

[0031] In the above method, the metal ion described in S2 is a zinc ion.

[0032] In the above method, the fragmentation in S2 is to fragment the mRNA into 100 nt in size.

[0033] In the above method, the fragmentation in S2 is carried out in reaction system A, which is a liquid composed of the following substances: mRNA, Tris-HCl, zinc chloride and water; the content of the mRNA is 20 μg, the content of Tris-HCl is 100 mM, the content of zinc chloride is 100 mM, and the balance is water.

[0034] In the above method, the fragmentation in S2 was performed at 94° C. for 5 min and then terminated by adding 2 μl of 0.5 M EDTA to obtain a fragmented mRNA solution.

[0035] In the above method, the in vitro incubation combination in S4 is to incubate the reaction system B on ice for 1 hour, and then incubate with magnetic beads with His tags at 4°C for 2 hours with rotation; the reaction system B is a liquid composed of the following substances: the target protein, the purified fragmented mRNA, Tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA, CAS No. 60-00-4), ethylphenyl polyethylene glycol (NP-40, Product No. N8032), dithiothreitol (DTT, CAS No. 27565-41-9), ribonuclease inhibitor (RNasin, Promega product, catalog number: N2511) and water; the content of the target protein is 20 μg / μl, the content of the purified fragmented mRNA is 1 μg / μl, the content of Tris-HCl is 50 mM, the content of sodium chloride (NaCl) is 250 mM, the content of ethylenediaminetetraacetic acid (EDTA) is 0.4 mM, the mass percentage of ethylphenyl polyethylene glycol (NP-40) is 0.1%, the content of dithiothreitol (DTT) is 1 mM, the content of the ribonuclease inhibitor (RNasin) is 0.4 U / μl, and the balance is water.

[0036] In the above method, the protein-RNA complex is eluted and separated by rotating the protein-RNA complex at 4°C using buffer solution X to remove non-specific fragments, then digesting it with PK buffer at 37°C for 20 minutes, and finally incubating it with 200 μl PK-urea buffer at 37°C for 20 minutes to obtain the target mRNA solution.

[0037] In the above method, the buffer solution X is a liquid composed of the following substances: Tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA, CAS No. 60-00-4), ethylphenyl polyethylene glycol (NP-40, Catalog No. N8032-4), dithiothreitol (DTT, CAS No. 27565-41-9), ribonuclease inhibitor (RNasin, Promega Company, Catalog No.: N2511) and water; the content of the sodium chloride (NaCl) is 250 mM, the content of the ethylenediaminetetraacetic acid (EDTA) is 0.4 mM, the mass percentage content of the ethylphenyl polyethylene glycol (NP-40) is 0.1%, the content of the dithiothreitol (DTT) is 1 mM, the content of the ribonuclease inhibitor (RNasin) is 0.4 U / μl, and the balance is water.

[0038] In the above method, the PK buffer is a liquid composed of the following substances: Tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA), proteinase K (Proteinase K, CAS No. 39450-01-6) and water; the content of the Tris-HCl is 100mM, the content of the sodium chloride (NaCl) is 50mM, the content of the ethylenediaminetetraacetic acid (EDTA) is 10mM EDTA, the content of the proteinase K is 4μg / μl, and the balance is water.

[0039] In the above method, the PK-urea buffer is a liquid composed of the following substances: Tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA), urea and water; the content of the Tris-HCl is 100mM, the content of the sodium chloride (NaCl) is 50mM, the content of the ethylenediaminetetraacetic acid (EDTA) is 10mM EDTA, the content of the urea is 7M, and the balance is water.

[0040] In the above method, the preparation of mononucleotides in S7 is carried out in reaction system C, which is a liquid composed of the following substances: RNA, adenosine deaminase inhibitor, tetrahydrouridine, antioxidant, 2,6-di-tert-butyl-p-methylphenol, nuclease P1, phosphodiesterase I, sodium acetate, ZnCl2 and water; the RNA content is 500ng / 300μl, the adenosine deaminase inhibitor content is 0.005μg / μl, the tetrahydrouridine content is 0.05μg / μl, the antioxidant content is 0.1mM, the 2,6-di-tert-butyl-p-methylphenol content is 0.1mM, the nuclease P1 content is 1U, the phosphodiesterase I content is 0.01U, the sodium acetate content is 0.1mM, the ZnCl2 content is 0.0067mM, and the balance is water.

[0041] In the above method, the preparation of single nucleotides as described in S7 is carried out at 37°C for 3 hours.

[0042] In the above method, the dephosphorylation in S7 is performed using alkaline phosphatase.

[0043] In the above method, the dephosphorylation of S7 was carried out at 37°C for 30 minutes.

[0044] In the above method, the purification of single nucleotides in S7 is performed using an ultrafiltration tube with a molecular weight cutoff of 10 kDa.

[0045] In the above method, in the liquid chromatography-mass spectrometry detection described in S8, the mobile phase used in the liquid chromatography is composed of phase A and phase B, wherein phase A is a 0.1% by volume formic acid aqueous solution (solvent is water, solute is formic acid), and phase B is an acetonitrile solution of formic acid with a volume percentage of 0.1% formic acid (solvent is acetonitrile, solute is formic acid); the elution program used in the liquid chromatography is as follows: from 0 min to 8 min, the volume ratio of phase A to the mobile phase decreases linearly from 100% to 99%; from greater than 8 min to 10 min, the volume ratio of phase A to the mobile phase decreases linearly from less than 99% to 94%; from greater than 10 min to 15 min, the volume ratio of phase A to the mobile phase decreases linearly from less than 94% to 0%; from greater than 15 min to 20 min, the volume fraction of phase A to the mobile phase increases linearly from 0% to 100%, and the chromatographic column used in the liquid chromatography is a Thermo Scientific Hypersil GOLD aQ reverse phase The column has a length of 100 mm, an inner diameter of 2.1 mm, and a filler particle size of 1.9 μm. The column temperature of the chromatographic column is 25° C., and the flow rate of the mobile phase is 0.3 ml / min. The retention time of the chromatographic peak of N6-methyladenosine is 10.5±1 min; the retention time of the chromatographic peak of adenine ribonucleotide is 5.5±1 min.

[0046] LC-MS / MS is considered to be an accurate and fast means of detecting RNA chemical modifications, and this technology has been widely used in the field of epigenetic biology. This technology detects the molecular weight and peak time of mononucleotide bases in the sample by comparing with the standard, and then identifies the relevant chemical modifications. Compared with the detection of specific chemical modification antibodies, LC-MS / MS has the advantages of fast time, low cost and high safety factor. The present invention discloses for the first time the detection of plant protein binding to mRNA on plant body by RIP combined with LC-MS. 6The detection method of the present invention can enrich more RNA modifications than the existing animal protein detection technology, greatly optimizes the existing method for detecting RNA modifications near the target protein and reduces the use of harmful reagents, and provides an excellent detection method for accurately detecting modifications near plant protein-bound RNA fragments, further improving the detection of plant protein-bound RNA mRNA. 6 A modification detection accuracy. m 6 A modification has been reported to play an important role in plant growth and development and response to adverse stress. 6 The detection method of A modification is not only of great value for studying plant RNA epigenetics, but also for RNA epitranscriptome breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is an electrophoresis diagram of mRNA nucleic acid after fragmentation in Example 1 and Example 2. The nucleic acid maker is 5000bp, 3000bp, 2500bp, 1500bp, 1000bp, 750bp, 500bp, 200bp, and 100bp from top to bottom.

[0048] FIG2 is a diagram of the in vitro purified proteins in Example 1 and Example 2, and the protein size is between 30-35 kd.

[0049] Figure 3 shows the detection of m in Example 1 6 The peak graph output by A, the horizontal axis is the retention time (min), that is, m 6 The peak time of A is 10.5 min.

[0050] FIG4 is a peak diagram of the output of rA in Example 1, where the abscissa represents the retention time (min), that is, the peak time of rA is 5.5 min.

[0051] Figure 5 shows the m in Example 2 6 The peak graph of A output, the horizontal axis is the retention time (min), that is, the peak time of rA is 10.5min.

[0052] FIG6 is a peak diagram of the output of rA in Example 2, where the abscissa represents the retention time (min), i.e., the peak time of rA is 5.5 min.

[0053] Figure 7 is a comparison of the changes in m6A enrichment in Example 1 and Example 2. Taking Example 1 before optimization as the control, the m6A enrichment in Example 2 after optimization increased by 40 times (P = 2.44495E -06 ).

[0054] Figure 8 is a comparison of rA enrichment changes in Example 1 and Example 2. Taking Example 1 before optimization as the control, rA enrichment in Example 2 after optimization increased by 51 times (P = 1.57716E -07 ). DETAILED DESCRIPTION

[0055] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and do not in any way limit the present invention.

[0056] Unless otherwise noted, the experimental methods in the following examples are conventional methods, performed according to the techniques and conditions described in literature in the field or according to product specifications. Materials and reagents used in the following examples are commercially available unless otherwise noted. Quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0057] In the following examples, N6-methyladenosine (m 6 A) is a product of Shanghai Yuanye Biotechnology Co., Ltd., catalog number 1867-73-8. Adenine ribonucleotide (rA) is a product of Shanghai Yuanye Biotechnology Co., Ltd., catalog number 58-61-7. The expression strain BL21 is a product of Nanjing Novozymes Biotechnology Co., Ltd., catalog number C504-02.

[0058] The use of mass spectrometry multiple reaction monitoring (MRM) technology is a technology that acquires data in a targeted manner based on known or assumed information and collects mass spectrometry signals. It has the advantages of sensitivity, accuracy and specificity.

[0059] Liquid chromatography-mass spectrometry was used for detection. The following examples specifically used an Agilent 1260 liquid chromatography-G6400 series triple quadrupole mass spectrometer. The target detection object is m 6 A and rA.

[0060] In mass spectrometry, N6-methyladenosine (m 6 A) m / z parameter was set to 282.1-150.1, and Collision Energy parameter was set to 16; adenine ribonucleotide (rA) m / z parameter was set to 268.1-136.1, and Collision Energy parameter was set to 10; specifically, the "m / z" parameter and "CID" parameter in the mass spectrometer were set according to Table 1.

[0061] Table 1 Mass spectrometry parameters

[0062]

[0063] In Table 1, m 6 A stands for N6-methyladenylic acid, and rA stands for adenine ribonucleotide.

[0064] The liquid chromatography detection column was a Thermo Scientific Hypersil GOLD aQ reverse phase column (100 x 2.1 mm, 1.9 μm). The column temperature was 25°C. The mobile phases A and B consisted of 0.1% (v / v) formic acid in water and 0.1% formic acid in acetonitrile. The mobile phase flow rate was 0.3 ml / min.

[0065] The elution process of liquid chromatography is shown in Table 2: from 0 min to 8 min, the volume ratio of phase A in the mobile phase linearly decreases from 100% to 99%; from 8 min to 10 min, the volume ratio of phase A in the mobile phase linearly decreases from 99% to 94%; from 10 min to 15 min, the volume ratio of phase A in the mobile phase linearly decreases from 94% to 0%; from 15 min to 20 min, the volume ratio of phase A in the mobile phase linearly increases from 0% to 100%.

[0066] Table 2 Elution process

[0067]

[0068] Set the "m / z" parameters and "CID" parameters in the mass spectrometer according to Table 1 and the elution process of the liquid chromatography in Table 2. 6 The retention time of the peak of standard A is 10.5 min. If the sample has a peak at 10.5 ± 1 min, it can be determined that the compound corresponding to the peak is m 6 A. The peak of the rA standard substance has a retention time of 5.5 min. If the sample has a peak at 5.5 ± 1 min, it can be determined that the compound corresponding to the peak is rA.

[0069] Relative abundance indicates the intensity of ions. The strongest peak (base peak) is set as 100, and the relative intensities of other peaks are determined based on this.

[0070] m 6 Relative abundance of A = m in the peak diagram 6 The peak area corresponding to A / the peak area corresponding to rA in the peak shape diagram.

[0071] Preparation of concentration gradient of N6-methyladenosine (m 6A), the concentrations were 1ng / ml, 10ng / ml, 50ng / ml, 100ng / ml, and the established m 6 A standard curve is linearly distributed from 1ng / ml to 100ng / ml, with a correlation coefficient R 2 The correlation coefficient R was 0.96 or higher, indicating that the prepared standard curve had a strong feasibility and linear relationship. A concentration gradient of adenine ribonucleoside (rA) was prepared, with concentrations of 1 ng / ml, 10 ng / ml, 50 ng / ml, and 100 ng / ml, respectively. The rA standard curve established was linearly distributed from 1 ng / ml to 100 ng / ml, with a correlation coefficient R 2 Above 0.95, it shows that the prepared standard curve has strong feasibility and linear relationship.

[0072] Example 1 Optimization of in vitro RIP combined with LC-MS / MS for the detection of target protein binding to mRNA 6 A modification

[0073] Z1. Extract plant mRNA;

[0074] The total RNA in corn leaves was enriched using a commercial plant RNA extraction kit (Nanjing Novozymes Biotech Co., Ltd., Product No.: rA101) to obtain corn leaf total RNA, and then mRNA was purified and enriched from the corn leaf total RNA using a commercial mRNA purification kit (Nanjing Novozymes Biotech Co., Ltd., Product No.: N403-01) to obtain purified mRNA.

[0075] Z2, fragmenting the purified mRNA with metal ions to obtain a fragmented mRNA solution;

[0076] The fragmentation is carried out in reaction system A, which is a liquid composed of the following substances: mRNA, Tris-HCl, zinc chloride and water; the content of the mRNA is 20 μg, the content of Tris-HCl is 100 mM, the content of zinc chloride is 100 mM, and the balance is water.

[0077] The fragmentation was carried out at 94°C for 5 minutes and then terminated by adding 2 μl of 0.5 M EDTA to obtain a fragmented mRNA solution. The nucleic acid electrophoresis diagram of the fragmented mRNA is shown in FIG1 .

[0078] Z3. expressing the target protein of the plant in vitro;

[0079] The constructed target protein zc1 (zc1 in this example is the protein encoded by the maize Zm00001d028655 gene) was conjugated to a His-tagged fusion protein encoding gene (SEQ ID No. 1, 5071-5973, is the coding sequence of the fusion protein, SEQ ID No. 1 is the vector sequence for expressing the fusion protein, and SEQ ID No. 2 is the amino acid sequence of the fusion protein). The fusion protein encoding gene was transformed into the expression strain BL21 and induced overnight at 16° C. using 1 M isopropyl-β-D-thiogalactopyranoside IPTG (product of Beijing Solebaud Technology Co., Ltd., catalog number: I8070). After induction, the supernatant was collected by sonication to obtain the protein, incubated with commercial His-tagged magnetic beads (product of Beijing Solebaud Technology Co., Ltd., catalog number: M2300), and the zc1 protein was eluted with imidazole to obtain the target protein after in vitro purification. The protein image is shown in Figure 2. The protein size is between 30 and 35 kd.

[0080] Z4, incubating the target protein described in Z3 with the fragmented mRNA described in Z2 in vitro to obtain a protein-RNA complex;

[0081] The in vitro purified target protein (see Z3) and fragmented mRNA solution (see Z2) were added to a buffer solution (50 mM Tris-HCl pH 7.5, 250 mM NaCl, 0.4 mM EDTA, 0.1% NP-40, 1 mM DTT, 0.4 U / μl RNasin (Promega, Catalog No.: N2511)) to form a reaction system B (100 μl system). The reaction system B is a liquid composed of the following substances: target protein, fragmented mRNA, Tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA, CAS No. 60-00-4), ethylphenyl polyethylene glycol (NP-40, Catalog No.: N8032-4), dithiothreitol (DTT, CAS No. 27565-41-9), ribonuclease inhibitor (RNasin, Promega, Catalog No.: N2511) and water. The content of the target protein is 20 μg / μl, the content of the fragmented mRNA is 1 μg / μl, the content of Tris-HCl is 50 mM, the content of sodium chloride (NaCl) is 250 mM, the content of ethylenediaminetetraacetic acid (EDTA) is 0.4 mM, the mass percentage of ethylphenyl polyethylene glycol (NP-40) is 0.1%, the content of dithiothreitol (DTT) is 1 mM, the content of ribonuclease inhibitor (RNasin) is 0.4 U / μl, and the balance is water.

[0082] The reaction system B was incubated on ice for 1 hour, and then incubated with commercial magnetic beads with a His tag at 4° C. for 2 hours under rotation to obtain a protein-RNA complex.

[0083] Z5, eluting and separating the protein-RNA complex to obtain a target mRNA solution;

[0084] The protein-RNA complex was eluted and separated by rotating the protein-RNA complex at 4°C to remove non-specific fragments using buffer solution X, then digested with PK buffer at 37°C for 20 minutes, and finally incubated with 200 μl PK-urea buffer at 37°C for 20 minutes to obtain the target mRNA solution.

[0085] The buffer solution X is a liquid composed of the following substances: Tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA, CAS No. 60-00-4), ethylphenyl polyethylene glycol (NP-40, Catalog No.: N80324), dithiothreitol (DTT, CAS No. 27565-41-9), ribonuclease inhibitor (RNasin, Promega Company, Catalog No.: N2511) and water; the content of the sodium chloride (NaCl) is 250 mM, the content of the ethylenediaminetetraacetic acid (EDTA) is 0.4 mM, the mass percentage of the ethylphenyl polyethylene glycol (NP-40) is 0.1%, the content of the dithiothreitol (DTT) is 1 mM, the content of the ribonuclease inhibitor (RNasin) is 0.4 U / μl, and the balance is water.

[0086] The PK buffer is a liquid composed of the following substances: Tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA), proteinase K (Proteinase K, CAS No. 39450-01-6) and water; the content of the Tris-HCl is 100mM, the content of the sodium chloride (NaCl) is 50mM, the content of the ethylenediaminetetraacetic acid (EDTA) is 10mM EDTA, the content of the proteinase K is 4μg / μl, and the balance is water.

[0087] The PK-urea buffer is a liquid composed of the following substances: Tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA), urea and water; the content of the Tris-HCl is 100mM, the content of the sodium chloride (NaCl) is 50mM, the content of the ethylenediaminetetraacetic acid (EDTA) is 10mM EDTA, the content of the urea is 7M, and the balance is water.

[0088] Z6.mRNA enrichment

[0089] 1 ml of phenol:chloroform mixture (Mock product, catalog number: P3803) was added to the target mRNA solution for extraction. The supernatant was removed by centrifugation at 12000 rpm at 4°C for 15 min, and 1 ml of anhydrous ethanol and a final concentration of 100 μg ml were added. -1 The glycogen was centrifuged again at 12000 rpm at 4°C for 15 min, and the supernatant was removed. The excess anhydrous ethanol was dried and the obtained precipitate was the enriched target mRNA.

[0090] Z7, preparing the enriched target mRNA described in Z6 into mononucleotides, further dephosphorylating, and then purifying the mononucleotides to obtain a sample for use in an apparatus;

[0091] Take the enriched target mRNA obtained by Z6, add 15 μl of enzyme-free sterile water to prepare a solution, and then prepare single nucleotides.

[0092] The preparation of the mononucleotide is carried out in reaction system C (300 μl system), which is a liquid composed of the following substances: RNA (provided by the nucleic acid sample), adenosine deaminase inhibitor, tetrahydrouridine, antioxidant, 2,6-di-tert-butyl-p-methylphenol, nuclease P1, phosphodiesterase I, sodium acetate, ZnCl2 and water; the RNA content is 500 ng / 300 μl, the adenosine deaminase inhibitor content is 0.005 μg / μl, the tetrahydrouridine content is 0.05 μg / μl, the antioxidant content is 0.1 mM, the 2,6-di-tert-butyl-p-methylphenol content is 0.1 mM, the nuclease P1 content is 1 U, the phosphodiesterase I content is 0.01 U, the sodium acetate content is 0.1 mM, the ZnCl2 content is 0.0067 mM, and the balance is water.

[0093] The specific adenosine deaminase inhibitor used in this example is erythro-9-(2-Hydroxy-3-nonyl)adenine (EHMA) hydrochloride, a product of Sigma, catalog number E114.

[0094] The specific antioxidant used in this example is deferoxamine, a product of Sigma-Aldrich (Shanghai) Trading Co., Ltd., product number Y0001937.

[0095] The specific phosphodiesterase I used in this example is snake venom phosphodiesterase I, a product of Shanghai Yuanye Biotechnology Co., Ltd., product number S10225.

[0096] When the nucleic acid sample is an RNA sample, the reaction conditions are: 37°C, 3 hours.

[0097] 10 U of alkaline phosphatase was added to the system for reaction.

[0098] The specific alkaline phosphatase used in this example is calf intestinal alkaline phosphatase (CIP), NEB, catalog number MO290S.

[0099] Reaction conditions: 37°C, 30 minutes.

[0100] The system was added to a 10KD ultrafiltration tube, centrifuged at 13,000 rpm for 10 min, and the filtrate was collected as the upper sample obtained from the corn leaves.

[0101] Z8, perform liquid chromatography-mass spectrometry detection, use external standard method to detect N6-methyladenosine (m 6 A) and adenine ribonucleoside (rA) contents were quantitatively analyzed.

[0102] The mass spectrometer "m / z" and "CID" parameters were set according to Table 1 and the elution process of the liquid chromatography in Table 2 to detect the sample obtained from the corn leaves in S7. 6 The peak shape diagram of A output is shown in Figure 5. In Figure 5, the horizontal axis is the retention time (min), i.e., m 6 The peak time of A is 10.5 min. The mass spectrum output by rA is shown in Figure 6, where the abscissa is the retention time (min), that is, the peak time of rA is 5.5 min.

[0103] Calculate the m of corn leaves 6 The relative abundance of A is 0.00249.

[0104] According to the established m 6 A standard curve and rA standard curve were used to obtain the m 6 The absolute content of A was 0.00249 ng / ml, and the absolute content of rA was 0.2848 ng / ml.

[0105] Example 2 Optimized in vitro RIP combined with LC-MS / MS to detect mRNA mRNA near the target protein 6 A modification

[0106] S1. Extract plant mRNA;

[0107] Total RNA in corn leaves was enriched using a commercial plant RNA extraction kit (Nanjing Novozymes Biotech Co., Ltd., catalog number: rA101), and then mRNA was purified and enriched from the total RNA in corn leaves using a commercial mRNA purification kit (Nanjing Novozymes Biotech Co., Ltd., catalog number: N403-01) to obtain purified mRNA.

[0108] S2. fragmenting the mRNA with metal ions to obtain a fragmented mRNA solution, and then adding a composition to promote mRNA precipitation to obtain purified fragmented mRNA;

[0109] The fragmentation is carried out in reaction system A, which is a liquid composed of the following substances: mRNA, Tris-HCl, zinc chloride and water; the content of the mRNA is 20 μg, the content of Tris-HCl is 100 mM, the content of zinc chloride is 100 mM, and the balance is water.

[0110] The fragmentation was carried out at 94° C. for 5 minutes and then terminated by adding 2 μl of 0.5 M EDTA to obtain a fragmented mRNA solution.

[0111] At this time, the mRNA is fragmented to a size of 100 nt. The nucleic acid electrophoresis diagram of the fragmented mRNA is shown in Figure 1.

[0112] Compared with Example 1, the optimization here is as follows: adding a composition (in the composition, the ratio of ethanol, sodium acetate and glycogen is 500ul: 3mol sodium acetate: 0.1g glycogen) to promote mRNA precipitation.

[0113] A composition was added to promote mRNA precipitation, wherein a reaction system was obtained by adding a composition consisting of ethanol (100% by volume), a 3M sodium acetate aqueous solution (pH 5.2) (Beijing Solebold Technology Co., Ltd., product number: A1070), and glycogen (product of Thermo Fisher Scientific, product number: AM9510) to the fragmented mRNA solution, wherein the ethanol content in the reaction system was 500 L, the sodium acetate content was 3 M, and the glycogen content was 100 μg ml -1 .

[0114] After overnight at -80°C, the mixture was further centrifuged at 15,000 g for 25 min at 4°C, the supernatant was discarded, and 1 ml of 75% ethanol was added to wash the precipitate. The mixture was centrifuged at 15,000 g for 15 min at 4°C, and the supernatant was discarded. The resulting precipitate was the purified fragmented mRNA.

[0115] S3. expressing the target protein of the plant in vitro;

[0116] The constructed target protein zc1 (zc1 in this example is the protein encoded by the maize Zm00001d028655 gene) was conjugated to a His-tagged fusion protein encoding gene (SEQ ID No. 1, 5071-5973, is the coding sequence of the fusion protein, SEQ ID No. 1 is the vector sequence for expressing the fusion protein, and SEQ ID No. 2 is the amino acid sequence of the fusion protein). The fusion protein encoding gene was transformed into the expression strain BL21 and induced overnight at 16° C. using 1 M isopropyl-β-D-thiogalactopyranoside IPTG (product of Beijing Solebaud Technology Co., Ltd., catalog number: I8070). After induction, the supernatant was collected by sonication to obtain the protein, and the supernatant was incubated with commercial His-tagged magnetic beads (product of Beijing Solebaud Technology Co., Ltd., catalog number: M2300). The zc1 protein was eluted with imidazole to obtain the target protein after in vitro purification. The protein image is shown in FIG2 . The protein size is between 30 and 35 kd.

[0117] S4, incubating the target protein described in S3 with the fragmented mRNA purified in S2 in vitro to obtain a protein-RNA complex;

[0118] When the purified fragmented mRNA is used, 15 μl of enzyme-free sterile water is added to obtain a purified fragmented mRNA solution.

[0119] The target protein (see S3) and purified fragmented mRNA solution after in vitro purification were added to a buffer solution (50 mM Tris-HCl pH 7.5, 250 mM NaCl, 0.4 mM EDTA, 0.1% NP-40, 1 mM DTT, 0.4 U / μl RNasin (Promega, Catalog No.: N2511)) to form reaction system B (1000 μl system). The reaction system B is a liquid composed of the following substances: target protein, purified fragmented mRNA, Tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA, CAS No. 60-00-4), ethylphenyl polyethylene glycol (NP-40, Catalog No.: N8032-4), dithiothreitol (DTT, CAS No. 27565-41-9), ribonuclease inhibitor (RNasin, Promega, Catalog No.: N2511) and water. The content of the target protein is 20 μg / μl, the content of the purified fragmented mRNA is 1 μg / μl, the content of Tris-HCl is 50 mM, the content of sodium chloride (NaCl) is 250 mM, the content of ethylenediaminetetraacetic acid (EDTA) is 0.4 mM, the mass percentage of ethylphenyl polyethylene glycol (NP-40) is 0.1%, the content of dithiothreitol (DTT) is 1 mM, the content of ribonuclease inhibitor (RNasin) is 0.4 U / μl, and the balance is water.

[0120] The reaction system B was incubated on ice for 1 hour, and then incubated with commercial magnetic beads with a His tag at 4° C. for 2 hours under rotation to obtain a protein-RNA complex.

[0121] S5, eluting and separating the protein-RNA complex to obtain a target mRNA solution;

[0122] The protein-RNA complex was eluted and separated by rotating the protein-RNA complex at 4°C to remove non-specific fragments using buffer solution X, then digested with PK buffer at 37°C for 20 minutes, and finally incubated with 200 μl PK-urea buffer at 37°C for 20 minutes to obtain the target mRNA solution.

[0123] The buffer solution X is a liquid composed of the following substances: Tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA, CAS No. 60-00-4), ethylphenyl polyethylene glycol (NP-40, Catalog No.: N8032), dithiothreitol (DTT, CAS No. 27565-41-9), ribonuclease inhibitor (RNasin, Promega Company, Catalog No.: N2511) and water; the content of the sodium chloride (NaCl) is 250 mM, the content of the ethylenediaminetetraacetic acid (EDTA) is 0.4 mM, the mass percentage of the ethylphenyl polyethylene glycol (NP-40) is 0.1%, the content of the dithiothreitol (DTT) is 1 mM, the content of the ribonuclease inhibitor (RNasin) is 0.4 U / μl, and the balance is water.

[0124] The PK buffer is a liquid composed of the following substances: Tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA), proteinase K (Proteinase K, CAS No. 39450-01-6) and water; the content of the Tris-HCl is 100mM, the content of the sodium chloride (NaCl) is 50mM, the content of the ethylenediaminetetraacetic acid (EDTA) is 10mM EDTA, the content of the proteinase K is 4μg / μl, and the balance is water.

[0125] The PK-urea buffer is a liquid composed of the following substances: Tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA), urea and water; the content of the Tris-HCl is 100mM, the content of the sodium chloride (NaCl) is 50mM, the content of the ethylenediaminetetraacetic acid (EDTA) is 10mM EDTA, the content of the urea is 7M, and the balance is water.

[0126] S6. Adding the composition to the target mRNA solution to promote mRNA precipitation, thereby obtaining purified target mRNA;

[0127] Compared with Example 1, the optimization here is as follows: adding a composition (in the composition, the ratio of ethanol, sodium acetate and glycogen is 500ul: 3mol sodium acetate: 0.1g glycogen) to promote mRNA precipitation.

[0128] The composition was added to promote mRNA precipitation, and ethanol (100% by volume), 3M sodium acetate aqueous solution (pH 5.2) (Beijing Solebold Technology Co., Ltd., catalog number: A1070), and glycogen (Thermo Fisher Scientific, catalog number: AM9510) were added to the target mRNA solution; the resulting composition was used to obtain a reaction system, wherein the ethanol content in the reaction system was 2.5 L, the sodium acetate content was 0.3 M, and the glycogen content was 100 μg ml -1 .

[0129] S7, preparing the purified target mRNA in S6 into mononucleotides, further dephosphorylating, and then purifying the mononucleotides to obtain a sample for use in an apparatus;

[0130] Take the purified target mRNA obtained in S6, add 15 μl of enzyme-free sterile water to prepare a solution, and then prepare the single nucleotide.

[0131] The preparation of the mononucleotide is carried out in reaction system C (300 μl system), which is a liquid composed of the following substances: RNA (provided by the nucleic acid sample), adenosine deaminase inhibitor, tetrahydrouridine, antioxidant, 2,6-di-tert-butyl-p-methylphenol, nuclease P1, phosphodiesterase I, sodium acetate, ZnCl2 and water; the RNA content is 500 ng / 300 μl, the adenosine deaminase inhibitor content is 0.005 μg / μl, the tetrahydrouridine content is 0.05 μg / μl, the antioxidant content is 0.1 mM, the 2,6-di-tert-butyl-p-methylphenol content is 0.1 mM, the nuclease P1 content is 1 U, the phosphodiesterase I content is 0.01 U, the sodium acetate content is 0.1 mM, the ZnCl2 content is 0.0067 mM, and the balance is water.

[0132] The specific adenosine deaminase inhibitor used in this example is erythro-9-(2-Hydroxy-3-nonyl)adenine (EHMA) hydrochloride, a product of Sigma, catalog number E114.

[0133] The specific antioxidant used in this example is deferoxamine, a product of Sigma-Aldrich (Shanghai) Trading Co., Ltd., product number Y0001937.

[0134] The specific phosphodiesterase I used in this example is snake venom phosphodiesterase I, a product of Shanghai Yuanye Biotechnology Co., Ltd., product number S10225.

[0135] When the nucleic acid sample is an RNA sample, the reaction conditions are: 37°C, 3 hours.

[0136] 10 U of alkaline phosphatase was added to the system for reaction.

[0137] The specific alkaline phosphatase used in this example is calf intestinal alkaline phosphatase (CIP), NEB, catalog number MO290S.

[0138] Reaction conditions: 37°C, 30 minutes.

[0139] The system was added to a 10KD ultrafiltration tube, centrifuged at 13,000 rpm for 10 min, and the filtrate was collected as the upper sample obtained from the corn leaves.

[0140] S8, perform liquid chromatography-mass spectrometry detection, and use external standard method to detect N6-methyladenosine (m 6 A) and adenine ribonucleoside (rA) contents were quantitatively analyzed.

[0141] The mass spectrometer "m / z" and "CID" parameters were set according to Table 1 and the elution process of the liquid chromatography in Table 2 to detect the sample obtained from the corn leaves in S7. 6 The peak shape diagram of A output is shown in Figure 5. In Figure 5, the horizontal axis is the retention time (min), i.e., m 6 The peak time of A is 10.5 min. The mass spectrum output by rA is shown in Figure 6, where the abscissa is the retention time (min), that is, the peak time of rA is 5.5 min.

[0142] Calculate the m of corn leaves 6 The relative abundance of A is 0.1050.

[0143] According to the established m 6 A standard curve and rA standard curve were used to obtain the m 6 The absolute content of A was 0.1050 ng / ml, and the absolute content of rA was 13.5550 ng / ml.

[0144] By comparing the enriched m before and after optimization of Example 1 and Example 2 6 The relative abundance of A (implementation 1: 0.00249 ng / ml, implementation 2: 0.1050 ng / ml) and the absolute content of rA (implementation 1: 0.2848 ng / ml, implementation 2: 13.5550 ng / ml), the optimized m 6 The A modification ratio was 40 times higher than before optimization (see Figure 7), and the absolute content of rA was 51 times higher after optimization (see Figure 8). In addition, the optimized experimental method replaced phenol and chloroform with sodium acetate and glycogen, which greatly reduced the risk during the experimental operation.

[0145] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific examples are provided herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made using conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

[0146] SEQ ID No. 1:

[0147]

[0148] SEQ ID No. 2:

[0149]

Claims

1. Use of a composition in detecting N6-adenosine methylation modification in mRNA bound to a protein, wherein the composition consists of ethanol, sodium acetate and glycogen, and in the composition, the ratio of ethanol, sodium acetate and glycogen is 500 μL: 3 mol sodium acetate: 0.1 g glycogen.

2. The composition according to claim 1.

3. A method for detecting the abundance of N6-adenosine methylation modification in mRNA bound to proteins, characterized in that: Comprising the following steps: S1. Extract the mRNA of a plant; S2. Fragment the mRNA with metal ions to obtain a fragmented mRNA solution, and then add the composition according to claim 1 to promote mRNA precipitation to obtain purified fragmented mRNA; S3. Express the target protein of the plant in vitro; S4. Incubate and bind the target protein in S3 with the purified fragmented mRNA in S2 in vitro to obtain a protein-RNA complex; S5. Elute and separate the protein-RNA complex to obtain a target mRNA solution; S6. Add the composition according to claim 1 to the target mRNA solution to promote mRNA precipitation to obtain purified target mRNA; S7. Prepare the purified target mRNA in S6 into mononucleotides, further dephosphorylate, and then purify the mononucleotides to obtain a sample for loading; S8. Perform liquid chromatography-mass spectrometry detection, and use the external standard method to quantify the contents of N6-methyladenosine and adenosine ribonucleoside in the sample for loading in S7, and divide the content of N6-methyladenosine by the content of adenosine ribonucleoside to obtain the abundance of protein-bound N6-adenosine methylation modification in the biological sample; The content of N6-methyladenosine is quantified according to the chromatographic peak corresponding to the ion peak with a collision energy of 16 V and m / z of 282.1 - 150.1; The content of adenosine ribonucleoside is quantified according to the chromatographic peak corresponding to the ion peak with a collision energy of 10 V and m / z of 268.1 - 136.

1.

4. Method for detecting the abundance of protein binding to N6-adenosine methylation modification by RIP combined with MS, characterized in that: Including S1, S2, S3, S4, S5, S6 and S7 of claim 1, and further including S8': S8'. Perform liquid chromatography detection, and use the external standard method to quantify the contents of N6-methyladenosine and adenosine riboside in the sample loaded onto the machine as described in S7. Divide the content of N6-methyladenosine by the content of adenosine riboside to obtain the protein-bound N6-adenosine methylation modification abundance of the biological sample. The mobile phase used in liquid chromatography consists of phase A and phase B. Phase A is an aqueous solution of formic acid with a volume percentage of 0.1%, and phase B is an acetonitrile solution of formic acid with a volume percentage of 0.1% formic acid. The elution program used in liquid chromatography is as follows: from 0 min to 8 min, the volume ratio of phase A in the mobile phase linearly decreases from 100% to 99%; from greater than 8 min to 10 min, the volume ratio of phase A in the mobile phase linearly decreases from less than 99% to 94%; from greater than 10 min to 15 min, the volume ratio of phase A in the mobile phase linearly decreases from less than 94% to 0%; from greater than 15 min to 20 min, the volume fraction of phase A in the mobile phase linearly increases from 0% to 100%. The chromatographic column used in the liquid chromatography is a Thermo Scientific Hypersil GOLD aQ reverse phase column, with a column length of 100 mm, a column inner diameter of 2.1 mm, and a filler particle size of 1.9 μm. The column temperature of the chromatographic column is 25 °C, and the flow rate of the mobile phase is 0.3 ml / min. The retention time of the chromatographic peak of N6-methyladenosine is 10.5 ± 1 min, and the retention time of the chromatographic peak of adenosine ribonucleotide is 5.5 ± 1 min.

5. The method according to claim 3 or 4, characterized in that: Adding the composition described in claim 1 as described in S2 or S6 promotes mRNA precipitation. A reaction system is obtained by adding the composition described in claim 1 to the fragmented mRNA solution or the target mRNA solution. The content of ethanol in the reaction system is 2.5 L, the content of sodium acetate is 0.3 M, and the content of glycogen is 100 μg / ml -1 .

6. The method according to any one of claims 3 to 5, characterized in that: The addition of the composition described in claim 1 as described in S2 or S6 promotes mRNA precipitation, and the precipitation is carried out at -80 °C.

7. The method according to any one of claims 3-6, characterized in that: The metal ion described in S2 is a zinc ion.

8. The method according to any one of claims 3-7, characterized in that: The fragmentation described in S2 is carried out in reaction system A, which is a liquid composed of the following substances: mRNA, Tris-Hcl, zinc chloride, and water. The content of mRNA is 20 μg, the content of Tris-Hcl is 100 mM, the content of zinc chloride is 100 mM, and the balance is water.

9. The method according to any one of claims 3-8, characterized in that: The in vitro incubation and binding described in S4 is to incubate reaction system B on ice for 1 hour and then rotate and incubate with magnetic beads with His tags at 4 °C for 2 hours. Reaction system B is a liquid composed of the following substances: the target protein, the purified fragmented mRNA, Tris-HCl with a pH value of 7.5, sodium chloride, ethylenediaminetetraacetic acid, octylphenoxypolyethoxyethanol, dithiothreitol, ribonuclease inhibitor, and water. The content of the target protein is 20 μg / … μl, the content of the purified fragmented mRNA is … μg / … μl, the content of Tris-HCl is 50 mM, the content of sodium chloride is 250 mM, the content of ethylenediaminetetraacetic acid is 0.4 mM, the mass percentage of octylphenoxypolyethoxyethanol is 0.1%, the content of dithiothreitol is 1 mM, the content of ribonuclease inhibitor is 0.4 U / μl, and the balance is water.

10. The method according to any one of claims 3-9, characterized in that: For the elution and separation of the protein-RNA complex described in S5, the protein-RNA complex was eluted with buffer solution X at 4°C with rotation to remove non-specific fragments, then digested with PK buffer at 37°C for 20 min, and finally incubated with 200 μl of PK-urea buffer at 37°C for 20 min to obtain the target mRNA solution; The buffer solution X is a liquid composed of the following substances: Tris-HCl with a pH value of 7.5, sodium chloride, ethylenediaminetetraacetic acid, ethyl phenyl polyethylene glycol, dithiothreitol, ribonuclease inhibitor, and water; the content of sodium chloride is 250 mM, the content of ethylenediaminetetraacetic acid is 0.4 mM, the mass percentage content of ethyl phenyl polyethylene glycol is 0.1%, the content of dithiothreitol is 1 mM, the content of ribonuclease inhibitor is 0.4 U / μl, and the balance is water; The PK buffer is a liquid composed of the following substances: Tris-HCl with a pH value of 7.5, sodium chloride, ethylenediaminetetraacetic acid, proteinase K, and water; the content of Tris-HCl is 100 mM, the content of sodium chloride is 50 mM, the content of ethylenediaminetetraacetic acid is 10 mM EDTA, the content of proteinase K is 4 μg / μl, and the balance is water; The PK-urea buffer is a liquid composed of the following substances: Tris-HCl with a pH value of 7.5, sodium chloride, ethylenediaminetetraacetic acid, urea, and water; the content of Tris-HCl is 100 mM, the content of sodium chloride is 50 mM, the content of ethylenediaminetetraacetic acid is 10 mM EDTA, the content of urea is 7 M, and the balance is water.

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