CN111154837A

CN111154837A - Method and kit for detecting RNA N6-methyladenine modification in single base resolution in full transcriptome range

Info

Publication number: CN111154837A
Application number: CN201910822658.3A
Authority: CN
Inventors: 刘建钊; 冯新华; 舒潇; 曹婕
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2019-09-02
Filing date: 2019-09-02
Publication date: 2020-05-15
Anticipated expiration: 2039-09-02
Also published as: CN111154837B; WO2021042883A1

Abstract

The invention provides a method for detecting RNA N with single base resolution in the full transcriptome range⁶-methyl adenine modification method and kit. The method is based on N of ribonucleic acid (RNA) adenine in vivo⁶Allyl labelling and chemical treatment to induce base mutation during reverse transcription into DNA, and identifying the mutation site by nucleic acid sequencing to obtain⁶A site, which is the original m in cellular RNA⁶A site of modification. The method realizes N in the cells for the first time⁶-a marker specific for allyladenine which can be used not only to replace N in cells⁶-methyladenine sites and can be localized by means of sequencing of mutations. The method of the invention and the existing application to m⁶Compared with the gene sequencing technology of A detection, the mutation site can be accurate to the resolution of a single base, so that the currently commonly adopted m-based gene sequencing technology is improved⁶Antibody A immunoprecipitation and large-scale parallel sequencing method detection m⁶The precision of the A site is a direct high-throughput single-base identification method.

Description

Method and kit for detecting RNA N6-methyladenine modification in single base resolution in full transcriptome range

Technical Field

The invention belongs to the field of gene sequencing, and particularly relates to a method for detecting RNA N in a full transcriptome range with single base resolution⁶-methyl adenine modification method and kit.

Background

RNA is composed of not only cytosine (C), thymine (U), guanine (G) and adenine (A) in combination. N is a radical of⁶-methyladenine (m)⁶A) Is an extremely important modified base on RNA, and plays a plurality of biological functions in biological processes, such as regulating the expression of genes and the like. Among them, the identification and sequencing method of the modification is a precondition for the research of the biological significance.

Because the physical and chemical properties of methyl modified adenine are close to those of common adenine, the detection can not be directly carried out by utilizing the existing one-generation or two-generation sequencing technology. At present, based on m⁶The antibody immunoprecipitation sequencing technology (MeRIP-seq) can make us know which transcripts and genomes contain m⁶A is modified, but the resolution is only limited to the range of 100 to 200 bases, which A is methylated cannot be distinguished, and whether the A is a single A or a cluster A is methylated cannot be distinguished. In order to increase the resolution, a method of immunoprecipitation (CLIP) after photocrosslinking of antibody/RNA, i.e. m on antibody and RNA, was also used⁶Optically active uracil or thio-congener adjacent to A is crosslinked, reverse transcription process causes the crosslinked uracil site to mutate or terminate near the crosslinking position, and further analysis of mutation or termination information indirectly identifies A adjacent to uracil as m⁶And (3) A site. Although the resolution of this method is improved, the location identification is indirect and m cannot be distinguished⁶And (4) clustering A.

From the above analysis, it was found that m is present on conventional RNA⁶The reason that the high-throughput analysis means of the A site does not obtain major breakthrough is mainly that the current antibody immunoprecipitation is richCollection techniques have always been based on m for antibody enrichment⁶A sequence fragment, or antibody and m⁶Cross-linking of photoactive uracils or thio-homologues on A sequence fragments to indirectly identify m⁶Site of A, not passing through m directly⁶The A site is mutated in a manner to identify its site.

We wish to replace m with an identifiable modification group by grafting the modification group to an amino acid, introducing the amino acid with the modification group into cellular metabolism⁶A is subjected to methylation modification, and then the site where the modification group is located is identified by means of mutation sequencing, so that m is identified⁶Purpose of A site. However, under the current culture conditions, amino acids with modification groups are at a disadvantage in competition with normal amino acids in cell metabolism and cannot be introduced into cells.

Therefore, a culture condition suitable for introducing a modified amino acid into cells is under urgent development.

Disclosure of Invention

In view of the disadvantages of the prior art, the present invention aims to provide a full transcriptome-wide single base resolution method for detecting RNA N⁶The method and the kit for modifying the-methyladenine are different from the traditional method for indirectly identifying the m⁶A site method for direct identification of m of cellular whole transcriptome RNA by means of mutation sequencing with single base resolution⁶A modification site.

The invention adopts the following technical scheme:

RNA N detection with single base resolution in full transcriptome range⁶-a method for methyladenine modification comprising the steps of:

(1) allyl-L-seleno/thiohomocysteine is involved in cellular metabolism, labeling nucleic acid adenine: treating cells with methionine analogue allyl-L-seleno/thio-homocysteine, the cells can introduce allyl group into specific adenine N of RNA in cells through natural metabolism under the action of a series of enzymes⁶On bit, form N⁶-allyladenine (a)⁶A) The site should originally be a natural methylation modification, i.e. N⁶-methyladenine (m)⁶A)；

(2) Containing a⁶Enrichment of a-modified RNA: extracting total RNA of cells, further extracting mRNA of the cells, then breaking the RNA of the whole transcriptome of the cells into fragmented RNA of 100-300 nt, and combining N with an antibody⁶-allyladenosine enrichment of a by immunoprecipitation⁶A modified RNA and elution from the antibody with an eluent a⁶A, modifying RNA, and purifying the eluted RNA;

(3)RNA a⁶n on A⁶Iodine addition and cyclization treatment of allyladenine: RNA a⁶N on A⁶Iodine addition reaction of allyl adenine and subsequent induction of RNA a under alkaline conditions⁶N on A⁶N of allyl adenine¹，N⁶Forming a cyclization structure at the site, and shielding base complementary pairing;

(4) reverse transcription mutation and sequencing recognition of circularized RNA: adding HIV reverse transcriptase, N on RNA to the circularized structure obtained in step (3)¹，N⁶In the process of reverse transcription of cyclic adenine into DNA under the action of in vitro HIV reverse transcriptase, errors are generated by introducing para-complementary base, and mutation sites are identified by means of nucleic acid sequencing, so that a is obtained⁶A site, which is the original m in cellular RNA⁶A site of modification.

In the above technical solution, further, in the step (1), the preparation method of allyl-L-seleno/thiohomocysteine comprises: under the protection of nitrogen, selenium powder Se and sodium borohydride NaBH are firstly mixed in a molar ratio of 1:1₄Taking ethanol as a solvent as a raw material, heating and refluxing at 80 ℃ for 6-24 hours to prepare Na₂Se₂Heating and refluxing the compound and hydrobromide (compound 1) of (S) - (+) -2-amino-4-bromobutyric acid with the molar weight of one half to one third of selenium powder at 80 ℃ for 6-24 hours, stopping the reaction with acid, filtering to remove insoluble substances, washing off by-products with diethyl ether, adjusting the pH value to be neutral to obtain seleno-homocystine (compound 2), adding sodium borohydride with the molar weight of 1-2 times of the selenium powder to reduce diselenide bond, and then adding sodium bicarbonate/sodium carbonate with the molar weight of 0.5-2 times of the selenium powder and sodium bicarbonate with the molar weight of 0.5-2 times of the selenium powder under alkaline conditionsReacting 0.5-1.5 times of allyl bromide in selenium powder/sulfur powder at room temperature for 6-24 times, and analyzing and purifying by High Performance Liquid Chromatography (HPLC) to obtain allyl-L-seleno-homocysteine (compound 3); sulfur S is used as raw material to prepare thiohomocystine (compound 4) and allyl-L-thiohomocysteine (compound 5).

The cells may be, without limitation, normal mammalian cells, mammalian cancer cells, mammalian stem cells, host cells for bacteria, viruses, and cells derived from various types of tissues and organs.

Further, the specific method of cell treatment in step (1) is to replace the normal culture medium of the cell culture system with a methionine-deficient culture medium, add 10% Fetal Bovine Serum (FBS), on the basis of 1% 100x penicillin-streptomycin double antibody, add cysteine (0.1-2 mM) for half an hour, then add allyl-L-seleno/thiohomocysteine (0.1-2 mM), and continue to culture for 12-24 hours, then N on RNA can be obtained⁶-methyl adenine modification to N⁶-allyladenine modification. The methionine-deficient medium is available directly from commercial sources.

Further, in the step (2), the sample cell whole transcriptome RNA fragmentation treatment is performed by Zn²⁺Heating at 70 deg.C for 4-10 min.

Further, in the step (2), for binding N⁶-allyladenosine antibody is N⁶An antibody of isopentenyl adenosine, wherein 10 μ g of the antibody is used for enriching 1-20 μ g of fragmented RNA, and the antibody is combined with the sample cell whole transcriptome fragmented RNA through the ProteinA of the magnetic beads.

Further, in the step (2), the eluent is N⁶-allyl adenosine triphosphate (a)⁶ATP) in a concentration of 5 to 10mM N⁶-allyl adenosine triphosphate (a)⁶ATP) from N⁶Competitive elution on antibodies to isopentenyl adenosine of the above cell whole transcriptome fragmentationRNA, the RNA obtained is purified by ethanol or isopropanol precipitation.

Further, in step (3), iodine (0.1-0.5M iodine dissolved in 0.2-1M potassium iodide) and N⁶Iodine addition reaction of allyl group of allyladenine, removal of excess iodine (0.1 to 0.5M sodium thiosulfate), and N addition under alkaline conditions (0.1 to 0.5M sodium carbonate, pH 9 to 10)¹，N⁶The site is self-closing, thus shielding the normal hydrogen bonding pairing of adenine.

Further, in step (4), the method for sequencing the mutation comprises the steps of: i) after the HIV reverse transcriptase is used for reverse transcription of the allyl-L-seleno/thiohomocysteine cell metabolic marker, the RNA cyclized by addition of the antibody and iodine is enriched; ii) adopting RNA library preparation technology and high-throughput sequencing means to carry out identification on the mutation sites of the whole transcriptome so as to obtain m with single base resolution of the whole transcriptome⁶And (3) distributing the A sites, or identifying and verifying the mutation sites on a specific transcript by adopting PCR and TA-cloning technologies.

Furthermore, the invention provides a full transcriptome range single base resolution detection RNA N⁶-methods of methyladenine modification, which may also have the following characteristics: amino acids for the cellular metabolism and in vitro enzyme assistance include, but are not limited to, allyl-L-selenohomocysteine (allyl-L-selenohomocysteine), allyl-L-thiohomocysteine (allyl-L-homocysteine), and derivatives and analogs thereof; n is a radical of⁶Antibodies to allyladenosine include, but are not limited to, N⁶Isopentenyl adenosine (anti-cytokine. RTM. N)⁶-isopentenyladenosines) and derivatives, analogues thereof; reverse transcriptase of reverse transcriptase mutations include but are not limited to HIV reverse transcriptase (Recombinant HIV reverse transcriptase enzyme).

In the step (1), the cells are cultured by adding methionine derivative and cysteine to the methionine-deficient medium, wherein the methionine derivative includes, but is not limited to, allyl-L-selenohomocysteine (allyl-L-selenohomocysteine), allyl-L-thiohomocysteine (allyl-L-homocysteine) and its derivatives and analogs; cysteine in the method includes, but is not limited to, downstream metabolites of methionine in cellular metabolism. The chemical structural formulas of allyl-L-seleno-homocysteine (allyl-L-selenohomocysteine) and allyl-L-thiohomocysteine (allyl-L-selenohomocysteine) are shown in the specification.

In the step (1), the method for extracting RNA includes a common purification method or a commercial purification kit. Methods include, but are not limited to: using TRIzol^TMReagent, chloroform-phenol extraction, protease K digestion, silica gel membrane centrifugal column method, magnetic bead method, ethanol and isopropanol precipitation. Purification kits include, but are not limited to: GeneElute^TMmRNA Miniprep Kit、RNeasy^TMMini Kit(Qiagen),RNA Clean&Concentrator(Zymo)。

In the step (2), the fragmentation of RNA in the immunoprecipitation method includes, but is not limited to, RNA fragmentation by a metal ion method, and RNA fragmentation by ultrasonication; antibody enrichment in immunoprecipitation methods includes, but is not limited to, the method of protein A/protein G beads; elution in immunoprecipitation methods includes, but is not limited to, N⁶-allyladenosine monophosphate, N⁶-allyl adenosine triphosphate (N)⁶-allyl adenosine-5' -triphosphatate) and TRIzol Reagent. N is a radical of⁶-allyl adenosine triphosphate (N)⁶-allyl adenosine-5' -triphosphatate) and the synthesis steps thereof are as follows: under the protection of nitrogen, ethanol is used as a solvent, 6-chloropurine nucleoside (compound 6), allyl amine and triethylamine are heated and refluxed at the temperature of 80 ℃ for 3-6 hours in a molar ratio of 1:3:3, the obtained product is subjected to vacuum rotary evaporation, ether precipitation is carried out, insoluble substances are removed through filtration, and recrystallization is carried out through methanol to obtain N⁶Allyladenosine (compound 7). Then, N is added under the protection of nitrogen⁶Allyladenosine (0.2mmol), phosphorus oxychloride (POCl)₃0.26mmol) was added 0.5mL of anhydrous trimethyl phosphate (MeO)₃PO, reacting at 0 deg.C for 1.5 hr, dissolving in water2mL of tributylammonium pyrophosphate (TBAPP, 1mmol) in anhydrous N, N-Dimethylformamide (DMF) was added to the above reaction system, reacted at 0 ℃ for 20 minutes, further reacted at room temperature for 5 minutes, finally the reaction was terminated with 2mL of triethylammonium bicarbonate (TEAB, 1mol/L), and purified by High Performance Liquid Chromatography (HPLC) analysis to obtain N⁶-allyl adenosine triphosphate (a)⁶ATP, compound 8). The amounts of the reactants may be adjusted proportionally.

In the step (3), the iodine addition method includes, but is not limited to, potassium iodide solution of iodine (0.125M I)₂0.25M KI), methods for removing excess iodine include, but are not limited to, sodium thiosulfate treatment (0.2M Na)₂S₂O₃) Methods for inducing cyclization under alkaline conditions include, but are not limited to, sodium carbonate, sodium bicarbonate solution (0.1M Na)₂CO₃pH 9.5) and the reaction formula is as follows. Under alkaline conditions, N¹，N⁶And (4) a self-closed loop is positioned, and base complementary pairing is shielded.

In the above step (4), the reverse transcription method includes, but is not limited to, a method of treating with HIV reverse transcriptase (Recombinant HIVreversperse enzyme).

In step (4) above, the sequencing method after reverse transcription includes, but is not limited to, high-throughput sequencing for constructing a library, and low-throughput sequencing based on TA-cloning. Wherein, the PCR enzyme in the TA-cloning method includes but is not limited to KOD-FXDNA polymerase, and the ligation of the plasmid vector in the TA-cloning method includes but is not limited to T vector; the library construction method includes, but is not limited to, the library construction of the illuma stranded, the library construction of NEB small RNA, eCIP, the improved library construction method and the like.

In the above steps (1) to (4), the nucleic acid purification step after each reaction may be carried out by a conventional purification method or a commercially available purification reagentAnd (5) a box. Methods include, but are not limited to: one or more of the techniques of silica gel membrane centrifugal column method, magnetic bead method, ethanol and isopropanol precipitation, etc. Purification kits include, but are not limited to: the AmpureXP beads are used,

PCRpurification Kit(Qiagen)，RNA Clean&Concentrator(Zymo)，DNA Clean&Concentrator(Zymo)。

the invention also provides a method for detecting RNA N by using single base resolution in the full transcriptome range⁶-methyladenine modified kit comprising allyl-L-seleno/thiohomocysteine, cysteine, N⁶Allyl adenosine triphosphate, RNA fragmentation solution containing divalent zinc ions, N⁶An isopentenyl adenosine antibody, a potassium iodide solution of iodine, a sodium thiosulfate solution, a sodium carbonate solution, an HIV reverse transcriptase, an HIV reverse transcription reaction solution, Tris-HCl, an RNase inhibitor, a sequencing linker and a sequencing primer.

In the present invention:

(1) iodine induced double bond addition after addition to the double bond, iodine has some dissociative properties, and the carbon atom in the immediate vicinity thereof can electrophilically attack the atom with higher electron cloud density, and for adenine, the nitrogen atom on the purine ring has the characteristic of higher electron cloud density, especially the nitrogen atom at the 1-position. Therefore we have designed N⁶Allyl adenine nucleoside as a molecule, and under the iodine addition and alkaline conditions, allyl iodine at the N6 position can induce carbon atoms to attack N1 at the adjacent position electrophilically after iodine atoms leave to form N1 and N6 ring-closing reactions, and hydrogen bonds used for base complementary pairing at the two positions originally are shielded, so that mutation is caused in the reverse transcription process, and N recognition is achieved by means of sequencing⁶-the purpose of the allyladenine site;

(2) to apply the above method of mutation sequencing to m⁶Recognition of A site, we designed a method for cell metabolism, which utilizes the methyl metabolic pathway modified by RNA methylation in cells to replace methyl with allyl, thereby m can be replaced⁶Replacement of A by N⁶-allyl glandPurines, identification of N by mutational sequencing⁶-site of allyladenine to achieve single base resolution detection of m⁶Purpose of A site. The intracellular RNA methylation modified metabolic pathway is methionine, S-adenosylmethionine, N⁶-methyladenine, we removed the methionine present in the cell itself and then replaced it with allyl-L-seleno/thiohomocysteine (seleno amino acids proved to be more susceptible to group transfer than thio) in culture conditions, successfully modifying the allyl group onto the RNA of the cellular whole transcriptome. Due to the identification of N by quantitative mass spectrometry⁶The modification efficiency of-allyladenosine is low, and we found that N can be specifically enriched⁶Antibodies to allyladenosine, enrichment of N by antibody immunoprecipitation⁶Allyl adenosine modified full transcriptome RNA, and identifying a majority of single bases m in the full transcriptome range by using two ways of high throughput and low throughput by mutation sequencing according to the iodine addition and cyclization method⁶A site, greatly promoting m⁶And (3) researching the biological function of the A modification.

The invention has the beneficial effects that:

the invention relates to a method for detecting RNA N with single base resolution in the full transcriptome range⁶The method and kit for modifying methyl adenine is based on the chemical marking and induced mutation of nucleic acid adenine in vivo and the application in m⁶Compared with the gene sequencing technology of A detection, the mutation site can be accurate to the resolution of a single base, so that the currently commonly adopted m-based gene sequencing technology is improved⁶Antibody A immunoprecipitation and large-scale parallel sequencing method detection m⁶The precision of the A site is a direct high-throughput single-base identification method.

The invention realizes N of ribonucleic acid (RNA) adenine in cells for the first time⁶Allyl labelling for the subsequent passage m⁶The way in which the A site is mutated to identify its site provides the possibility.

Due to N⁶The modification efficiency of-allyladenosine is low, so that N is used in the present invention⁶Antibodies to isopentenyl adenosineSpecific enrichment of N⁶Antibodies to allyladenosine. Because currently there is no N temporarily⁶Antibodies specific for allyladenosine, analyzed and screened for some commercial antibodies, we found that N⁶Antibodies to isopentenyl adenosine to N⁶-allyladenosine, the antibody having a specificity for N⁶Allyl adenosine has better binding capacity, and the commercial antibody is more easily obtained, thereby greatly improving the practicability of the method.

The invention adopts N⁶-allyl adenosine triphosphate (a)⁶ATP) as eluent due to a⁶A on ATP and RNA⁶The main structure of the A modification is the same, and the water solubility of the A modification is increased by the triphosphate, so that the A modification has stronger binding capacity with the antibody, and the A modification is similar to the a modification⁶The A modified RNA is in competition in binding to the antibody, so a can be bound to the antibody through competition⁶Elution of A-modified RNA. Therefore, compared with the common RNA extraction method for eluting RNA, the method enables a⁶The RNA modified by A is almost completely separated from the antibody, and the yield of the eluted RNA is greatly improved.

In the present invention, HIV reverse transcriptase is used for reverse transcription of RNA subjected to iodine addition and cyclization induction, because it is found in our research that HIV reverse transcriptase has a stronger recognition ability for hydrogen bond-shielded cyclization structures of adenine for base complementary pairing, we have selected a number of commercial reverse transcriptases, including HIV reverse transcriptase (ReverseTranscriptase Recombinant HIV, Worthington Biochemical Corporation), M-MLV reverse transcriptase (PROMEGA, M170A), AMV reverse transcriptase (PROMEGA, M510F), Reversaid reverse transcriptase (Thermofisher, EP0441), SuperScript II reverse transcriptase (Invitrogen,100004925), SuperScript III reverse transcriptase (Invitrogen,55549), etc., and finally discovered that HIV reverse transcriptase is found at a⁶The corresponding site is mutated during the reverse transcription of the A iodine addition cyclization RNA.

The invention can be applied to various analysis methods based on gene sequencing on the basis of mutation sequencing, such as m on various types of nucleic acids⁶Detection of A modification sites, and based on N⁶Dynamic sequencing of cellular RNA of allyladenosine, etc.

Drawings

FIG. 1 is a single base resolution detection of RNA N in the full transcriptome range⁶-a schematic representation of a method for methyladenine modification;

FIG. 2 shows N in mRNA obtained by introducing allyl-L-seleno/thiohomocysteine into HeLa cells for metabolism⁶Content of allyl adenosine, N in mRNA obtained after introduction of methionine into HeLa cells for metabolism⁶Content of-methyladenine nucleosides and N in mRNA obtained after HeLa cells were cultured normally⁶-the content of methyladenosine;

FIG. 3 shows RNA bands obtained by fragmenting mRNA of a sample cell with divalent zinc ions;

FIG. 4 is N⁶Antibodies to isopentenyl adenosine to normal A, m⁶A modified RNA, a⁶A dot blot hybridization of modified RNA showing binding ability of corresponding RNA to antibody, methylene blue showing RNA loading;

FIG. 5 shows N before and after mRNA was enriched in HeLa (human cancer cells) and H2.35 (mouse common cells) cells by antibody immunoprecipitation⁶-content of allyladenosine;

FIG. 6 shows a in HeLa cells⁶A modified RNA, the result of gene sequencing before and after iodine addition induced cyclization;

FIG. 7 shows m on transcriptome from high throughput sequencing of HeLa cells and H2.35 cells⁶Conserved sequence of A site;

FIG. 8 shows m on transcriptome from high throughput sequencing of HeLa cells⁶Site A, exemplified by three mRNA gene sequences;

FIG. 9 shows m on transcriptome from high throughput sequencing of H2.35 cells⁶Site A, exemplified by four mRNA gene sequences;

FIG. 10 is m on transcriptome from low throughput sequencing of HeLa cells⁶Site A, exemplified by three mRNA gene sequences;

FIG. 11 is a high resolution mass spectrum of allyl-L-selenohomocysteine;

FIG. 12 is allylProcess for preparing L-seleno-homocysteine¹H nuclear magnetic resonance spectroscopy;

FIG. 13 is a photograph of allyl-L-seleno-homocysteine¹³C nuclear magnetic resonance spectrum;

FIG. 14 is a high resolution mass spectrum of allyl-L-thiohomocysteine;

FIG. 15 is N⁶-high resolution mass spectrometry of allyl triphosphate.

Detailed Description

The present invention will be described in detail with reference to the drawings and specific examples, but they should not be construed as limiting the scope of the present invention.

FIG. 1 shows a single base resolution method for detecting RNA N in the full transcriptome range according to the present invention⁶Schematic representation of the process for the modification of methyladenine. The method comprises the following steps:

(1) allyl-L-seleno/thiohomocysteine is involved in cellular metabolism, labeling nucleic acid adenine: the cell can take in the methionine analogue allyl-L-seleno-homocysteine, and the allyl group can be introduced into the specific adenine N of RNA in the cell under the action of a series of enzymes through the natural metabolism of the cell⁶On bit, form N⁶-allyladenine (a)⁶A) The site should originally be a natural methylation modification, i.e. N⁶Methyl adenine (m)⁶A)；

(2) Containing a⁶Enrichment of a-modified RNA: extracting total RNA of cells, further extracting mRNA of the cells, then breaking the RNA of the sample cell complete transcriptome into fragmented RNA of 100-300 nt, and utilizing N⁶-antibodies to allyladenosine enrichment of a by immunoprecipitation⁶A modified RNA with N⁶-allyl adenosine triphosphate (a)⁶ATP) elution from the antibody a⁶A, modifying RNA, and purifying the eluted RNA;

(3) iodine-added RNA a⁶N on A⁶Allyl induced generation of N¹，N⁶Site cyclization: RNA a⁶N on A⁶Iodine addition reaction of allyl adenine and subsequent induction of RNA a under alkaline conditions⁶N on A⁶N of allyl adenine¹，N⁶Forming a cyclization structure at the site, and shielding base complementary pairing;

(4) reverse transcription mutation and sequencing recognition of circularized RNA: n on RNA¹，N⁶In the process of reverse transcription of cyclic adenine into DNA under the action of in vitro HIV reverse transcriptase, errors are generated by introducing para-complementary base, mutation sites can be identified by means of nucleic acid sequencing, and then a is obtained⁶A site, which is the original m in cellular RNA⁶A site of modification.

In the invention, after sample cells are normally cultured to about eighty percent of fusion degree, 1mM cysteine is added into a cell culture medium without methionine for pretreatment for 30 minutes to remove residual methionine in the cells, 1mM allyl-L-seleno/thiohomocysteine is added into the culture medium, and after the cells are cultured for 12-24 hours, total RNA of the sample cells is firstly extracted, and then the total transcriptome RNA of the sample cells is further extracted. The present invention is not particularly limited in the kind of sample cells that can be used, and the cells may be, but are not limited to, general mammalian cells, mammalian cancer cells, mammalian stem cells, host cells of viruses, bacteria, and cells derived from various types of tissues and organs. In the present invention, the method for collecting, lysing and extracting total RNA and total transcriptome RNA from cells and tissues can be performed by RNA extraction methods conventional in the art without any special requirement, for example, TRIzol Reagent is used for extracting total RNA in the specific implementation process of the present invention, GenElute^TMmRNA was extracted with the mRNA Miniprep Kit.

After the sample cell full transcriptome RNA is obtained, the sample cell full transcriptome RNA is fragmented into 100-300 nt RNA. The fragmentation treatment described in the present invention is preferably achieved using divalent zinc ions which can cleave phosphodiester bonds on RNA without destroying the linked bases. A preferred fragmentation buffer in the present invention is (1. mu.L of 1M ZnCl)₂1 μ L of Tris-HCl pH 7.0,8 μ L of RNase-free water), 400-700 ng/μ L of the sample cell whole transcriptome RNA and fragmentation buffer were heated at 70 ℃ for 4-10 minutes at a volume ratio of 9: 1. The inventionAfter obtaining the fragmented RNA, the method preferably further comprises a step of purifying the fragmented RNA, wherein the purification method adopted in the invention is an RNA isopropanol precipitation method which is conventional in the field, namely an RNA sample, a 3M sodium acetate solution, isopropanol and glycogen have a volume ratio of 100:10:110:1, after precipitation overnight, the RNA sample is centrifuged at 15000rpm for 45 minutes at 4 ℃, and after washing with 80% ethanol, the RNA solution is dissolved to obtain the fragmented RNA solution of the sample cell complete transcriptome.

After the fragmented RNA of the sample cell complete transcriptome is obtained, the RNA is enriched by an antibody immunoprecipitation method. The antibody Immunoprecipitation method (IP) adopted by the invention is a conventional ProteinA magnetic bead method in the field, and the antibody used is preferably commercialized N⁶Antibodies to isopentenyl adenosine. Every 1-20 mu g of the fragmented RNA, preferably 3-10 mu g, is incubated and combined with 1-20 mu g of antibody, preferably 5-10 mu g, for 2-6 hours, preferably 3-4 hours, and preferably at 4 ℃. Binding the fragmented RNA to the antibody, binding the antibody to ProteinA magnetic beads, and magnetically separating ProteinA magnetic beads and N⁶Antibodies to isopentenyladenosine, N⁶After the product of the combination of allyl adenosine-modified RNA and N⁶-elution with allyl triphosphate of RNA. N is a radical of⁶The concentration of the eluent of allyl adenosine triphosphate is 5 to 10mM, preferably 6.67 mM. After enriching RNA, the invention preferably further comprises a step of purifying RNA to obtain N⁶Allyl adenosine modified RNA was purified by the above-described isopropanol precipitation method.

As shown in FIG. 4, three RNAs were obtained by in vitro transcription, each being normal RNA (A-RNA), with substitution of A for m⁶RNA (m) of A⁶A-RNA), and replacement of A with a⁶RNA of A (a)⁶A-RNA), three RNAs and N⁶Immunodot hybridization dot-blot of the antibodies to isopentenyl adenosine revealed that only a⁶A-RNA (compare A-RNA to m)⁶A-RNA) has a relatively specific binding ability to the antibody, and can be derived from the normal A sequence of cellular mRNA and m⁶A in the modified sequence⁶A modified mRNA is identified, thereby providing an enrichment function, byFor comparison, Methylene blue methyl blue shows the loading amount of the corresponding RNA, demonstrating a under the same amount of RNA⁶The specific binding ability of A-RNA to antibodies.

The invention obtains the sample cell complete transcriptome N⁶Allyl adenosine-modified RNA was then subjected to iodine addition and cyclization under alkaline conditions. In the present invention, 26. mu.L of the above RNA and 4. mu.L of a 0.125M iodine solution (dissolved in 0.25M potassium iodide) are treated at 4 to 50 ℃ for 15 to 60 minutes, preferably at 37 ℃ for 30 minutes. Then, 2 to 4. mu.L of 0.2M sodium thiosulfate was added until the solution became colorless, and 6. mu.L of 0.1M sodium carbonate (pH 9 to 10, preferably 9.5) was added and the mixture was treated at 4 to 50 ℃ for 15 to 60 minutes, preferably 37 ℃ for 30 minutes. In the present invention, after the iodine addition and cyclization treatment of RNA, it is preferable to further include a step of purifying RNA, and the obtained RNA product is purified by the above-mentioned isopropanol precipitation method.

After immunoprecipitated cyclized and unclycled products are obtained, fragmented RNA is subjected to HIV reverse transcription and then connected with a sequencing joint, and a sequencing primer is used for PCR amplification to construct a high-throughput sequencing library. In the invention, the fragmented RNA is subjected to HIV reverse transcription, then is connected with a sequencing joint, and is subjected to PCR amplification by using a sequencing primer to construct a high-throughput library, preferably an illumina Troseq stranded mRNA LT kit, wherein the specific library construction method is carried out according to an instruction.

After the construction of the sequencing library is completed, the sequencing library is sequenced to obtain sequencing data. In the present invention, the sequencing is preferably next generation sequencing, more preferably Illumina double-ended sequencing, and the read length of the sequencing is preferably 150 bp.

The invention analyzes the mutation rate of the samples with cyclization and non-cyclization at the adenylic acid sites after obtaining the sequencing data, if the mutation rate of the samples with cyclization is more than three times of the samples with non-cyclization, and the mutation rate is a⁶The site is considered as m in the antibody enrichment region A⁶And (3) A site. The analysis includes quality control of the sequencing data, removal of adapter sequences from the data, removal of low quality bases, alignment of the low quality and adapter removed data to genomic sequences and enrichment statistics, and comparison of the sequence with the sequence of the genomic sequencesData from the low mass and linker were removed and aligned to the transcriptome sequences and mutation statistics were performed. In a specific embodiment of the present invention, the analysis comprises the steps of: quality control is carried out on the original sequencing data through fastqc default parameters, a linker sequence and a sequence with less than 25 bases (fastp-F10-F10-x- -detect _ adapter _ for _ pe-l 25-I Raw-R1. fq-I Raw-R2. fq-O Clean-R1. fq-O Clean-R2.fq) are removed through fastqc software, the obtained sequence is subjected to quality control through the fastqc software default parameters again, then the default parameters of hisat2 are used for alignment to a transcriptome sequence, SAM obtained by alignment is converted into a BAM file through samtools, repeated sequences are removed through samtools, finally mutation information in the BAM file is counted through sampleap, the mutation rates of cyclized samples and uncyclized samples at the same position are compared, and the fold change is more than three, namely, the m is considered as the position⁶And (3) A site.

After the immunoprecipitated cyclized and unclycled products are obtained, PCR and TA-cloning technology can be used for low-throughput sequencing of a certain section of transcriptome to identify mutation sites. In the present invention, the DNA polymerase for PCR is preferably KOD-FXDNA polymerase, and the plasmid vector for ligation of the PCR product is preferably a T-vector. As shown in fig. 6, a certain segment passes through a⁶The result of low-flux sequencing of A-labeled RNA by PCR and TA-cloning technology, wherein X ═ a⁶A indicates that the site is determined to have a⁶A is modified, X ═ cyc-A represents a⁶The A modified site is processed by iodine addition and induced cyclization, and the result shows that HIV reverse transcriptase generates mutation at X ═ cyc-A site and X ═ a⁶The absence of mutations at the A site demonstrates the necessity for iodine addition and induction of cyclization as well as the necessity for HIV reverse transcriptase. Comparing the mutation rate of cyclized and uncyclized samples at the same locus, finding that the fold change is more than three, and confirming that m can be accurately detected by adopting the method of the invention⁶And (3) A site.

The invention also provides a method for detecting RNA N by using single base resolution in the full transcriptome range⁶-methyladenine modified kit comprising allyl-L-seleno/thiohomocysteine, cysteine, N⁶Allyl adenosine triphosphate, RNA fragmentation solution containing divalent zinc ions,N⁶isopentenyl adenosine antibody, 0.125M iodine in potassium iodide, 0.2M sodium thiosulfate, 0.1M sodium carbonate (pH 9.5), HIV reverse transcriptase, HIV reverse transcription reaction solution, Tris-HCl, rnasinhibitor, sequencing linker, sequencing primer.

The synthesis steps of the allyl-L-seleno/thiohomocysteine are as follows: under the protection of nitrogen, selenium powder Se (4mmol) and sodium borohydride NaBH are firstly added₄(4mmol) as a raw material and ethanol as a solvent, and heating and refluxing the raw material and the ethanol at the temperature of 80 ℃ for 6 to 24 hours to obtain Na₂Se₂Heating and refluxing the mixture and hydrobromide (2mmol) of (S) - (+) -2-amino-4-bromobutyric acid at 80 ℃ for 6-24 hours, stopping the reaction by using acid, filtering to remove insoluble substances, washing off by-products by using diethyl ether, and adjusting the pH value to be neutral to obtain seleno-homocystine (1 mmol); adding sodium borohydride (6mmol) to reduce diselenide bond, reacting with allyl bromide (3.8mmol) under alkaline condition (sodium bicarbonate, 3mmol) for 6-24, and purifying by high performance liquid chromatography to obtain allyl-L-seleno-homocysteine; in the same way, the allyl-L-thiohomocysteine is prepared by taking the sulfur S as a raw material.

FIG. 11 shows a high resolution mass spectrum of allyl-L-selenohysteine prepared according to the present invention (M/z is 224.0183, [ M + H ]]⁺Theoretical calculation is 224.0184); FIG. 12 is a drawing thereof¹An H nuclear magnetic resonance spectrum, wherein the H nuclear magnetic resonance spectrum,¹H NMR(500MHz,D₂o) δ 5.84(ddt, J ═ 17.6,9.9,7.7Hz,1H), 5.10-4.90 (m,2H),3.72(dd, J ═ 6.7,5.7Hz,1H), 3.29-3.06 (m,2H), 2.62-2.41 (m,2H), 2.24-1.93 (m, 2H); FIG. 13 is the same¹³A C nuclear magnetic resonance spectrum of a nuclear magnetic resonance,¹³C NMR(126MHz,D₂o) δ 174.02(s),134.73(s),116.75(s),54.78(s),31.19(s),25.33(s), 17.17(s); as can be seen from FIGS. 11-13, the present invention successfully prepares allyl-L-selenohomocysteine. For the same reason, refer to FIG. 14 (C)₇H₁₃NO₂S, M/z is 176.0729, [ M + H ]]⁺Theoretical calculation is 176.0740), it is known that allyl-L-thiohomocysteine is successfully prepared according to this method.

N⁶The synthesis of allyl adenosine triphosphate comprises the following steps: under the protection of nitrogen, ethanol is used as a solvent, 6-chloropurine nucleoside and allylHeating and refluxing amine and triethylamine with the molar weight of 1:3:3 at 80 ℃ for 3-6 hours, carrying out vacuum rotary evaporation on the obtained product, precipitating with diethyl ether, filtering to remove insoluble substances, and recrystallizing with methanol to obtain N⁶-allyladenosine. Then, N is added under the protection of nitrogen⁶Allyladenosine (0.2mmol), phosphorus oxychloride (POCl)₃0.26mmol) was added 0.5mL of anhydrous trimethyl phosphate (MeO)₃PO, 0 deg.C for 1.5 hr, adding tributyl ammonium pyrophosphate (TBAPP, 1mmol) dissolved in 2mL anhydrous N, N-Dimethylformamide (DMF) into the reaction system, reacting at 0 deg.C for 20 min, further reacting at room temperature for 5 min, terminating the reaction with 2mL triethylammonium bicarbonate (TEAB, 1mol/L), and purifying by High Performance Liquid Chromatography (HPLC) analysis to obtain N⁶-allyl adenosine triphosphate (a)⁶ATP)。

FIG. 15 shows that N prepared by the present invention⁶-allyl adenosine triphosphate (a)⁶ATP) associated characterization data (high resolution Mass Spectrometry, C₁₃H₂₀N₅O₁₃P₃M/z is 546.0192, [ M-H ]]^-Theoretical calculation 546.0198), it was found that a was successfully prepared⁶ATP。

Example 1m on transcriptome mRNA in HeLa cells⁶Detection of A modification sites

1.HeLa cell culture, allyl labeling and mRNA extraction

(1) Culturing HeLa cells under normal culture conditions until the fullness is eighty percent, sucking out the culture medium, and washing away the residual culture medium by Phosphate Buffered Saline (PBS);

(2) adding 10% Fetal Bovine Serum (FBS), 1% 100 Xpenicillin-streptomycin double antibody and 1mM cysteine into methionine-free culture medium, culturing the above HeLa cells in the culture medium for 30 min, and removing methionine remaining in the cells;

(3) removing methionine, adding 1mM allyl-L-seleno/thiohomocysteine into the culture medium for culturing the HeLa cells, and continuously culturing for 16 hours;

(4) after the cells were cultured, the medium was aspirated, the residual medium was washed with PBS, and T was added to the petri dishRIzol^TMReagent to cover the bottom of the culture dish, eluting all the adherent HeLa cells into the solution, transferring the HeLa cells into a centrifuge tube, and cracking the HeLa cells for 5 minutes at room temperature;

(5) each 1mL of TRIzol^TMReagent, adding 0.2mL of chloroform into the centrifuge tube, violently shaking the centrifuge tube for 15 seconds, extracting at room temperature for 3 minutes, centrifuging at 4 ℃ for 15 minutes under the condition of 12000g of RCF, layering the solution, wherein the supernatant is RNA, the middle white precipitate is DNA, and the lower red liquid is protein;

(6) transferring the upper aqueous phase to a new centrifuge tube, adding isopropanol (isoprostanol) with the same volume, incubating on ice for 10 minutes, and centrifuging at 4 ℃ at RCF 15000g for 15 minutes to obtain total RNA white precipitate;

(7) removing the supernatant to leave white particles of total RNA, washing the precipitate with 1mL of 75% ethanol (ethanol)/1mL of TRIZOL, centrifuging at 4 ℃ for 15 minutes at RCF 15000g, removing the supernatant again, air-drying for 3 minutes, dissolving the total RNA with 250. mu.L of RNase-free water, heating at 70 ℃ for 10 minutes to dissolve the total RNA sufficiently, and measuring the RNA concentration with Bio Drop to be 1000 ng/. mu.L;

(8) mRNA was further extracted with GenElute mRNA miniprep Kipp. Taking 250 mu L of total RNA (1000 ng/. mu.L) into a 1.5mL centrifuge tube, adding 250 mu L of 2x Binding Solution, and slightly shaking the centrifuge tube to mix the Solution;

(9) adding 20 mu L of evenly dispersed oligo (dT) beads, shaking to fully and evenly mix the system, heating at 70 ℃ for 3 minutes to denature RNA, then incubating at room temperature for 10 minutes to fully combine oligo (dT) with RNA, centrifuging the mixed system at RCF 15000g for 2 minutes to obtain oligo (dT) beads combined with mRNA, carefully removing supernatant, and leaving about 50 mu L of solution to prevent the beads from losing;

(10) adding 500 mu L of Wash Solution into a centrifuge tube to fully suspend oligo (dT), transferring the oligo (dT) into a GenElute centrifugal filter column/collection tube, centrifuging at the RCF 15000g for 2 minutes, and discarding the liquid in the collection tube;

(11) adding 500 mu L of Wash Solution into the filter column again, centrifuging for 2 minutes at the speed of 15000g of RCF, and discarding the liquid in the collecting pipe;

(12) finally, the filtration column was transferred to a new collection tube, 50. mu.L of Elutionsolution preheated at 70 ℃ was added to the center of the filtration membrane of the centrifugal filtration column, brought into full contact with the mRNA complex in the well of the column, incubated at 70 ℃ for 5 minutes, and centrifuged at RCF 15000g for 1 minute to obtain an mRNA solution in the collection tube, and the mRNA concentration was measured to be 200 ng/. mu.L by Bio Drop. In addition, can also absorb 50 u L70 degrees of Elution Solution repeated above Elution process, so that the filter column hole in the mRNA fully dissolved, will get the mRNA placed at-80 degrees C storage.

2. Containing adenine N⁶Site allyl modified RNA enrichment

(1) Mixing 200 μ L of the above RNA sample (200 ng/. mu.L), 20 μ L of 3M sodium acetate solution, 220 μ L of isopropanol, and 2 μ L of glycogen, pumping, mixing, precipitating overnight, centrifuging at 15000rpm for 45 minutes at 4 ℃, washing the precipitate with 440 μ L of 80% ethanol, centrifuging at 15000rpm for 15 minutes at 4 ℃, removing the supernatant again, air drying for 3 minutes, and dissolving RNA with 70 μ L of RNase-free water to a concentration of 550 ng/. mu.L;

(2) mu.L of the above RNA sample (550 ng/. mu.L) was mixed with 5. mu.L of Zn²⁺Mixing fragmentation buffer (10x Fragmentbuffer), beating, mixing, heating at 70 deg.C for 7 min, adding 10 μ L0.5M EDTA solution to terminate fragmentation, and adding Zn²⁺The fragmentation buffer (10x Fragment buffer) composition is shown in table 1;

TABLE 1 Zn²⁺Fragmentation buffer (10X Fragment buffer)

(3) Mixing the 60 μ L solution with 6 μ L3M sodium acetate solution, 66 μ L isopropanol, 1 μ L glycogen, blowing, mixing, precipitating overnight, centrifuging at 15000rpm for 45 min at 4 deg.C, washing the precipitate with 132 μ L80% ethanol, centrifuging at 15000rpm for 15 min at 4 deg.C, removing the supernatant again, air drying for 3 min, dissolving RNA with 200 μ L RNase-freewater to concentration of 100ng/μ L, and obtaining RNA fragment (fragments) with fragment length of 100-300 nt as shown in FIG. 3;

(4) 50 μ L of the above-described fragmented RNA (100 ng/. mu.L) was mixed with 80 μ L of 5 XP buffer (5 XP buffer) and 2 μ L of LRNA enzyme inhibitorPreparation (RNase inhibitor) 1 μ L N⁶Mixing the antibody of isopentenyl adenosine and 267 μ L of RNase-free water, beating, mixing, rotating and shaking at 4 deg.C for 4 hr to make N⁶Allyl adenosine modified RNA with N⁶Antibodies to isopentenyl adenosine bind well, 5 × IP buffer composition is shown in table 2;

TABLE 25 XIP buffer (5XIP buffer)

(5) Meanwhile, 40. mu.L of ProteinA magnetic beads (ProteinA beads) were separated on a magnetic frame, washed three times with 200. mu.L of 1 XP buffer (1 XP buffer), the remaining clear solution was aspirated, mixed with 80. mu.L of 5 XP buffer (5 XP buffer), 10. mu.L of 20 mg/. mu.L Bovine Serum Albumin (BSA), 310. mu.L of RNase-free water, and then whipped to mix well, and vortexed at 4 ℃ for 2 hours to prevent non-specific adsorption of the magnetic beads when they bind to the antibody, and the composition of 1 XP buffer (1 XP) is shown in Table 3;

TABLE 31 XIP buffer (1 XIP buffer)

(6) After RNA and antibody are fully combined, separating the mixed solution of the ProteinA magnetic beads and BSA on a magnetic frame, washing the magnetic beads with 200 mu L of 1x IP buffer solution (1x IP buffer) for three times, sucking the residual clear solution, mixing the ProteinA magnetic beads with the mixed solution of the RNA and the antibody, blowing and uniformly mixing the mixture, rotating and uniformly mixing the mixture at 4 ℃ and incubating the mixture for 2 hours to ensure that the antibody combined with the RNA is combined with the ProteinA on the magnetic beads;

(7) the mixture was placed on a magnetic rack to separate the magnetic beads, washed three times with 200. mu.L of 1 XP buffer (1 XP buffer), the remaining supernatant was aspirated, and 100. mu. L N was added⁶Mixing allyl adenosine triphosphate eluate (Elution buffer) with ProteinA magnetic beads, pumping, shaking at 4 deg.C for 3 hr, and adding N⁶-allyladenosine modified RNA eluted from antibodies bound to ProteinA magnetic beads into solution, N⁶-allyl adenosine triphosphateThe glycoside eluent composition is shown in Table 4;

TABLE 4N⁶Eluate of allyl adenosine triphosphate (Elution buffer)

(8) mu.L of the eluate after immunoprecipitation of 50. mu.L of fragmented RNA (100 ng/. mu.L) antibody was mixed to give 400. mu.L of solution, mixed with 40. mu.L of 3M sodium acetate solution, 440. mu.L of isopropanol, 2. mu.L of glycogen, whipped and mixed, after overnight precipitation, centrifuged at 15000rpm for 45 minutes at 4 ℃ and 880. mu.L of 80% ethanol, the precipitate was washed at 15000rpm for 15 minutes at 4 ℃, the supernatant was removed again, air dried for 3 minutes and RNA was dissolved at 15 ng/. mu.L with 20. mu.L of RNase-free water. Carrying out enzymolysis on the obtained RNA to obtain single nucleoside, and carrying out liquid mass spectrometry quantification to obtain a before and after IP⁶The content of A in RNA is shown in FIG. 5, HeLa Input is a in mRNA of HeLa cells before IP⁶A content, HeLa IP as a after IP⁶Content of A, H2.35Input is a in mRNA of H2.35 cell of pre-IP mouse⁶A content, H2.35 IP is a after IP⁶A content, the results show that IP is indeed a⁶The a modified mRNA was enriched.

3. N of RNA⁶Iodine addition reaction and cyclization treatment of-allyladenine

(1) mu.L of the above antibody-enriched treated RNA (15 ng/. mu.L) was transferred to a PCR tube, diluted to 26. mu.L with RNase-freewater, added with 4. mu.L of a 0.125M iodine solution (dissolved in 0.25M potassium iodide), the solution turned brown, and treated at 37 ℃ for 30 minutes;

(2) the brown solution was transferred to a new PCR tube, 4 μ L of 0.2M sodium thiosulfate was added until the solution was colorless, 6 μ L of 0.1M sodium carbonate (pH 9.5) was added, and treated at 37 ℃ for 30 minutes;

(3) mu.L of the above solution was mixed with 40. mu.L of isopropanol and 1. mu.L of glycogen, and then the mixture was shaken and mixed, and after overnight precipitation, the mixture was centrifuged at 4 ℃ at 15000rpm for 45 minutes, and the precipitate was washed with 880. mu.L of 80% ethanol, and centrifuged at 4 ℃ at 15000rpm for 15 minutes, and the supernatant was removed again, air-dried for 3 minutes, and RNA was dissolved with 15. mu.L of RNase-free water to give a concentration of 10 ng/. mu.L.

4. Constructing a library by using an illumina mRNA library construction kit for RNA fragments obtained without immunoprecipitation and immunoprecipitation (including cyclization treatment and non-cyclization treatment);

(1) configuring a reverse transcription system according to a reaction system shown in the table 5;

TABLE 5 mRNA library construction reverse transcription reaction System

(2) The program was run in a PCR instrument after mixing as shown in Table 6;

TABLE 6 mRNA library construction reverse transcription PCR run program

(3) Adding 5 μ L of resuspension buffer and 20 μ L of Second Strand MarkingMaster Mix into the system, mixing uniformly, and incubating at 16 ℃ for 1 h;

(4) purifying the double-stranded DNA fragment obtained above: adding 100 mu L of AMPure XP beads which are restored to the room temperature in advance into a reaction system, blowing and beating the mixture uniformly for 6 to 10 times, then incubating the mixture for 15 minutes at the room temperature, separating the mixture by using a magnetic frame, discarding the supernatant, washing the beads twice by using 80 percent ethanol, standing the mixture for 30 seconds each time for 5 to 10 minutes to volatilize the ethanol, eluting a double-stranded DNA fragment by using 17.5 mu L of resuspension buffer, incubating the mixture for 2 minutes at the room temperature, then separating the mixture by using the magnetic frame, and taking 15 mu L of the supernatant for the next reaction;

(5) the adenylic acid tail was added to the double-stranded DNA obtained above according to the following reaction system configuration of Table 7;

TABLE 7 mRNA library construction with adenylate tail addition reaction System

(6) The system is blown, beaten and mixed uniformly, and then the running program in a PCR instrument is shown in Table 8;

table 8 mRNA library construction with addition of adenylate tail PCR run program

(7) Add the adaptor according to the following table 9 reaction system configuration;

TABLE 9 mRNA library construction configuration addition linker reaction System

(8) Incubating the reaction system at 30 ℃ for 10 minutes;

(9) adding 5 mu L of connection termination mixture into the reaction, and uniformly mixing by blowing;

(10) purification of the adaptor-ligated DNA fragment: adding 42.5 mu L of AMPure XP beads which are restored to room temperature in advance into a reaction system, blowing and uniformly mixing for 6-10 times, then incubating for 15 minutes at room temperature, separating by using a magnetic frame and discarding the supernatant, washing the beads twice by using 80% ethanol, standing for 30 seconds each time, volatilizing the ethanol for 5-10 minutes, eluting a double-stranded DNA fragment by using 52.5 mu L of resuspension buffer, separating by using the magnetic frame after incubating for 2 minutes at room temperature, taking 50 mu L of supernatant, adding 50 mu L of AMPure XPbeads, repeating the purification step, eluting by using 22.5 mu L of resuspension buffer, and taking 20 mu L of supernatant for next PCR library amplification;

(11) configuring a library amplification system according to the following reaction system shown in Table 10;

TABLE 10 mRNA library construction amplification reaction System

(12) The system is blown, beaten and mixed evenly, and then the running program in a PCR instrument is shown in a table 11;

(13) purification of the amplified library: adding 55 mu L of AMPureXP beads which are restored to room temperature in advance into a reaction system, blowing and beating the mixture uniformly for 6-10 times, then incubating the mixture for 15 minutes at room temperature, separating the mixture by using a magnetic frame, discarding the supernatant, washing the beads twice by using 80% ethanol, standing the mixture for 30 seconds each time for 5-10 minutes to volatilize the ethanol, eluting a double-stranded DNA fragment by using 22.5 mu L of resuspension buffer, incubating the mixture for 2 minutes at room temperature, then separating the mixture by using the magnetic frame, taking 20 mu L of supernatant, wherein 1 mu L of the supernatant is used for determining the concentration of the Qubit;

TABLE 11 mRNA library construction amplification PCR run program

(14) Establishing a library of the obtained mRNA, performing double-end 150 sequencing by using an illumina X-10 platform, comparing the obtained data with a transcriptome of a cell source species to be tested after low-quality filtration and joint filtration, and obtaining m on the transcriptome of the HeLa cell and the mouse H2.35 cell by analyzing sequence characteristics before and after a mutation site⁶Conserved sequence of A site, which is related to the common m⁶The antibody A immunoprecipitates and the conserved sequences obtained by sequencing technology are approximately the same, which indicates that the site identified by the method is m⁶A site (as in FIG. 7).

(15) The high throughput sequencing results are shown in FIG. 8, and the sequencing results of the mutations show that LATS1 (NM-001350339) has m at the two sites 2970 and 2991⁶Modification A, ZNF445 (NM-181489) has m at 3451 and 3462 sites⁶A modification, OTUD1 (NM-001145373) has m at position 1479⁶And (C) modifying.

5. Performing low-flux sequencing on the RNA fragment subjected to iodine addition and cyclization treatment by using a TA cloning technology, verifying mutation of a specific site on the mRNA of the HeLa cell, and further identifying m⁶A site

(1) Mixing 1 μ L of the above RNA (10 ng/. mu.L) subjected to iodine addition and cyclization treatment with 1 μ L of 10 μ M reverse primer (R-primer) and 12 μ L of RNase-free water, pipetting, mixing, heating at 65 deg.C for denaturation for 5 min, wherein the sequence of the reverse primer (R-primer) is shown in Table 12;

TABLE 12 Forward primer (F-primer) and reverse primer (R-primer) sequences

(2) After cooling on ice, 4. mu.L of 5 XTRT Reaction Buffer (5 XT Reaction Buffer, Thermofisiher, EP0441), 1. mu.L of 10mM dNTP, 1. mu.L of 10U/. mu.L HIV reverse transcriptase (ReverseTranscriptase Recombinant HIV, Worthington Biochemical Corporation) was added, and the reverse transcriptase was inactivated by heating at 70 ℃ for 10 minutes;

(3) mu.L of the reverse transcription reaction solution was transferred to a new PCR tube, PCR amplification was performed with KOD-FX (TOYOBO, KFX-101) enzyme, 10. mu.L of 2mM dNTP, 1.5. mu.L of 10. mu.M forward primer (F-primer) and reverse primer (R-primer), 25. mu.L of 2 XKOD-FX reaction buffer, and 1. mu.L of KOD-FX enzyme were added, mixed and blown to mix, the sequences of the forward primer (F-primer) and reverse primer (R-primer) are shown in Table 5, and the PCR reaction program is shown in Table 13;

TABLE 13 post reverse transcription PCR amplification reaction procedure

(4) After the PCR reaction, the product DNA was purified with 1.8-fold volume of AMPure XP beads (BECKMAN COULTER, A63881), and the DNA was dissolved with 15. mu.L of LRNase-free water to a concentration of 50 ng/. mu.L;

(5) t Vector ligation of 1. mu.L of the above DNA (50 ng/. mu.L) product was performed using pUCM-T Vector (Sangon Biotech, B522213) and reacted at 16 ℃ for 6 hours, and the pUCM-T Vector ligation system is shown in Table 14;

TABLE 14 pUCm-T Vector ligation System

(6) Thawing 100 μ L DH5 α competent cells on ice for 5 min, suspending the cells uniformly, adding 5 μ L of the above connecting solution, beating gently, mixing well, and standing on ice for 25 min;

(7) performing heat shock in 42 ℃ water bath for 45 seconds, then placing on ice for 2 minutes, adding 700 mu L of SOC culture medium, and performing shake culture at 37 ℃ and 220rpm for 1 hour;

(8) centrifuging at 5000rpm for 1 min, sucking out 600 μ L of supernatant with a pipette tip, and suspending the cells with the rest of the culture medium;

(9) preparing 50mL LB plate containing 50. mu.L IPTG (100mM), 100. mu. L X-gal (20mg/mL), 50. mu.L 1000 Xampicillin, 15mL each, and uniformly spreading the bacterial suspension on the LB plate;

(10) firstly, culturing the plate at 37 ℃ for 1 hour, then culturing the plate in an inverted mode for 16 hours, screening white single colony extraction plasmids in a blue-white spot, and carrying out Sanger sequencing to obtain a low-pass sequencing result;

(11) the low-throughput sequencing results are identical to the high-throughput sequencing results. As shown in FIG. 10, the results of the mutation showed that LATS1 (NM-001350339) has m at two sites 2970 and 2991⁶Modification A, ZNF445 (NM-181489) has m at 3451 and 3462 sites⁶A modification, OTUD1 (NM-001145373) has m at position 1479⁶And (C) modifying.

Example 2m on transcriptome mRNA in mouse H2.35 cells⁶Detection of A modification sites

1. Culture of mouse H2.35 cells, allyl labeling and mRNA extraction

(1) Mouse H2.35 cells were cultured under normal culture conditions (with the addition of 200nM DEX, dexamethasone) to sixty percent fullness, the medium was aspirated, and the residual medium was washed away with Phosphate Buffered Saline (PBS);

(2) adding 10% Fetal Bovine Serum (FBS), 1% 100 Xpenicillin-streptomycin double antibody, 200nM DEX (dexamethasone), and 1mM cysteine to a methionine-free medium, culturing the above mouse H2.35 cells in the medium for 30 minutes, and removing methionine remaining in the cells;

(3) after methionine was removed, 1mM allyl-L-seleno/thiohomocysteine was added to the medium for culturing the above mouse H2.35 cells, and the culture was continued for 16 hours;

(4) - (12) culturing HeLa cells, allyl labeling and mRNA extraction as in example 1.

2. The same as example 1, which contains adenine N⁶And enriching the RNA modified by allyl.

3. N of RNA as in example 1⁶Iodine addition reaction to allyladenine and cyclizationAnd (6) processing.

4. In the same example 1, RNA fragments obtained by non-immunoprecipitation and immunoprecipitation (including cyclization treatment and non-cyclization treatment) were used to construct a library using the illumina mRNA library construction kit.

5. The high throughput sequencing results are shown in FIG. 9, and based on the mutation sequencing results, Xist (NR _001463) has m at both positions 11956 and 11964⁶Modification A, Usp42 (NM-029749) with m at position 2973⁶Modification A, Ice1 (NM-144837) has m at position 3777⁶A modification, Eppk1 (NM-144848) has m at two positions 2899 and 2924⁶And (C) modifying.

6. Or performing low-throughput sequencing on the RNA fragment subjected to iodine addition and cyclization treatment by using TA cloning technology in the same way as in example 1, verifying mutation of specific site on HeLa cell mRNA, and further identifying m⁶And (3) A site.

Example 3 culturing HeLa cells and mouse H2.35 cells under Normal cell culture conditions

Culture of HeLa cells

Adding 10% Fetal Bovine Serum (FBS) and 1% 100x penicillin-streptomycin double antibody into normal cell culture medium, culturing HeLa cell in the culture medium for 16-24 hr to extract mRNA of HeLa cell⁶The A content is shown as Ctrl in FIG. 2.

Simultaneously, HeLa cells were cultured under the above-mentioned allyl-labeling method, 1mM allyl-L-seleno, allyl-L-thiohomocysteine, or methionine (methyl-L-thiohomocysteine) was added thereto, and the cells were cultured for 16 hours to obtain m of mRNA⁶The content of A is shown in FIG. 2 as Se-allyl-L-selenohomocysteine, S-allyl-L-homocysteine, and L-Methionine. In FIG. 2, Se-allyl-L-selenohomocysteine/S-allyl-L-homocysteine is obtained by introducing allyl-L-seleno/thiohomocysteine into HeLa cell for metabolism⁶Content of allyl adenosine, L-Methionine Methionine introduced into HeLa cell for metabolism to obtain N in mRNA⁶-methyladenine content, Ctrl is N in mRNA obtained after HeLa cells are normally cultured⁶The content of-methyladenine nucleoside indicates that allyl-L-selenohomocysteine has a labeling efficiency higher than that of thio-cysteineHigh, with a much lower level of labeling than m in normal cultured cells⁶A level, and after half an hour of the treatment to remove intracellular methionine, methionine is added again, m of the cell⁶The level of A was nearly normal, indicating that the treatment to remove methionine in a short time did not have an excessive effect on the cell status.

2. Culture of mouse H2.35 cells

The mRNA of H2.35 cells was extracted by culturing H2.35 cells in a normal cell culture medium containing 10% Fetal Bovine Serum (FBS), 1% 100 Xpenicillin-streptomycin diabody, 200nM DEX (dexamethasone) for 16-24 hours.

HeLa cells were cultured under the same conditions as described above for the allyl labeling method, and 1mM of allyl-L-seleno, allyl-L-thiohomocysteine, or methionine (methyl-L-thiohomocysteine) was added thereto and cultured for 16 hours to obtain m of each group of mRNA⁶A. The results were similar to those of HeLa cells, and the labeling efficiency of allyl-L-seleno-homocysteine was higher than that of thio-cysteine, and the labeling level was much lower than that of m in normal cultured cells⁶A level, and after half an hour of the treatment to remove intracellular methionine, methionine is added again, m of the cell⁶The level of A was nearly normal, indicating that the treatment to remove methionine in a short time did not have an excessive effect on the cell status.

Claims

1. RNA N detection with single base resolution in full transcriptome range⁶-a method for methyladenine modification, characterized in that it comprises the following steps:

(1) allyl-L-seleno/thiohomocysteine labeling nucleic acid adenine: treatment of cells with the methionine analogue allyl-L-seleno/thiohomocysteine, which introduces an allyl group into specific adenine N of intracellular RNA by natural metabolism⁶At the position of methyladenine to form N⁶-allyladenine a⁶A；

(2) Containing a⁶Enrichment of a-modified RNA: extracting total RNA from cells, further extracting mRNA from the cells, and then breaking the whole transcriptome RNA of the cells intoFragmented RNA of 100-300 nt, binding N with antibody⁶-allyladenosine enrichment of a by immunoprecipitation⁶A modified RNA and elution from the antibody with an eluent a⁶A, modifying RNA, and purifying the eluted RNA;

(4) reverse transcription mutation and sequencing recognition of circularized RNA: adding HIV reverse transcriptase, N on RNA to the circularized structure obtained in step (3)¹，N⁶In the process of reverse transcription of cyclic adenine into DNA under the action of HIV reverse transcriptase, errors are generated by introducing para-complementary base, and mutation sites are identified by means of nucleic acid sequencing, so that a is obtained⁶A site, which is the original m in cellular RNA⁶A site of modification.

2. The full transcriptome-wide single base resolution detection of RNA N of claim 1⁶The method for modifying methyl adenine is characterized in that in the step (1), the preparation method of allyl-L-seleno/thiohomocysteine comprises the following steps: under the protection of nitrogen, taking selenium powder/sulfur powder and sodium borohydride with a molar ratio of 1:1 as raw materials, taking ethanol as a solvent, and heating and refluxing at 80 ℃ for reaction for 6-24 hours; adding hydrogen bromate of (S) - (+) -2-amino-4-bromobutyric acid with the molar weight of one half to one third of selenium powder/sulfur powder, continuously heating and refluxing for 6-24 hours at 80 ℃, adding acid to stop the reaction, filtering to remove insoluble substances, washing off by-products by using ether, and adjusting the pH value to be neutral; then adding sodium borohydride with the molar weight 1-2 times that of the selenium powder/sulfur powder to reduce diselenide/disulfide bonds, adding sodium bicarbonate/sodium carbonate with the molar weight 0.5-2 times that of the selenium powder/sulfur powder and allyl bromide with the molar weight 0.5-1.5 times that of the selenium powder/sulfur powder to react at room temperature for 6-24 hours, and purifying through high performance liquid chromatography to obtain the allyl bromide-L-seleno/thiohomocysteine.

3. The full transcriptome-wide single base resolution detection of RNA N of claim 1⁶-methyladenine modification, characterized in that, in step (1), said cells are normal mammalian cells, mammalian cancer cells, mammalian stem cells, host cells of bacteria, viruses, or are taken from various types of tissues and organs.

4. The full transcriptome-wide single base resolution detection of RNA N of claim 1⁶The method for modifying the methyl adenine is characterized in that the specific method for treating the cells in the step (1) is to culture the cells by adopting a methionine-deficient culture medium, add 0.1-2 mM of cysteine for half an hour, then add 0.1-2 mM of allyl-L-seleno/thiohomocysteine and culture for 12-24 hours.

5. The full transcriptome-wide single base resolution detection of RNA N of claim 1⁶-methyladenine modification method, characterized in that in step (2), said cellular whole transcriptome RNA fragmentation is by Zn²⁺The ions are heated for 5-10 minutes at 70 ℃.

6. The full transcriptome-wide single base resolution detection of RNA N of claim 1⁶-methyladenine modification method, characterized in that, in step (2), it is used to bind N⁶-allyladenosine antibody is N⁶Antibodies to isopentenyl adenosine, 10 μ g of antibodies were used to enrich for 1-20 μ g of fragmented RNA.

7. The full transcriptome-wide single base resolution detection of RNA N of claim 1⁶-methyladenine modification method, wherein in step (2), the eluent is N⁶Adenosine triphosphate, at a concentration of 5 to 10 mM.

8. The full transcriptome-wide single base resolution detection of RNA N of claim 1⁶-a method for modifying methyladenine, characterized in that said step (3) is specifically: dissolving 0.1-0.5M iodine simple substance in 0.2-1M potassium iodide to obtain potassium iodide solution of iodine, and mixing the potassium iodide solution of iodine with RNA a⁶N on A⁶-allylic reaction of allyladenine followed by removal of excess iodine with 0.1-0.5M sodium thiosulfate; adding 0.1-0.5M sodium carbonate, adjusting the pH value to 9-10, and inducing RNA a⁶N on A⁶N of allyl adenine¹，N⁶The site forms a cyclic structure, thus shielding the normal hydrogen bonding pairing of adenine.

9. The full transcriptome-wide single base resolution detection of RNA N of claim 1⁶-a method for methyladenine modification, characterized in that said step (4) is in particular: i) reverse transcribing the iodine addition-cyclized RNA using HIV reverse transcriptase; ii) adopting RNA library preparation technology and high-throughput sequencing means to carry out identification on the mutation sites of the whole transcriptome so as to obtain m with single base resolution of the whole transcriptome⁶And (3) distributing the A sites, or identifying and verifying the mutation sites on a specific transcript by adopting PCR and TA-cloning technologies.

10. RNA N detection with single base resolution in full transcriptome range⁶-methyladenine modified kit characterized in that it comprises allyl-L-seleno/thiohomocysteine, cysteine, N⁶Allyl adenosine triphosphate, RNA fragmentation solution containing divalent zinc ions, N⁶An isopentenyl adenosine antibody, a potassium iodide solution of iodine, a sodium thiosulfate solution, a sodium carbonate solution, an HIV reverse transcriptase, an HIV reverse transcription reaction solution, Tris-HCl, an RNase inhibitor, a sequencing linker and a sequencing primer.