WO2023116490A1 - 小rna的新型检测方法及其应用 - Google Patents

小rna的新型检测方法及其应用 Download PDF

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WO2023116490A1
WO2023116490A1 PCT/CN2022/138474 CN2022138474W WO2023116490A1 WO 2023116490 A1 WO2023116490 A1 WO 2023116490A1 CN 2022138474 W CN2022138474 W CN 2022138474W WO 2023116490 A1 WO2023116490 A1 WO 2023116490A1
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srna
seq
linker sequence
rna
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翟琦巍
赖和劲
冯宁
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中国科学院上海营养与健康研究所
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    • C12Q1/6869—Methods for sequencing
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the present invention claims the priority right of the patent application number 202111571180.5 submitted to the State Intellectual Property Office of China on December 21, 2021, and the title of the invention is "a new detection method for small RNA and its application”.
  • the invention belongs to the field of biotechnology, and more specifically, the invention relates to a novel detection method and application of small RNA.
  • sRNA Small RNA
  • sRNA small RNA
  • miRNA small RNA
  • piRNA tsRNA
  • tRNA-derived small RNA tRNA-derived small RNA
  • srRNA small rDNA-derived RNA
  • miRNA is a kind of sRNA with an important biological regulation function in the range of 15-30nt in length, and participates in various biological processes through complementary pairing with target RNA.
  • the current conventional sRNA high-throughput sequencing library construction strategy is mainly by adding a linker sequence to the 3' end of the sRNA, and the ligation reaction of adding the linker sequence is based on T4 RNA ligase 2.
  • T4 RNA ligase 2 can specifically ligate the linker sequence to the 3' end-OH of sRNA.
  • the purpose of the present invention is to provide a novel detection method and application of small RNA.
  • a method for detecting terminal modifications of sRNA comprising:
  • sequences of the 3'-OH-labeled linker sequence, the 3'-cP-labeled linker sequence, and the sRNA-labeled 5'-linker sequence are different.
  • the linker sequence is a linker sequence of appropriate length, such as 5-100nt, such as but not limited to 10, 12, 16, 18, 20, 25, 30nt etc., preferably like 16nt.
  • the specific length is the length of the sRNA of interest depending on the research object, such as 10-150nt, such as but not limited to 12, 16, 18, 20, 25, 30 , 50, 60, 80, 100, 120nt, etc., preferably sRNA such as 16nt, preferably 15-46nt.
  • T4 RNA ligase 2 is used to ligate the linker sequence labeled 3'-OH.
  • dephosphorylation is performed with alkaline phosphatase (AP).
  • AP alkaline phosphatase
  • the oxidation treatment is performed with sodium periodate (NaIO 4 ).
  • phosphorylation is performed with T4 polynucleotide kinase (3' phosphatase deletion) (preferably T4 Pnk 3' phosphatase minus).
  • the linker sequence labeled 3'-cP is ligated in an RtcB ligation reaction.
  • T4 Rnl1 T4 RNA ligase 1
  • the linker sequence labeled 3'-OH and the linker sequence labeled 3'-cP it is determined to carry 3'- The presence or amount of OH and sRNA carrying 3'-cP; preferably, the linker sequence labeled 3'-OH, the linker sequence labeled 3'-cP, and the 5'-linker sequence labeled sRNA are scaled Sequenced adapter sequences (such as P5 and P7 high-throughput sequencing sequences carried by conventional sRNA sequencing).
  • the methods for identifying the product include (but are not limited to): quantitative PCR, sequencing, and Northern blot.
  • the sRNA to be tested in (a) forms a library; after determination by the method, according to the identification result of (f), Form 3'-OH sRNA sub-library, 3'-cP modified sRNA sub-library.
  • the reaction system of (a) includes: adding a linker sequence labeled 3'-OH (such as App-DNA12-ddC linker containing the 3'-OH sub-library barcode) to the sRNA to be tested sequence), T4 RNA ligase reaction buffer, PEG8000, T4 RNA ligase 2, ribonuclease inhibitor.
  • a linker sequence labeled 3'-OH such as App-DNA12-ddC linker containing the 3'-OH sub-library barcode
  • T4 RNA ligase reaction buffer Preferably, the sample is incubated at 16 ⁇ 3°C or 16 ⁇ 2°C or 16 ⁇ 1°C for 10-20 hours (such as 16 hours).
  • a step of enzyme inactivation is included; preferably, heat inactivation is performed, such as heat inactivation at 70 ⁇ 10°C.
  • the reaction system of (b) includes: CutSmart buffer, ribonuclease inhibitor, and alkaline phosphatase.
  • the sample is incubated at 37 ⁇ 3°C or 37 ⁇ 2°C or 37 ⁇ 1°C for 30 ⁇ 10 minutes or 30 ⁇ 5 minutes.
  • a step of enzyme inactivation is included; preferably, heat inactivation is performed, such as heat inactivation at 70 ⁇ 10°C.
  • the alkaline phosphatase comprises shrimp alkaline phosphatase.
  • the reaction system of (c) includes: NaIO 4 , ribonuclease inhibitor.
  • the samples are placed on ice and incubated in the dark for 40 ⁇ 20 minutes or 40 ⁇ 10 minutes.
  • the sRNA of 15-46 nt is recovered after precipitation with ethanol-sodium acetate and separation by PAGE.
  • the reaction system of (d) includes: T4 Pnk 3'phosphatase minus; after that, the nucleic acid is extracted, and precipitated with ethanol-sodium acetate.
  • the reaction system of (e) includes: a linker sequence for marking 3'-cP (such as an OH-RNA13-ddC linker sequence comprising a 3'-cP sublibrary barcode), RtcB reaction buffer solution, Mn 2+ ions, GTP, RtcB ligase, ribonuclease inhibitors.
  • a linker sequence for marking 3'-cP such as an OH-RNA13-ddC linker sequence comprising a 3'-cP sublibrary barcode
  • RtcB reaction buffer solution Mn 2+ ions
  • GTP GTP
  • RtcB ligase ribonuclease inhibitors.
  • the sample is incubated at 37 ⁇ 3°C or 37 ⁇ 2°C or 37 ⁇ 1°C for 120 ⁇ 30 minutes or 120 ⁇ 15 minutes.
  • a step of enzyme inactivation is included; preferably, heat inactivation is performed, such as heat inactivation at 70 ⁇ 10°C.
  • the reaction system after the reaction in (e) the reaction system includes separating the ligation products, selecting sRNAs with adapter sequences with a length in the range of 31-46 nt, and recovering.
  • the reaction system of the RNA 5' linker sequence ligation reaction includes: a 5'-linker sequence (such as, OH-RNA14-OH linker sequence) of the labeled sRNA, T4 RNA ligase Reaction buffer, PEG8000, ATP, T4 RNA ligase 1, ribonuclease inhibitor.
  • a 5'-linker sequence such as, OH-RNA14-OH linker sequence
  • the sample is reacted at 25 ⁇ 3°C or 25 ⁇ 2°C for 2 ⁇ 1 hours or 2 ⁇ 0.5 hours.
  • a step of enzyme inactivation is included; preferably, heat inactivation is performed, such as heat inactivation at 70 ⁇ 10°C.
  • the reverse transcription system includes: reverse transcription primers, SSIV buffer, reverse transcriptase, DTT, dNTP, ribonuclease inhibitor.
  • the sample is incubated at 50 ⁇ 3°C or 50 ⁇ 2°C for 1 ⁇ 0.5 hours or 1 ⁇ 0.2 hours.
  • a step of enzyme inactivation is included; preferably, heat inactivation is performed, such as heat inactivation at 80 ⁇ 10°C.
  • reverse transcription is followed by PCR amplification.
  • a method for detecting terminal modifications of sRNA comprising:
  • sRNA to be tested divide it into three groups, and treat with solvent, alkaline phosphatase and T4 polynucleotide kinase respectively:
  • T4 polynucleotide kinase (non-"T4 Pnk 3'phosphatase minus"; preferably specifically T4 Pnk) group the 3' ends of both the original 3'-P modification and the original 3'-cP modification of the sRNA are converted is 3'-OH;
  • PAP Polyadenylation reaction
  • the 3'-polyadenylated products of the original 3'-OH and the original 3'-P modified sRNA form cDNA
  • the 3'-end polyadenylated products of the original 3'-OH, the original 3'-P modification and the original 3'-cP modification sRNA form cDNA;
  • a reverse transcription primer such as OH-DNA15-OH
  • oligo dT a reverse transcription primer
  • the solvent includes (but not limited to): water, buffer.
  • the quantitative PCR is real-time fluorescent quantitative PCR; more preferably, quantitative by determining the Ct values of different groups: In the solvent group, the Ct value reflects the amount of 3'-OH sRNA; in the alkaline phosphatase group, the Ct value reflects the amount of 3'-OH and 3'-P modified sRNA; in the T4 polynucleotide kinase group , the Ct value reflects the amount of 3'-OH, 3'-P and 3'-cP modified sRNA.
  • the relative content of 3'-OH, 3'-P and 3'-cP is calculated according to the following formula:
  • (3'-OH)% 100%-(3'-P)%-(3'-cP)%.
  • it also includes further subdividing the sRNA product obtained by the method; preferably, the sRNA product obtained by the method is compared with the sRNA information base or database, thereby performing Segmentation of sRNA.
  • the sRNA to be tested in (1) forms a library; after the determination by the method, according to the determination result of (4), A 3'-OH sRNA sub-library and a 3'-cP modified sRNA sub-library were formed.
  • the sRNA products are further subdivided into molecules including but not limited to: tsRNA, miRNA, piRNA, snoRNA, snRNA, sinRNA, sgmRNA, smRNA, slncRNA, smcRNA, srpRNA, etc.
  • the information bases or databases include but are not limited to: rRNA databases from different species sources, GtRNAdb and mitotRNAdb databases, miRNA databases from miRbase and Ensembl, piRNA databases from piRNAdb, from Ensembl The snRNA, snoRNA, lncRNA, miscRNA and other ncRNA databases from Ensembl intron database, mature mRNA from Ensembl, IG gene, TR gene, Pseudogene, repeat sequence database from UCSC Genome Browser (RepeatMasker, GRCm38 /mm10 or GRCh38/hg38), genome database (GRCm38/mm10 or GRCh38/hg38), mitochondrial genome database (NC_005089.1 or NC_012920.1), etc.
  • rRNA databases from different species sources GtRNAdb and mitotRNAdb databases
  • miRNA databases from miRbase and Ensembl miRNA databases from miRbase and Ensembl
  • the sRNA to be detected includes (but is not limited to): total sRNA from cells, locally obtained or isolated sRNA.
  • the sRNA includes: sRNA derived from animals, plants or microorganisms.
  • the animals include (but are not limited to): mammals, fish, birds, amphibians, insects.
  • the plants include (but are not limited to): monocotyledonous plants, dicotyledonous plants.
  • the sRNA to be tested includes, but is not limited to, biological samples from cells (culture), tissues, blood, serum, plasma, urine, saliva, and cerebrospinal fluid.
  • the application of any of the methods described above is provided, including for detecting the terminal modification of sRNA; the terminal modification of the sRNA includes: (a) 3'-OH and 3'- cP modification; or, (b) 3'-OH, 3'-P and 3'-cP modification.
  • the application of any of the methods described above is provided, including the expression profile/profile/library of sRNA modified at the 3'-OH and 3'-cP ends of the sRNA for establishing the cell, or establishing Expression profiles/maps/libraries of 3'-OH, 3'-P and 3'-cP end-modified sRNAs.
  • discrimination of sRNA terminal modifications is further used to determine specific biological states (including physiological changes or pathological changes).
  • the distinction of sRNA terminal modifications is further used to screen for effector molecules (such as genes, proteins, compounds, etc.) that regulate sRNA terminal modifications, specifically such as Ang and/or RNase 4 significantly increased in vitro or in cells sRNA-cPs produced).
  • effector molecules such as genes, proteins, compounds, etc.
  • sRNA terminal modifications specifically such as Ang and/or RNase 4 significantly increased in vitro or in cells sRNA-cPs produced.
  • a test kit for detecting the terminal modification of sRNA which includes the following reagents: a reagent for detecting 3'-OH and 3'-cP terminal modification of sRNA: T4 RNA ligase 2 , alkaline phosphatase, sodium periodate, T4 polynucleotide kinase (3' phosphatase deletion, preferably T4 Pnk 3' phosphatase minus), RtcB ligase, linker sequence labeled 3'-OH, The linker sequence labeled 3'-cP, the linker sequence labeled 5'.
  • T4 RNA ligase 2 alkaline phosphatase, sodium periodate
  • T4 polynucleotide kinase (3' phosphatase deletion, preferably T4 Pnk 3' phosphatase minus
  • RtcB ligase linker sequence labeled 3'-OH
  • the linker sequence labeled 3'-cP the
  • a kit for detecting terminal modifications of sRNA which includes the following reagents: reagents for detecting 3'-OH, 3'-P and 3'-cP terminal modifications of sRNA: Solvent, alkaline phosphatase and T4 polynucleotide kinase (not "T4 Pnk 3' phosphatase minus"; preferably it is specifically T4 Pnk).
  • the T4 RNA ligase 2 comprises KQ truncated T4 RNA ligase 2 (T4 Rn12).
  • the alkaline phosphatase comprises shrimp alkaline phosphatase.
  • the T4 polynucleotide kinase is a 3' phosphatase mutated enzyme.
  • each reagent is packaged in different containers/packages and placed in the kit.
  • reagents selected from the group consisting of: spike-in mixture, T4 RNA ligase reaction buffer, water (including nuclease-free water), PEG8000, ribonucleic acid Enzyme inhibitor, CutSmart buffer, nucleic acid precipitation reagent (such as ethanol-sodium acetate), nucleic acid extraction reagent (such as phenol-chloroform), RtcB reaction buffer, Mn 2+ ion solution, GTP, ATP, reverse transcription primer, SSIV buffer, SuperScript IV reverse transcriptase, DTT, dNTP, DNA polymerase.
  • reagents selected from the group consisting of: spike-in mixture, T4 RNA ligase reaction buffer, water (including nuclease-free water), PEG8000, ribonucleic acid Enzyme inhibitor, CutSmart buffer, nucleic acid precipitation reagent (such as ethanol-sodium acetate), nucleic acid extraction reagent (such as phenol-chlor
  • the application of Ang and/or RNase 4 is provided to promote the formation of sRNA-cP; preferably, the promotion includes in vitro or intracellular promotion.
  • any of the foregoing methods or uses of the present invention are non-diagnostic/non-therapeutic methods or uses.
  • Figure 1 The 15-30nt sRNA in mouse liver is mainly sRNA-cP.
  • TANT-seq sequencing shows that there are multiple high-abundance sRNA types in mammalian 15-30nt sRNA-OH and sRNA-cP.
  • TE-qPCR detection showed that the expression of some sRNA-cP was significantly increased in RNH1 knockout Hepa 1-6 cells transfected with Ang or RNase 4 protein.
  • Mouse and human 15-30nt sRNA is mainly sRNA-cP.
  • RtcB ligase specifically ligates sRNA with 3'-P or 3'-cP at the 3' end.
  • TANT-seq detects the distribution of exogenously added spike-in in samples.
  • Figure 9 Sequence overlap analysis of sRNA-OH and sRNA-cP.
  • Figure 13A Representative 15-30nt sRNA-OH identified by TANT-seq in mouse hepatocytes or Hepa1-6 cells.
  • Figure 13B Representative 15-30nt sRNA-cP identified by TANT-seq in mouse hepatocytes or Hepa1-6 cells.
  • Figure 13C representative 15-30nt sRNA-OH identified by TANT-seq in human Hep G2 cells.
  • Figure 13D representative 15-30nt sRNA-cP identified by TANT-seq in human Hep G2 cells.
  • sRNA is extensively modified.
  • sRNA sequencing when there is chemical modification at the 3' end of sRNA, it will seriously hinder the ligation reaction.
  • the inventors revealed a novel method for detecting sRNA 3' end modification, including TANT-seq and TE-qPCR methods.
  • the detection method disclosed in the present invention provides a new technical means for the identification of sRNA-cP, and also provides a new idea for one-time qualitative/semi-quantitative/quantitative identification of various sRNA terminal modifications.
  • sRNA small molecule RNA, small RNA
  • sRNA refers to a short RNA expressed in an organism, usually referring to an RNA with a length less than 200nt (such as 5-199nt, specifically 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 150, 160, 180 or 190).
  • sRNA is usually not translated into protein and effectively inhibits the translation of specific mRNA through complementary binding, including but not limited to participating in the induction of gene silencing, participating in many regulatory processes such as cell growth, development, gene transcription and translation.
  • sRNA to be tested is not particularly limited, and it can be total sRNA from cells, locally obtained or isolated sRNA; it can be sRNA from animal, plant or microbial sources.
  • RNA-seq means RNA sequencing;
  • sRNA-seq means sRNA sequencing.
  • sRNA-seq involves enrichment and sequencing of sRNA species such as microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), transfer RNA-derived small RNAs (tsRNAs) , small RNA fragments derived from ribosomal RNA (rsRNA), RNA derived from small rRNA (srRNA), etc.
  • sRNA-seq can be combined with high-throughput sequencing platforms.
  • sRNA-Seq library preparation involves isolation of total RNA from samples, size fractionation, ligation of sequencing junction sequences, reverse transcription and PCR amplification, and DNA sequencing.
  • 5'-P is the abbreviation of 3' phosphate modification of sRNA
  • 3'-OH is the abbreviation of 3' hydroxyl modification
  • 3'-cP is the abbreviation of 3' ring phosphate modification of sRNA.
  • srRNA is the abbreviation of small rDNA-derived RNA
  • tsRNA is the abbreviation of tRNA-derived small RNA
  • snoRNA is the abbreviation of nucleolus small RNA (small nucleolar RNA)
  • snRNA is small nuclear RNA (small nuclear RNA, snRNA)
  • SinRNA is the abbreviation of small intron RNA
  • sgmRNA is the abbreviation of small genome-derived RNA
  • smRNA is the abbreviation of small mature mRNA
  • slncRNA is the abbreviation of small lncRNA
  • piRNA is the RNA (Piwi-interactiing smcRNA is the abbreviation of small miscRNA
  • srpRNA is the abbreviation of small repeat element derived RNA.
  • TANT-seq is an sRNA high-throughput sequencing method proposed in the present invention, which is based on the sRNA-seq method of T4 Rnl2/AP/NaIO 4 /T4 Pnk/RtcB.
  • TE-qPCR is a qPCR method for simultaneously detecting three 3' ends of RNA proposed by the present invention.
  • the present invention firstly provides a method for distinguishing the 3'-OH and 3'-cP terminal modifications of sRNA, called TANT-seq, this method can simultaneously realize the simultaneous qualitative and quantitative analysis of the two terminal modifications, and can cooperate with high
  • the high-throughput sequencing platform can meet the needs of high-throughput sequencing.
  • This method can realize the distinction of terminal modifications from the established sRNA total library, and then build sub-libraries according to the differences in modifications.
  • the TANT-seq high-throughput sequencing method is mainly based on the processing of multiple steps, including: (a) connecting the 3' of the sRNA with 3'-OH in the sRNA to be tested to a linker sequence marked with 3'-OH; ( b) dephosphorylate the product of (a); (c) oxidize the product of (b); (d) phosphorylate the product of (c); (e) make the product of (d)
  • the sRNA with 3'-cP in the sRNA is connected to the linker sequence labeled 3'-cP; and (f) according to the linker sequence labeled 3'-OH, the linker sequence labeled 3'-cP, identify 3 in the product of (f) '-OH, 3'-cP modified sRNA.
  • various enzymes/chemical substances are used in combination in each step, including: T4 Rnl2, AP, NaIO 4 , T4 Pnk and RtcB.
  • the T4 Rn12 is a KQ truncated enzyme.
  • the T4 Pnk is an enzyme with a 3' phosphatase mutation.
  • the present invention also provides a method for distinguishing 3'-OH, 3'-cP and 3'-P terminal modifications of sRNA, called TE-qPCR.
  • This method can simultaneously realize the determination of three terminal modifications, and is suitable for Refined analysis. It can quantitatively detect the ratio of different end-modified sRNAs in a sample at one time, including sRNA-cP, sRNA-P and sRNA-OH.
  • the sRNA modification assay method of TE-qPCR is mainly based on the processing of multiple steps, including: (1) provide sRNA to be tested, divide it into three groups, and use solvent, alkaline phosphatase and T4 polynucleotide kinase respectively Processing; (2) polyadenylation reaction (PAP) is performed on the product of (1) to polyadenylate the 3' end of the sRNA of 3'-OH; (3) reverse transcription is carried out on the product of (2) reaction; and (4) qualitative or quantitative determination of the product of the reverse transcription reaction of (3).
  • PAP polyadenylation reaction
  • This method uses RNA samples to form different modifications after water, AP or T4 Pnk treatment, polyadenylation reaction and reverse transcription reaction are performed on the treated RNA, followed by qPCR detection with specific primers.
  • the enzymes and biologically active substances used also include their variants, truncations, fusion proteins with other functional molecules, homologous molecules, and derivatives , isomers, etc.
  • the sRNA to be tested can be obtained from a variety of biological samples and detected by the method of the present invention, including solid tissues and/or biological fluids, which are not particularly limited in the present invention.
  • sRNA can be obtained in prokaryotic or eukaryotic organisms, including animals (e.g., vertebrates and invertebrates), plants, microorganisms (e.g., bacteria and yeast), or in some embodiments, including Cultured cells derived from these sources.
  • the test sample is a biological fluid sample, such as blood, serum, plasma, urine, saliva, or cerebrospinal fluid, from a human or animal subject (eg, a mammalian subject).
  • Methods for extracting and isolating all RNA (such as total cellular RNA) or partial RNA (such as RNA from specific organelles) in a given sample are well known in the art.
  • a variety of methods can be used to extract RNA, among which the guanidine isothiocyanate method and the Trizol method are more commonly used methods for the extraction of total RNA from animal tissues and animal cells.
  • Trizol contains guanidine isothiocyanate, which can rapidly break the cells, and at the same time denature the protein in the nucleoprotein complex and release the nucleic acid.
  • RNA can be purified using a variety of standard methods. In addition, there are various methods and products commercially available for isolating small molecular weight RNA.
  • RNA size fractionation can be performed. For example, small RNAs are isolated by running the isolated RNAs on denaturing polyacrylamide gels (or using any of a variety of commercially available kits). In some embodiments, sRNAs of 15-46 nt in length are considered sRNAs of interest.
  • junction sequences which serve as primer binding sites during reverse transcription and PCR amplification, can be added to both ends of small RNAs by a ligation step.
  • a pre-adenylated single-stranded DNA 3'-junction sequence followed by a 5'-junction sequence is ligated to a small RNA using a ligase such as T4 RNA ligase 2 (KQ truncated).
  • Linker sequences are utilized to label/isolate specifically modified small RNAs (eg, microRNAs).
  • the sRNA library was then reverse transcribed and amplified by PCR. This step converts the adapter-ligated RNA into cDNA clones that serve as templates for sequencing reactions.
  • Primers designed with unique nucleotide tags can also be used in this step to generate ID tags (ie, barcodes) in multiplexed sequencing of pooled libraries.
  • the detection platform can use reverse transcription, amplification and/or hybridization of probes, including quantitative or qualitative PCR, or real-time PCR.
  • the detection platform includes polymerase-based sequencing-by-synthesis (for example, Illumina), such as pyrosequencing (for example, 454Life Sciences), or sequencing-by-ligation (for example, ABI solid-state sequencing platform), etc.
  • the adapter sequence for sequencing that matches the specific platform also referred to as "barcode (sequence)" in the embodiments.
  • PCR detection can use RT-PCR primers, as well as fluorescently labeled probes matched therewith.
  • qPCR real-time polymerase chain reaction
  • Real-time PCR monitors the amplification of target DNA molecules during PCR (ie, in real time). Real-time PCR can be used both quantitatively and semiquantitatively.
  • Two common methods for detecting PCR products in real-time PCR are: (i) non-specific fluorescent dyes (e.g., SYBR Green (I or II)) that intercalate into any double-stranded DNA, and (ii) oligos labeled with fluorescent reporters. Sequence-specific DNA probes composed of nucleotides, the fluorescent reporter can only be detected after the probe hybridizes to its complementary sequence (eg TAQMAN).
  • non-specific fluorescent dyes e.g., SYBR Green (I or II)
  • the defined adapter sequence for sequencing is removed from the sRNA sequence after tagging, complete sequencing.
  • the adapter sequence is generally defined according to the sequencing platform employed.
  • Sub-libraries are created according to the different modifications of the assayed modified sRNAs after assaying by the method of the invention, preferably after removal of the linker sequence. For sub-libraries, different types of sRNAs can be further identified and quantified, subdivided.
  • the specific modified sRNA determined by the specific method of the present invention can be further compared with the data/database generated and/or provided by those skilled in the art in the research, and based on the comparison results.
  • the data/databases described are, for example, those listed in the embodiments of the present invention, or some data/databases that have been disclosed or are being developed in this field, or data that are established by themselves for some specific purposes /database.
  • Modified forms of sRNA can be identified to understand various utility in cellular or biological states, including in human and animal health and agriculture.
  • the present invention can be used for drug discovery, drug toxicology and therapeutic research, and individualized research of animals and plants.
  • the modified form of sRNA can be further subdivided; preferably, the sRNA product obtained by the method and The sRNA information base or database is compared to perform sRNA subdivision.
  • Software program quantification can also be used to achieve large-scale segmentation through software program identification.
  • the linker sequence can be removed first, so as to identify a single sRNA, including tsRNA, miRNA, piRNA, snoRNA, snRNA, sinRNA, sgmRNA, smRNA, slncRNA, smcRNA, srpRNA and the like.
  • the method of the present invention can be applied to detect the terminal modification of sRNA, and the terminal modification of the sRNA includes: (a) 3'-OH and 3'-cP modification; or, (b) 3'-OH, 3' -P modification and 3'-cP modification.
  • the 15-30nt sRNA in animals is mainly sRNA-cP, rather than sRNA-OH (as known in the art as miRNA) which is generally considered before.
  • sRNA-cP sRNA-OH
  • miRNA miRNA
  • sRNA-OH 3'-OH terminal
  • the present invention has developed two new methods for detecting sRNA modification, TANT-seq and TE-qPCR, both of which can simultaneously detect sRNA-OH and sRNA-cP.
  • TANT-seq in the present invention can simultaneously qualitatively and quantitatively analyze sRNA-OH and sRNA-cP, and will not convert the 2' end to oxygen Methyl-modified (2'-OMe) sRNAs are misidentified as sRNA-cP (RtcB ligase contributes to this effect).
  • sRNA-cP sRNA-cP
  • sRNA-P sRNA-P
  • sRNA-OH sRNA-OH
  • sRNA-OH and sRNA-cP from more than 10 different biotypes of mouse and human origin have been discovered and verified, and related nomenclature and sRNA-cP are also provided. details.
  • different types of sRNA could only be sequenced based on sRNA-OH.
  • smRNA-Seq sequencing it was found that miRNA in mouse brain tissue was about 16.6% of the total sRNA-OH.
  • TANT-seq was used to find that miRNAs in mouse liver only accounted for 10.5% of the total sRNA-OH, which was also consistent with previous reports. Based on the importance of miRNA in various physiological and pathological states, the inventors speculate that sRNA-cP with a higher proportion may also play a key role in some biological processes.
  • RNA 3'-cP modified RNA There are a variety of biological processes in organisms related to the production of terminal 3'-cP modified RNA, such as by endonuclease or ribozyme cleavage, by RNA splicing, by exonuclease cleavage, or by using RNA 3' terminal phosphocyclase for de novo synthesis, etc.
  • endonucleases-Ang and RNase 4 can mediate the generation of a part of sRNA-cP in mammalian cells.
  • the present invention provides a new source of sRNA, which also provides a reasonable basis for the different sRNA sequences at different ends.
  • the present invention finds that there are a large number of 3'-cP modified sRNAs that have not been reported before in mammals, which expands the sRNA library, provides two powerful sRNA research methods, promotes the understanding of sRNA, and greatly expands the sRNA research field.
  • the sRNA to be tested may be from a sample with a specific indication/disease state.
  • indications/disease states include neurodegenerative diseases, cardiovascular diseases, inflammatory and/or immune diseases, and cancer, among others.
  • the source of the sRNA to be tested can be further defined and subdivided based on advanced or early disease, or disease progression, treatment received and patient response to treatment. Nevertheless, the main purpose of the method of the present invention is to analyze the modification state of sRNA, and can determine some factors/factors (such as genes, proteins, small molecular substances), signaling pathways that affect sRNA modification from the analysis results, and determine some meaningful sRNA with specific modifications.
  • the main purpose of the method of the present invention may not be the direct diagnosis and treatment of diseases; however, people can carry out further analysis, research and screening on the basis of the technical solutions provided by the present invention, so as to have Significant sRNA or its presence (such as ratio) is truly associated with the disease, thereby advancing people's understanding of the disease, or advancing people's development of new programs for diagnosing or treating diseases.
  • the reagents used in the method of the present invention can be combined to form a kit suitable for detecting the terminal modification of sRNA.
  • the reagent kit for detecting the terminal modification of sRNA includes reagents for detecting 3'-OH and 3'-cP terminal modification of sRNA: T4 RNA ligase 2, alkaline phosphate Enzyme, sodium periodate, T4 polynucleotide kinase, RtcB ligase, 3'-OH labeled linker sequence, 3'-cP labeled linker sequence, 3'-OH labeled linker sequence.
  • the kit for detecting the terminal modification of sRNA includes reagents for detecting 3'-OH, 3'-P and 3'-cP terminal modification of sRNA: solvent, alkaline Phosphatase and T4 polynucleotide kinase.
  • each reagent is divided into different containers/packages and placed in the kit.
  • each reagent is mixed in an appropriate solvent or buffer, packed in a container/package and placed in the kit.
  • the kit may also contain a variety of solvents, buffers, auxiliary reagents and the like that are compatible with the implementation of the method of the present invention.
  • the kit may also contain instructions for use, which illustrate the method for implementing the technical solution of the present invention, so as to facilitate the application of those skilled in the art.
  • Oligonucleotides for small RNA analysis sequence P-RNA1-OH P-aacgacaagacacaccaccacgagagcaauaagcag (SEQ ID NO: 1)-OH P-RNA2-OH P-gucaucugaaacuugcucucgaaaa (SEQ ID NO: 2)-OH
  • App-DNA3-ddC App-gtagcacgacaagacacaccaccacgagagcaataagcagatgac (SEQ ID NO: 3)-ddC P-RNA4-OH P-aucgauucgaucguaaucguaugccuaa (SEQ ID NO: 4)-OH P-RNA5-P P-ucacugcagaugaugaguagacacguaga (SEQ ID NO: 5)-P P-RNA6-cP P-augccuauggauucgucguc (SEQ ID NO: 6)-cP P-RNA7-cP P-caguacaguaugcagccucaucaaagugaaga (SEQ ID NO: 7)-cP OH-RNA8-OH OH-aacgacaagacacaccaccacgagagcaauaagcagaugac (SEQ ID NO: 8)-OH P-RNA9-OH P-guucucgaguaaaaguguc (S
  • Mouse RNase 4 sgRNA-sense caccgggtgttgaagcgtttgcac (SEQ ID NO: 116) Mouse RNase 4 sgRNA-antisense aaatgtgcaaacgcttcaacaccc (SEQ ID NO: 117) Mouse RNH1 sgRNA-sense caccgcaatacgaagtggtcaggt (SEQ ID NO: 118) Mouse RNH1 sgRNA-antisense aaatacctgaccacttcgtattgc (SEQ ID NO: 119) Mouse Ang sgRNA1-sense caccgtggcatacatagtgctgggtc (SEQ ID NO: 120) Mouse Ang sgRNA1-antisense aaacgacccagcactatgatgccac (SEQ ID NO: 121) Mouse Ang sgRNA2-sense caccgatcgctggtacatgcca
  • mice 8-week-old male C57BL/6 mice were randomly divided into two groups, raised in standard cages with or without food for 24 hours, and had free access to water.
  • mice 8-week-old male C57BL/6 mice were randomly divided into two groups, fed with a high-fat diet containing 60kcal% fat (research diet) and normal diet for 16 weeks.
  • mice 16-week-old male mice were used.
  • mice After mice were anesthetized and dissected, the collected tissues were quickly frozen with liquid nitrogen and stored in a -80°C refrigerator.
  • Hepa 1-6 Mouse hepatoma cells (Hepa 1-6), mouse hepatocytes (AML12), mouse embryonic fibroblasts (NIH/3T3) and human hepatoma cells (Hep G2) were cultured in DMEM containing 10% fetal bovine serum. Incubate at 37°C, 5% CO 2 . Total RNA was extracted when the cells were 80-90% grown, and sRNA was isolated.
  • RNA or DNA oligomer samples were mixed with an equal volume of 2 ⁇ RNA loading buffer (95% formamide, 18mM EDTA, 0.025% SDS, 0.025% bromophenol blue, 0.025% xylene cyanol), and incubated at 70°C for 10min Afterwards, immediately place the samples on ice. The samples were separated by 15% denatured PAGE gel containing 7M urea, and the PAGE gel after electrophoresis was stained with SYBR Gold staining solution (Invitrogen), photographed under ultraviolet light, and the gel images were quantitatively analyzed by ImageJ software.
  • 2 ⁇ RNA loading buffer 95% formamide, 18mM EDTA, 0.025% SDS, 0.025% bromophenol blue, 0.025% xylene cyanol
  • the 20 ⁇ l reaction system is as follows: 100pmol of 5' phosphorylated DNA oligonucleotide, 2 ⁇ l of 10 ⁇ 5' DNA adenylation reaction buffer, 2 ⁇ l of 1mM ATP, 100pmol of Mth RNA ligase, mix the sample evenly and place it at 65°C for reaction 1 hr, followed by 5 min incubation at 85°C to inactivate the enzyme. After the reaction, the samples were separated with 15% denatured PAGE gel containing 7M urea, and then stained with SYBR Gold to recover the adenylated product from the gel.
  • Mth RNA ligase has the activity of converting 3'-P to 3'-cP, and this property is used to prepare 3'-cP sRNA standards.
  • the reaction system was as follows: 100 pmol of synthesized sRNA with 5'-OH and 3'-P structures, 2 ⁇ l of 10 ⁇ 5' DNA adenylation reaction buffer, 2 ⁇ l of 1 mM ATP, 100 pmol of Mth RNA ligase, supplemented with H2O to 20 ⁇ l, After mixing evenly, place it at 65°C for 1 hour and inactivate the enzyme at 85°C for 5 minutes.
  • RNAs containing 5'-OH and 3'-cP were separated from the gel and extracted.
  • the reaction system is as follows: 300pmol structure of 5'-OH, 3'-cP sRNA, 5 ⁇ l 10 ⁇ T4 Pnk reaction buffer, 5 ⁇ l ATP (10 ⁇ M), 40U recombinant ribonucleic acid Enzyme inhibitor, 10U T4 Pnk (3' phosphatase mutation) (NEB), add nuclease-free water to a final volume of 50 ⁇ l, react the sample at 37°C for 30 minutes, then incubate at 65°C for 20 minutes for heat inactivation Enzyme, 5'-P, 3'-cP sRNA was recovered with QIAquick Nucleotide Removal Kit (Qiagen), and dissolved in nuclease-free water for later use.
  • Qiagen QIAquick Nucleotide Removal Kit
  • the supernatant contained sRNA less than 200nt, and washed with ethanol - Precipitate with sodium acetate, dissolve the final sRNA in nuclease-free water, store at -80°C or use for reaction. 15-30nt sRNA was separated with 15% denatured PAGE gel, stained with SYBR Gold, and then sRNA of corresponding length was recovered from the gel.
  • T4 RNA ligase 2 connects sRNA-OHs
  • the ligation reaction system is as follows: 50ng sRNA, 20pmol adenylated DNA linker, 2 ⁇ l 10 ⁇ T4 RNA ligase reaction buffer, 4 ⁇ l 50% PEG8000, 20U recombinant ribonuclease inhibitor, 200U truncated KQ type T4 RNA ligase 2( T4 Rnl2, KQ) (NEB), add nuclease-free water to make up to 20 ⁇ l. The samples were incubated at 16°C for 16 hours, and the ligation products were analyzed by 15% denaturing PAGE containing 7M urea.
  • RNA polyadenylation experimental system is as follows: 50ng sRNA, 2 ⁇ l 10 ⁇ poly(A) polymerase reaction buffer, 2 ⁇ l 10mM ATP, 20U recombinant ribonuclease inhibitor, 5U poly(A) polymerase, add nuclease-free water Make up to 20 ⁇ l. Samples were incubated at 37°C for 30 minutes and reaction products were analyzed using 15% denaturing PAGE with 7M urea.
  • Guanosine 5'-monophosphate 13 C 10 , 15 N 5
  • 140 ⁇ g/mL 5'-guanosine monophosphate 13 C 10 , 15 N 5
  • RNA processing method and LC-MS/MS analysis are briefly described as follows: 100ng sRNA was added to 60 ⁇ l 50mM NH 4 OAc, pH 5.3 solution, added 0.2U nuclease P1 (Sigma-Aldrich), incubated at 50°C for 3 hours, and then added 0.04U Phosphodiesterase I (USB) was treated at 37°C for 2 hours. Subsequently, RNA samples were treated with 2 U alkaline phosphatase (Sigma-Aldrich) for 2 hours at 37°C. Protein was removed by centrifugation through Nanosep 3K Omega membrane (Pall). Nucleoside mixtures were analyzed by an API 4000 Q-TRAP mass spectrometer (Applied Biosystems).
  • rSAP Shrimp alkaline phosphatase (abbreviated as AP) (NEB) reaction system
  • 50ng sRNA 1 ⁇ l 10 ⁇ CutSmart buffer
  • 20U recombinant ribonuclease inhibitor 1U rSAP
  • nuclease-free water 10 ⁇ l.
  • the samples were incubated at 37°C for 30 minutes, then incubated at 65°C for 5 minutes to heat inactivate the enzyme, and placed on ice after the reaction for subsequent ligation or RNA polyadenylation (PAP).
  • PAP RNA polyadenylation
  • the T4 Pnk reaction system is as follows: 50ng sRNA, 1 ⁇ l 10 ⁇ T4 Pnk reaction buffer, 20U recombinant ribonuclease inhibitor, 10U T4 Pnk, add nuclease-free water to 10 ⁇ l, incubate the sample at 37°C for 30 minutes, and continue to incubate at 65°C Heat inactivate the enzyme for 5 minutes, and place on ice after the reaction for subsequent ligation or RNA polyadenylation reactions.
  • the 15-30nt sRNA used in the RtcB ligation reaction was first treated with T4 Pnk (3' phosphatase mutation) to convert the 5'-OH of the sRNA to 5'-P, then extracted by phenol-chloroform and precipitated by ethanol-sodium acetate , dissolved in nuclease-free water.
  • the RtcB (NEB) ligation reaction system is as follows: 50ng sRNA, 20pmol linker (OH-RNA8-OH), 2 ⁇ l 10 ⁇ RtcB reaction buffer, 2 ⁇ l MnCl 2 (10mM), 1 ⁇ l GTP (10mM), 15pmol RtcB ligase, 20U recombinant Ribonuclease inhibitor, add nuclease-free water to 20 ⁇ l. The samples were mixed well and incubated at 37°C for 2 hours, then the ligation products were analyzed by 15% denaturing PAGE containing 7M urea.
  • TANT-seq sRNA library preparation process and related reaction system are as follows: 1 ⁇ g sRNA less than 200nt (9 ⁇ l volume) and 1 ⁇ l spike-in mixture (P-RNA2-OH, P-RNA10-P and P-RNA6-cP each 8nM; P- RNA9-OH, P-RNA5-P and P-RNA18-cP each 0.8nM; P-RNA16-OH, P-RNA17-P and P-RNA11-cP each 0.08nM) incubate at 70°C for 5 minutes and place on ice immediately Cool on the refrigerator, then add 20pmol App-DNA12-ddC linker sequence (Applied Biosystems) containing 3'-OH sub-library barcode, 4 ⁇ l 10 ⁇ T4 RNA ligase reaction buffer, 8 ⁇ l 50% PEG8000, 400U KQ truncated T4 RNA Ligase 2 (T4 Rnl2) (purchased from NEB), 40U recombinant ribonuclease
  • the ligation system is as follows: 10 pmol OH-RNA13-ddC linker sequence containing 3'-cP sub-library barcode, 2 ⁇ l 10 ⁇ RtcB reaction buffer, 2 ⁇ l MnCl 2 (10 mM), 1 ⁇ l GTP (10 mM), 15 pmol RtcB ligase, 20U recombinant ribonuclease inhibitor, and add nuclease-free water to a final volume of 20 ⁇ l.
  • the reaction system is as follows: 5pmol OH-RNA14-OH linker sequence, 3 ⁇ l 10 ⁇ T4 RNA ligase reaction buffer, 6 ⁇ l 50% PEG8000, 1 ⁇ l ATP (10mM), 30U T4 RNA ligase 1 ( T 4 Rnl1 ) (NEB), 20U recombinant ribonuclease inhibitor, and add nuclease-free water to a final volume of 30 ⁇ l. After mixing evenly, the sample was reacted at 25°C for 2 hours, and then incubated at 70°C for 5 minutes to heat inactivate the enzyme.
  • ligation product was then subjected to reverse transcription experiment (RT-PCR), and the system was as follows: 3 pmol OH-DNA15-OH reverse transcription primer, 4 ⁇ l 5 ⁇ SSIV buffer, 200U SuperScript IV reverse transcriptase (Invitrogen), 2.5 ⁇ l DTT ( 100mM), 2.5 ⁇ l dNTP (10mM), 20U recombinant ribonuclease inhibitor, and added nuclease-free water to a final volume of 50 ⁇ l. After mixing well, the samples were incubated at 50°C for 1 hour, and then incubated at 80°C for 10 minutes to heat inactivate the enzyme.
  • RT-PCR reverse transcription experiment
  • Phusion high-fidelity DNA polymerase (NEB) was used for PCR amplification.
  • the PCR primers and PCR index sequences used in the experiment are shown in Table 1.
  • the amplified products were separated by 7.5% non-denaturing PAGE gel, and the products with a length of 130-160 bp were selected for recovery by gel cutting.
  • the recovered samples were subjected to high-throughput sequencing using the Xten platform of Illumina.
  • High-throughput sequencing raw sequences (sequences obtained in the TANT method) first use fastp (v0.20.0) to remove linker sequences, low-quality sequences (Phred score ⁇ 30), and sequences shorter than 21nt or longer than 46nt are also removed.
  • the GACGTA barcode sequence (Applied Biosystems) in App-DNA12-ddC and the CTATCG barcode sequence (Applied Biosystems) in OH-RNA13-ddC were then classified into the 3'-OH sub-library and the 3'-cP sub-library, respectively , and remove the corresponding barcode sequence.
  • sequences were sequentially aligned to the corresponding reference databases using ncbi-BLAST+2.11.0.
  • first modify the relevant database as follows: add a CCA sequence to the 3' end of all tRNA sequences in GtRNAdb and mitotRNAdb.
  • the mouse rRNA database includes: mmu-5S rRNA (NR_030686. 1), mmu-5.8S rRNA(NR_003280.2), mmu-12S rRNA(NC_005089.1), mmu-16S rRNA(NC_005089.1), mmu-18S rRNA(NR_003278.3), mmu-28S rRNA(NR_003279 .1), mmu-45S rRNA (NR_046233.2) and the mouse rRNA sequence in the Ensembl database;
  • the human rRNA database includes: has-5S rRNA (NR_023363.1), has-5.8S rRNA (NR_145821.1), has -12S rRNA (NC_012920.1), has-16S rRNA (NC_012920.1), has-18S rRNA sequence in the Ensembl database;
  • the human rRNA database includes: has-5S rRNA (NR_023363.1), has-5.
  • Sequences that completely match the sequences in the database are assigned to the corresponding aligned categories, and then the unaligned sequences are aligned to the above database again, but the number of mismatches + skipped bases is allowed in this alignment ⁇ 2. Assign the aligned sequences to the corresponding categories again. Sequences that did not align twice were discarded.
  • sequences classified as pseudogene repeat sequences in the Dfam database were selected and classified as pseudogenes.
  • each type of highly abundant sRNA was manually checked using the nucleotide-nucleotide BLAST software in the NCBI website.
  • sequences classified into repetitive sequences but could be aligned to snRNA, C/D box 118 (Snord118) (NR_028566.3) or Gm25313 (XR_004935972.1) were reassigned to snoRNA or snRNA;
  • the sequence in the mRNA sequence that can be compared to Snord14c is assigned to snoRNA.
  • sequences that are classified as repetitive sequences but can be compared to RNU5A-1 (NR_002756.2) and RNU5B-1 (NR_002757.3) are assigned to snRNA; sequences that are classified as repetitive sequences but can be compared To snRNA, the sequence of C/D box 118 (SNORD118) (NR_033294.1) was assigned to snoRNA.
  • sRNAs mapped to rRNA and tRNA are classified as srRNA and tsRNA, respectively.
  • sRNAs mapped to introns, lncRNAs, genomes, mature mRNAs, repeats, and miscRNAs were named small intron RNAs (sinRNAs), small lncRNAs (slncRNAs), genome-derived small RNAs (sgmRNAs), and small mature mRNAs ( smRNA), small repeat-derived RNA (srpRNA), and small miscRNA (smcRNA).
  • the sRNA with an average sequencing read of more than 2 is uniformly named.
  • the naming principle is based on the sRNA category, matching sequence name, starting position, sequence length and 3' end modification; at the same time, in order to simplify the naming of small RNAs, according to the sRNA category, according to The abundance is named sequentially from high to low. Representative examples of specific sRNA nomenclature are shown in Figures 13A-D.
  • Figure 13A representative 15-30nt sRNA-OH identified by TANT-seq in mouse hepatocytes or Hepa1-6 cells
  • Figure 13B representative 15nt sRNA-OH identified by TANT-seq in mouse hepatocytes or Hepa1-6 cells -30nt sRNA-cP
  • Figure 13C representative 15-30nt sRNA-OH identified by TANT-seq in human Hep G2 cells
  • Figure 13D representative 15-30nt identified by TANT-seq in human Hep G2 cells sRNA-cP.
  • tsRNA can be further divided into 5'-tsRNA (tRNA-derived sRNA containing 5'-end) and 3'-CCA-tsRNA (tRNA-derived sRNA containing CCA at 3'-end) according to its position in the full-length tRNA. ), 3'-CC-tsRNA (tRNA-derived sRNA containing the additional CC at 3'-terminal) and internal-tsRNA.
  • the relative content of 15-30nt sRNA or various types of sRNA containing 3'-OH and 3'-cP is calculated as follows: Considering the strong reactivity of Poly(A) polymerase, almost all sRNA-OH can be polyadenylated, Use the reaction results in Figure 1e and Figure 1f to calculate the ligation efficiency of T4 RNA ligase 2 to sRNA-OH in 15-30 nt. The ligation efficiency of RtcB ligase to sRNA-cP in 15-30nt was calculated using the reaction results in Figure 7a and Figure 1f.
  • Venn diagram is generated by VennDiagram. Conservative analysis was generated by ggseqlogo in R language.
  • edgeR was used to calculate the fold change and P value, and the adjusted P value was calibrated by the Benjamini-Yekutieli method.
  • sRNAs with adjusted P ⁇ 0.01 and log2(fold change) ⁇ 2 or ⁇ 2 were considered to have significant changes.
  • sRNA-seq with an average of ⁇ 2 reads is shown in the volcano plot.
  • Quantitative PCR was performed using FastStart Universal SYBR Green Master from Roche Company.
  • the qPCR reverse and forward primers are listed in Table 1, and the experiment was performed according to the product instructions.
  • Figure 3a shows the principle of qPCR quantitative detection of three RNA 3' ends (3'-OH, 3'-P and 3'-cP).
  • the reaction system is as follows: 1 ⁇ l spike-in RNA (P-RNA2-OH, P-RNA10-P and P-RNA6-cP, each 8nM; P-RNA9-OH, P-RNA5-P and P-RNA18-cP, each 0.8nM; P-RNA16-OH, P-RNA17-P and P-RNA11-cP, each 0.08nM) or 1 ⁇ g of total RNA were treated with water, shrimp alkaline phosphatase and T4 Pnk, respectively, and the reaction volume was 10 ⁇ l.
  • nuclease-free water 1 ⁇ l 10 ⁇ poly(A) polymerase reaction buffer, 2 ⁇ l ATP (10mM), 5U recombinant ribonuclease inhibitor, 5U poly(A) polymerase, add nuclease-free water to a final volume of 20 ⁇ l, and incubate at 37 Incubate at °C for 60 minutes, then incubate at 70°C for 5 minutes for heat inactivation.
  • 3pmol reverse transcription primer (table 1), 200U SuperScript IV reverse transcriptase, 4 ⁇ l 5 ⁇ SSIV damping fluid, 2.5 ⁇ l DTT (100mM), 2.5 ⁇ l dNTP (10mM), 20U recombinant ribonucleic acid inhibitor, and Add nuclease-free water to a final volume of 40 ⁇ l. After mixing evenly, incubate at 50°C for 60 minutes, and continue to incubate at 80°C for 10 minutes for heat inactivation.
  • RNA 20 ⁇ g of total RNA or 100-200ng of 15-30nt sRNA was separated by 15% denaturing PAGE and transferred to a positively charged nylon membrane (Roche). Subsequently, ultraviolet crosslinking was performed, and DIG Easy Hyb buffer (Roche) was added for prehybridization. Then, add 10nM 3'-digoxigenin-labeled oligonucleotide probes and incubate overnight at 45-55°C, the temperature mainly depends on the annealing temperature of each probe, and the probe information is attached in Table 1.
  • the Ang and RNase 4 gene sequences were amplified from mouse liver cDNA, and inserted into the Nde I and Xho I sites of the pET28a plasmid to obtain the prokaryotic expression of pET28a-Ang and pET28a-RNase 4 plasmid.
  • Recombinant Ang and RNase 4 were induced and expressed in Escherichia coli BL21 Rosetta (DE3) cells, and purified using His-Tag purification resin (Roche) and ion-exchange columns (Hitrap Q HP, GE Healthcare).
  • Ang and RNase 4 proteins were analyzed for purity by SDS-PAGE and Coomassie blue staining, and finally dialyzed into storage buffer (20mM Tris-HCl pH 7.5, 200mM NaCl, 5% glycerol) and stored in a -80°C refrigerator .
  • RNA 4 ⁇ g mouse liver total RNA was treated with 0.5ng RNase A (Thermo), 1U RNase T1 (Thermo), 0.32 ⁇ g Ang or 0.08 ⁇ g RNase4 respectively, and the reaction was carried out at 37°C for 30 minutes. Then the RNA in the sample was extracted with phenol-chloroform, and after ethanol-sodium acetate precipitation, the final RNA was dissolved in nuclease-free water for TE-qPCR or Northern blot detection.
  • RNase A Thermo
  • 1U RNase T1 Thermo
  • RNA-seq data for analyzing the expression levels of submembers of the RNase A family in mouse liver were obtained from the GEO database (accession code: GSE164819).
  • the expression level is represented by FPKM (Fragments Per Kilobase of exon model per Million mapped fragments).
  • the two complementary oligonucleotides encoding gRNA in Table 1 were annealed to form a double strand, and then inserted into the BsmBI site of the lentiCRISPR v2 (Addgene plasmid 52961) vector, and the constructed plasmid was verified by first-generation sequencing.
  • the lentiCRISPR-mRNH1-sgRNA, lentiCRISPR-mANG-sgRNA1, lentiCRISPR-mRNase4-sgRNA, lentiCRISPR-hAng-sgRNA1, lentiCRISPR-hRNase 4-sgRNA plasmids were respectively obtained.
  • the px330-mp50 plasmid system containing mCherry was selected, and the sgRNA was inserted into the Bbs I site of the px330-mp50 plasmid to obtain the px330-mp-mAng-sgRNA2 and px330-mp-hAng-sgRNA2 plasmids.
  • the constructed plasmid containing sgRNA was transfected into Hepa 1-6 or Hep G2 cells by Lipofectamine 3000 transfection reagent (Thermo).
  • the transfected cells were screened with 2 ng/ ⁇ tl puromycin, and after 48 hours of screening, the cells were digested and diluted to obtain monoclonal cells.
  • px330-mp-mAng-sgRNA2 and px330-mp-hAng-sgRNA2 were transfected into RNase 4 knockout Hepa 1-6 and Ang knockout Hep G2 cells with Lipofectamine 3000 transfection reagent, respectively. After 48 hours of transfection, red fluorescent cells were sorted into 96-well plates by flow cytometry, with 1 cell per well, and then monoclonal cells were collected, and gene knockout was confirmed by immunoblotting and/or sequencing to obtain Ang and RNase 4 double knockout (DKO) Hepa 1-6 and Hep G2 cells.
  • DKO double knockout
  • Xfect Protein Transfection Reagent (Takara) to transfect 2 ⁇ g Ang or RNase 4 protein into transfected WT or RNH1 KO Hepa 1-6 cells in a 6-well plate. After 6 hours of transfection, wash 2 times with PBS, followed by Trizol Reagents to extract total RNA.
  • the data are expressed as the mean ⁇ SD of at least three independent experiments, and the statistical significance was assessed by Student's t test or one-way analysis of variance. Unless otherwise specified, the difference was considered statistically significant when P ⁇ 0.05. * or a means P ⁇ 0.05, ** or b means P ⁇ 0.01, *** or c means P ⁇ 0.001.
  • the original sequencing data is stored in NCBI Sequence Read Archive, BioProject number: PRJNA725316.
  • Embodiment 1 mouse and human 15-30nt sRNA end are mainly 3'-cP
  • the T4 RNA ligase 2 ligation reaction in the conventional sRNA library construction method was used to detect the proportion of sRNA with -OH at the 3' end in the 15-30nt sRNA as a whole.
  • Mouse liver 15-30nt sRNA was recovered by gel cutting, and then ligated using pre-adenylated linker sequences. The results showed that only about 10% of the sRNA could be ligated, meanwhile, the synthetic sRNA-OH standard could be almost completely ligated under the same conditions (Fig. 1a).
  • RNA non-template In order to eliminate the experimental error that may be caused by a single enzymatic reaction, and using the characteristic of E.coli Poly(A) polymerase that can continuously add ATP to the 3'-OH end of RNA non-template, the recovered mouse liver 15-30nt RNA polyadenylation experiments were carried out on sRNA, and it was found that about 90% of sRNA could not be polyadenylated. Similarly, the synthetic sRNA-OH standard could be completely polyadenylated (Fig. 1b).
  • LC-MS/MS method that can characterize and quantify more than 40 different types of nucleosides and nucleotides at one time was used for analysis Modified content of 15-30nt sRNA.
  • LC-MS/MS results showed that there were a large number of modifications on sRNA, such as ⁇ , m 1 A, Um, m 6 A, m 3 C, m 2 2 G, m 5 C, I.
  • sRNA-cP 3'-cP
  • Fig. 1c,d Fig. 5a-d
  • 3'-cP is a specific modification present at the 3' end of RNA, which can block the ligation of sRNA by T4 RNA ligase 2.
  • LC-MS/MS was used to detect different sources of tissue and cellular sRNA, and found that about 80% of small RNAs in mouse spleen tissue, Hepa 1-6 and Hep G2 cells also carried 3'-cP modification (Fig. 5e,f).
  • the above experiments show that the 15-30nt sRNA ends of mouse and human are mainly modified by 3'-cP.
  • the sRNA was treated with T4 polynucleotide kinase (T4 Pnk) to remove the 3' phosphate (3'-P) and 3'-cP of the sRNA, or alkaline phosphatase (AP) Treatment was used to remove the 3'-P group, and then the content of sRNA was analyzed by T4 RNA ligase 2 ligation reaction or polyadenylation reaction. The results showed that there was no obvious change after AP treatment, while after T4 Pnk treatment, sRNA Almost completely ligated or polyadenylated (Fig. 1e,f).
  • TANT-seq reveals the complete expression profile of 15-30nt sRNA, indicating that sRNA-OH and sRNA-cP usually have different sequences
  • this method can simultaneously detect sRNA-OH and sRNA-cP separately in one library.
  • the relative content of 3'-OH and 3'-cP sub-libraries can be obtained by combining the TANT-seq sequencing data with the ligation efficiency of T4 RNA ligase 2 and RtcB ligase, the results show that sRNA-OH and sRNA-cP in the mouse liver , Hepa 1-6 and Hep G2 cells were 11.7% and 88.3%, 11.4% and 88.6%, 12.4% and 87.6% respectively (Fig. The results of adenylation reaction and LC-MS/MS were similar.
  • TANT-seq comprehensively revealed the expression profiles of sRNA-OH and sRNA-cP, and divided sRNA into more than 10 categories according to the source (Fig. 2c).
  • srRNA small intron RNA
  • slncRNA small lncRNA
  • snRNA small genome-derived RNA
  • sgmRNA small genome-derived RNA
  • smRNA small mature mRNA
  • srpRNA small repetitive element-derived RNA
  • smcRNA small miscRNA
  • piRNA was 33.8, 21.9, 5.4, 1.8, 1.8, 1.4, 1.3, 0.71, 0.68, 0.64 and 0.38 times (Fig.
  • miRNAs in the sRNA-OH sub-library have an obvious The enrichment peak of sRNA-cP is different from it (Fig. 2e, Fig. 8i, j).
  • TANT-seq an sRNA high-throughput sequencing method called TANT-seq was successfully established, whose main advantage is that sRNA-OH and sRNA-cP can be detected simultaneously in one library.
  • TANT-seq we revealed a complete map of mouse and human 15-30nt sRNA-OH and sRNA-cP and found that they often have different sequences.
  • a qPCR method for the simultaneous detection of three 3' ends of RNA was established.
  • This method can simultaneously detect RNA samples containing 3'- OH, 3'-P and 3'-cP sRNAs (Fig. 3a).
  • the specific operation method is to divide the RNA sample into 3 parts, treat them with water, AP or T4 Pnk respectively, then perform polyadenylation reaction and reverse transcription reaction on the treated RNA, and then perform qPCR detection with specific primers (Fig.
  • the quantitative analysis of the sRNA-OH, sRNA-P and sRNA-cP content of the specific sequence in the sample can be carried out.
  • the TE-qPCR system was first verified using synthetic 3'-OH, 3'-P and 3'-cP standards, and the results showed that TE-qPCR can well distinguish different 3' ends Modified sRNA (Fig. 3b).
  • TE-qPCR method to detect mouse liver tissue, Hepa 1-6 and Hep G2 cell samples, the results showed that in the detected sRNA, almost 100% of the 3' end of miRNA was 3'-OH, which further illustrated that TE - The reliability of the qPCR method, while at the same time the sRNA-cP 3' end found in TANT-seq is 3'-cP (Fig. 3c, d, Fig. 10a, b).
  • the inventors also detected some sRNA-cPs found from TANT-seq in mouse brain tissue, mouse white adipose tissue, NIH/3T3 and AML12 cells (Fig. d, Fig. 10a), which indicated that sRNA -cP is ubiquitously expressed in different tissues or cultured cells.
  • TE-qPCR was used to explore the expression of some newly discovered sRNA-cPs under different physiological and pathological conditions.
  • rsR-67-cP, tsR-2-cP, sn-7-cP, sinR-7-cP and smR-26-cP The expression levels of rsR-67-cP, sn-7-cP, snR-7-cP, sinR-7-cP, smR-26 were significantly increased in the liver of high-fat diet mice or db/db mice -cP, slncR-106-cP and smR-13-cP expression levels were significantly decreased.
  • Embodiment 4 angiopoietin and RNase 4 participate in the generation of small RNA-cPs
  • Ang and RNase 4 protein was transfected into Hepa 1-6 cells, and the expression of some sRNA-cP was found to be moderately increased by TE-qPCR (Fig. 12d). It is known that RNH1 protein in cells can bind to Ang or RNase 4 and inhibit its activity.
  • a stable RNH1 knockout (HKO) Hepa 1-6 cell line was established using CRISPR/Cas9 technology, and was sequenced and immunoblotted Validation was performed on knockout cell lines (Fig. 12e, Fig. 4f).
  • Ang or RNase 4 protein was transfected into RNH1 knockout Hepa 1-6 cells, and the expression of some sRNA-cP was found to be significantly increased by TE-qPCR and Northern blot detection (Fig. 4f, g, Fig. 12f). These data suggest that Ang and RNase 4 can lead to the production of sRNA-cPs in vitro or in cells.
  • CRISPR/Cas9 technology was further used to construct stable Ang knockout (AKO), RNase 4 knockout (RKO) and Ang/RNase 4 double knockout (DKO) Hepa 1-6 cell lines, which were sequenced and immunoassayed. Blots were validated against knockout cell lines (Fig. 12g, Fig. 4i). Through TE-qPCR detection, it was found that the expression of some sRNA-cP in RKO and DKO Hepa 1-6 cells decreased to a certain extent (Fig. 12h).
  • a recombinant ribonuclease inhibitor is added to the Hepa 1-6 cell culture medium to block the activity of exogenous RNase.
  • a recombinant ribonuclease inhibitor is added to the Hepa 1-6 cell culture medium to block the activity of exogenous RNase.

Abstract

本发明提供了一种小RNA的新型检测方法及其应用。本发明揭示了新型的检测sRNA 3'端修饰的方法，包括TANT-seq和TE-qPCR方法。本发明所披露的检测方法为sRNA-cP的检测和鉴定提供了新的技术手段，同时也为一次性定性/半定量/定量鉴定多种末端修饰的sRNA提供了新思路。

Description

小RNA的新型检测方法及其应用
本发明要求2021年12月21日向中国国家知识产权局提交的，专利申请号为202111571180.5，发明名称为“小RNA的新型检测方法及其应用”的优先权。
技术领域
本发明属于生物技术领域，更具体地，本发明涉及小RNA的新型检测方法及其应用。
背景技术
小RNA(sRNA)是生物体内一类重要的特殊分子，目前认为此类特殊分子能够诱导基因沉默，参与细胞生长、发育、基因转录和翻译等诸多生命活动的调控过程。
高通量测序技术的发展极大地促进了小RNA(sRNA)的发现，例如miRNA，piRNA，tsRNA(tRNA-derived small RNA)，srRNA(small rDNA-derived RNA)等。miRNA是一类长度在15-30nt范围内的具有重要生物学调控作用的sRNA，通过和靶标RNA互补配对参与多种生物学过程。
本领域中，目前常规sRNA高通量测序文库构建策略主要是通过在sRNA的3’末端加上接头序列，而添加接头序列的连接反应是基于T4 RNA连接酶2。T4 RNA连接酶2可特异性地将接头序列连接到sRNA的3’末端-OH上。
然而，鉴于目前关于sRNA 3’端修饰的研究尚不明确，这样的测定方法是否能够反映细胞内所存在的sRNA的全貌，在本领域中是不够清楚的。
发明内容
本发明的目的在于提供小RNA的新型检测方法及其应用。
在本发明的第一方面，提供一种检测sRNA的末端修饰的方法，所述末端修饰包括3’-OH和3’-cP修饰，所述方法包括：
(a)提供待测sRNA，使其中存在3’-OH的sRNA的3’端连接上标记3’-OH的接头序列；
(b)对(a)的产物进行脱磷酸化处理，获得5’端和3’端脱去磷酸的产物；
(c)对(b)的产物进行氧化处理，使3’-OH发生氧化，分离(回收)sRNA；
(d)对(c)的产物进行磷酸化处理，获得携带5’-P的产物；
(e)使(d)的产物中存在3’-cP的sRNA连接上标记3’-cP的接头序列；
(f)根据标记3’-OH的接头序列、标记3’-cP的接头序列，鉴定(f)的产物中含有3’-OH和3’-cP的sRNA。
在一个或多个实施方式中，(a)中，所述标记3’-OH的接头序列、标记3’-cP的接头序列、标记sRNA的5’-接头序列的序列各不相同。
在一个或多个实施方式中，(a)或(e)中，所述接头序列为适当长度的接头序列，如5-100nt，例如但不限于10、12、16、18、20、25、30nt等，优选地如16nt。
在一个或多个实施方式中，(c)中，特定长度为根据研究对象的不同而感兴趣的sRNA的长度，如10-150nt，例如但不限于12、16、18、20、25、30、50、60、80、100、120nt等，优选地如16nt，优选 地如15-46nt的sRNA。
在一个或多个实施方式中，(a)中，利用T4 RNA连接酶2连接标记3’-OH的接头序列。
在一个或多个实施方式中，(b)中，以碱性磷酸酶(AP)进行脱磷酸化处理。
在一个或多个实施方式中，(c)中，以高碘酸钠(NaIO 4)进行氧化处理。
在一个或多个实施方式中，(d)中，以T4多聚合核苷酸激酶(3’磷酸酶缺失)(较佳地为T4 Pnk 3’phosphatase minus)进行磷酸化处理。
在一个或多个实施方式中，(e)中，以RtcB连接反应连接标记3’-cP的接头序列。
在一个或多个实施方式中，在(e)和(f)之间，还包括：在(e)的产物的5’端连接上标记sRNA的5’-接头序列；较佳地，利用T4 RNA连接酶1(T4 Rnl1)连接标记sRNA的5’-接头序列。
在一个或多个实施方式中，(f)中，根据标记3’-OH的接头序列、标记3’-cP的接头序列的存在情况(定性)/存在量(定量)，确定携带3’-OH和携带3’-cP的sRNA的存在情况或存在量；较佳地，标记3’-OH的接头序列、标记3’-cP的接头序列、标记sRNA的5’-接头序列为配合规模化测序的接头序列(如携带常规sRNA测序的P5和P7高通量测序上机序列)。
在一个或多个实施方式中，(f)中，对产物进行鉴定的方法包括(但不限于)：定量PCR法，测序法，Northern blot法。
在一个或多个实施方式中，(f)后，还包括：通过高通量测序，去除所述的标记3’-OH的接头序列、标记3’-cP的接头序列，根据鉴定结果，建立子文库。
在一个或多个实施方式中，规模化检测(如全细胞sRNA检测)时，(a)中所述待测sRNA形成一个文库；经由所述方法进行测定后，根据(f)的鉴定结果，形成3’-OH的sRNA子文库、3’-cP修饰的sRNA子文库。
在一个或多个实施方式中，(a)的反应体系中包括：在待测sRNA中加入标记3’-OH的接头序列(如，含有3’-OH子文库条形码的App-DNA12-ddC接头序列)，T4 RNA连接酶反应缓冲液，PEG8000，T4 RNA连接酶2，核糖核酸酶抑制剂。较佳地，样品置于16±3℃或16±2℃或16±1℃孵育10-20小时(如16小时)。
在一个或多个实施方式中，在(a)的反应体系反应之后，包括酶失活的步骤；较佳地，进行热失活，如在70±10℃热失活。
在一个或多个实施方式中，(b)的反应体系中包括：CutSmart缓冲液，核糖核酸酶抑制剂，碱性磷酸酶。较佳地，样品置于37±3℃或37±2℃或37±1℃孵育30±10分钟或30±5分钟。
在一个或多个实施方式中，在(b)的反应体系反应之后，包括酶失活的步骤；较佳地，进行热失活，如在70±10℃热失活。
在一个或多个实施方式中，所述碱性磷酸酶包括虾碱性磷酸酶。
在一个或多个实施方式中，(c)的反应体系中包括：NaIO 4，核糖核酸酶抑制剂。较佳地，样品置于冰上，黑暗条件下孵育40±20分钟或40±10分钟。
在一个或多个实施方式中，(c)中，利用乙醇-乙酸钠进行沉淀、PAGE分离后回收15-46nt的sRNA。
在一个或多个实施方式中，(d)的反应体系中包括：T4 Pnk 3’phosphatase minus；之后抽提核酸，乙醇-乙酸钠沉淀。
在一个或多个实施方式中，(e)的反应体系中包括：标记3’-cP的接头序列(如，包含3’-cP子文库 条形码的OH-RNA13-ddC接头序列)，RtcB反应缓冲液，Mn 2+离子，GTP，RtcB连接酶，核糖核酸酶抑制剂。较佳地，样品置于37±3℃或37±2℃或37±1℃孵育120±30分钟或120±15分钟。
在一个或多个实施方式中，在(e)的反应体系反应之后，包括酶失活的步骤；较佳地，进行热失活，如在70±10℃热失活。
在一个或多个实施方式中，在(e)的反应体系反应之后，包括分离连接产物，选择长度在31-46nt范围内的带有接头序列sRNA，回收。
在一个或多个实施方式中，(e)之后，RNA 5’接头序列连接反应的反应体系包括：标记sRNA的5’-接头序列(如，OH-RNA14-OH接头序列)，T4 RNA连接酶反应缓冲液，PEG8000，ATP，T4 RNA连接酶1，核糖核酸酶抑制剂。较佳地，样品在25±3℃或25±2℃条件下反应2±1小时或2±0.5小时。较佳地，反应之后，包括酶失活的步骤；较佳地，进行热失活，如在70±10℃热失活。
在一个或多个实施方式中，反转录体系包括：反转录引物，SSIV缓冲液，反转录酶，DTT，dNTP，核糖核酸酶抑制剂。较佳地，样品在50±3℃或50±2℃条件下孵育1±0.5小时或1±0.2小时。较佳地，反应之后，包括酶失活的步骤；较佳地，进行热失活，如在80±10℃热失活。
在一个或多个实施方式中，反转录之后进行PCR扩增。
在本发明的另一方面，提供一种检测sRNA的末端修饰的方法，所述末端修饰包括3’-OH、3’-cP和3’-P，所述方法包括：
(1)提供待测sRNA，将其分成三组，分别以溶剂、碱性磷酸酶和T4多聚合核苷酸激酶处理：
溶剂组不变，
碱性磷酸酶组中，原3’-P修饰的sRNA的3’末端转变为3’-OH，
T4多聚合核苷酸激酶(非“T4 Pnk 3’phosphatase minus”；较佳地具体为T4 Pnk)组中，原3’-P修饰和原3’-cP修饰的sRNA的3’末端均转变为3’-OH；
(2)对(1)的产物进行聚腺苷化反应(PAP)，使3’-OH的sRNA的3’端聚腺苷化(形成PolyA尾巴：
溶剂组中，仅原3’-OH的sRNA的3’端聚腺苷化，
碱性磷酸酶组中，原3’-OH和原3’-P修饰的sRNA的3’端聚腺苷化，
T4多聚合核苷酸激酶组中，原3’-OH、原3’-P修饰和原3’-cP修饰的sRNA的3’端聚腺苷化；
(3)对(2)的产物进行反转录反应：
溶剂组中，仅原3’-OH的sRNA的经3’端聚腺苷化的产物形成cDNA，
碱性磷酸酶组中，原3’-OH和原3’-P修饰的sRNA的经3’端聚腺苷化的产物形成cDNA，
T4多聚合核苷酸激酶组中，原3’-OH、原3’-P修饰和原3’-cP修饰的sRNA的经3’端聚腺苷化的产物形成cDNA；
(4)对(3)的反转录反应的产物进行定性或定量测定。
在一个或多个实施方式中，(3)中，进行反转录反应时，采用含有oligo dT的反转录引物(如OH-DNA15-OH)。
在一个或多个实施方式中，(1)中，所述的溶剂包括(但不限于)：水，缓冲液。
在一个或多个实施方式中，(4)中，通过定量PCR进行定性或定量测定；较佳地，所述定量PCR为实时荧光定量PCR；更佳地，通过测定不同组的Ct值定量：溶剂组中，Ct值反映3’-OH的sRNA的量；碱性磷酸酶组中，Ct值反映3’-OH和3’-P修饰的sRNA的量；T4多聚合核苷酸激酶组中，Ct值反 映3’-OH、3’-P和3’-cP修饰的sRNA的量。
在一个或多个实施方式中，(4)中，根据下列公式计算3’-OH，3’-P和3’-cP的相对含量：
Figure PCTCN2022138474-appb-000001
Figure PCTCN2022138474-appb-000002
(3′-OH)％＝100％-(3′-P)％-(3′-cP)％。
在一个或多个实施方式中，还包括，将所述方法获得的sRNA产物进行进一步的细分；较佳地，所述方法获得的sRNA产物与sRNA的信息库或数据库进行比对，从而进行sRNA的细分。
在一个或多个实施方式中，规模化检测(如全细胞sRNA检测)时，(1)中所述待测sRNA形成一个文库；经由所述方法进行测定后，根据(4)的测定结果，形成3’-OH的sRNA子文库、和3’-cP修饰的sRNA子文库。
在一个或多个实施方式中，所述sRNA产物进一步细分为包括但不限于以下分子：tsRNA，miRNA，piRNA，snoRNA，snRNA，sinRNA，sgmRNA，smRNA，slncRNA，smcRNA，srpRNA等。
在一个或多个实施方式中，所述信息库或数据库包括但不限于：不同物种来源的rRNA数据库，GtRNAdb和mitotRNAdb数据库，来自miRbase和Ensembl中的miRNA数据库，来自piRNAdb中的piRNA数据库，来自Ensembl中的snRNA，snoRNA，lncRNA，miscRNA和other ncRNA数据库，来自Ensembl中的内含子数据库，来自Ensembl中的mature mRNA，IG gene，TR gene，Pseudogene，来自UCSC Genome Browser的重复序列数据库(RepeatMasker，GRCm38/mm10或GRCh38/hg38)，基因组数据库(GRCm38/mm10或GRCh38/hg38)，线粒体基因组数据库(NC_005089.1或NC_012920.1)等。
在一个或多个实施方式中，根据形态，所述待测sRNA包括(但不限于)：来自细胞的总sRNA，局部获取或分离的sRNA。
在一个或多个实施方式中，根据物种，所述sRNA包括：动物、植物或微生物来源的sRNA。
在一个或多个实施方式中，所述动物包括(但不限于)：哺乳动物、鱼类、禽类、两栖动物、昆虫。
在一个或多个实施方式中，所述植物包括(但不限于)：单子叶植物，双子叶植物。
在一个或多个实施方式中，所述待测sRNA包括但不限于来自细胞(培养物)、组织、血液、血清、血浆、尿液、唾液、脑脊髓液的生物样品。
在本发明的另一方面，提供前面任一所述的方法的应用，包括用于检测sRNA的末端修饰；所述的sRNA的末端修饰包括选自：(a)3’-OH和3’-cP修饰；或，(b)3’-OH、3’-P和3’-cP修饰。
在本发明的另一方面，提供前面任一所述的方法的应用，包括用于建立细胞的sRNA的3’-OH和3’-cP末端修饰的sRNA的表达谱/图谱/文库，或建立3’-OH、3’-P和3’-cP末端修饰的sRNA的表达谱/图谱/文库。
在一个或多个实施方式中，sRNA末端修饰的区分进一步被用于测定特定的生物状态(包括生理变化或病理变化)。
在一个或多个实施方式中，sRNA末端修饰的区分进一步被用于筛选调节sRNA末端修饰的效应分子(如基因、蛋白、化合物等，具体如Ang和/或RNase 4在体外或细胞内显著增加sRNA-cPs产生)。
在本发明的另一方面，提供一种用于检测sRNA的末端修饰的试剂盒，其中包括下组试剂：检测sRNA的3’-OH和3’-cP末端修饰的试剂：T4 RNA连接酶2、碱性磷酸酶、高碘酸钠、T4多聚合核苷酸激酶(3’磷酸酶缺失，较佳地为T4 Pnk 3’phosphatase minus)、RtcB连接酶、标记3’-OH的接头序列、标记3’-cP的接头序列、标记5’的接头序列。
在本发明的另一方面，提供一种用于检测sRNA的末端修饰的试剂盒，其中包括下组试剂：检测sRNA的3’-OH、3’-P和3’-cP末端修饰的试剂：溶剂、碱性磷酸酶和T4多聚合核苷酸激酶(非“T4 Pnk 3’phosphatase minus”；较佳地其具体为T4 Pnk)。
在一个或多个实施方式中，所述T4 RNA连接酶2包括KQ截短型T4 RNA连接酶2(T4 Rnl2)。
在一个或多个实施方式中，所述碱性磷酸酶包括虾碱性磷酸酶。
在一个或多个实施方式中，T4多聚合核苷酸激酶为3’磷酸酶突变的酶。
在一个或多个实施方式中，各个试剂分装于不同的容器/包装中，置于所述试剂盒中。
在一个或多个实施方式中，其中还包括(但不限于)选自下组的试剂：spike-in混合物，T4 RNA连接酶反应缓冲液，水(包括无核酸酶水)，PEG8000，核糖核酸酶抑制剂，CutSmart缓冲液，核酸沉淀试剂(如乙醇-乙酸钠)，核酸抽提试剂(如苯酚-氯仿)，RtcB反应缓冲液，Mn 2+离子溶液，GTP，ATP，反转录引物，SSIV缓冲液，SuperScript IV反转录酶，DTT，dNTP，DNA聚合酶。
在本发明的另一方面，提供Ang和/或RNase 4的应用，用于促进sRNA-cP的形成；较佳地，所述的促进包括体外或胞内的促进。
在一个或多个实施方式中，本发明前面任一所述的方法或应用为非诊断性/非治疗性的方法或应用。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、小鼠肝脏中15-30nt sRNA主要为sRNA-cP。
(a)T4 RNA连接酶2连接反应表明只有少数15-30nt sRNA能被App-DNA3-ddC连接。
(b)聚腺苷化反应显示绝大多数sRNA不能加上聚腺苷化尾。
(c)LC-MS/MS质谱检测发现小鼠肝脏15-30nt sRNA存在大量修饰。数字标识分别代表如下修饰1，m 1A；2，2’，3’-cUMP；3，2’，3’-cGMP；4，2’，3’-cAMP；5，2’，3’-cCMP；6，m 6A；7，m 3C；8，ψ；9，I；10，m 5C；11，Um；12，m 2 2G。
(d)LC-MS/MS检测结果中15-30nt sRNA末端环磷酸修饰的定量统计。
(e-f)通过T4 RNA连接酶2连接反应(e)和聚腺苷化反应(f)分析15-30nt sRNA末端修饰。
(g)定量统计e，f中sRNA-OH和sRNA-cP比例。
图2、TANT-seq测序表明哺乳动物15-30nt sRNA-OH和sRNA-cP存在多种高丰度sRNA类型。
(a)TANT-seq流程示意图。
(b)TANT-seq检测小鼠肝脏sRNA-OH和sRNA-cP相对含量。
(c)小鼠肝脏中不同类型sRNA-OH和sRNA-cP相对miRNA-OH的丰度。
(d)哺乳动物不同类型15-30nt sRNA中不同类别sRNA含量分析。#表示3’-OH和3’-cP子文库之间存在显著变化。
(e)小鼠肝脏中15-30nt sRNA的长度分布情况。
(f)在3’-OH和3’-cP子文库中tsRNA种类存在显著差异。
(g)3’-OH和3’-cP子文库中的的全部sRNA或丰度排名前1000的sRNA序列重叠性分析。
图3、TE-qPCR和Northern blot验证TANT-seq数据。
(a)TE-qPCR检测3’-OH，3’-P或3’-cP sRNA示意图。
(b)合成标准品sRNA验证TE-qPCR效果。
(c-d)分别用TE-qPCR检测小鼠肝脏(c)，Hepa 1-6和NIH/3T3细胞(d)中特定sRNA。
(e)小鼠肝脏15-30nt sRNA用AP或T4 Pnk处理后，通过T4 RNA连接酶2连接反应后，用Northern blot检测特定sRNA末端修饰。
(f)TE-qPCR检测禁食、高脂饮食和db/db小鼠肝脏中特定sRNA含量。
图4、哺乳动物sRNA-cP产生部分依赖于Ang和RNase 4。
(a)小鼠肝脏15-30nt sRNA-cP 3’和5’端碱基保守性分析。
(b)RNase A或RNase T1处理小鼠肝脏总RNA后对应RNA变化情况。a，P＜0.05；b，P＜0.01；c，P＜0.001。
(c)小鼠肝脏中RNase A家族成员的mRNA表达量分析。
(d)RNase A、Ang或RNase 4处理小鼠肝脏总RNA后对应RNA变化情况。
(e)Ang或RNase 4处理小鼠肝脏总RNA后，Northern blot分析特定sRNA表达量。箭头表示预期序列大小。
(f)TE-qPCR检测显示，转染Ang或RNase 4蛋白到RNH1敲除的Hepa 1-6细胞中部分sRNA-cP表达量显著升高。
(g)Northern blot结果表明RNH1敲除的Hepa 1-6细胞中转染Ang或RNase 4蛋白后部分sRNA-cP表达量显著升高。
(h-i)TE-qPCR(h)和Northern blot(i)检测重组核糖核酸酶抑制剂预处理的Hepa 1-6细胞中部分sRNA表达量。
(j)火山图展示AKO、RKO和DKO Hepa 1-6细胞中sRNA-OH和sRNA-cP变化情况。
(k)TE-qPCR检测部分sRNA在AKO、RKO和DKO Hep G2敲除细胞系中的变化情况。
图5、LC-MS/MS分析RNA修饰。
(a-d)2’，3’-cAMP(a)，2’，3’-cUMP(b)，2’，3’-cCMP(c)and 2’，3’-cGMP(d)四种环磷酸修饰的电喷雾电离质谱图。
(e)LC-MS/MS质谱分析组织和细胞中sRNA修饰。
(f)定量统计组织和细胞中sRNA环磷酸修饰。
图6、小鼠和人15-30nt sRNA主要为sRNA-cP。
(a)聚腺苷化实验分析多种组织和细胞中15-30nt sRNA末端修饰。
(b)定量分析图2a中聚腺苷化实验结果。
(c)定量比较多种组织和细胞中15-30nt sRNA-OH和sRNA-cP含量。
图7、sRNA文库构建策略验证。
(a)RtcB连接酶特异性连接3’末端为3’-P或3’-cP的sRNA。
(b)sRNA文库构建中的的关键步骤汇总表。
(c)15％变性PAGE分析经T4 RNA连接酶2连接、AP去磷酸、NaIO 4氧化、乙醇沉淀后连接产物，表明仅sRNA-OH与App-DNA12-ddC连接。矩形框内的产物通过胶回收用于下一步反应。
(d)15％变性PAGE分析经T4 Pnk(3’磷酸酶缺失)处理，苯酚-氯仿抽提，RtcB连接酶连接的连接产物，表明不同建库策略可将末端为3’-P和3’-cP的sRNA与OH-RNA13-ddC连接。矩形框内的产物通过胶回收用于下一步反应。
(e)7.5％非变性PAGE分析最终扩增的文库。
(f)qPCR检测不同文库构建策略对合成RNA标准品的扩增效率。
图8、Hepa 1-6和Hep G2细胞TANT-seq高通量测序数据分析。
(a)TANT-seq检测样品中外源添加spike-in分布情况。
(b)小鼠肝脏15-30nt sRNA TANT-seq高通量测序结果重复性分析。Pearson相关系数表明独立重复实验高度重叠。
(c，e)Hepa 1-6和Hep G2细胞中15-30nt sRNA-OH和sRNA-cP相对丰度。
(d，f)TANT-seq揭示Hepa 1-6和Hep G2细胞中含有大量高丰度的不同类型sRNA。
(g，h)Hepa 1-6和Hep G2细胞中15-30nt sRNA的长度分布情况。
(i，j)Hepa 1-6和Hep G2细胞中3’-OH和3’-cP子文库中tsRNA种类存在显著差异。
图9、sRNA-OH和sRNA-cP序列重叠性分析。
(a-c)小鼠肝脏(a)、Hepa 1-6(b)或Hep G2(c)细胞中，3’-OH和3’-cP子文库之间的sRNAs仅存在少量重叠。
图10、TE-qPCR和Northern blot验证TANT-seq测序结果。
(a，b)小鼠(a)和人源(b)样品中，TE-qPCR验证TANT-seq数据。
(c，d)小鼠肝脏组织中，通过T4 RNA连接酶2连接反应(c)和聚腺苷化反应(d)结合Northern blot验证sRNA末端修饰。
图11、15-30nt sRNA-OH和sRNA-cP碱基保守性分析。
(a)小鼠肝脏15-30nt sRNA-OH 3’和5’端碱基保守性分析。
(b)小鼠肝脏中不同类型sRNA 3’端碱基保守性分析。
(c)Hepa 1-6细胞15-30nt sRNA碱基保守性分析。
(d)Hep G2细胞15-30nt sRNA碱基保守性分析。
图12、Ang和RNase 4是部分sRNA-cPs产生必需的。
(a，b)重组Ang(a)和RNase 4(b)蛋白纯度鉴定。
(c)定量分析图4e中Ang或RNase 4酶切小鼠肝脏总RNA后的sRNA相对表达量。
(d)Hepa 1-6细胞中转染Ang或RNase 4蛋白后增加部分sRNA表达量。
(e)RNH1敲除Hepa 1-6细胞DNA测序结果。
(f)定量分析图4g中Ang或RNase 4酶切后的sRNA相对表达量。
(g)Ang和RNase 4单敲除Hepa 1-6细胞和Ang/RNase 4双敲除Hepa 1-6细胞DNA测序结果。
(h)TE-qPCR检测AKO、RKO和DKO Hepa 1-6细胞中部分sRNA表达量。
(i)重组核糖核酸酶抑制剂预处理的Hepa 1-6细胞中15-30nt sRNA含量检测和定量。15-30nt RN A 定量统计用5S rRNA表达水平归一化。
(j)Northern blot检测WT、AKO、RKO和DKO Hepa 1-6细胞中部分sRNA表达量。
(k)定量分析图4i和图12j中Northern blot检测结果。
(l)Ang和RNase 4单敲除Hep G2细胞和Ang/RNase 4双敲除Hep G2细胞DNA测序结果。
图13A、鼠肝细胞或Hepa1-6细胞中TANT-seq鉴定到的代表性15-30nt sRNA-OH。
图13B、鼠肝细胞或Hepa1-6细胞中TANT-seq鉴定到的代表性15-30nt sRNA-cP。
图13C、人Hep G2细胞通过TANT-seq中鉴定到的代表性15-30nt sRNA-OH。
图13D、人Hep G2细胞通过TANT-seq中鉴定到的代表性15-30nt sRNA-cP。
具体实施方式
sRNA广泛存在修饰，在sRNA测序中，当sRNA 3’端存在化学修饰时会严重阻碍连接反应进行。本发明人在深入研究后，揭示了新型的检测sRNA 3’端修饰的方法，包括TANT-seq和TE-qPCR方法。本发明所披露的检测方法为sRNA-cP的鉴定提供了全新的技术手段，也为多种sRNA末端修饰的一次性定性/半定量/定量鉴定提供了新思路。
术语
如本发明所用，术语“sRNA(小分子RNA，small RNA)”是指在生物体中表示的短的RNA，通常指长度小于200nt的RNA(如5-199nt，具体如10、15、20、25、30、40、50、60、80、100、120、140、150、160、180或190)。sRNA通常不翻译成蛋白质且通过互补结合有效抑制特定mRNA的翻译，包括但不限于参与诱导基因沉默，参与细胞生长、发育、基因转录和翻译等诸多调控过程。
如本发明所用，“待测sRNA”没有特别的限制，其可以是来自细胞的总sRNA，局部获取或分离的sRNA；其可以为来自动物、植物或微生物来源的sRNA。
如本发明所用，“seq”为测序的简称。“RNA-seq”表示RNA测序；“sRNA-seq”表示sRNA测序。sRNA-seq包括使sRNA物质富集并对其进行测序，小RNA物质诸如微小RNA(miRNA)、Piwi相互作用RNA(piRNA)、小核仁RNA(snoRNA)、转运RNA来源的小RNA(tsRNA)、核糖体RNA来源的小RNA片段(rsRNA)、小rRNA来源的RNA(srRNA)等。sRNA-seq可结合高通量测序平台来进行。通常，sRNA测序文库制备包括从样品中分离总RNA，大小分级，测序连接序列的连接，逆转录和PCR扩增，以及DNA测序。
如本发明所用，除非另外说明，5’-P为sRNA的3’磷酸修饰的简称，3’-OH为3’羟基修饰的简称，3’-cP为sRNA的3’环磷酸修饰的简称。
如本发明所用，srRNA为small rDNA-derived RNA的简称；tsRNA为tRNA-derived small RNA的简称；snoRNA为核仁小RNA(small nucleolar RNA)的简称；snRNA为小核RNA(small nuclearRNA，snRNA)的简称；sinRNA为小内含子RNA的简称；sgmRNA为小基因组衍生RNA的简称；smRNA为小成熟mRNA的简称；slncRNA为小lncRNA的简称；piRNA为与Piwi蛋白相作用的RNA(Piwi-interactiing RNA)的简称；smcRNA为小miscRNA的简称；srpRNA为小重复元件衍生RNA的简称。
如本发明所用，“TANT-seq”为本发明提出的一种sRNA高通量测序方法，其是基于T4 Rnl2/AP/NaIO 4/T4 Pnk/RtcB的sRNA-seq方法。
如本发明所用，“TE-qPCR”为本发明提出的一种同时检测RNA三种3’末端的qPCR方法。
检测sRNA的末端修饰的方法
本发明首先提供了一种区分sRNA的3’-OH和3’-cP末端修饰的方法，称为TANT-seq，这一方法可同时实现两种末端修饰的同时定性和定量分析，可配合高通量测序平台，可满足高通量测序的需求。这一方法可实现从建立的sRNA总文库中进行末端修饰的区分、进而根据修饰的不同建立子文库。
所述TANT-seq高通量测序方法主要基于多个步骤的处理，包括：(a)使待测sRNA中存在3’-OH的sRNA的3’连接上标记3’-OH的接头序列；(b)对(a)的产物进行脱磷酸化处理；(c)对(b)的产物进行氧化处理；(d)对(c)的产物进行磷酸化处理；(e)使(d)的产物中存在3’-cP的sRNA连接上标记3’-cP的接头序列；以及(f)根据标记3’-OH的接头序列、标记3’-cP的接头序列，鉴定(f)的产物中3’-OH、3’-cP修饰修饰的sRNA。作为本发明的优选方式，所述的各个步骤中，将多种酶/化学物质的联合运用，它们包括：T4 Rnl2，AP，NaIO 4，T4 Pnk以及RtcB。优选地，所述T4 Rnl2为KQ截短型的酶。优选地，所述T4 Pnk为3’磷酸酶突变的酶。
本发明也提供了一种区分sRNA的3’-OH、3’-cP和3’-P末端修饰的方法，称为TE-qPCR，这一方法可同时实现三种末端修饰的测定，适用于精细化的分析。可以一次定量检测一个样品中不同的末端修饰的sRNA比例，包含sRNA-cP，sRNA-P和sRNA-OH。
所述TE-qPCR的sRNA修饰测定方法主要基于多个步骤的处理，包括：(1)提供待测sRNA，将其分成三组，分别以溶剂、碱性磷酸酶和T4多聚合核苷酸激酶处理；(2)对(1)的产物进行聚腺苷化反应(PAP)，使3’-OH的sRNA的3’端聚腺苷化；(3)对(2)的产物进行反转录反应；以及(4)对(3)的反转录反应的产物进行定性或定量测定。这一方法利用RNA样品在经由水、AP或T4 Pnk处理后形成不同的修饰，对处理后的RNA进行聚腺苷化反应和反转录反应，随后用特异性的引物进行qPCR检测。
应理解，本发明的上述两种检测方法中，所用的酶、生物活性物质也包括了它们的同功能的变异体、截短体、与其它功能性分子的融合蛋白、同源分子、衍生物、异构体等。
多种多样的生物样品中可以获取待测sRNA并应用本发明的方法进行检测，包括固体组织和/或生物液体，本发明中没有特别的限制。可以在原核或真核生物中获取sRNA，所述原核或真核生物包括动物(例如，脊椎动物和无脊椎动物)、植物、微生物(例如，细菌和酵母)，或在一些实施方案中，包括源自这些来源的培养的细胞。作为一些实施方式，待测样品是来自人或动物受试者(例如，哺乳动物受试者)的生物流体样品，诸如血液、血清、血浆、尿液、唾液或脑脊髓液。
在sRNA处理之前从样品中提取RNA用于检测。在给定样品中，提取并分离所有RNA(如细胞总RNA)或部分RNA(如特定细胞器来源的RNA)的方法是本领域公知的方法。多种方法可提取RNA，其中异硫氰酸胍法和Trizol法是动物组织及动物细胞总RNA提取较为常用的方法。Trizol方法中，Trizol内含异硫氰酸胍能迅速破碎细胞，同时使核蛋白复合体中的蛋白变性并释放出核酸，由于释放出的DNA和RNA在特定pH值下的溶解度不同，且分别位于中间相和水相， 从而使DNA和RNA得到分离，取出水相后，通过有机溶剂(如氯仿)抽提及异丙醇沉淀，可得到纯净RNA。RNA可以使用多种标准方法纯化。另外，还存在各种商业上可用于分离小分子量RNA的方法以及产品。
为了获得适当大小的含有sRNA的样品，可进行RNA的大小分级。例如，通过使分离的RNA在变性聚丙烯酰胺凝胶上跑胶(或使用各种市售试剂盒中的任一种)来分离小RNA。在一些实施方案中，考虑将长度为15-46nt sRNA作为感兴趣的sRNA。
可以通过连接步骤将连接序列添加到小RNA的两个末端，所述连接序列在逆转录和PCR扩增期间可作为引物结合位点。例如，使用连接酶诸如T4 RNA连接酶2(KQ截短型)将后面是5’-连接序列的预腺苷酸化单链DNA 3’-连接序列连接至小RNA。利用连接序列以标记/分离特定修饰的小RNA(例如，微RNA)。然后通过PCR使sRNA文库逆转录和扩增。该步骤将连接序列连接的RNA转化为作为测序反应的模板的cDNA克隆。经设计具有独特核苷酸标签的引物也可用于该步骤，以在合并的文库多重测序中产生ID标签(即条形码)。
sRNA的规模化测序列的测序平台可以是多种，所述检测平台可以利用逆转录、扩增和/或探针的杂交，包括定量或定性PCR，或实时PCR。例如包括基于聚合酶的边合成边测序(例如，Illumina)、如焦磷酸测序(例如，454Life Sciences)、或连接法测序(例如，ABI固体测序平台)等。运用不同的测序平台时，与之相应地，可运用与特定平台相配合的测序用接头序列(如本发明所定义的标记3’-OH的接头序列、标记3’-cP的接头序列、标记3’-OH的接头序列)，在实施例中也称为“条形码(序列)”。一些实施方案中，PCR检测可以应用RT-PCR引物，以及应用与之相配合的荧光标记探针。通常，实时聚合酶链反应(qPCR)在PCR期间(即实时)监测靶DNA分子的扩增。实时PCR可以定量和半定量地使用。在实时PCR中检测PCR产物的两种常用方法是：(i)嵌入任何双链DNA的非特异性荧光染料(例如，SYBR Green(I或II))，和(ii)由荧光报告因子标记的寡核苷酸组成的序列特异性DNA探针，所述荧光报告因子只有在探针与其互补序列(例如TAQMAN)杂交后才能检测到。
在一些实施方案中，在标记、完成测序后，从sRNA序列中去除所定义的测序用接头序列。所述接头序列通常根据所运用的测序平台来定义。
在经由本发明的方法进行测定后，优选地，在去除了接头序列后，根据所测定的修饰的sRNA的不同修饰，建立子文库。对于子文库，还可以进一步鉴定和量化、细分的不同类型的sRNA。
利用本发明的特定的方法确定的特定修饰的sRNA，其可进一步与本领域技术人员的研究中生成和/或提供的数据/数据库进行比对，并根据比对结果来。所述的数据/数据库例如被列举于本发明的实施例中的那些，也可以是本领域已经揭示或正在发展中的一些数据/数据库，也可以是出于一些特定的目的而自行建立的数据/数据库。
可以鉴定sRNA的修饰形式，籍此了解细胞或生物状态的各种效用，包括人类和动物健康以及农业中的效用。例如，本发明可用于药物发现、药物的毒理学和治疗学研究，动植物体的个性化研究。
鉴于sRNA可进一步细分为多种类型的功能性RNA分子，本发明的方法所测定后，确定了修饰形式的sRNA，可以进行进一步的细分；较佳地，所述方法获得的sRNA产物与sRNA的信 息库或数据库进行比对，从而进行sRNA的细分。也可通过软件程序鉴定，利用软件程序量化来实现大规模的细分。优选地，可先将接头序列进行去除，以便鉴定单个sRNA，包括tsRNA，miRNA，piRNA，snoRNA，snRNA，sinRNA，sgmRNA，smRNA，slncRNA，smcRNA，srpRNA等。
应用
本发明的方法可应用于检测sRNA的末端修饰，所述的sRNA的末端修饰包括选自：(a)3’-OH和3’-cP修饰；或，(b)3’-OH、3’-P修饰和3’-cP修饰。
在本发明中，发现动物15-30nt sRNA中主要是sRNA-cP，而不是之前通常认为的sRNA-OH(如本领域所熟知的miRNA)。同时，检测到sRNA-OH大约占到10％，而sRNA-cP约占85％。对于sRNA-OH和sRNA-cP含量的检测是在本发明中首次提出。
本发明人深入研究后确定，本领域中以末端为3’-OH的sRNA(sRNA-OH)作为15-30nt范围内最主要的sRNA是存在技术偏见的，针对多种细胞的测定显示，15-30nt sRNA中主要是sRNA-cP，而不是之前通常认为的sRNA-OH。
本发明开发了两种新的检测sRNA修饰的方法，TANT-seq和TE-qPCR，均可以同时检测sRNA-OH和sRNA-cP。之前本领域存在至检测sRNA-cP的cP-RNA-seq方法，与其相比，本发明中TANT-seq可以同时定性和定量分析sRNA-OH和sRNA-cP，而且不会将2’端为氧甲基修饰(2’-OMe)的sRNA错误的当作sRNA-cP(RtcB连接酶有贡献于实现这一效果)。以往有的研究中仅能获得sRNA-OH、sRNA-P和sRNA-cP混合在一起的信息、而不能精准的区分每一单独序列的特定末端修饰，本发明解决了这一问题。在以往的一些研究中，通过对RNA进行AlkB处理，可以克服某些RNA甲基化对反转录抑制的影响，从而提高sRNA-seq完整性，但AlkB处理RNA会增加RNA降解的风险，所以在本发明中，并未将AlkB处理过程加入其中。对于开发的TE-qPCR，可以一次定量检测一个样品中不同的末端修饰的sRNA比例，包含sRNA-cP，sRNA-P和sRNA-OH，这为sRNA-cP的鉴定提供了新的技术手段。
根据本发明的实施例，利用本发明的两种新方法，发现和验证了超过10种不同生物型类的小鼠和人来源的sRNA-OH和sRNA-cP，同时也提供了相关的命名和详细信息。以往不同型类的sRNA仅能基于sRNA-OH测序，比如利用smRNA-Seq测序得到小鼠大脑组织中miRNA约为sRNA-OH总量的16.6％。在本发明中，利用TANT-seq发现小鼠肝脏中miRNA大约只占sRNA-OH总量的10.5％，这也和之前的报道相吻合。基于miRNA在多种生理病理状态下的重要性，本发明人推测占比更高的sRNA-cP也很有可能在一些生物过程中扮演关键角色。
生物体内有多种生物学过程和末端3’-cP修饰的RNA产生有关，例如通过内切酶或核酶酶切产生，RNA剪接时产生，通过外切酶酶切产生，或利用RNA 3’末端磷酸环化酶进行从头合成等。本发明中，发现两种内切酶-Ang和RNase 4可以介导一部分sRNA-cP在哺乳动物细胞中的生成。参考熟知的miRNA或piRNA这些已被发现和证明分别通过Drosha/Dicer和Zucchini依赖的特定通路产生，本发明提供了新的sRNA的来源途径，这也为不同末端sRNA序列不相同提供了合理依据。
因此，本发明发现哺乳动物中存在大量之前未被报道的3’-cP修饰sRNA，扩充了sRNA库， 同时提供了两个强大的sRNA研究方法，推进了对sRNA的认识，极大的拓展了sRNA研究领域。
本发明中，所述的待测sRNA可以是来自具有特定适应症/疾病状态的样品。例如，适应症/疾病状态包括神经退行性疾病、心血管疾病、炎性和/或免疫性疾病和癌症等。可以基于晚期或早期疾病、或疾病进展过程、接受的治疗和患者对治疗的响应来进一步定义、细分待测sRNA的来源。尽管如此，本发明的方法的主要目的在于分析sRNA的修饰状态，可以从分析结果中确定一些影响sRNA修饰的因素/因子(如基因、蛋白、小分子物质)、信号通路，确定一些有意义的具有特定修饰的sRNA。由此可见，本发明的方法的主要目的可以不在于进行疾病的直接诊断和治疗；然而，人们可在本发明所提供的技术方案的基础上，进行进一步的分析、研究、筛选，以将有意义的sRNA或其存在情况(如比例)与疾病实现真正的关联，进而推进人们对于疾病的认识，或推进人们开发诊断或治疗疾病的新方案。
试剂盒
本发明的方法中所应用的试剂，可被组合在一起，形成适用于检测sRNA的末端修饰的试剂盒。
作为本发明的一种优选方式，所述的用于检测sRNA的末端修饰的试剂盒中包括检测sRNA的3’-OH和3’-cP末端修饰的试剂：T4 RNA连接酶2、碱性磷酸酶、高碘酸钠、T4多聚合核苷酸激酶、RtcB连接酶、标记3’-OH的接头序列、标记3’-cP的接头序列、标记3’-OH的接头序列。
作为本发明的一种优选方式，所述的用于检测sRNA的末端修饰的试剂盒中包括检测sRNA的3’-OH、3’-P和3’-cP末端修饰的试剂：溶剂、碱性磷酸酶和T4多聚合核苷酸激酶。
作为本发明的一种优选方式，各个试剂分装于不同的容器/包装中，置于所述试剂盒中。
作为本发明的一种优选方式，各个试剂被混合于适当的溶剂或缓冲液中，装于容器/包装中并置于所述试剂盒中。
所述的试剂盒中还可含有多种配合本发明的方法的实施的溶剂、缓冲液、辅助试剂等。
所述试剂盒中还可含有使用说明书，其中说明了实施本发明的技术方案的方法，从而便于本领域技术人员应用。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，中所述的条件，或按照制造厂商所建议的条件。
材料与方法
1、寡核苷酸
本发明所涉及的DNA和RNA寡核苷酸序列附于表1中。
表1
small RNA分析用寡核苷酸 序列
P-RNA1-OH P-aacgacaagacacacaccacgagagcaauaagcag(SEQ ID NO：1)-OH
P-RNA2-OH P-gucaucugaaacuugcucucgaaaa(SEQ ID NO：2)-OH
App-DNA3-ddC App-gtagcacgacaagacacacaccacgagagcaataagcagatgac(SEQ ID NO：3)-ddC
P-RNA4-OH P-aucgauucgaucguaaucguaugccuaa(SEQ ID NO：4)-OH
P-RNA5-P P-ucacugcagaugaugaguagacacguaga(SEQ ID NO：5)-P
P-RNA6-cP P-augccuauggauucgucguc(SEQ ID NO：6)-cP
P-RNA7-cP P-caguacaguaugcagccucaucaaagugaaga(SEQ ID NO：7)-cP
OH-RNA8-OH OH-aacgacaagacacacaccacgagagcaauaagcagaugac(SEQ ID NO：8)-OH
P-RNA9-OH P-guucucgaguaaaaguguguc(SEQ ID NO：9)-OH
P-RNA10-P P-cucaguugaucacacaugauccuaugu(SEQ ID NO：10)-P
P-RNA11-cP P-cacguagagccuucuuagcgcag(SEQ ID NO：11)-cP
small RNA文库制备用寡核苷酸 序列
App-DNA12-ddC App-gacgtatggaattct(SEQ ID NO：12)-ddC
OH-RNA13-ddC OH-cuaucguggaauucu(SEQ ID NO：13)-ddC
OH-RNA14-OH OH-guucagaguucuacaguccgacgauc(SEQ ID NO：14)-OH
OH-DNA15-OH OH-gccttggcacccgagaattcca(SEQ ID NO：15)-OH
Spike-ins 序列
P-RNA2-OH P-gucaucugaaacuugcucucgaaaa(SEQ ID NO：16)-OH
P-RNA5-P P-ucacugcagaugaugaguagacacguaga(SEQ ID NO：17)-P
P-RNA6-cP P-augccuauggauucgucguc(SEQ ID NO：18)-cP
P-RNA9-OH P-guucucgaguaaaaguguguc(SEQ ID NO：19)-OH
P-RNA10-P P-cucaguugaucacacaugauccuaugu(SEQ ID NO：20)-P
P-RNA11-cP P-cacguagagccuucuuagcgcag(SEQ ID NO：21)-cP
P-RNA16-OH P-uggaauucucgggcaccaaggu(SEQ ID NO：22)-OH
P-RNA17-P P-guggaacucagaccuaaacgg(SEQ ID NO：23)-P
P-RNA18-cP P-uccaguuaugaucaggaauuucaguu(SEQ ID NO：24)-cP
RNA PCR引物和RNA PCR index引物 序列
RNA PCR引物 aatgatacggcgaccaccgagatctacacgttcagagttctacagtccga(SEQ ID NO：25)
RNA PCR引物，Index 1 caagcagaagacggcatacgagatcgtgatgtgactggagttccttggcacccgagaattcca(SEQ ID NO：26)
RNA PCR引物，Index 2 caagcagaagacggcatacgagatacatcggtgactggagttccttggcacccgagaattcca(SEQ ID NO：27)
RNA PCR引物，Index 3 caagcagaagacggcatacgagatgcctaagtgactggagttccttggcacccgagaattcca(SEQ ID NO：28)
RNA PCR引物，Index 4 caagcagaagacggcatacgagattggtcagtgactggagttccttggcacccgagaattcca(SEQ ID NO：29)
RNA PCR引物，Index 5 caagcagaagacggcatacgagatcactgtgtgactggagttccttggcacccgagaattcca(SEQ ID NO：30)
RNA PCR引物，Index 6 caagcagaagacggcatacgagatattggcgtgactggagttccttggcacccgagaattcca(SEQ ID NO：31)
RNA PCR引物，Index 7 caagcagaagacggcatacgagatgatctggtgactggagttccttggcacccgagaattcca(SEQ ID NO：32)
RNA PCR引物，Index 8 caagcagaagacggcatacgagattcaagtgtgactggagttccttggcacccgagaattcca(SEQ ID NO：33)
RNA PCR引物，Index 9 caagcagaagacggcatacgagatctgatcgtgactggagttccttggcacccgagaattcca(SEQ ID NO：34)
反向转录和qPCR引物 序列
Universal反向转录引物 gcgagcacagaattaatacgactcactataggttttttttttttvn(SEQ ID NO：35)
Universal qPCR反向引物1 caagcagaagacggcatacg(SEQ ID NO：36)
Universal qPCR反向引物2 gcgagcacagaattaatacgac(SEQ ID NO：37)
P-RNA2-OH qPCR正向引物 tcatctgaaacttgctctcgaaaa(SEQ ID NO：38)
P-RNA5-P qPCR正向引物 gcagatgatgagtagacacgtaga(SEQ ID NO：39)
P-RNA6-cP qPCR正向引物 atgcctatggattcgtcgtc(SEQ ID NO：40)
P-RNA9-OH qPCR正向引物 gttctcgagtaaaagtgtgtc(SEQ ID NO：41)
P-RNA10-P qPCR正向引物 agttgatcacacatgatcctatgt(SEQ ID NO：42)
P-RNA11-cP qPCR正向引物 cacgtagagccttcttagcgcag(SEQ ID NO：43)
P-RNA16-OH qPCR正向引物 tggaattctcgggcaccaaggt(SEQ ID NO：44)
P-RNA17-P qPCR正向引物 gtggaactcagacctaaacgg(SEQ ID NO：45)
P-RNA18-cP qPCR正向引物 cagttatgatcaggaatttcagtt(SEQ ID NO：46)
5S RNA qPCR正向引物 gtctacggccataccaccctg(SEQ ID NO：47)
5S RNA qPCR反向引物 aaagcctacagcacccggtat(SEQ ID NO：48)
Mouse actin qPCR正向引物 tgtccaccttccagcagatgt(SEQ ID NO：49)
Mouse actin qPCR反向引物 agctcagtaacagtccgcctaga(SEQ ID NO：50)
Mouse rsR-67-cP qPCR正向引物 agcggaggaaaagaaactaaaa(SEQ ID NO：51)
Mouse tsR-2-cP qPCR正向引物 gtttccgtagtgtagtggttaaaa(SEQ ID NO：52)
Mouse snoR-7-cP qPCR正向引物 ctgcgatgatgtcatatcttaaaa(SEQ ID NO：53)
Mouses snR-7-cP qPCR正向引物 acctggcaggggagataccaaaa(SEQ ID NO：54)
Mouse sinR-7-cP qPCR正向引物 agaagtttgggatatactaaaa(SEQ ID NO：55)
Mouse smR-26-cP qPCR正向引物 aattccacctttcgggcgccaaaa(SEQ ID NO：56)
Mouse slncR-106-cP qPCR正向引物 gcgcggcgcctcgcctcaaaa(SEQ ID NO：57)
Mouse srpR-5-cP qPCR正向引物 tttgaaacacgggaaacctcaaaa(SEQ ID NO：58)
Mouse smcR-13-cP qPCR正向引物 gcgcggtggcgcacgcctgtaaaa(SEQ ID NO：59)
Mouse srR-4-cP qPCR正向引物 acggccataccaccctgaacaaaa(SEQ ID NO：60)
Mouse srR-15-cP qPCR正向引物 gactcttagcggtggatcaaaa(SEQ ID NO：61)
Mouse srR-43-cP qPCR正向引物 aggggaatccgactgtttaaaa(SEQ ID NO：62)
Mouse tsR-4-cP qPCR正向引物 tcaggatggccgagcggtctaaaa(SEQ ID NO：63)
Mouse tsR-17-cP qPCR正向引物 ggggaattagctcaaatggtaaaa(SEQ ID NO：64)
Mouse snoR-3-cP qPCR正向引物 acacaacttttttccccatcaaaa(SEQ ID NO：65)
Mouse snoR-12-cP qPCR正向引物 agtgatgtgatgattctgccaaaa(SEQ ID NO：66)
Mouse snR-3-cP qPCR正向引物 gtgtagtatctgttcttatcaaaa(SEQ ID NO：67)
Mouse snR-22-cP qPCR正向引物 gcgggaaactcgactgcaaaa(SEQ ID NO：68)
Mouse sinR-1-cP qPCR正向引物 gagttcggttcccagcacccaaaa(SEQ ID NO：69)
Mouse sinR-136-cP qPCR正向引物 ggttcagtggtagaattctcaaaa(SEQ ID NO：70)
Mouse smR-2-cP qPCR正向引物 agggaacgtgagctgggattaaaa(SEQ ID NO：71)
Mouse smR-12-cP qPCR正向引物 tccttttgtatagtggtgagtaaaa(SEQ ID NO：72)
Mouse slncR-2-cP qPCR正向引物 ggagagatggctcagccgttaaaa(SEQ ID NO：73)
Mouse slncR-6-cP qPCR正向引物 aggctcacaaccaaaaatataaaa(SEQ ID NO：74)
Mouse srpR-11-cP qPCR正向引物 gttaatgtgcttaataacaaaa(SEQ ID NO：75)
Mouse smcR-2-cP qPCR正向引物 gttctgggctgtagtgcgctaaaa(SEQ ID NO：76)
Mouse miR-122 qPCR正向引物 tggagtgtgacaatggtgtttgaaaa(SEQ ID NO：77)
Mouse miR-21a qPCR正向引物 agcttatcagactgatgttgaaaa(SEQ ID NO：78)
Mouse miR-152 qPCR正向引物 tcagtgcatgacagaacttggaaaa(SEQ ID NO：79)
Mouse miR-181a qPCR正向引物 attcaacgctgtcggtgagtaaaa(SEQ ID NO：80)
Human miR-19a qPCR正向引物 gcaaatctatgcaaaactgaaaa(SEQ ID NO：81)
Human miR-148a qPCR正向引物 tcagtgcactacagaactttgtaaaa(SEQ ID NO：82)
Human srR-12-cP qPCR正向引物 cgactcttagcggtggatcaaaa(SEQ ID NO：83)
Human srR-51-cP qPCR正向引物 gcgccgaatccccgccccgcaaaa(SEQ ID NO：84)
Human srR-20-cP qPCR正向引物 agtgaaactgcgaatggctcaaaa(SEQ ID NO：85)
Human tsR-4-cP qPCR正向引物 gtttccgtagtgtagtggtcaaaa(SEQ ID NO：86)
Human tsR-15-cP qPCR正向引物 gccgctggtgtagtggtatcaaaa(SEQ ID NO：87)
Human snoR-18-cP qPCR正向引物 actggtctgcagctgttcttaaaa(SEQ ID NO：88)
Human snR-3-cP qPCR正向引物 ccggatgtgctgacccctgcaaaa(SEQ ID NO：89)
Human snR-31-cP qPCR正向引物 aaattcgtgaagcgttccaaaa(SEQ ID NO：90)
Human sinR-20-cP qPCR正向引物 ttccgtagtgtagtggtatcaaaa(SEQ ID NO：91)
Human sinR-90-cP qPCR正向引物 acttgaacgcgcccgatctcaaaa(SEQ ID NO：92)
Human smR-44-cP qPCR正向引物 ctggcgctctcgctcccgacaaaa(SEQ ID NO：93)
Human smR-96-cP qPCR正向引物 cttagtagagcagccaccacaaaa(SEQ ID NO：94)
Human slncR-2-cP qPCR正向引物 gccgatcgggtgtccgcactaaaa(SEQ ID NO：95)
Human slncR-13-cP qPCR正向引物 gggccagaggcggccctaacaaaa(SEQ ID NO：96)
Human smcR-7-cP qPCR正向引物 tggtccgaaggtagtgagttaaaa(SEQ ID NO：97)
Human srpRNA-2-cP qPCR正向引物 cagtggagagcattgactgcaaaa(SEQ ID NO：98)
Northern blot探针 序列
Mouse miR-152探针 ccaagttctgtcatgcactga(SEQ ID NO：99)
Mouse miR-122探针 caaacaccattgtcacactcca(SEQ ID NO：100)
Mouse rsR-67-cP探针 agtttcttttcctccgct(SEQ ID NO：101)
Mouse snR-7-cP探针 ggtatctcccctgccaggt(SEQ ID NO：102)
Mouse sinR-7-cP探针 agtatatcccaaacttct(SEQ ID NO：103)
Mouse smR-26-cP探针 ggcgcccgaaaggtggaatt(SEQ ID NO：104)
Mouse slncR-106-cP探针 gaggcgaggcgccgcgc(SEQ ID NO：105)
Mouse smcR-13-cP探针 acaggcgtgcgccaccgcgcccggc(SEQ ID NO：106)
App-DNA12-ddC探针 gagaattccatacgtc(SEQ ID NO：107)
扩增Ang和RNase4引物 序列
Mouse Ang正向引物 catatgcaggatgactccaggtacac(SEQ ID NO：108)
Mouse Ang反向引物 gctcgagctatagactgaaaaacgactc(SEQ ID NO：109)
Mouse RNase 4正向引物 catatgcaggatcgaatgtaccaacgg(SEQ ID NO：110)
Mouse RNase 4反向引物 gctcgagctatctgtcaaagtgcactg(SEQ ID NO：111)
gRNA寡核苷酸 序列
Mouse Ang sgRNA1-sense caccgatgtcaacacctttatcca(SEQ ID NO：112)
Mouse Ang sgRNA1-antisense aaattggataaaggtgttgacatc(SEQ ID NO：113)
Mouse Ang sgRNA2-sense caccgcttgatcttcgtgctgggtc(SEQ ID NO：114)
Mouse Ang sgRNA2-antisense aaacgacccagcacgaagatcaagc(SEQ ID NO：115)
Mouse RNase 4 sgRNA-sense caccgggtgttgaagcgtttgcac(SEQ ID NO：116)
Mouse RNase 4 sgRNA-antisense aaatgtgcaaacgcttcaacaccc(SEQ ID NO：117)
Mouse RNH1 sgRNA-sense caccgcaatacgaagtggtcaggt(SEQ ID NO：118)
Mouse RNH1 sgRNA-antisense aaatacctgaccacttcgtattgc(SEQ ID NO：119)
Mouse Ang sgRNA1-sense caccgtggcatcatagtgctgggtc(SEQ ID NO：120)
Mouse Ang sgRNA1-antisense aaacgacccagcactatgatgccac(SEQ ID NO：121)
Mouse Ang sgRNA2-sense caccgatcgctggtacatgccatcc(SEQ ID NO：122)
Mouse Ang sgRNA2-antisense aaacggatggcatgtaccagcgatc(SEQ ID NO：123)
Mouse RNase 4 sgRNA-sense caccgatcgctggtacatgccatcc(SEQ ID NO：124)
Mouse RNase 4 sgRNA-antisense aaacggatggcatgtaccagcgatc(SEQ ID NO：125)
2、实验动物
本实验涉及动物均按照动物护理和使用委员会的指导方针进行饲养和使用。
小鼠禁食实验，8周龄雄性C57BL/6小鼠随机为两组，在有或没有食物的标准饲养笼中饲养24h，自由饮水。
小鼠高脂饮食实验，8周龄雄性C57BL/6小鼠随机分为两组，分别饲喂含60kcal％脂肪的高脂饮食(研究饮食)和正常饲料，共喂食16周。
db/db小鼠则选用16周龄雄性小鼠。
小鼠麻醉解剖后，立即收集组织用液氮快速冷冻，保存在-80℃冰箱。
3、细胞培养
小鼠肝癌细胞(Hepa 1-6)，小鼠肝细胞(AML12)，小鼠胚胎成纤维细胞(NIH/3T3)和人肝癌细胞(Hep G2)在含10％的胎牛血清的DMEM，于37℃，5％的CO 2条件下培养。当细胞长到80-90％时提取总RNA，并分离sRNA。
4、变性PAGE分析
RNA或DNA寡聚物样品与等体积的2×RNA加样缓冲液(95％甲酰胺，18mM EDTA，0.025％SDS，0.025％溴酚蓝，0.025％二甲苯氰)混合，于70℃孵育10min后，立即将样本置于冰上。样品通过含有7M尿素的15％变性PAGE胶进行分离，电泳结束的PAGE胶用SYBR Gold染色液(Invitrogen)进行染色，在紫外灯下拍照，用ImageJ软件对凝胶图像进行定量分析。
5、RNA或DNA胶回收
用注射器挤压粉碎含有RNA或DNA的PAGE凝胶，将粉碎的凝胶块在0.3M NaCl中4℃恒定旋转浸泡约2小时，用乙醇-乙酸钠(3倍体积无水乙醇和0.1倍体积3M乙酸钠，pH 5.2)沉淀，沉淀样品置于-80℃冰箱过夜。样品用预先冷却到4℃的离心机在12000g条件下离心15分钟，最后RNA或DNA寡聚体溶解在无核酸酶水中保存备用。
6、预腺苷化DNA寡核苷酸制备
使用5’DNA腺苷化试剂盒(NEB)对5’为磷酸的单链DNA进行腺苷化。20μl反应体系如下：5’磷酸化DNA寡核苷酸100pmol，2μl 10×5’DNA腺苷化反应缓冲液，2μl 1mM ATP，100pmol Mth RNA连接酶，样品混合均匀后置于65℃条件下反应1小时，然后在85℃孵育5分钟灭活酶。反应后样品用含7M尿素的15％变性PAGE胶进行分离，然后用SYBR Gold染色，将腺苷化的产物从凝胶中回收。
7、制备5’-P，3’-cP sRNA标准品
Mth RNA连接酶具有将3’-P转化为3’-cP的活性，利用该特性制备3’-cP sRNA标准品。反应体系如下：100pmol合成的结构为5’-OH和3’-P sRNA，2μl 10×5’DNA腺苷化反应缓冲液，2μl 1mM ATP，100pmol Mth RNA连接酶，补充H 2O至20μl，混合均匀后置于65℃反应1小时，85℃下5分钟失活酶。然后添加1μl 10×CutSmart缓冲液，18μl无核酸酶水和1μl虾碱性磷酸酶(NEB)，样品在37℃条件下孵 育30分钟，将未反应的3’-P转化为3’-OH，然后在65℃条件下孵育10分钟热失活酶，反应结束置于冰上。之后添加1μl 10×poly(A)聚合酶反应缓冲液，加入4μl ATP(10μM)，10U重组核糖核酸酶抑制剂(RRI)，10U poly(A)聚合酶(NEB)并加入无核酸酶水至终体积40μl，混合均匀后样品置于37℃下孵育30分钟，将3’末端未转换为3’-cP的剩余sRNA加上poly(A)尾，用含7M尿素的15％变性PAGE胶进行分离，将含有5’-OH和3’-cP的小RNA从凝胶中分离并提取。
为获得结构为P-sRNA-cP标准品，反应体系如下：300pmol结构为5’-OH，3’-cP的sRNA，5μl 10×T4 Pnk反应缓冲液，5μl ATP(10μM)，40U重组核糖核酸酶抑制剂，10U T4 Pnk(3’磷酸酶突变)(NEB)，补加无核酸酶水至终体积50μl，样品在37℃下反应30分钟，然后于65℃条件下孵育20分钟热失活酶，用QIAquick Nucleotide Removal Kit(Qiagen)回收5’-P，3’-cP sRNA，并溶解于无核酸酶水中备用。
8、总RNA提取和sRNA分离
按照Trizol试剂(Invitrogen)使用说明提取组织或细胞的总RNA，具体步骤参照说明书。总RNA用5％聚乙二醇8000(PEG8000)和0.5M NaCl冰上孵育45分钟，将大分子量RNA(大于200nt)沉淀出来，通过离心去除沉淀，上清中包含小于200nt的sRNA，用乙醇-乙酸钠进行沉淀，最终sRNA用无核酸酶水溶解，-80℃保存或用于反应。15-30nt sRNA则用15％变性PAGE胶进行分离，SYBR Gold染色，然后从凝胶中切胶回收相应长度sRNA。
9、T4 RNA连接酶2连接sRNA-OHs
连接反应体系如下：50ng sRNA，20pmol腺苷化DNA linker，2μl 10×T4 RNA连接酶反应缓冲液，4μl 50％PEG8000，20U重组核糖核酸酶抑制剂，200U的截断KQ型T4 RNA连接酶2(T4 Rnl2，KQ)(NEB)，加无核酸酶水补齐至20μl。样品在16℃条件下孵育16小时，用含7M尿素的15％变性PAGE分析连接产物。
10、RNA聚腺苷化实验
RNA聚腺苷化实验体系如下：50ng sRNA，2μl 10×poly(A)聚合酶反应缓冲液，2μl 10mM ATP，20U重组核糖核酸酶抑制剂，5U poly(A)聚合酶，加无核酸酶水补齐至20μl。样品在37℃孵育30分钟然后利用含7M尿素的15％变性PAGE分析反应产物。
11、LC-MS/MS分析RNA修饰
腺苷酸(A)，尿苷(U)，胞嘧啶核苷(C)，鸟苷(G)，2’-O-甲基腺苷(Am)，1-甲基腺苷(m 1A)，N 6-甲基腺苷(m 6A)，N 6-异戊烯腺苷(i 6A)，2’-O-甲基尿苷(Um)，5-甲基-2-硫代尿苷(m 5s 2U)，5-甲氧基尿苷(mo 5U)，5-甲基尿苷(m 5U)，4-硫代尿苷(s 4U)，3’-O-甲基尿苷(3-OMeU)，2’-O-甲基胞苷(Cm)，5-甲基胞苷(m 5C)，N 4-乙酰胞苷(ac 4C)，2’-O-甲基鸟苷(Gm)，N 2-甲基鸟苷(m 2G)，7-甲基鸟苷(m 7G)，肌苷(I)，腺苷2’，3’-环磷酸(2’，3’-cAMP)，尿苷2’，3’-环磷酸(2’，3’-cUMP)，胞苷2’，3’-环磷酸(2’，3’-cCMP)和鸟苷2’，3’-环磷酸(2’，3’-cGMP)购自Sigma-Aldrich。5，2’-O-二甲基尿苷(m 5Um)，3-甲基尿苷(m 3U)，2-硫代胞苷(s 2C)，5-羟甲基尿苷(hm 5C)，N 2-二甲基鸟苷(m 2 2G)，假尿苷(ψ)，1-甲基尿苷(m 1ψ)和2’-O-甲基肌苷(Im)，3’-O-甲基胞苷(3’-OMeC)，5，2’-O-二甲基胞苷(m 5Cm)，N 4-乙酰-2’-O-甲基胞苷(ac 4Cm)，1-甲基鸟苷(m 1G)，3’-O-甲基鸟苷(3’-OMeG)，N 2，N 2，7-三甲基鸟苷(m 2，2，7G)，5’-O-甲基胸腺嘧啶(5’-OMeT)和3’-O-甲基肌苷(3’-OMeI)均购自Carbosynth。鸟苷5’-单磷酸( 13C 10， 15N 5)购自剑桥同位素实验室。140μg/mL 5’-单磷酸鸟苷( 13C 10， 15N 5)用牛碱性磷酸酶(Takara)水解24小时得到鸟苷( 13C 10， 15N 5)。
RNA处理方法和LC-MS/MS分析简述如下：100ng sRNA在60μl 50mM NH 4OAc，pH 5.3溶液中， 加入0.2U核酸酶P1(Sigma-Aldrich)，50℃孵育3小时，然后加入0.04U磷酸二酯酶I(USB)在37℃下处理2小时。随后，RNA样品在37℃下用2U碱性磷酸酶(Sigma-Aldrich)处理2小时。通过Nanosep 3K Omega膜(Pall)离心去除蛋白。核苷混合物通过API 4000 Q-TRAP质谱仪(Applied Biosystems)进行分析。
12、虾碱性磷酸酶或T4 Pnk反应
虾碱性磷酸酶(rSAP)(简称AP)(NEB)反应体系如下：50ng sRNA，1μl 10×CutSmart缓冲液，20U重组核糖核酸酶抑制剂，1U rSAP，加无核酸酶水至10μl。样品在37℃孵育30分钟，65℃继续孵育5分钟热失活酶，反应结束置于冰上用于后续的连接反应或RNA聚腺苷化反应(PAP)。
T4 Pnk反应体系如下：50ng sRNA，1μl 10×T4 Pnk反应缓冲液，20U重组核糖核酸酶抑制剂，10U T4 Pnk，加无核酸酶水至10μl，样品在37℃孵育30分钟，65℃继续孵育5分钟热失活酶，反应结束置于冰上用于后续的连接反应或RNA聚腺苷化反应。
13、RtcB连接反应
用于RtcB连接反应的15-30nt sRNA先用T4 Pnk(3’磷酸酶突变)处理，使sRNA的5’-OH转变为5’-P，然后通过苯酚-氯仿抽提，乙醇-乙酸钠沉淀，溶解于无核酸酶水中。RtcB(NEB)连接反应体系如下：50ng sRNA，20pmol linker(OH-RNA8-OH)，2μl 10×RtcB反应缓冲液，2μl MnCl 2(10mM)，1μl GTP(10mM)，15pmol RtcB连接酶，20U重组核糖核酸酶抑制剂，加无核酸酶水至20μl。样品混合均匀后在37℃下孵育2小时，然后用含7M尿素的15％变性PAGE分析连接产物。
14、TANT-seq sRNA文库制备和高通量测序
TANT-seq sRNA文库制备流程和相关反应体系如下：1μg小于200nt sRNA(9μl体积)与1μl spike-in混合物(P-RNA2-OH，P-RNA10-P和P-RNA6-cP各8nM；P-RNA9-OH，P-RNA5-P和P-RNA18-cP各0.8nM；P-RNA16-OH，P-RNA17-P和P-RNA11-cP各0.08nM)于70℃孵育5分钟后立即置冰上冷却，然后加入20pmol含有3’-OH子文库条形码的App-DNA12-ddC接头序列(Applied Biosystems)，4μl 10×T4 RNA连接酶反应缓冲液，8μl 50％PEG8000，400U KQ截短型T4 RNA连接酶2(T4 Rnl2)(购自NEB)，40U重组核糖核酸酶抑制剂，加无核酸酶水至40μl，样品置于16℃孵育16小时。70℃继续孵育5分钟热失活酶。
随后，加入1μl 10×CutSmart缓冲液，20U重组核糖核酸酶抑制剂，2.5U虾碱性磷酸酶(AP)，并补加无核酸酶水至终体积50μl，混合均匀于37℃下孵育30分钟，65℃继续孵育5分钟热失活酶。
随后，加入20μl 100mM NaIO 4，40U重组核糖核酸酶抑制剂，并补加无核酸酶水至终体积为200μl，样品置于冰上，黑暗条件下孵育40分钟。之后利用乙醇-乙酸钠进行沉淀，RNA用15％变性PAGE进行分离，选择长度在15-46nt范围内的sRNA进行切胶回收，并溶解于无核酸酶水中备用。
回收的样品经T4 Pnk(3’磷酸酶突变)(NEB)处理，然后用苯酚-氯仿抽提，乙醇-乙酸钠沉淀，溶解在无核酸酶水中。
之后进行RtcB连接反应，连接体系如下：10pmol包含3’-cP子文库条形码的OH-RNA13-ddC接头序列，2μl 10×RtcB反应缓冲液，2μl MnCl 2(10mM)，1μl GTP(10mM)，15pmol RtcB连接酶，20U重组核糖核酸酶抑制剂，并补加无核酸酶水至终体积为20μl。样品在37℃下孵育2小时，然后70℃孵育5分钟热失活酶，用15％变性PAGE分离连接产物，选择长度在31-46nt范围内的带有接头序列sRNA进行切胶回收，回收产物溶解在无核酸酶水中。
之后进行RNA 5’接头序列连接反应，反应体系如下：5pmol OH-RNA14-OH接头序列，3μl 10×T4  RNA连接酶反应缓冲液，6μl 50％PEG8000，1μl ATP(10mM)，30U T4 RNA连接酶1( T4 Rnl1)(NEB)，20U重组核糖核酸酶抑制剂，并补加无核酸酶水至终体积为30μl。混合均匀后样品在25℃条件下反应2小时，然后70℃孵育5分钟热失活酶。
连接产物随后进行反转录实验(RT-PCR)，体系如下：3pmol OH-DNA15-OH反转录引物，4μl 5×SSIV缓冲液，200U SuperScript IV反转录酶(Invitrogen)，2.5μl DTT(100mM)，2.5μl dNTP(10mM)，20U重组核糖核酸酶抑制剂，并补加无核酸酶水至终体积为50μl。混合均匀后样品在50℃条件下孵育1小时，然后80℃孵育10分钟热失活酶。
接着用Phusion高保真DNA聚合酶(NEB)进行PCR扩增，实验用到的PCR引物和PCR index序列见表1。扩增产物用7.5％的非变性PAGE胶进行分离，选择长度在130-160bp的产物进行切胶回收。回收的样品利用Illumina公司的Xten平台进行高通量测序。本发明人将这种基于T4 Rnl2/AP/NaIO 4/T4 Pnk(3’磷酸酶突变)/RtcB的sRNA-seq方法称为TANT-seq。
15、TANT-seq sRNA测序数据分析
高通量测序原始序列(TANT方法中获得的序列)首先使用fastp(v0.20.0)去除接头序列，低质量序列(Phred score＜30)，长度短于21nt或长于46nt的序列也被去除。然后将App-DNA12-ddC中带有GACGTA条形码序列(Applied Biosystems)和OH-RNA13-ddC中带有CTATCG条形码序列(Applied Biosystems)分别分类到3’-OH子文库和3’-cP子文库中，并去除相应的条形码序列。
随后，使用ncbi-BLAST+2.11.0将序列按顺序比对到相应的参考数据库。比对之前，首先对相关数据库就行修改，具体如下：在GtRNAdb和mitotRNAdb中的所有tRNA序列的3’末端添加CCA序列。
首先将序列和spike-in序列(表1)进行比对，然后将剩余的序列按照种属分别比对到小鼠rRNA数据库或人rRNA数据库，小鼠rRNA数据库包括：mmu-5S rRNA(NR_030686.1)，mmu-5.8S rRNA(NR_003280.2)，mmu-12S rRNA(NC_005089.1)，mmu-16S rRNA(NC_005089.1)，mmu-18S rRNA(NR_003278.3)，mmu-28S rRNA(NR_003279.1)，mmu-45S rRNA(NR_046233.2)和Ensembl数据库中的小鼠rRNA序列；人rRNA数据库包括：has-5S rRNA(NR_023363.1)，has-5.8S rRNA(NR_145821.1)，has-12S rRNA(NC_012920.1)，has-16S rRNA(NC_012920.1)，has-18S rRNA(NR_146146.1，NR_145820.1)，has-28S rRNA(NR_003287.4，NR_146118.1，NR_146154.1，NR_146148.1，NR_145822.1)，has-45S rRNA(NR_046235.3，NR_146117.1，NR_146151.1，NR_146144.1)和Ensembl数据库中的人rRNA序列。然后再依次比对到小鼠或人的添加了3’-CCA的GtRNAdb和mitotRNAdb数据库，来自miRbase和Ensembl中的miRNA数据库，来自piRNAdb(https：//www.pirnadb.org)中的piRNA数据库，来自Ensembl中的snRNA，snoRNA，lncRNA，miscRNA和other ncRNA数据库，来自Ensembl中的内含子数据库，来自Ensembl中的mature mRNA，IG gene，TR gene，Pseudogene数据库，来自UCSC Genome Browser(http：//genome.ucsc.edu)的重复序列数据库(RepeatMasker，GRCm38/mm10或GRCh38/hg38)，基因组数据库(GRCm38/mm10或GRCh38/hg38)，线粒体基因组数据库(NC_005089.1或NC_012920.1)。和数据库中序列完全匹配的序列被分配到对应比对到的类别中，然后将未比对上的序列再次比对到上述数据库，但此次比对时允许错配+跳跃碱基个数≤2，将比对上的序列再次分配到对应类别中。两次都未比对上的序列被舍弃。
将Dfam数据库中归类为假基因的重复序列的序列进行挑选，归类于假基因。同时利用NCBI网站中的核苷酸-核苷酸BLAST软件对每个类型的高丰富的sRNA进行手动检查。在小鼠样本中，将分类到重 复序列中但可以比对到snRNA，C/D box 118(Snord118)(NR_028566.3)或Gm25313(XR_004935972.1)的序列重新分配给snoRNA或snRNA；分类到mRNA序列中但可以比对到Snord14c(NR_028276.2)的序列分配给snoRNA。在人源样本中，将分类到重复序列中但可以比对到RNU5A-1(NR_002756.2)和RNU5B-1(NR_002757.3)的序列分配给snRNA；将分类到重复序列中但可以比对到snRNA，C/D box 118(SNORD118)(NR_033294.1)的序列分配给snoRNA。
将比对到rRNA和tRNA上的sRNA分别分类到为srRNA和tsRNA。比对到内含子、lncRNA、基因组、成熟mRNA、重复序列和miscRNA的sRNA分别命名为小内含子RNA(sinRNA)、小lncRNA(slncRNA)、基因组衍生小RNA(sgmRNA)、小成熟mRNA(smRNA)、重复序列衍生小RNA(srpRNA)、小miscRNA(smcRNA)。对平均测序读数大于2的sRNA进行了统一性的命名，命名原则是依据sRNA类别，匹配序列名称，起始位置、序列长度和3’末端修饰；同时为了简化小RNA命名，依据sRNA类别，按照丰度从高到底的排列进行顺序命名。具体sRNA命名的代表性举例如图13A-D。其中，图13A、鼠肝细胞或Hepa1-6细胞中TANT-seq鉴定到的代表性15-30nt sRNA-OH；图13B、鼠肝细胞或Hepa1-6细胞中TANT-seq鉴定到的代表性15-30nt sRNA-cP；图13C、人Hep G2细胞通过TANT-seq中鉴定到的代表性15-30nt sRNA-OH；图13D、人Hep G2细胞通过TANT-seq中鉴定到的代表性15-30nt sRNA-cP。
此外，tsRNA依据其在全长tRNA中的位置可以进一步分为5’-tsRNA(tRNA-derived sRNA containing 5’-end)、3’-CCA-tsRNA(tRNA-derived sRNA containing CCA at 3’-end)、3’-CC-tsRNA(tRNA-derived sRNA containing the additional CC at 3’-terminal)和internal-tsRNA。
对于测序结果重现性分析，采用GraphPad Prism 7.0计算任意两个独立重复样品Pearson相关系数，用来比较TANT-seq方法的可重现性。
15-30nt sRNA或各种类别sRNA含有3’-OH和3’-cP相对含量计算如下：考虑到Poly(A)聚合酶反应活性强，几乎可以使得所有的sRNA-OH被聚腺苷化，使用图1e和图1f中的反应结果计算T4 RNA连接酶2对15-30nt中sRNA-OH的连接效率。用图7a和图1f中的反应结果计算RtcB连接酶对15-30nt中sRNA-cP的连接效率。然后根据T4 RNA连接酶2和RtcB连接酶的连接效率校准TANT-seq测序数据中sRNA-OH和sRNA-cP的相对含量，或计算各种类别sRNA的相对含量。
维恩图是由VennDiagram制作生成。保守性分析是通过R语言中的ggseqlogo制作生成。
对于TNAT-seq测序结果的差异表达分析，用edgeR计算变化倍数和P值，调整的P值用Benjamini-Yekutieli方法进行校准。调整后的P＜0.01和log2(fold change)≥2或≤-2的sRNA被认为有显著变化。火山图中显示了平均读值≥2的sRNA序列。
16、定量PCR
采用Roche公司FastStart Universal SYBR Green Master进行定量PCR。qPCR反向和正向引物在表1中列出，实验按照产品说明书进行。
17、3’-OH，3’-P或3’-cP sRNA定量分析
图3a中展示了qPCR定量检测3种RNA 3’末端(3’-OH，3’-P和3’-cP)的原理。反应体系如下：1μl spike-in RNA(P-RNA2-OH，P-RNA10-P和P-RNA6-cP，各8nM；P-RNA9-OH，P-RNA5-P和P-RNA18-cP，各0.8nM；P-RNA16-OH，P-RNA17-P和P-RNA11-cP，各0.08nM)或1μg总RNA分别用水、虾碱性磷酸酶和T4 Pnk处理，反应体积为10μl。然后加入1μl 10×poly(A)聚合酶反应缓冲液，2μl ATP(10mM)，5U重组核糖核酸酶抑制剂，5U poly(A)聚合酶，补加无核酸酶水至终体积20μl，在37℃条件下孵育60 分钟，然后70℃继续孵育5分钟进行热失活。
随后，加入3pmol反转录引物(表1)，200U SuperScript IV反转录酶，4μl 5×SSIV缓冲液，2.5μl DTT(100mM)，2.5μl dNTP(10mM)，20U重组核糖核酸抑制剂，并补加无核酸酶水至终体积为40μl。混合均匀后再50℃条件下孵育60分钟，80℃继续孵育10分钟进行热失活。
随后采用FastStart Universal SYBR Green Master进行定量PCR，qPCR引物附于表1。三组处理分别得到Ct(3’-OH)，Ct[3’-(OH+P)]和Ct[3’-(OH+P+cP)]，然后根据下列公式计算3’-OH，3’-P和3’-cP的相对含量：
Figure PCTCN2022138474-appb-000003
Figure PCTCN2022138474-appb-000004
(3′-OH)％＝100％-(3′-P)％-(3′-cP)％
18、Northern blot
20μg总RNA或100-200ng 15-30nt sRNA通过15％变性PAGE分离，并转移到带正电的尼龙膜(Roche)上。随后进行紫外交联，加入DIG Easy Hyb buffer(Roche)进行预杂交。然后，加入10nM 3’端地高辛标记的寡核苷酸探针在45-55℃条件下孵育过夜，温度主要取决于每个探针的退火温度，探针信息附于表1中。杂交结束后进行洗涤和封闭膜，然后加入Anti-Digoxigenin-AP Fab fragments(Roche)室温孵育45分钟，并用CSPD ready-to-use试剂(Roche)检测sRNA的表达。
19、重组蛋白的表达和纯化
利用表1中所列引物，从小鼠肝脏cDNA中扩增获得Ang和RNase 4基因序列，并将其插入pET28a质粒中的Nde I和Xho I位点，得到pET28a-Ang和pET28a-RNase 4原核表达质粒。重组Ang和RNase 4在大肠杆菌BL21 Rosetta(DE3)细胞中诱导表达，并用His-Tag纯化树脂(Roche)和离子交换柱(Hitrap Q HP，GE Healthcare)纯化。纯化得到的Ang和RNase 4蛋白经SDS-PAGE和考马斯蓝染色分析纯度，最后透析到储存缓冲液(20mM Tris-HCl pH 7.5，200mM NaCl，5％甘油)中，-80℃冰箱中保存。
20、RNA酶消化肝脏总RNA
4μg小鼠肝脏总RNA分别用0.5ng RNase A(Thermo)，1U RNase T1(Thermo)，0.32μg Ang或0.08μg RNase4处理，反应在37℃下进行30分钟。然后用苯酚-氯仿提取样品中RNA，经过乙醇-乙酸钠沉淀，最终RNA溶解于无核酸酶水中，用于TE-qPCR或Northern blot检测。
21、分析RNase A家族成员的表达水平
分析小鼠肝脏RNase A家族亚成员的表达水平的RNA-seq数据来源于GEO数据库(登录代码：GSE164819)。表达水平采用FPKM(Fragments Per Kilobase of exon model per Million mapped fragments)表示。
22、CRISPR/Cas9基因编辑
将表1中编码gRNA的两条互补寡核苷酸进行退火，形成双链，然后将其插入lentiCRISPR v2(Addgene plasmid 52961)载体的BsmBI位点，构建好的质粒通过一代测序进行验证。分别得到lentiCRISPR-mRNH1-sgRNA，lentiCRISPR-mANG-sgRNA1，lentiCRISPR-mRNase4-sgRNA，lentiCRISPR-hAng-sgRNA1，lentiCRISPR-hRNase 4-sgRNA质粒。为了构建双敲除细胞系，选择含有 mCherry的px330-mp50质粒系统，将sgRNA插入到px330-mp50质粒的Bbs I位点，得到px330-mp-mAng-sgRNA2和px330-mp-hAng-sgRNA2质粒。
根据转染试剂说明书，将构建好的包含有sgRNA的质粒通过Lipofectamine 3000转染试剂(Thermo)转染进入Hepa 1-6或Hep G2细胞。并用2ng/μtl嘌呤霉素对转染后的细胞进行筛选，筛选48小时后，对细胞进行消化稀释，分离获得单克隆细胞。通过免疫印迹和/或一代测序确认基因敲除效果，获得对照Hepa 1-6或Hep G2细胞(WT)，Ang敲除Hepa 1-6或Hep G2细胞(AKO)，RNase 4敲除Hepa 1-6或Hep G2细胞(RKO)和RNH1敲除(RNH1 KO)Hepa 1-6细胞。为了进一步构建双敲除细胞系，用Lipofectamine 3000转染试剂分别转染px330-mp-mAng-sgRNA2和px330-mp-hAng-sgRNA2到RNase 4敲除Hepa 1-6和Ang敲除Hep G2细胞，转染48小时后，用流式细胞仪将红色荧光细胞分选到96孔板中，每孔1个细胞，随后收集单克隆细胞，通过免疫印迹和/或测序确认基因敲除，获得Ang和RNase 4双敲除(DKO)的Hepa 1-6和Hep G2细胞。
23、蛋白转染
使用Xfect蛋白转染试剂(Takara)在6孔板中转染2μg Ang或RNase 4蛋白进入转染WT或RNH1 KO Hepa 1-6细胞，转染6小时后，用PBS洗涤2次，随后用Trizol试剂提取总RNA。
24、核糖核酸酶抑制剂处理
将重组核糖核酸酶抑制剂(Takara)加入到WT、AKO、RKO或DKO Hepa 1-6或Hep G2细胞的细胞培养基中，最终浓度为2000U/ml。孵育48小时后，用Trizol试剂提取总RNA，进行TE-qPCR，Northern blot或TANT-seq检测。
25、免疫印迹
用含有50mM Tris-HCl，pH 7.6，150mM NaCl，1％NP-40，1％脱氧胆酸钠，0.1％SDS的RIPA裂解缓冲液裂解细胞。免疫印迹检检测中的抗体和稀释比例如下：anti-RNH1(abclone，A4079，1∶500)，anti-Angiogenin(Abcam，ab189207，1∶1000)，anti-RNase 4(Abcam，ab200717，1∶1000)或anti-Tubulin(Sigma-Aldrich，T6074，1∶1000)。
26、定量检测15-30nt sRNA
收集经重组核糖核酸酶抑制剂预处理48小时的WT，AKO，RKO或DKO Hepa 1-6细胞，Trizol试剂提取总RNA。4μg总RNA，分别用H 2O或T4 Pnk处理，37℃条件下孵育30分钟，然后65℃继续孵育20分钟进行热失活。随后加入20pmol App-DNA12-ddC，1μl 10×T4 RNA连接缓冲液，2μl 50％PEG8000，20U重组核糖核酸酶抑制剂，200U KQ截短型T4 RNA连接酶2，补加无核酸酶水至终体积为20μl，样品置于16℃下孵育16小时。反应结束后，用15％变性PAGE分离，然后转移到尼龙膜上进行Northern blot，加入带地高辛标记的与App-DNA12-ddC反向互补的寡核苷酸探针进行杂交。ImageJ分析长度在31-46nt连接产物sRNA(对应初始sRNA范围是15-30nt)，并通过5S rRNA进行归一化。
27、统计分析
除有特殊说明外，数据以至少三次独立实验的均值±SD表示，采用Student’s t检验或单因素方差分析评估统计学意义。除有特殊说明外，在P＜0.05时认为差异有统计学意义。*或a表示P＜0.05、**或b表示P＜0.01，***或c表示P＜0.001。
28、数据可用性
测序原始序列数据保存于NCBI Sequence Read Archive中，BioProject编号：PRJNA725316。
实施例1、小鼠和人15-30nt sRNA末端主要是3’-cP
首先，利用了常规的sRNA建库方法中的T4 RNA连接酶2连接反应从整体上检测3’末端为-OH的sRNA在15-30nt sRNA中的比重。通过切胶回收小鼠肝脏15-30nt sRNA，然后利用预腺苷化的接头序列进行连接。结果显示，大约只有10％的sRNA能被连接，与此同时，合成的sRNA-OH标准品在相同条件下几乎可以被完全连接(图1a)。
为排除单一酶促反应可能带来的实验误差，又利用E.coli Poly(A)聚合酶可以在RNA 3’-OH末端非模板性连续添加ATP的特性，对回收的小鼠肝脏15-30nt sRNA进行了RNA聚腺苷化实验，结果发现大约90％的sRNA不能被聚腺苷化，同样，合成的sRNA-OH标准品可以被完全聚腺苷化(图1b)。
上述两个实验的结果表明，小鼠肝脏中的15-30nt的sRNA中只有一小部分是3’-OH，且占比约为10％，并提示在此范围内还有大量的未被发现的sRNA，而这些sRNA可能含有特定修饰，进而阻止3’末端的连接反应或聚腺苷化反应。
为探究sRNA上可能存在的修饰，采用之前报道(PMID：24261999)过的一种可以一次性定性和定量超过40种不同类型的核苷和核苷酸的LC-MS/MS方法，用于分析15-30nt sRNA的修饰含量。LC-MS/MS结果表明，sRNA上存在大量的修饰，如ψ，m 1A，Um，m 6A，m 3C，m 2 2G，m 5C，I，与此同时，还发现大量的3’-cP修饰(2’，3’-cCMP，2’，3’-cUMP，2’，3’-cAMP和2’，3’-cGMP)，定量计算表明大约80％的15-30nt sRNA末端含有3’-cP(sRNA-cP)(图1c，d；图5a-d)。3’-cP是存在于RNA 3’末端的特定修饰，可以封闭T4 RNA连接酶2对sRNA的连接作用。为了探究这一现象是否是广泛存在，利用LC-MS/MS检测了不同来源的组织和细胞sRNA，发现小鼠脾脏组织、Hepa 1-6和Hep G2细胞中大约80％的小RNA也带有3’-cP修饰(图5e，f)。上述实验表明，小鼠和人的15-30nt sRNA末端主要是3’-cP修饰。
为进一步证实上述实验结果，将sRNA用T4多核苷酸激酶(T4 Pnk)处理，用以除去sRNA的3’磷酸(3’-P)和3’-cP，或用碱性磷酸酶(AP)处理用以去除3’-P基团，然后再通过T4 RNA连接酶2连接反应或聚腺苷化反应分析sRNA的含量，结果表明AP处理后没有明显的变化，而当T4 Pnk处理后，sRNA几乎完全被连接或聚腺苷化(图1e，f)。这些结果进一步证实，15-30nt sRNA3’末端主要是3’-cP修饰(图1g)。同样，在小鼠脾脏、脑组织、白色脂肪组织、Hepa 1-6细胞、AML12细胞、NIH/3T3细胞和Hep G2细胞中也观测到了相似的现象(图6a-b)。
以上实验数据表明，小鼠和人15-30nt sRNA中3’-OH序列约为10％，其余主要是末端为3’-cP序列。
实施例2、TANT-seq揭示15-30nt sRNA完整表达谱，表明sRNA-OH和sRNA-cP通常具有不同序列
前面的结果表明哺乳动物15-30nt sRNA中最主要的是sRNA-OH和sRNA-cP，为系统揭示15-30nt sRNA完整表达谱，选择特异性连接3’-P/cP的RtcB连接酶，开发了新的sRNA文库构建策略(图7a，b)。
通过对关键步骤的PAGE分析和对构建文库进行qPCR检测(图7c-f)，最终选择了T4 Rnl2/AP/NaIO 4/T4 Pnk(3’磷酸酶突变)/RtcB-依赖的sRNA-seq(TANT-seq)方法构建sRNA高通量测序文库(图2a)，该方法可以在一个文库中同时分开检测sRNA-OH和sRNA-cP。
TANT-seq建库样品中外源添加了合成的sRNA-OH和sRNA-cP标准品(spike-in)，数据分析时表明外源添加的sRNA的丰度与预期一致(图8a)，并且不同末端的外源sRNA在3’-OH和3’-cP子文库之间没有 明显的交叉污染，这充分说明了TANT-seq建库数据的可靠性和准确性。重复性分析结果发现3’-OH和3’-cP子文库的各自的Pearson相关系数超过0.94(图8b)，这表明TANT-seq具有较高的可重现性。此外，结合TANT-seq测序数据和T4 RNA连接酶2和RtcB连接酶的连接效率可以得到3’-OH和3’-cP子文库相对含量，结果表明sRNA-OH和sRNA-cP在小鼠肝脏、Hepa 1-6和Hep G2细胞中的相对含量分别为11.7％和88.3％，11.4％和88.6％，12.4％和87.6％(图2b，图8c，e)，这和之前的连接反应，聚腺苷化反应和LC-MS/MS结果相近。
TANT-seq全面揭示了sRNA-OH和sRNA-cP的表达谱，依据来源，将sRNA分成10余个类别(图2c)。其中，在小鼠肝脏3’-cP子文库中的srRNA，tsRNA，小内含子RNA(sinRNA)，小lncRNA(slncRNA)，snRNA，小基因组衍生RNA(sgmRNA)，小成熟mRNA(smRNA)，小重复元件衍生RNA(srpRNA)，snoRNA，小miscRNA(smcRNA)和piRNA的丰度和3’-OH子文库中的miRNA相比，分别为33.8、21.9、5.4、1.8、1.8、1.4、1.3、0.71、0.68、0.64和0.38倍(图2c)，同样，在Hepa 1-6和Hep G2细胞中也观察到了这些具有3’-cP修饰的高丰度sRNA(图8d，f)，这些结果表明哺乳动物组织或细胞中存在大量不同来源的sRNA-cP。
接着通过对3’-OH和3’-cP子文库中各种类型sRNA的相对含量分析，发现小鼠肝脏、Hepa 1-6和Hep G2细胞中的3’-OH子文库中有10.5％、5.5％和8.2％的sRNA能与miRNAs匹配，而在3’-cP子文库中分别只有0.15％、0.40％和0.19％的sRNA与miRNAs匹配(图2d)。同时，也分析了小鼠肝脏、Hepa 1-6和Hep G2细胞中sRNA-OH或sRNA-cP的长度分布，结果如图2e和图8g，h所示。与之前miRNA的报道一致(Lau，N.C.等，An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans.Science 294，858，(2001))，sRNA-OH子文库中miRNAs在21-24nt有一个明显的富集峰，而sRNA-cP则与其不同(图2e，图8i，j)。
为了进一步揭示sRNA-OH和sRNA-cP之间的差异，分析了两种文库间序列的相似程度。结果表明小鼠肝脏、Hepa 1-6和Hep G2细胞中，3’-OH和3’-cP子文库中的sRNA序列并不相同，在丰度排名前1000的sRNA中，只有不到3％的sRNA重叠度(图2f-g)。此外，进一步分析每个型类的sRNA，结果也显示只有极少量重叠序列(图9a-c)。这些结果表明，sRNA-OH和sRNA-cP通常具有不同的序列。
综合以上结果，成功地建立了一种称为TANT-seq的sRNA高通量测序方法，其主要优势是可以在一个文库中同时检测sRNA-OH和sRNA-cP。利用TANT-seq，揭示了小鼠和人15-30nt sRNA-OH和sRNA-cP的完整图谱，并发现它们通常具有不同的序列。
实施例3、TE-qPCR建立和TANT-seq数据验证
为了验证TANT-seq检测得到的大量新发现的sRNA-cP，又建立了一种同时检测RNA三种3’末端的qPCR方法(TE-qPCR)，此方法可同时检测RNA样品中含有3’-OH，3’-P和3’-cP的sRNA(图3a)。具体操作方法是将RNA样品分为3份，分别用水、AP或T4 Pnk处理，然后对处理后的RNA进行聚腺苷化反应和反转录反应，随后用特异性的引物进行qPCR检测(图3a)，最后通过计算公式(材料部分)，便可以的对样品中特定序列的sRNA-OH、sRNA-P和sRNA-cP含量进行定量分析。为了验证方法的可行性，首先利用合成的3’-OH，3’-P和3’-cP标准品对TE-qPCR体系进行了验证，结果显示TE-qPCR可以很好的区分不同3’末端修饰的sRNA(图3b)。
利用TE-qPCR方法，对小鼠肝脏组织、Hepa 1-6和Hep G2细胞样本进行检测，结果显示在检测的 sRNA中，miRNA 3’末端几乎100％是3’-OH，这进一步说明了TE-qPCR方法的可靠性，而与此同时TANT-seq中所发现的sRNA-cP 3’末端则是3’-cP(图3c，d，图10a，b)。此外，本发明人也在小鼠脑组织，小鼠白色脂肪组织，NIH/3T3和AML12细胞中也检测到了从TANT-seq中发现的一些sRNA-cPs(图d，图10a)，这表明sRNA-cP在不同的组织或培养细胞中普遍表达。
接下来，进一步利用Northern blot验证TANT-seq中发现的sRNA的表达和末端修饰(图3e，图10c，d)。本发明人发现阴性对照(miRNA)可以直接被T4 RNA连接酶2连接或者被聚腺苷化，但只有用T4 Pnk而不是AP处理后，sRNA-cP才可以被T4 RNA连接酶2连接或聚腺苷化(图3e，图6c)，这些数据进一步证明TANT-seq中发现的sRNA-cP的表达和大小是可靠的。
此外，还利用TE-qPCR探究了部分新发现sRNA-cP在不同生理和病理条件下的表达情况。如图3f所示，当小鼠禁食24小时后，在小鼠肝脏中，rsR-67-cP、tsR-2-cP、sn-7-cP、sinR-7-cP和smR-26-cP的表达水平显著提高；在高脂饮食的小鼠或db/db小鼠的肝脏中，rsR-67-cP、sn-7-cP、snR-7-cP、sinR-7-cP、smR-26-cP、slncR-106-cP和smR-13-cP表达水平显著降低。这些结果提示，部分sRNA-cP与生理和病理变化有关，并潜在地可作为某些特定的生物状态测定的标记物。
实施例4、血管生成素和RNase 4参与小RNA-cPs的生成
通过对TANT-seq测序结果的核苷酸富集分析，发现sRNA-cP的3’端嘧啶碱基具有强烈富集信号，而其5’端嘌呤碱基有一定程度的富集(图4a)；而对sRNA-OH，仅在一些特定的类型中观察到3’端核苷酸的显著富集，如tsRNA，snoRNA，snRNA，sgmRNA，piRNA和smcRNA(图11a-d)，这表明sRNA-cP和sRNA-OH很可能具有不同的产生方式。通过体外酶切反应实验，发现小鼠总RNA经过RNase A酶切后会导致actin和miR-122显著下降，但sRNA-cP的表达量却有显著增加(图4b)。与此相反的是，用G碱基后酶切RNA生成3’-cP的RNase T1酶切小鼠总RNA，所检测的sRNA均明显减少(图4b)。RNase A酶是一个大家族酶类，小鼠中包含十几个亚成员，有趣的是，在小鼠肝脏中RNase A家族中只有血管生成素(Ang)和RNase 4具有较高的表达量(图4c)，因此接下来将Ang和RNase 4作为主要研究目标。Ang和RNase 4的体外酶切实验结果表明，两者均可导致actin和miR-122显著减少，但可以显著增加sRNA-cP的表达(图4d，图12a，b)。Northern blotting实验也进一步证实这一结果，且sRNA-cP的表达量呈现出对Ang和RNase 4剂量依赖关系(图4e，图12c)。
为了探究Ang和RNase 4在细胞内是否和sRNA-cP产生有关，转染Ang或RNase 4蛋白到Hepa 1-6细胞内，通过TE-qPCR检测发现部分sRNA-cP表达量有适度的增加(图12d)。已知细胞中的RNH1蛋白可以与Ang或RNase 4结合并抑制其活性，对此，利用CRISPR/Cas9技术建立了稳定的RNH1敲除(HKO)Hepa 1-6细胞系，并通过测序和免疫印迹对敲除细胞系进行了验证(图12e，图4f)。然后将Ang或RNase 4蛋白转染到RNH1敲除的Hepa 1-6细胞中，利用TE-qPCR和Northern blot检测发现部分sRNA-cP表达量有显著增加(图4f，g，图12f)。这些数据表明，Ang和RNase 4可以在体外或细胞内导致sRNA-cPs产生。
接下来进一步利用CRISPR/Cas9技术构建了稳定的Ang敲除(AKO)、RNase 4敲除(RKO)和Ang/RNase 4双敲除(DKO)的Hepa 1-6细胞系，并通过测序和免疫印迹对敲除细胞系进行了验证(图12g，图4i)。通过TE-qPCR检测，发现RKO和DKO的Hepa 1-6细胞中部分sRNA-cP表达量一定程度降低(图12h)。由于细胞培养基中含有血清，而血清中含有的RNase可以进入细胞，因此在Hepa 1-6细胞培养基中加入重 组核糖核酸酶抑制剂阻断外源RNase活性。重组核糖核酸酶抑制剂预处理后，在RKO和DKO Hepa 1-6细胞中，srR-67-cP、snR-7-cP、sinR-7-cP、smR-26-cP和slncR-106-cP水平显著降低(图4h，i，图12j，k)。此外，利用T4 RNA连接酶2连接反应结合Northern blot方法系统检测野生型(WT)、AKO、RKO和DKO Hepa 1-6细胞中15-30nt sRNA 3’为-OH末端和3’为-(OH+P+cP)末端的sRNA相对表达量，结果表明RKO和DKO Hepa 1-6细胞中，sRNA-OH含量无明显变化，但是T4 Pnk处理后得到的3’为-(OH+P+cP)末端的sRNA含量显著降低，而之前的结果表明此范围内sRNA-cP占大多数的，进一步说明sRNA-cP在RKO和DKO Hepa 1-6细胞中显著减少(图12i)。
为了系统研究Ang、RNase 4在Hepa 1-6细胞中对15-30nt sRNA生成的作用，利用TANT-seq对上述敲除细胞系进行了高通量测序。结果表明在重组核糖核酸酶抑制剂预处理后，AKO，RKO和DKO的Hepa 1-6细胞中sRNA-OH显著降低的分别有367、346、222个，而sRNA-cP显著降低分别有96、3598、3630个(图4j)，这些数据表明，RNase 4在小鼠sRNA-cP的生物发生中具有重要作用。
此外，为更进一步探究Ang和RNase 4对人源sRNA-cP生成的作用，又建立了稳定的AKO、RKO和DKO的Hep G2敲除细胞系，并通过测序验证敲除效果(图12l)。通过TE-qPCR检测，发现重组核糖核酸酶抑制剂预处理后AKO、RKO和DKO Hep G2细胞中，srR-12-cP、srR-20-cP、snR-31-cP、sinR-20-cP、sinR-90-cP、smR-44和slncR-13表达水平显著降低(图4k)。这些数据说明Ang和RNase 4在人源细胞中和sRNA-cP的生物发生密切相关。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (16)

  1. 一种检测sRNA的末端修饰的方法，其特征在于，所述末端修饰包括3’-OH和3’-cP修饰，所述方法包括：
    (a)提供待测sRNA，使其中存在3’-OH的sRNA的3’端连接上标记3’-OH的接头序列；
    (b)对(a)的产物进行脱磷酸化处理，获得5’端和3’端脱去磷酸的产物；
    (c)对(b)的产物进行氧化处理，使3’-OH发生氧化，分离sRNA；
    (d)对(c)的产物进行磷酸化处理，获得携带5’-P的产物；
    (e)使(d)的产物中存在3’-cP的sRNA连接上标记3’-cP的接头序列；
    (f)根据标记3’-OH的接头序列、标记3’-cP的接头序列，鉴定(f)的产物中含有3’-OH和3’-cP的sRNA。
  2. 如权利要求1所述的方法，其特征在于，(a)中，利用T4 RNA连接酶2连接标记3’-OH的接头序列；和/或
    (b)中，以碱性磷酸酶进行脱磷酸化处理；和/或
    (c)中，以高碘酸钠进行氧化处理；和/或
    (d)中，以T4多聚合核苷酸激酶进行磷酸化处理；较佳地该T4多聚合核苷酸激酶3’磷酸酶缺失，更佳地其为T4 Pnk 3’phosphatase minus；和/或
    (e)中，以RtcB连接反应连接标记3’-cP的接头序列。
  3. 如权利要求1所述的方法，其特征在于，在(e)和(f)之间，还包括：在(e)的产物的5’端连接上标记sRNA的5’-接头序列；较佳地，利用T4 RNA连接酶1连接标记sRNA的5’-接头序列。
  4. 如权利要求1所述的方法，其特征在于，(f)中，根据标记3’-OH的接头序列、标记3’-cP的接头序列的存在情况/存在量，确定携带3’-OH和携带3’-cP的sRNA的存在情况或存在量；较佳地，标记3’-OH的接头序列、标记3’-cP的接头序列、标记sRNA的5’-接头序列为配合规模化测序的接头序列。
  5. 如权利要求1或4所述的方法，其特征在于，(f)中，对产物进行鉴定的方法包括：定量PCR法，测序法，Northern blot法；或
    (f)后，还包括：通过高通量测序，去除所述的标记3’-OH的接头序列、标记3’-cP的接头序列，根据鉴定结果，建立子文库。
  6. 一种检测sRNA的末端修饰的方法，其特征在于，所述末端修饰包括3’-OH、3’-cP和3’-P，所述方法包括：
    (1)提供待测sRNA，将其分成三组，分别以溶剂、碱性磷酸酶和T4多聚合核苷酸激酶处理：
    溶剂组不变，
    碱性磷酸酶组中，原3’-P修饰的sRNA的3’末端转变为3’-OH，
    T4多聚合核苷酸激酶组中，原3’-P修饰和原3’-cP修饰的sRNA的3’末端均转变为3’-OH；较佳地该T4多聚合核苷酸激酶不是T4 Pnk 3’phosphatase minus；
    (2)对(1)的产物进行聚腺苷化反应，使3’-OH的sRNA的3’端聚腺苷化：
    溶剂组中，仅原3’-OH的sRNA的3’端聚腺苷化，
    碱性磷酸酶组中，原3’-OH和原3’-P修饰的sRNA的3’端聚腺苷化，
    T4多聚合核苷酸激酶组中，原3’-OH、原3’-P修饰和原3’-cP修饰的sRNA的3’端聚腺苷化；
    (3)对(2)的产物进行反转录反应：
    溶剂组中，仅原3’-OH的sRNA的经3’端聚腺苷化的产物形成cDNA，
    碱性磷酸酶组中，原3’-OH和原3’-P修饰的sRNA的经3’端聚腺苷化的产物形成cDNA，
    T4多聚合核苷酸激酶组中，原3’-OH、原3’-P修饰和原3’-cP修饰的sRNA的经3’端聚腺苷化的产物形成cDNA；
    (4)对(3)的反转录反应的产物进行定性或定量测定。
  7. 如权利要求6所述的方法，其特征在于，(3)中，进行反转录反应时，采用含有oligo dT的反转录引物；较佳地如OH-DNA15-OH。
  8. 如权利要求7所述的方法，其特征在于，(1)中，所述的溶剂包括：水，缓冲液；和/或
    (4)中，通过定量PCR进行定性或定量测定；较佳地，所述定量PCR为实时荧光定量PCR；更佳地，通过测定不同组的Ct值定量：
    溶剂组中，Ct值反映3’-OH的sRNA的量；
    碱性磷酸酶组中，Ct值反映3’-OH和3’-P修饰的sRNA的量；
    T4多聚合核苷酸激酶组中，Ct值反映3’-OH、3’-P和3’-cP修饰的sRNA的量。
  9. 如权利要求8所述的方法，其特征在于，(4)中，根据下列公式计算3’-OH，3’-P和3’-cP的相对含量：
    Figure PCTCN2022138474-appb-100001
    Figure PCTCN2022138474-appb-100002
    (3′-OH)％＝100％-(3′-P)％-(3′-cP)％。
  10. 如权利要求1或6所述的方法，其特征在于，还包括，将所述方法获得的sRNA产物进行进一步的细分；较佳地，所述方法获得的sRNA产物与sRNA的信息库或数据库进行比对，从而进行sRNA的细分。
  11. 如权利要求1或6所述的方法，其特征在于，根据形态，所述待测sRNA包括：来自细胞的总sRNA，局部获取或分离的sRNA；或
    根据物种，所述sRNA包括：动物、植物或微生物来源的sRNA。
  12. 权利要求1-11任一所述的方法的应用，包括：
    用于检测sRNA的末端修饰；所述的sRNA的末端修饰包括选自：(a)3’-OH和3’-cP修饰；或，(b)3’-OH、3’-P和3’-cP修饰；或
    用于建立细胞的sRNA的3’-OH和3’-cP末端修饰的sRNA的表达谱/图谱/文库，或建立3’-OH、3’-P和3’-cP末端修饰的sRNA的表达谱/图谱/文库。
  13. 如权利要求12所述的应用，其特征在于，sRNA末端修饰的区分进一步被用于：
    测定特定的生物状态；
    筛选调节sRNA末端修饰的效应分子。
  14. 一种用于检测sRNA的末端修饰的试剂盒，其特征在于，其中包括下组试剂：
    检测sRNA的3’-OH和3’-cP末端修饰的试剂：T4 RNA连接酶2、碱性磷酸酶、高碘酸钠、T4多聚合核苷酸激酶、RtcB连接酶、标记3’-OH的接头序列、标记3’-cP的接头序列、标记5’的接头序列；较佳地该T4多聚合核苷酸激酶3’磷酸酶缺失，更佳地其为T4 Pnk 3’phosphatase minus；或
    检测sRNA的3’-OH、3’-P和3’-cP末端修饰的试剂：溶剂、碱性磷酸酶和T4多聚合核苷酸激酶。
  15. 如权利要求14所述的用于检测sRNA的末端修饰的试剂盒，其特征在于，其中还包括选自下组的试剂：spike-in混合物，T4 RNA连接酶反应缓冲液，水，PEG8000，核糖核酸酶抑制剂，CutSmart缓冲液，核酸沉淀试剂，核酸抽提试剂，RtcB反应缓冲液，Mn 2+离子溶液，GTP，ATP，反转录引物，SSIV缓冲液，SuperScript IV反转录酶，DTT，dNTP，DNA聚合酶。
  16. Ang和/或RNase 4的应用，用于促进sRNA-cP的形成；较佳地，所述的促进包括体外或胞内的促进。
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