WO2021042883A1 - Procédé et kit de détection de modification de l'arn n6-méthyladénine à une résolution de base unique dans la plage d'un transcriptome entier - Google Patents

Procédé et kit de détection de modification de l'arn n6-méthyladénine à une résolution de base unique dans la plage d'un transcriptome entier Download PDF

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WO2021042883A1
WO2021042883A1 PCT/CN2020/102643 CN2020102643W WO2021042883A1 WO 2021042883 A1 WO2021042883 A1 WO 2021042883A1 CN 2020102643 W CN2020102643 W CN 2020102643W WO 2021042883 A1 WO2021042883 A1 WO 2021042883A1
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rna
allyl
adenine
modification
cell
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刘建钊
冯新华
舒潇
曹婕
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浙江大学
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
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    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

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  • the present invention belongs to the field of gene sequencing, and particularly to a full range of single-base resolution transcriptome detecting RNA N 6 - methyl adenine modification methods and kits.
  • RNA is not only composed of the four bases of cytosine, thymine, guanine, and adenine.
  • N 6 -Methyladenine (m 6 A) is an extremely important modified base on RNA, which plays a variety of biological functions in biological processes, such as regulating gene expression. Among them, modification identification and sequencing methods are prerequisites for studying its biological significance.
  • the method of immunoprecipitation after antibody/RNA light cross-linking can also be used, that is, the antibody and the photoactive uracil or thio homologue of the m 6 A neighbor on the RNA are cross-linked.
  • the reverse transcription process leads to cross-linking. Uracil site mutation or termination near the cross-linking position, and then by analyzing the mutation or termination information to indirectly identify the A near the uracil as the m 6 A site.
  • the resolution of this method has been improved, its position identification is indirect, and m 6 A clusters cannot be distinguished.
  • the inventor hopes to use the recognizable modification group to introduce the modified group into the cell metabolism by grafting the modification group to the amino acid, replace the methylation modification of m 6 A, and then use the mutation sequencing method. In this way, the site of the modified group is identified, so as to achieve the purpose of identifying the m 6 A site.
  • amino acids with modified groups and normal amino acids are at a disadvantage in the competition of cell metabolism and cannot be introduced into cells.
  • the purpose of the present invention is to provide a method and kit for detecting RNA N 6 -methyladenine modification with single base resolution in the whole transcriptome range.
  • a method for detecting RNA N 6 -methyladenine modification with single-base resolution in the whole transcriptome range including the following steps:
  • Allyl-L-seleno/thiohomocysteine participates in cell metabolism and labeling nucleic acid adenine: use methionine analogue allyl-L-seleno/thiohomocysteine Acid-treated cells, cells undergo natural metabolism, and under the action of a series of enzymes, allyl groups can be introduced into the specific adenine N 6 position of RNA in the cell to form N 6 -allyl adenine (a 6 A), the site should be a natural methylation modification, that is, N 6 -methyladenine (m 6 A);
  • RNA containing a 6 A modification extract the total cell RNA, and then further extract the cell mRNA, and then fragment the whole transcriptome RNA of the cell into 100-300 nt fragmented RNA, and use the antibody to bind a 6 A , With the help of immunoprecipitation method, enrich a 6 A modified RNA, and use eluent to elute the a 6 A modified RNA from the antibody, and purify the eluted RNA;
  • Reverse transcription mutation and sequencing identification of circularized RNA Add HIV reverse transcriptase to the circularized structure obtained in step (3), and N 1 , N 6 cyclized adenine on RNA acts on HIV reverse transcriptase in vitro In the process of reverse transcription into DNA, an error occurs in the introduction of complementary bases. Nucleic acid sequencing is used to identify the mutation site, and then obtain the a 6 A site, which is the original m 6 A modified site in the cellular RNA. point.
  • step (1) the preparation method of allyl-L-seleno/thiohomocysteine is: under the protection of nitrogen, first use selenium powder with a molar ratio of 1:1 Se and sodium borohydride NaBH 4 are used as raw materials, ethanol is used as the solvent, and Na 2 Se 2 is prepared by heating and refluxing at 80°C for 6 to 24 hours, and then combining it with selenium powder with a molar amount of one-half to one-third (S )-(+)-2-amino-4-bromobutyric acid hydrobromide (compound 1) was heated and refluxed at 80°C for 6-24 hours, the reaction was terminated with acid, the insoluble matter was removed by filtration, and the by-products were washed away with ether , And then adjust the pH to neutral to obtain selenohomocysteine (compound 2).
  • the cells can be, but are not limited to, common mammalian cells, mammalian cancer cells, mammalian stem cells, bacteria, virus host cells, and cells derived from various types of tissues and organs.
  • the specific method of cell treatment in step (1) is to replace the normal medium of the cell culture system with a medium lacking methionine, and then add 10% FBS, 1% 100x penicillin-streptomycin double On the basis of resistance, add cysteine (0.1 ⁇ 2mM) for 0.5h, then add allyl-L-seleno/thiohomocysteine (0.1 ⁇ 2mM) and continue to incubate for 12-24h After that, the m 6 A modification on the RNA can be replaced with a 6 A modification. Media lacking methionine can be purchased directly.
  • step (2) the whole transcriptome RNA fragmentation treatment of the sample cell is realized by heating Zn 2+ at 70° C. for 4-10 min.
  • the antibody used to bind a 6 A is an antibody of N 6 -prenyladenosine, and 10 ⁇ g of antibody is used to enrich 1-20 ⁇ g of fragmented RNA. Binding antibody, the antibody binds to the fragmented RNA of the whole transcriptome of the above-mentioned sample cell.
  • the eluent in step (2) is N 6 -allyl adenosine triphosphate (a 6 ATP), and 5-10 mM a 6 ATP is used to compete for washing from N 6 -isopentenyl adenosine antibody.
  • the fragmented RNA of the whole transcriptome of the above-mentioned cells is removed, and the resulting RNA is purified by ethanol or isopropanol precipitation.
  • the method of mutation sequencing in step (4) is: i) After reverse transcription of the above-mentioned allyl-L-seleno/thiohomocysteine cell metabolism marker using HIV reverse transcriptase, the antibody is enriched with iodine Add circularized RNA; ii) Use RNA library preparation technology combined with high-throughput sequencing to identify mutation sites in the whole transcriptome to obtain a single-base resolution m 6 A site distribution in the whole transcriptome, or use PCR Identify and verify mutation sites on specific transcripts with TA-cloning technology.
  • the method of the present invention may also have the following characteristics: the amino acids assisted by cell metabolism and in vitro enzymes include but are not limited to allyl-L-selenohomocysteine (allyl-L-selenohomocysteine), allyl -L-thiohomocysteine (allyl-L-homocysteine) and its derivatives and analogues; N 6 -allyl adenine nucleoside antibodies include but are not limited to N 6 -isopentenyl adenosine Antibodies of its derivatives and analogs; reverse transcription mutant reverse transcriptases include but are not limited to HIV reverse transcriptase.
  • the amino acids assisted by cell metabolism and in vitro enzymes include but are not limited to allyl-L-selenohomocysteine (allyl-L-selenohomocysteine), allyl -L-thiohomocysteine (allyl-L-homocysteine) and its
  • the method for culturing cells in the above step (1) is to add a methionine derivative and cysteine to a medium lacking methionine, wherein the methionine derivative includes, but is not limited to, allyl-L-selenohomocysteine , Allyl-L-homocysteine and its derivatives and analogs; Cysteine includes but is not limited to the downstream metabolites of methionine in cell metabolism.
  • the chemical structural formulas of allyl-L-selenohomocysteine and allyl-L-homocysteine are as follows
  • the method for extracting RNA includes commonly used purification methods or commercial purification kits.
  • Purification methods include, but are not limited to: using TRIzol TM Reagent, chloroform-phenol extraction, Proteinase K digestion, silica gel membrane spin column method, magnetic bead method, ethanol, isopropanol precipitation and other techniques or a combination of one or more.
  • Purification kits include but are not limited to: GeneElute TM mRNA Miniprep Kit, RNeasy TM Mini Kit (Qiagen), RNA Clean & Concentrator (Zymo).
  • RNA fragmentation includes but is not limited to RNA fragmentation by metal ion method and RNA fragmentation by ultrasonic disruption; antibody enrichment includes but is not limited to the method of ProteinA/ProteinG beads; elution includes but is not limited to N 6 -allyl adenosine monophosphate, a 6 ATP competitive elution, TRIzol Reagent extraction elution.
  • the chemical structure of a 6 ATP and its synthesis steps are as follows: under the protection of nitrogen, ethanol is the solvent, 6-chloropurine nucleoside (compound 6), allylamine, and triethylamine are in a molar ratio of 1:3:3 in 80 After heating and refluxing at °C for 3-6 hours, the product obtained was evaporated in vacuum and then precipitated with ether, filtered to remove insoluble matter, and then recrystallized from methanol to obtain N 6 -allyl adenosine (compound 7).
  • the method of reverse transcription includes but is not limited to the method of HIV reverse transcriptase (Recombinant HIV reverse transcriptase enzyme) treatment.
  • the sequencing method includes, but is not limited to, high-throughput sequencing for library construction, and low-throughput sequencing based on TA-cloning.
  • the PCR enzymes in the TA-cloning method include but are not limited to KOD-FX DNA polymerase;
  • the library building methods include, but are not limited to, illuminated stranded library building, NEB small RNA library building, eCLIP and improved library building methods, etc.
  • the nucleic acid purification steps after each reaction can use common purification methods or commercial purification kits.
  • Purification methods include, but are not limited to: one or more combinations of silica gel membrane spin column method, magnetic bead method, ethanol, isopropanol precipitation and other technologies.
  • Purification kits include but are not limited to: AmpureXP beads, PCR purification Kit (Qiagen), RNA Clean&Concentrator (Zymo), DNA Clean&Concentrator (Zymo).
  • RNA N 6 -methyladenine modification with single-base resolution in the whole transcriptome range including allyl-L-seleno/thiohomocysteine, cysteine Amino acid, N 6 -allyl adenosine triphosphate, RNA
  • Iodine-induced double bond addition After being added to the double bond, iodine has a certain degree of leaving, and its adjacent carbon atoms can electrophilically attack atoms with higher electron cloud density.
  • the nitrogen atom on the purine ring has the characteristic of higher electron cloud density, especially the nitrogen atom at position 1.
  • the allyl iodide at the N6 position can induce the carbon atom to electrophilically attack the adjacent position N1 after the iodine atom leaves.
  • RNA methylation modification in the cell are methionine, S-adenosylmethionine, and N 6 -methyladenine, which remove the methionine that exists in the cell itself, and then use it in the culture conditions.
  • Allyl-L-seleno/thiohomocysteine replaced methionine and successfully modified the allyl group to the RNA of the whole transcriptome of the cell. Since the modification efficiency of N 6 -allyl adenine nucleoside is identified by quantitative mass spectrometry is low, the present invention finds an antibody that can specifically enrich N 6 -allyl adenine nucleoside, and uses the technique of antibody immunoprecipitation.
  • the method of the present invention is based on the chemical labeling of nucleic acid adenine in vivo and inducing mutations. Compared with the existing gene sequencing technology applied to m 6 A detection, the mutation site can be accurate to the single-base resolution, which improves the current universal It is a direct high-throughput single-base identification method based on m 6 A antibody immunoprecipitation and massively parallel sequencing to detect the accuracy of m 6 A sites.
  • the present invention realizes the N 6 -allyl labeling of adenine in the inner ribonucleic acid of the cell for the first time, which provides the possibility to identify its site by means of mutation of the m 6 A site.
  • the present invention uses N 6 -prenyl adenosine antibodies to specifically enrich N 6 -allyl adenosine antibodies. Because there is currently no N 6 -allyl adenosine specific antibody, after analysis and screening of some commercial antibodies, it was found that the N 6 -isopentenyl adenosine antibody is against N 6 -allyl gland Purine nucleoside has a certain specificity, the antibody has good binding ability with N 6 -allyl adenosine nucleoside, and commercial antibodies are easier to obtain, which greatly improves the practicability of the method.
  • the present invention uses a 6 ATP as the eluent, because a 6 ATP has the same main structure as the a 6 A modification on RNA, and the triphosphate increases its water solubility and has stronger binding ability with antibodies, making it modified with a 6 A
  • the RNA of ⁇ is at a competitive advantage, so the ⁇ 6 A modified RNA can be eluted by competing with the antibody. Therefore, compared with the common RNA extraction method for eluting RNA, this method makes a 6 A modified RNA and antibody almost completely separated, which greatly improves the yield of eluted RNA.
  • the present invention uses HIV reverse transcriptase to perform reverse transcription processing on iodine addition and inducing circularization of RNA, because the inventors have found that HIV reverse transcriptase has a shielded circular structure for the hydrogen bond of adenine for base complementary pairing.
  • HIV reverse transcriptase Reverse Transcriptase Recombinant HIV, Worthington Biochemical Corporation
  • M-MLV reverse transcriptase PROMEGA, M170A
  • AMV reverse transcriptase PROMEGA, M510F
  • RevertAid reverse transcriptase Thermofisher, EP0441
  • SuperScript II reverse transcriptase Invitrogen, 18064022
  • SuperScript III reverse transcriptase Invitrogen, 18080093
  • the present invention can be applied to a variety of analysis methods based on gene sequencing, such as detection of m 6 A modification sites on various types of nucleic acids, and cells based on N 6 -allyl adenosine RNA dynamic sequencing, etc.
  • Figure 1 is a schematic diagram of the modification method of the present invention
  • Figure 2 shows the content of N 6 -allyl adenine nucleoside in mRNA after allyl-L-seleno/thiohomocysteine is introduced into HeLa cells for metabolism;
  • Figure 3 is an RNA band obtained by fragmentation treatment of sample cell mRNA by divalent zinc ions
  • Figure 4 is a dot-hybrid diagram of N 6 -prenyladenosine antibody and normal A, m 6 A modified RNA, a 6 A modified RNA, which shows the binding ability of the corresponding RNA and antibody, and methylene blue shows the amount of RNA loaded ;
  • Figure 5 shows the N 6 -allyl adenine nucleoside content before and after mRNA enrichment in HeLa and H2.35 cells by antibody immunoprecipitation method
  • Figure 6 shows the results of gene sequencing of a 6 A modified RNA in HeLa cells before and after iodine addition-induced circularization
  • Figure 7 is the conservative sequence of the m 6 A site on the transcriptome obtained by high-throughput sequencing of HeLa cells and H2.35 cells;
  • Figure 8 is the m 6 A site on the transcriptome obtained by high-throughput sequencing of HeLa cells, taking three mRNA gene sequences as examples;
  • Figure 9 is the m 6 A site on the transcriptome obtained by high-throughput sequencing of H2.35 cells, taking four mRNA gene sequences as examples;
  • Figure 10 is the m 6 A site on the transcriptome obtained by low-throughput sequencing of HeLa cells, taking three mRNA gene sequences as examples;
  • Figure 11 is the high resolution mass spectrum of allyl-L-selenohomocysteine
  • Figure 12 is the 1 H NMR spectrum of allyl-L-selenohomocysteine
  • Figure 13 is the 13 C NMR spectrum of allyl-L-selenohomocysteine
  • Figure 14 is the high resolution mass spectrum of allyl-L-homocysteine
  • Figure 15 is a high resolution mass spectrum of a 6 ATP.
  • RNA As shown in Figure 4, three types of RNA were obtained by in vitro transcription, namely normal RNA (A-RNA), A was replaced by m 6 A RNA (m 6 A-RNA), and A was replaced by a 6 A RNA (a 6 A-RNA), it can be seen from the figure that only a 6 A-RNA (compared to A-RNA and m 6 A-RNA) has a relatively specific binding ability with antibodies, which can be derived from cellular mRNA
  • the normal A sequence and the m 6 A modified sequence identify the a 6 A modified mRNA to provide an enrichment function.
  • Methylene blue shows the loading amount of the corresponding RNA, which proves that the same amount of RNA is under a 6
  • the specific binding ability of A-RNA and antibody As shown in vitro transcription, namely normal RNA (A-RNA), A was replaced by m 6 A RNA (m 6 A-RNA), and A was replaced by a 6 A RNA (a 6 A-RNA), it can be seen from the figure that only a 6 A
  • the fragmented RNA is subjected to HIV reverse transcription and then connected to a sequencing adapter, and the sequencing primers are used for PCR amplification to construct a high-throughput sequencing library.
  • the high-throughput library is preferably illumina Truseq stranded mRNA LT kit. Sequencing the constructed sequencing library can obtain sequencing data.
  • the sequencing is preferably next-generation sequencing, more preferably Illumina paired-end sequencing, and the read length of sequencing is preferably 150 bp.
  • the sequencing data After obtaining the sequencing data, analyze the mutation rate of the circularized and uncyclized samples at the adenylate sites. If the mutation rate of the circularized sample relative to the uncircularized sample is greater than 3, and in the a 6 A antibody-rich region, It is considered that this site is the m 6 A site.
  • the analysis methods are: quality control of sequencing data, removal of adapter sequences and low-quality bases, alignment of the data with low-quality and adapters removed to the genome sequence and enrichment statistics, and comparison of data with low-quality and adapters removed Go to the transcriptome sequence and perform mutation statistics; specifically include the following steps: quality control of the original sequencing data through fastqc default parameters, and fastp software to remove linker sequences and sequences less than 25 bases (fastp -f 10 -F 10- x --detect_adapter_for_pe-l 25 –i Raw-R1.fq –I Raw-R2.fq –o Clean-R1.fq –O Clean-R2.fq), the obtained sequence is again used for quality control with the default parameters of the fastqc software, Then use hisat2 default parameters to align to the transcriptome sequence, use samtools to convert the aligned SAM file into a BAM file, and use samtools rmdup to remove repetitive sequences. Finally, use samtools mp
  • PCR and TA-cloning technology can also be used to perform low-throughput sequencing of a certain transcriptome to identify mutation sites.
  • the DNA polymerase for PCR is preferably KOD-FX DNA polymerase
  • the plasmid vector for connecting the PCR product is preferably a T vector.
  • Figure 11 shows the high resolution mass spectrum of allyl-L-selenohomocysteine prepared by the present invention (m/z is 224.0183, [M+H] + theoretical calculation is 224.0184);
  • Figure 12 shows the 1 H nuclear magnetic resonance spectrum, 1 H NMR ( 500MHz, D 2 O)
  • Figure 13 shows the 13 C nuclear magnetic resonance spectrum, 13 C NMR (126MHz, D 2 O); combined with Figures 11-13, it can be seen that allyl-L-selenohomocysteine was successfully prepared.
  • a 6 ATP 15 of the present invention is prepared as shown in the relevant characterization data (high resolution mass spectrum, C 13 H 20 N 5 O 13 P 3, m / z was 546.0192, [MH] - theoretical 546.0198), found successfully prepared a 6 ATP.
  • RNA hydrolysis obtained for individual nucleosides After quantitative liquid chromatography mass, to give a 6 A longitudinal IP RNA content is shown in FIG. 5, HeLa Input IP prior to the mRNA in HeLa cells in a 6 A content, HeLa IP is a 6 a content after IP, H2.35Input front H2.35 IP cells in mice in the mRNA content of a 6 a, H2.35IP as a 6 a content IP, the results show that, indeed a 6 a IP modified mRNA was enriched.
  • RNA fragments obtained without immunoprecipitation and immunoprecipitation are used to construct a library using the Illumina mRNA library construction kit;
  • connection termination mixture (9) Add 5 ⁇ L of the connection termination mixture to the above reaction, and mix by pipetting;
  • Table 11 The operating program of PCR for mRNA library construction and library amplification
  • the high-throughput sequencing results are shown in Figure 8.
  • the mutation sequencing results show that LATS1 (NM_001350339) has m 6 A modification at 2970 and 2991, and ZNF445 (NM_181489) has m 6 A modification at 3451 and 3462.
  • m 6 A modification, OTUD1 (NM_001145373) has m 6 A modification at position 1479.
  • RNA fragments treated with iodine addition and circularization were subjected to low-throughput sequencing using TA cloning technology to verify the mutation of specific sites on the mRNA of HeLa cells, and to further identify the m 6 A site
  • the product DNA is purified with 1.8 times the volume of AMPure XP beads (BECKMAN COULTER, A63881), and the DNA is dissolved in 15 ⁇ LRNase-free water to obtain a concentration of 50ng/ ⁇ L;
  • LATS1 (NM_001350339) has m 6 A modification at two positions 2970 and 2991
  • ZNF445 (NM_181489) has m 6 A modification at two positions 3451 and 3462
  • OTUD1 (NM_001145373) has m 6 A modification at position 1479. There is m 6 A modification.
  • Mouse H2.35 cells are cultured under normal culture conditions (additional 200nM DEX, dexamethasone is required), after culturing to a fullness of 60%, aspirate the medium and wash away the residual medium with PBS;
  • the high-throughput sequencing results are shown in Figure 9.
  • the mutation sequencing results show that Xist (NR_001463) has m 6 A modification at two positions 11956 and 11964, and Usp42 (NM_029749) has m 6 A modification at 2973 position. Ice1 (NM_144837) has m 6 A modification at position 3777, and Eppk1 (NM_144848) has m 6 A modification at two positions 2899 and 2924.
  • RNA fragments processed by iodine addition and circularization were subjected to low-throughput sequencing using TA cloning technology to verify the mutation of specific sites on the HeLa cell mRNA, and to further identify the m 6 A site, as in step 5 in Example 1. .
  • Se-allyl-L-selenohomocysteine/S-allyl-L-homocysteine is Se-allyl-L-selenohomocysteine/S-allyl-L-homocysteine introduced into HeLa cells for metabolism to obtain N 6 -allyl adenine in mRNA
  • L-Methionine is the N 6 -methyl adenine nucleoside content in the mRNA obtained after methionine is introduced into HeLa cells for metabolism
  • Ctrl is the N 6 -methyl adenine in the mRNA obtained after normal culture of HeLa cells
  • the content of nucleoside shows that the labeling efficiency of allyl-L-selenohomocysteine is higher than that of allyl-L-homocysteine, but it is much lower than the m 6 A level in normal cultured cells, and after 0.5h of removal of intracellular methionine After the treatment, methi
  • the HeLa cells were cultured according to the treatment conditions of the above-mentioned allyl labeling method to obtain m 6 A of each group of mRNA.
  • the result is similar to the above-mentioned HeLa cells.
  • the labeling efficiency of allyl-L-selenohomocysteine is higher than that of allyl-L-homocysteine, and its labeling level is much lower than the m 6 A level in normal cultured cells.
  • methionine methionine was added again, and the m 6 A level of the cells was almost normal.

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Abstract

L'invention concerne un procédé et un kit pour détecter une modification d'ARN N6-méthyladénine à une résolution de base unique dans la plage d'un transcriptome entier. Selon le procédé, sur la base d'une étiquette N6-allyle d'adénine d'acide ribonucléique (ARN) in vivo et d'un traitement chimique, une mutation de base adénine de l'ARN in vivo dans le processus de transcription inverse en ADN est induite, et un site de mutation est ensuite reconnu au moyen d'un séquençage d'acide nucléique, de telle sorte qu'un site a6A est obtenu, le site étant un site modifié à l'origine par m6A dans l'ARN cellulaire. Au moyen du procédé, l'étiquette spécifique de N6-allyladénine dans une cellule est obtenue pour la première fois, et l'étiquette peut non seulement être utilisée pour remplacer un site de N6-méthyladine dans la cellule, mais peut également être positionnée au moyen d'un séquençage de mutation.
PCT/CN2020/102643 2019-09-02 2020-07-17 Procédé et kit de détection de modification de l'arn n6-méthyladénine à une résolution de base unique dans la plage d'un transcriptome entier WO2021042883A1 (fr)

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