WO2020098806A1 - 鉴定rna分子中2'-o-甲基化修饰的方法及其应用 - Google Patents

鉴定rna分子中2'-o-甲基化修饰的方法及其应用 Download PDF

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WO2020098806A1
WO2020098806A1 PCT/CN2019/119168 CN2019119168W WO2020098806A1 WO 2020098806 A1 WO2020098806 A1 WO 2020098806A1 CN 2019119168 W CN2019119168 W CN 2019119168W WO 2020098806 A1 WO2020098806 A1 WO 2020098806A1
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mirna
rna
methylation modification
methylation
terminal nucleotide
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陈奇涵
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南京迈西可生物科技有限公司
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Priority to EP19883631.4A priority patent/EP3896170A4/en
Priority to JP2021526740A priority patent/JP2022534146A/ja
Priority to US17/294,065 priority patent/US20220372543A1/en
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Definitions

  • the present invention relates to methods for identifying whether RNA molecules have 2'-O-methylation modification and determining 2'-O-methylation modification sites, and also relates to these methods in determining disease diagnosis targets and determining whether subjects have Application of 2'-O-methylation modification related diseases.
  • RNA modifications Since the first pseudouracil nucleoside was discovered more than 50 years ago, more than 100 different RNA modifications have been identified in the biological world. These modifications mainly occur in the four nucleosides of newly generated precursor RNA It is on bases and widely exists in various RNA molecules, including transfer RNA, ribosomal RNA, messenger RNA, and various small RNAs. A large number of studies have shown that plant small RNAs (miRNAs) also have various modifications such as uridine and methylation, and participate in the regulation of miRNA production, stability and other aspects.
  • miRNAs plant small RNAs
  • the 2'-OH and 3'-OH of the 3 'terminal nucleotide on the substrate RNA are very important for the methylation activity of the Hen1 protein. These two sites are likely to play an important role in the substrate recognition process.
  • Studies on the crystal structure of the Arabidopsis Hen1 protein indicate that the Hen1 protein binds to the miRNA / miRNA dimer substrate as a monomer.
  • the substrate forms an A-fold conformation in its tertiary structure, and the end of each chain can be specifically recognized by the Hen1 protein.
  • Hen1 homologous protein Henmt1 in mammals can use 21 to 24 nt single-stranded small RNA (ssRNA) as a substrate and adenosylmethionine (AdoMet) as a methyl donor Methylation modifies small RNA.
  • ssRNA single-stranded small RNA
  • AdoMet adenosylmethionine
  • RNA-DNA-RNA sequence designed a single nucleotide of an RNA-DNA-RNA sequence to recognize the methylation site and guide RNAse H to degrade the recognized site. If the site is protected by methylation, it will not be degraded by RNAse H, so that it can Sensitively distinguish between methylated and non-methylated. At the same time, some studies have found that different sources of RNAseH have different recognition of cleavage sites. However, the above methods for detecting miRNA methylation still have the disadvantages of low sensitivity and large sample requirements.
  • the present invention provides a method for identifying whether an RNA molecule has a 2'-O-methylation modification on a 3 'terminal nucleotide, including:
  • RNA molecule if the RNA molecule is degraded, it indicates that the RNA molecule has no 2'-O-methylation modification on the 3 'terminal nucleotide.
  • the ribonuclease Rnase has the amino acid sequence shown in SEQ ID NO: 3 or 4, or, compared with the amino acid sequence shown in SEQ ID NO: 3 or 4, the ribonuclease Rnase R has substitution, deletion, addition of one or several amino acid residues, or has at least 95% sequence identity, and has exonuclease activity.
  • step (2) includes performing the detection using gel electrophoresis or RT-PCR.
  • the RNA molecule is a miRNA.
  • the present invention provides a method for screening miRNA that can be used as a diagnostic target for diseases, which includes:
  • the miRNA with 3 'terminal nucleotide 2'-O-methylation modification in the patient and normal person without 3' terminal nucleotide 2'-O-methylation modification was taken as the disease's Diagnosis target.
  • the disease is cancer, genetically disordered genetic disease, or developmental error.
  • the disease is lung adenocarcinoma.
  • the present invention provides a method for determining a disease associated with a 2'-O-methylation modification of the 3 'terminal nucleotide of miRNA in a subject, including:
  • the miRNA has a 3 ′ terminal nucleotide 2′-O-methylation modification, and the normal human miRNA does not have a 3 ′ terminal nucleotide 2′-O-methylation modification, it is considered that The subject has the disease associated with the 2'-O-methylation modification of the 3 'terminal nucleotide of the miRNA.
  • the miRNA is extracted from the serum of the subject.
  • the disease is cancer, genetically disordered genetic disease, or developmental error.
  • the disease is lung adenocarcinoma.
  • the present invention provides the application of the miRNAs listed in Table 2 as disease diagnosis targets.
  • the disease is lung adenocarcinoma.
  • the present invention provides a kit for detecting a disease associated with a 2'-O-methylation modification of a 3 'terminal nucleotide of miRNA in a subject, which includes a ribonuclease Rnase .
  • the ribonuclease Rnase has the amino acid sequence shown in SEQ ID NO: 3 or 4, or, compared with the amino acid sequence shown in SEQ ID NO: 3 or 4, the ribonuclease Rnase R has substitution, deletion, addition of one or several amino acid residues, or has at least 95% sequence identity, and has exonuclease activity.
  • the kit further includes the miRNA as a standard without the 3 'terminal nucleotide 2'-O-methylation modification.
  • the present invention provides a method for determining a 2'-O-methylation modification site in an RNA molecule, which includes:
  • the small RNA produced in step (1) is 60 to 200 nucleotides in length.
  • the ribonuclease Rnase has the amino acid sequence shown in SEQ ID NO: 3 or 4, or, compared with the amino acid sequence shown in SEQ ID NO: 3 or 4, the ribonuclease Rnase R has substitution, deletion, addition of one or several amino acid residues, or has at least 95% sequence identity, and has exonuclease activity.
  • the RNA molecule is an rRNA or mRNA molecule.
  • RNA sequences do not depend on specific RNA sequences, have universal applicability, and have high sensitivity, mild reaction conditions, and simple operation.
  • Fig. 1 is a schematic diagram of an enzymatic hydrolysis reaction for identifying whether a single-stranded RNA has a 3 'terminal 2'-O-methylation modification.
  • Figure 2 is a photograph of gel electrophoresis results of different RNA substrates treated with Rnase R enzyme.
  • Fig. 3 is a schematic diagram of using Rnase R enzyme to determine the 2'-O-methylation modification site in RNA.
  • Figure 4 shows the statistical results of the degradation termination sites near 868 nucleotides obtained by high-throughput sequencing of 18s RNA after random interruption and Rnase R enzyme treatment.
  • Figure 5 shows an 18s rRNA as an example to show the calculated ratio after high-throughput detection in rRNA using the method of the present invention.
  • Figure 6 is a photograph of gel electrophoresis results of different RNA substrates treated with truncated Rnase R enzyme (82-725aa).
  • Random nuclease refers herein to a nuclease capable of catalyzing the hydrolysis of RNA into a single nucleotide or small fragment, which is mainly divided into endonuclease and exonuclease.
  • Ribonuclease Rnase R has a well-known meaning in the art, and refers to a class of exonuclease, including many family members. Members of the RNase R family are widely distributed in species, and have the highest similarity to the RNase II family, usually exozymes that degrade RNA molecules from the 3 ’end to the 5’ end. Previous studies have suggested that RNase R family members may be involved in degrading foreign RNA fragments or participating in post-transcriptional modification of their own RNA under specific conditions.
  • Rnase R is an exonuclease isolated from Mycoplasma genitalium (may be referred to simply as "MgR").
  • Mycoplasma genitalium has a very small genome, and the ribonuclease Rnase R is also the only exonuclease identified in it.
  • the 3 'terminal nucleotide of the substrate single-stranded RNA has a 2'-O-methylation modification, it cannot be exerted Its RNase hydrolysis activity; and if the 3 'end nucleotide of the single-stranded RNA of the substrate is not modified, it can degrade the substrate like many other RNase R family members (see Figure 1).
  • the RNA exonuclease activity of the RnaseR is not limited to the sequence and secondary structure of the RNA itself, and has universality, and the reaction conditions are mild and simple, and the reaction speed is also very rapid.
  • the ribonuclease Rnase herein may also include its natural mutants or homologues in other species.
  • the inventors found that removing the amino terminus of ribonuclease MgR up to 81 amino acids does not affect the exonuclease activity of the enzyme. Therefore, in this article, it is mentioned that the ribonuclease RNase R (or MgR) also includes these truncated forms of the enzyme (also known as truncated body enzymes).
  • the inventors also found that for the case where the 2'-O-methylation modification is located in the RNA molecule, the degradation of the RNA molecule by the ribonuclease Rnase R will stop at the 2'-O-methylation modification site The latter nucleotide (ie, the adjacent nucleotide in the 3 'direction) (degradation termination site).
  • the RNA molecule When the RNA molecule is numerous (for example, 100, 1000, or 10,000 copies or more), when RNase Rn is degraded, a significant amount of fragments will be found to terminate at the 2'-O-methylation modification site It is located one nucleotide after the spot, so this site is also referred to herein as the "concentrated degradation termination site.” If there are multiple 2'-O-methylation modifications in the RNA molecule, the RNA molecule can be first fragmented into fragments of, for example, 60 to 200 nucleotides, and then degraded by RNase Rnase, then Correspondingly, multiple concentrated degradation termination sites are generated.
  • RNA refers to small non-coding RNA, usually a single-stranded RNA molecule of about 20 to 25 nucleotides. Its precursor pre-miRNA (hairpin-like) is transcribed in the nucleus and then cleaved by Dicer enzyme in the cytoplasm to form miRNA. It can form a complex with RISC (RNA-induced silencing complex), and then bind to the target mRNA through base pairing to prevent translation of the mRNA, thereby participating in the regulation of gene expression. Previous studies have shown that miRNA 3 'end 2'-O-methylation modification is related to the occurrence and development of tumors and other diseases.
  • RISC RNA-induced silencing complex
  • subject refers to an individual (preferably a human) suffering from or suspected of having a certain disease, which may also be a healthy individual.
  • the term is generally used interchangeably with “patient”, “test subject”, “treatment subject”, and the like.
  • Invitrogen was commissioned to synthesize the Rnase R gene sequence and the truncated gene sequence (SEQ ID NO: 1 and 2) with the first 81 amino acids removed, and introduce the Nco I endonuclease site at the 5 'end of the gene fragment and the 3' end Hind III endonuclease site.
  • the synthesized gene fragment and pET28a (+) vector were digested by Nco I and Hind III, respectively, and the gene fragment and vector fragment were connected by T4 DNA ligase, and then transformed into DH5 ⁇ competent cells (Tiangen Biochemical Technology (Beijing) Co., Ltd. ). Screen positive clones based on kanamycin resistance and extract plasmids.
  • the recombinant plasmid was identified by Nco I and Hind III double digestion and agarose gel electrophoresis, and Invitrogen was commissioned to sequence the recombinant plasmid, and the sequencing results were analyzed using BioEdit software. The results were the same as the designed sequence, indicating that the recombinant bacteria were successfully constructed.
  • the cells were lysed by ultrasonic method (6W output for 8 minutes, 20 seconds on and 20 seconds off), and the supernatant was separated by centrifugation at 25000g.
  • the supernatant and nickel resin Nickel resin, ThermoFisher
  • the recombinant protein was eluted with a lysis buffer solution containing 200 mM imidazole, diluted to 0.1 M NaCl and concentrated to 2 mg / ml in a centrifuge tube.
  • the quality and concentration of recombinant protein were determined by SDS-PAGE.
  • sequence of the recombinant protein is shown in SEQ ID NO: 3 and 4 respectively (6his tag is not shown). Unless otherwise stated, the following examples are carried out using 6his-tagged full-length Rnase R enzyme.
  • Example 2 Similar to the procedure of the enzyme reaction in Example 3, 10 ⁇ g, 5 ⁇ g, or 2.5 ⁇ g of the recombinant fusion protein obtained in Example 1 was mixed with 7 ⁇ L of the 100-fold dilution (1 nmol) of the RNA substrate obtained in Example 2, and the appropriate amount The buffer solution and water were brought to a final volume of 10 ⁇ L (20 mM Tris pH 8.5, 100 mM KCl, 0.01 mM ZnCl 2 ), reacted at 37 ° C for 1 hour, and then treated at 85 ° C for 5 minutes to inactivate the enzyme.
  • 10 ⁇ g, 5 ⁇ g, or 2.5 ⁇ g of the recombinant fusion protein obtained in Example 1 was mixed with 7 ⁇ L of the 100-fold dilution (1 nmol) of the RNA substrate obtained in Example 2, and the appropriate amount The buffer solution and water were brought to a final volume of 10 ⁇ L (20 mM Tris pH 8.5, 100 mM KCl, 0.
  • the CT value of the RNA substrate (miR-21) amplified without the 3 'end 2'-O-methylation modification was at three different enzyme concentrations Both are around 27, and the CT value of the control group using Rnase R enzyme buffer solution instead of enzyme reaction is around 18, and the difference between the two is very obvious; and after Rnase R enzyme treatment, there are 3 'end 2'-O-A
  • the CT value of the amplified RNA substrate (miR-21-ch3) amplification was about 19 at three different enzyme concentrations, and the CT value of the reaction of the control group using Rnase R buffer solution instead of the enzyme was also 19 There is no obvious difference between the two.
  • Rnase R enzyme can selectively hydrolyze RNA without 2'-O-methylation modification at the 3 'end, but not hydrolyze RNA with 2'-O-methylation modification at the 3' end.
  • qRT-PCR it is possible to identify whether the target RNA has a 3 'end 2'-O-methylation modification at a concentration of nmol level 100 times lower than that of gel electrophoresis.
  • RNA After measuring the total amount of RNA, a 10-fold amount of the Rnase R enzyme obtained in Example 1 (the buffer solution of the enzyme itself is 50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 0.5 mM TCEP), 5 ⁇ L of 10X Rnase R buffer (200 mM Tris) -Cl (pH 8.5), 1000 mM KCl, 0.1 mM ZnCl 2 ), and the final volume was made up to 50 mL with DEPC treated water. The reaction was performed at 37 ° C for 30 minutes, and then treated at 85 ° C for 10 minutes to inactivate the Rnase R enzyme.
  • the solution can be divided into three layers: the lower layer is an organic solvent, the middle layer is a protein, and the upper layer is a water-soluble substance. Transfer the upper water-soluble substance layer to a new centrifuge tube, add an equal volume of isopropyl alcohol, mix well, and let stand at -20 ° C for 1 hour. Subsequently, it was centrifuged at 16000g for 20 minutes at 4 ° C, and the supernatant was removed after completion. Add 1mL of 75% ethanol, wash the bottom of the tube and the wall sediment, and transfer to a new 1.5mL centrifuge tube. Centrifuge at 16000g for 20 minutes at 4 ° C, and remove the supernatant after completion. Treat RNA with 25 ⁇ L of DEPC to dissolve the RNA pellet and let stand for 10 minutes. This sample can be used directly for sequencing.
  • Lung adenocarcinoma and normal samples were sent to BGI for miRNA low-density chip sequencing (Applied Biosystems).
  • the chip will detect 384 miRNAs that are more common and less common in the sample, a total of 768, the principle is equivalent to doing 768 sets of qRT-PCR at the same time.
  • the analysis of the results is similar to the general qRT-PCR.
  • the CT value should be as low as 30 and the miRNA with a CT value of 30 or greater in normal people, that is, 2'-O-A exists at the 3 'end in the blood sample of lung adenocarcinoma
  • the miRNA modified without normal modification in normal humans some results are shown in Table 2 below.
  • this part of miRNA showed 3 'terminal 2'-O-methylation modification in the blood sample of lung adenocarcinoma patients (CT ⁇ 30), while in normal human blood sample was Rnase R enzyme Completely hydrolyzed (CT> 30).
  • CT ⁇ 30 the blood sample of lung adenocarcinoma patients
  • Rnase R enzyme Completely hydrolyzed CT> 30.
  • the 2'-O-methylation modification at the 3 'end of these miRNAs can be potential diagnostic targets for lung adenocarcinoma.
  • RNA acquisition RNA fragmentation
  • Rnase R enzyme processing fragment tailing and high-throughput sequencing
  • sequencing result comparison and modification site reduction RNA fragmentation and Rnase R enzyme processing.
  • fragment tailing fragment tailing and high-throughput sequencing
  • sequencing result comparison and modification site reduction RNA fragmentation and Rnase.
  • the RNA to be tested can be fragmented into fragments with a length of about 60 to 200 nt, and then the product is processed with exonuclease Rnase.
  • the inventors found that RNA fragments without 2'-O-methylation modification will be degraded, while the site (Nm) with 2'-O-methylation modification will be recognized by Rnase R enzyme, and the degradation will stop at Nm + 1 site. Subsequently, the degraded RNA samples were purified and sequenced for library construction.
  • Rnase R enzyme will produce a concentrated degradation termination site at the nucleotide (Nm + 1 site) downstream of each 2'-O-methylation modification site, by comparing the sequencing sequence with the genome Analysis can identify 2'-O-methylation sites.
  • RNA was extracted from Hela cell line using total RNA purification kit (Qiagen). Dissolve the total RNA obtained by dissolving 100 ⁇ L of DEPC in water. A 1% agarose gel was prepared, and 1XTAE running buffer was prepared using DEPC-treated water. The obtained total RNA was subjected to electrophoresis detection, 18S rRNA band was cut into gel, and recovered using RNA agarose recovery kit (zymoresearch). Use 100 ⁇ L of DEPC to dissolve RNA in water to obtain 50 ⁇ L of 18S RNA. Determination A260: 280 1.99, which proved to be a relatively pure 18S product. A portion of the sample may be electrophoresed on a 1% agarose gel to check the integrity of 18S.
  • 1.5 ⁇ g of the 18S rRNA (45 ⁇ L) obtained in the previous step was mixed with 45 ⁇ L of 100 mM Na 2 CO 3 (pH 9.0) and treated with a 95 ° C water bath for 10 min. Subsequently, 10 ⁇ L of 3 mol / L Na-oAC (pH 5.2) was added to terminate the reaction. Add 24 ⁇ L of 5 ⁇ g / ⁇ L glycogen to the system and mix well. Add 372 ⁇ L of 96% ethanol to the system, mix well, and place in liquid nitrogen. Centrifuge for 20 minutes at 12000g and 4 ° C.
  • the supernatant was discarded from the obtained centrifugal product, and 1 mL of 80% ethanol was added to wash the precipitated RNA, which was centrifuged again at 12000 g and 4 ° C for 20 minutes.
  • the supernatant was discarded from the obtained centrifugal product, the residual ethanol was completely evaporated in an ultra-clean workbench, and 25 ⁇ L of DEPC-treated water was added to dissolve the remaining RNA.
  • 0.1 ⁇ g of the processed product was used to generate a cDNA library using a tailing / reversal kit (bioengineering), and high-throughput sequencing was used to obtain the sequence information of the fragments.
  • high-throughput sequencing was used to obtain the sequence information of the fragments.
  • the statistics of the stopping point near the 850th nucleotide shows that about 70% of the fragments terminate at the 868th nucleotide, indicating that most of the 18s RNA molecules have a 2 'on the 867th nucleotide -O-methylation modification.
  • the other fragment termination sites 894, 915, 941, 988 may be a product of incomplete enzymatic hydrolysis after random interruption or a small amount of 2'-O-methylated sites with low modification ratios after hydrolysis product.
  • all 2'-O-methylation sites on single-stranded RNA can be identified, and these sites may be related to diseases such as cancer, genetic disorder genetic diseases, developmental errors, etc.
  • the method can be used for in vitro diagnosis of these diseases.
  • Example 6 Taking the Hela cell line as an example, the principle and operation are the same as in Example 6. After obtaining the sequencing results, based on the ratio of the number of fragments terminated at each site to the number of fragments terminated at the previous site, based on the principles described in Example 6, we can determine whether it may be 2'-O-methylated Location.
  • Figure 5 shows the ratio of each site using 18s rRNA as an example.
  • Table 3 and Table 4 list the distribution of some sites with higher ratios in 18s rRNA and 28s rRNA.

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Abstract

本发明提供了一种鉴定RNA分子是否在核苷酸上具有2'-O-甲基化修饰的方法，其包括(1)让所述RNA分子与核糖核酸酶Rnase R接触；以及(2)检测所述RNA分子是否被降解或降解后终止水解的位置。其中，如果所述RNA分子被降解，则表明所述RNA分子在3'末端核苷酸上不具有2'-O-甲基化修饰；而多个随机打断后的片段均终止水解于同一位点，则表明所述RNA分子在终止位点上一位的核苷酸上具有2'-O-甲基化修饰。本发明还提供了采用该方法来筛选疾病诊断靶点以及确认受试者是否具有与2'-O-甲基化修饰相关的疾病的应用。

Description

鉴定RNA分子中2’-O-甲基化修饰的方法及其应用 技术领域
本发明涉及鉴定RNA分子是否具有2’-O-甲基化修饰以及确定2’-O-甲基化修饰位点的方法，还涉及这些方法在确定疾病诊断靶点以及确定受试者是否具有2’-O-甲基化修饰相关疾病方面的应用。
背景技术
自从50多年前第一个假尿嘧啶核苷被发现，到目前为止，在生物界己经鉴定出100多种不同的RNA修饰，这些修饰主要发生在新生成的前体RNA的四种核苷碱基上，并且广泛存在于各种RNA分子中，包括转移RNA、核糖体RNA、信使RNA以及各种小RNA等。大量研究表明，植物小RNA(miRNA)也存在诸如尿苷化、甲基化等多种修饰，并参与调控了miRNA生成、稳定性等多个方面。近期的研究表明昆虫和哺乳动物体内多种小分子RNA也存在3’末端甲基化，如siRNA、hc-siRNA、ta-siRNA、nat-siRNA和piRNA。这些小分子RNA的3’末端甲基化可以使其免受细胞中多种核酸外切酶、连接酶、末端转移酶、聚合酶等可作用于核酸3’末端羟基的酶攻击，从而保持小分子RNA的稳定。
已有研究表明，很多植物的miRNA在形成过程中会经历由HEN1酶甲基化的调控，这是保护其免受尿苷化的一种机制。体外活性试验表明，重构的拟南芥Hen1蛋白能作用于21至24nt的miRNA/miRNA二聚体，在每条链的3’末端核苷酸的2’-OH位点添加一个甲基基团(2’-O-methylated group)。另外还发现dsRNA底物3'端2个碱基的突出和3’末端核苷酸2’-OH和3’-OH是拟南芥Hen1蛋白作用底物2个非常重要的特征。底物RNA上3’末端核苷酸的2’-OH和3’-OH对于Hen1蛋白的甲基化活性十分重要，这2个位点很可能在底物识别过程中发挥重要作用。对拟南芥Hen1蛋白的晶体结构研究表明，Hen1蛋白以单体的形式与miRNA/miRNA二聚体底物结合。该底物在其三级结构上形成一个A折叠构象，每条链的末端都能被Hen1蛋白特异识别。对Henmt1进一步研究表明，在试管实验中，哺乳动物中Hen1同源蛋白Henmt1可以以21至24nt单链小RNA(ssRNA)为底物，以腺苷甲硫氨酸(AdoMet)为甲基供体甲基化修饰小RNA。
有研究显示miRNA存在多种修饰，并且多种修饰丰度在肿瘤样品中升高。LC-MS/MS分析结果发现，在miRNA样品中一共发现12种甲基化修饰，其中包括3’端2’-O-甲基腺苷(Am)，2’-O-甲基尿苷(Um)，2’-O-甲基鸟苷(Gm)，2’-O-甲基胞苷(Cm)， 更有意思的是3’端2’-O-甲基化修饰丰度在肿瘤样品中显著升高。这些结果提示哺乳动物miRNA存在多种多样的修饰，并且这些修饰很可能参与了肿瘤等疾病的发生和发展。而这些修饰，也许具备了成为肿瘤诊断的标志物的潜力。
目前已有多种方法应用于2’-O-甲基化的检测。陈雪梅课题组利用miRNA末端2’-O-甲基化对于羟基的保护作用，高碘酸钠氧化，β消除，RNA纯化等一系列操作，检测出甲基化与非甲基化的miRNA。董志伟等通过特殊设计的逆转录引物，识别甲基化的位点，在低浓度dNTP的情况下有2’-O-甲基化修饰时，将不能进行RNA的逆转录，而非甲基化的位点则能正常逆转录。这一方法在人与酵母菌的核糖体RNA，小鼠的piRNA中均得到了验证。有报道发现，由于植物miRNA甲基化的存在，颈环法相对于加尾法，在检测植物miRNA时效率更高。同时，Andreas Marx等设计出对于甲基化位点无差异扩增的逆转录酶，与对甲基化不敏感的酶比较，通过不同检测方法的差异用于检测甲基化。王向东设计三种等温引物反应方式，使其更加灵敏的检测3’末端2’-O-甲基化的植物miRNA。JOAN A.STEITZ等开发了以一种更加灵敏的方法检测甲基化的位点。他们通过设计一段RNA-DNA-RNA序列的单核苷酸识别甲基化位点，引导RNAse H降解识别的位点，如果位点有甲基化保护，则不会被RNAse H降解，从而可以灵敏的区分甲基化与非甲基化。同时，也有研究发现不同来源的RNAseH，对于切割位点的识别也不相同。然而，以上多种检测miRNA甲基化的方法，仍存在灵敏性较低，对样本要求量大的缺点。
发明内容
在一方面，本发明提供了一种鉴定RNA分子是否在3’末端核苷酸上具有2’-O-甲基化修饰的方法，包括：
(1)让所述RNA分子与核糖核酸酶Rnase R接触；以及
(2)检测所述RNA分子是否被降解；
其中，如果所述RNA分子被降解，则表明所述RNA分子在3’末端核苷酸上不具有2’-O-甲基化修饰。
在一些实施方案中，所述核糖核酸酶Rnase R具有SEQ ID NO：3或4所示的氨基酸序列，或者，与SEQ ID NO：3或4所示的氨基酸序列相比，所述核糖核酸酶Rnase R具有一个或几个氨基酸残基的取代、缺失、添加，或者具有至少95％序列一致性，并具有外切核糖核酸酶活性。
在一些实施方案中，步骤(2)包括使用凝胶电泳或RT-PCR进行所述检测。
在一些实施方案中，所述RNA分子为miRNA。
另一方面，本发明提供了一种筛选能够作为疾病的诊断靶点的miRNA的方法，其包括：
(1)分别从患有所述疾病的患者和正常人获取所述miRNA；以及
(2)以上述方法鉴定所述miRNA是否具有3’末端核苷酸2’-O-甲基化修饰，
其中，将所述患者中具有3’末端核苷酸2’-O-甲基化修饰而正常人不具有3’末端核苷酸2’-O-甲基化修饰的miRNA作为所述疾病的诊断靶点。
在一些实施方案中，所述疾病为癌症、基因紊乱性遗传疾病、或发育错误。优选地，所述疾病为肺腺癌。
另一方面，本发明提供了一种在受试者中确定与miRNA的3’末端核苷酸2’-O-甲基化修饰相关的疾病的方法，包括：
(1)从来自所述受试者的生物样品中获取所述miRNA；以及
(2)以上述方法鉴定所述miRNA是否具有3’末端核苷酸2’-O-甲基化修饰，
其中，如果所述miRNA具有3’末端核苷酸2’-O-甲基化修饰，而正常人所述miRNA不具有3’末端核苷酸2’-O-甲基化修饰，则认为所述受试者具有所述与miRNA的3’末端核苷酸2’-O-甲基化修饰相关的疾病。
在一些实施方案中，所述miRNA提取自所述受试者的血清。
在一些实施方案中，所述疾病为癌症、基因紊乱性遗传疾病、或发育错误。优选地，所述疾病为肺腺癌。
另一方面，本发明提供了表2中列出的miRNA作为疾病诊断靶点的应用。
在一些实施方案中，所述疾病为肺腺癌。
另一方面，本发明提供了一种用于在受试者中检测与miRNA的3’末端核苷酸2’-O-甲基化修饰相关的疾病的试剂盒，其包括核糖核酸酶Rnase R。
在一些实施方案中，所述核糖核酸酶Rnase R具有SEQ ID NO：3或4所示的氨基酸序列，或者，与SEQ ID NO：3或4所示的氨基酸序列相比，所述核糖核酸酶Rnase R具有一个或几个氨基酸残基的取代、缺失、添加，或者具有至少95％序列一致性，并具有外切核糖核酸酶活性。
在一些实施方案中，所述试剂盒还包括作为标准品的不具有3’末端核苷酸2’-O-甲基化修饰的所述miRNA。
另一方面，本发明提供了一种在RNA分子中确定2’-O-甲基化修饰位点的方法，其包括：
(1)将所述RNA分子片段化为多个小分子RNA；
(2)用核糖核酸酶Rnase R降解所述小分子RNA，当所述小分子RNA内具有2’-O-甲基化修饰位点时，会形成集中的降解终止位点；
(3)将经步骤(2)处理得到的消化产物进行高通量测序；
(4)比对测序结果，并将所述集中的消化终止位点的前一个核苷酸确定为2’-O-甲基化修饰位点。
在一些实施方案中，步骤(1)产生的所述小分子RNA的长度为60至200个核苷酸。
在一些实施方案中，所述核糖核酸酶Rnase R具有SEQ ID NO：3或4所示的氨基酸序列，或者，与SEQ ID NO：3或4所示的氨基酸序列相比，所述核糖核酸酶Rnase R具有一个或几个氨基酸残基的取代、缺失、添加，或者具有至少95％序列一致性，并具有外切核糖核酸酶活性。
在一些实施方案中，所述RNA分子为rRNA或mRNA分子。
本发明提供的这些方法不依赖于特定RNA序列，具有普遍适用性，并且灵敏度高、反应条件温和，操作简单。
附图说明
图1为鉴别单链RNA是否具有3’末端2’-O-甲基化修饰的酶解反应示意图。
图2为不同RNA底物经Rnase R酶处理后的凝胶电泳结果照片。
图3为采用Rnase R酶确定RNA内2’-O-甲基化修饰位点的示意图。
图4为18s RNA经过随机打断和Rnase R酶处理后，通过高通量测序获得的在868位核苷酸附近的降解终止位点统计结果。
图5以18s rRNA为例显示采用本发明方法在rRNA中高通量检测后通过计算得到的比值。
图6为不同RNA底物经截短的Rnase R酶(82-725aa)处理后的凝胶电泳结果照片。
具体实施方式
除非另有说明，本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。
“核糖核酸酶(Ribonuclease，Rnase)”在本文中指能够催化RNA水解为单个核苷酸或小片段的核酸酶，主要分为内切酶与外切酶两大类。
核糖核酸酶Rnase R具有本领域公知的含义，指的是一类核糖核酸外切酶，包括众多家族成员。RNase R家族成员在物种中分布广泛种类繁多，与RNase II家族相似度最高，通常为从3’端向5’端降解RNA分子的外切酶。之前的研究认为，RNase R家族成员可能与降解外源RNA片段或者在特定条件下参与自身RNA的转录后修饰有关。
在特别优选的实施方案中，Rnase R是从生殖支原体(Mycoplasma genitalium)分离出的外切核糖核酸酶(可以简称为“MgR”)。生殖支原体具有非常小的基因组，该核糖核酸酶Rnase R也是目前在其中唯一鉴定出的外切核糖核酸酶。本发明人发现，该核糖核酸酶MgR的活性与许多其他RNase R家族成员有所不同，如果底物单链RNA的3’末端核苷酸具有2’-O-甲基化修饰，就无法发挥其RNA酶的水解活性；而如果底物单 链RNA的3’末端核苷酸不具有修饰，则可以和其它众多RNase R家族成员一样降解底物(见图1)。该Rnase R的RNA外切酶活性不受限于RNA本身的序列和二级结构，具有普适性，而且反应条件温和简单，反应速度也非常迅速。
本文中核糖核酸酶Rnase R还可以包括其天然突变体或者其它物种中的同源物。另外，本领域技术人员已知，可以在基本保留酶学活性的情况下，对酶的氨基酸序列进行一些保守性改动或修饰，例如一个或几个氨基酸残基的取代、缺失、添加，这些改动或修饰也包括在本发明的范围内。
例如，本发明人发现，去除核糖核酸酶MgR的氨基端多至81个氨基酸，并不影响该酶的外切酶活性。因此，在本文中，提到核糖核酸酶RNase R(或MgR)也包括该酶的这些截短形式(也称为截短体酶)。
本发明人还发现，对于2’-O-甲基化修饰位于RNA分子内的情况，核糖核酸酶Rnase R对该RNA分子的降解会终止在该2’-O-甲基化修饰位点的后一个核苷酸(即3’方向的相邻核苷酸)处(降解终止位点)。当该RNA分子为数众多(例如100个、1000个、或10000个拷贝以上)，则以核糖核酸酶Rnase R降解时，会发现显著量的片段终止在该2’-O-甲基化修饰位点的后一个核苷酸处，因此本文也将该位点称为“集中的降解终止位点”。如果所述RNA分子内具有多个2’-O-甲基化修饰，可以先将所述RNA分子片段化成例如60至200个核苷酸的片段，再以核糖核酸酶Rnase R降解，则会相应地产生多个集中的降解终止位点。
“miRNA(microRNA)”指小的非编码RNA，通常为约20至25个核苷酸的单链RNA分子。其前体pre-miRNA(发夹状)在细胞核中转录产生，随后在细胞质中被Dicer酶切割而形成miRNA。它可以与RISC(RNA诱导的沉默复合物)形成复合体，随后通过碱基配对与靶标mRNA结合，阻止mRNA的翻译，从而参与基因表达的调控。已有研究显示，miRNA的3’末端2’-O-甲基化修饰与肿瘤等疾病的发生和发展相关。
在提及疾病时，本文所用的“受试者”指患有或者怀疑患有某种疾病的个体(优选人)，该个体也可能为健康个体。该术语通常可以和与“患者”、“检测对象”、“治疗对象”等互换使用。
以下通过具体实施例来进一步详细说明本发明。应当理解，具体实施例仅用于解释本发明，并不用于限定本发明的保护范围。实施例中所用到的仪器、设备、试剂、方法等如未特别指明，均为本领域常用的仪器、设备、试剂及方法。
实施例1重组蛋白表达及纯化
委托Invitrogen公司合成Rnase R基因序列及去除前81个氨基酸的截短体基因序列(SEQ ID NO：1和2)，并在基因片段5’端引入Nco Ⅰ内切酶位点，3’端引入Hind Ⅲ内切酶位点。将合成的基因片段和pET28a(+)载体分别经Nco Ⅰ与Hind Ⅲ双酶切，使 用T4 DNA连接酶连接基因片段和载体片段，常规转化DH5α感受态细胞(天根生化科技(北京)有限公司)。根据卡那霉素抗性筛选阳性克隆，提取质粒。重组质粒经Nco Ⅰ与Hind Ⅲ双酶切和琼脂糖凝胶电泳鉴定，委托Invitrogen公司对重组质粒进行序列测定，使用BioEdit软件对测序结果进行分析，结果与设计序列相同，说明重组菌构建成功。
将获得的阳性克隆质粒转化到E.Coli BL21(DE3)感受态细胞(天根生化科技(北京)有限公司)内，在含有100μg/mL卡纳霉素的LB培养基中37℃培养过夜，之后转移到1L的同样的LB培养基中37℃培养到OD＝0.6左右。之后培养基被降温到4℃，加入0.5mM的IPTG诱导表达约16小时。菌体通过4000g离心收集，用裂解缓冲液(50mM Tris pH＝7.0,1M NaCl,20％甘油,10mM TCEP)重新悬浮。菌体由超声方法裂解(6W输出8分钟，20秒开20秒关)，上清液通过25000g离心分离。将上清液和镍树脂(Nickel resin，ThermoFisher)在4℃下共置1小时，然后通过重力柱并用40mL裂解缓冲溶液洗涤。重组蛋白用含有200mM咪唑的裂解缓冲溶液洗脱，稀释至0.1M的NaCl并用离心管浓缩至2mg/ml。重组蛋白的质量和浓度通过SDS-PAGE确定。
重组蛋白的序列分别如SEQ ID NO：3和4所示(未显示6his标签)。除非另有说明，以下实施例均采用带6his标签的全长Rnase R酶进行。
Figure PCTCN2019119168-appb-000001
Figure PCTCN2019119168-appb-000002
Figure PCTCN2019119168-appb-000003
Figure PCTCN2019119168-appb-000004
Figure PCTCN2019119168-appb-000005
Figure PCTCN2019119168-appb-000006
实施例2小分子RNA底物的制备
委托Invitrogen公司分别合成序列相同的单链小RNA(miR-21)(SEQ ID NO：5)：一种不添加任何额外的修饰(miR-21)，一种在3’末端具有2’-O-甲基化修饰(miR-21-ch3)。将它们稀释至100nmol，于-20℃避光保存。
SEQ ID NO：5人工合成的单链小RNA
UAGCUUAUCAGACUGAUGUUGA
实施例3 Rnase R酶降解反应及电泳检测
将实施例1获得的重组融合蛋白10μg、5μg、或2.5μg，与实施例2中获得的7μL稀释RNA底物混合，加入适量缓冲溶液及水至终体积10μL(20mM Tris pH 8.5,100mM KCl,0.01mM ZnCl 2)，在37℃下反应1小时，然后以85℃处理5分钟使酶失去活性。之后，将产物在10％的TBE-尿素胶上分离。
结果见图2。采用酶缓冲液的对照组，无论3’端修饰与否，RNA底物均清晰地呈现在凝胶上。与三种不同浓度的Rnase R酶反应后，3’端有2’-O-甲基化修饰的RNA底物也清晰地呈现在凝胶上。而与三种不同浓度的Rnase R酶反应后，3’端没有修饰的RNA底物已经被完全降解。
实施例4 Rnase R酶反应及qRT-PCR鉴定
与实施例3酶反应的步骤类似，将实施例1获得的重组融合蛋白10μg、5μg、或2.5μg，与7μL实施例2中获得的RNA底物的100倍稀释液(1nmol)混合，加入适量缓冲溶液及水至终体积10μL(20mM Tris pH 8.5,100mM KCl,0.01mM ZnCl 2)，37℃下反应1小时，然后在85℃处理5分钟使酶失去活性。
从ThermoFisher订购针对miR-21序列的qRT-PCR TaqMan探针试剂盒，按照试剂盒的操作手册完成逆转录和PCR的过程。由表1的结果可以看到，经过Rnase R酶处理后，没有3’端2’-O-甲基化修饰的RNA底物(miR-21)扩增的CT值在三种不同酶浓度下均为27左右，其对照组用Rnase R酶的缓冲溶液代替酶的反应CT值在18左右，两者的差别非常明显；而经过Rnase R酶处理后，有3’端2’-O-甲基化修饰的RNA底物(miR-21-ch3)扩增的CT值在三种不同酶浓度下均为19左右，其对照组用Rnase R酶的缓冲溶液代替酶的反应CT值也在19左右，两者没有明显差别。由此可见，Rnase R酶可以选择性水解3’端没有2’-O-甲基化修饰的RNA，而不水解3’端有2’-O-甲基化修饰的RNA。利用qRT-PCR可以在比凝胶电泳法低100倍的nmol级的浓度下鉴定目标RNA是否具有3’端2’-O-甲基化修饰。
表1底物RNA经过Rnase R酶处理后的qRT-PCR结果
  Rnase R CT值
miR-21 10μg 27.67
  5μg 27.49
  2.5μg 26.56
  缓冲液对照 18.03
miR-21-ch3 10μg 20.03
  5μg 18.84
  2.5μg 18.77
  缓冲液对照 18.36
实施例5 miRNA的3’末端2’-O-甲基化修饰在肺癌诊断中的应用
收集一期肺腺癌患者及正常人各4人的血液，每人5mL，混合为肺癌组和正常人组血样各20mL，加入柠檬酸钠抗凝。以转速150g室温离心20分钟，吸取上清；继续用转速10000g室温离心20分钟，吸取上清血浆约10mL。在10mL血浆中加入20mL Trizol，震荡摇匀。再加入4mL氯仿，震荡摇匀。以16000g室温离心20分钟。离心结束后可看到溶液分为三层：下层为有机溶剂，中层为蛋白质，上层为水溶性物质。将上层水溶性物质层转移到新的离心管中，加入等体积的异丙醇，充分混匀之后，于-20℃静置过夜。第二天，在4℃以16000g离心20分钟，结束后去除上清。加入1mL 75％乙醇，洗涤管底及管壁沉淀，并转移到新的1.5mL离心管内。在4℃以16000g离心20分钟，结束后去除上清。用40μL DEPC处理水溶解RNA沉淀，静置10分钟。
测定RNA总量后加入10倍量的实施例1中获得的Rnase R酶(酶本身的缓冲溶液为50mM Tris-HCl，pH 8.0，250mM NaCl，0.5mM TCEP)，5μL 10XRnase R缓冲液(200mM Tris-Cl(pH 8.5),1000mM KCl,0.1mM ZnCl 2)，并将最终体积用DEPC处理水补足至50mL。在37℃反应30分钟，之后在85℃处理10分钟以灭活Rnase R酶。
加入1mL Trizol，震荡摇匀。再加入200μL氯仿，震荡摇匀。以16000g室温离心20分钟，结束后可看到溶液分为三层：下层为有机溶剂，中层为蛋白质，上层为水溶性物质。将上层水溶性物质层转移到新的离心管中，加入等体积的异丙醇，充分混匀之后，于-20℃静置1小时。随后在4℃以16000g离心20分钟，结束后去除上清。加入1mL 75％乙醇，洗涤管底及管壁沉淀，并转移到新的1.5mL离心管内。在4℃以16000g离心20分钟，结束后去除上清。用25μL DEPC处理水溶解RNA沉淀，静置10分钟。此样本可直接用于测序。
分别将肺腺癌和正常人样本送交华大基因进行miRNA低密度芯片测序(Applied Biosystems)。该芯片会检测样本中比较常见的和不太常见的miRNA各384个，总计768个，其原理相当于同时做了768组qRT-PCR。其结果的分析与一般的qRT-PCR类似，CT值越小说明含量越高，而CT值大于等于30可以认为基本不存在。因此，我们关注的是在肺腺癌的样本中CT值尽可能小于30同时在正常人中CT值大于等于30的miRNA，即在肺腺癌患者血样中3’末端存在2’-O-甲基化修饰而在正常人中没有同样修饰的miRNA，部分结果如下表2所示。
表2肺腺癌及正常人miRNA在Rnase R酶处理后的低密度芯片测序结果(部分)
Figure PCTCN2019119168-appb-000007
如表2所示，这部分miRNA均在肺腺癌患者的血样中呈现出3’末端2’-O-甲基化修饰(CT<30)，而在正常人的血样中则被Rnase R酶完全水解(CT>30)。这些miRNA的3’末端的的2’-O-甲基化修饰均可成为潜在的肺腺癌诊断靶点。
实施例6 Rnase R酶处理在鉴定18s rRNA中2’-O-甲基化位点中的应用
原理：总体流程如图3所示，大致分为RNA获取、RNA片段化、Rnase R酶处 理、片段加尾及高通量测序、测序结果比对和修饰位点还原几个过程。具体地，可以将待测RNA片段化为长度约为60至200nt的片段，然后用核酸外切酶Rnase R对产物进行处理。本发明人发现，不具有2’-O-甲基化修饰的RNA片段会被降解，而具有2’-O-甲基化修饰的位点(Nm)会被Rnase R酶识别，降解终止在Nm+1位点。随后，对降解后的RNA样品进行纯化并进行建库测序。由于Rnase R酶会在每个2’-O-甲基化修饰位点下游的核苷酸处(Nm+1位点)产生一个集中的降解终止位点，通过将测序序列与基因组进行比对分析就能鉴定出2’-O-甲基化位点。
操作：使用总RNA纯化试剂盒(Qiagen)从Hela细胞系中提取总RNA。使用100μL DEPC处理水溶解获得的总RNA。制作1％琼脂糖凝胶，使用DEPC处理水配制1XTAE电泳缓冲液。将获得的总RNA进行电泳检测，对18S rRNA条带切胶，使用RNA琼脂糖回收试剂盒(zymo research)回收。使用100μL DEPC处理水溶解RNA，获得50μL 18S RNA。测定A260:280＝1.99，证明为较纯的18S产物。可取一部分样品用1％琼脂糖凝胶电泳，检测18S的完整性。
将1.5μg上一步获得的18S rRNA(45μL)与45μL 100mM的Na 2CO 3(pH 9.0)混匀，95℃水浴处理10min。随后加入10μL 3mol/L Na-oAC(pH5.2)，终止反应。向体系中加入24μL 5μg/μL的糖原，混匀。向体系中加入372μL 96％乙醇，混匀，置于液氮中。通过12000g、4℃的条件离心20分钟。所获得的离心产物弃除上清，加入1mL 80％的乙醇清洗沉淀的RNA，再次通过12000g，4℃的条件离心20分钟。所获得的离心产物弃除上清，在超净工作台中让残留的乙醇完全挥发，加入25μL DEPC处理水溶解剩下的RNA。
在25μL上一步获得的片段化后的18s rRNA中加入100μg实施例1中获得的Rnase R酶(酶本身的缓冲溶液为50mM Tris-HCl，pH 8.0，250mM NaCl，0.5mM TCEP)，6.1μL 10X Rnase R缓冲液(200mM Tris-Cl(pH 8.5),1000mM KCl,0.1mM ZnCl 2)。在37℃反应30分钟，之后以85℃处理10分钟，灭活Rnase R酶。再次利用总RNA纯化试剂盒(Qiagen)提纯反应后的RNA片段。
将处理后的产物0.1μg利用加尾/反转试剂盒(生工)，生成cDNA文库，利用高通量测序获得片段的序列信息。通过比对高通量测序获得的片段与18s rRNA的序列信息，可发现大部分的片段终止在特定的一些位置(图4)，而这些位置的上一位核苷酸即发生2’-O-甲基化的位置。图4中横坐标表示核苷酸位点，纵坐标表示3’末端位于相应氨基酸位点处的片段的相对含量。例如，在第850位核苷酸附近的停点统计可看出，约70％的片段终止于第868位核苷酸，说明大部分的18s RNA分子在第867位核苷酸上具有2’-O-甲基化修饰。而其它的几个片段终止的位点894、915、941、988则可能是随机打断后酶水解不完全的产物或者修饰比例不高的2’-O-甲基化位点水解后少量的产物。
采用本实施例的方法，可以鉴定单链RNA上所有2’-O-甲基化位点，而这些位点可能在动物中与癌症、基因紊乱性遗传疾病、发育错误等疾病相关，因此该方法可以用于这些疾病的体外诊断。
实施例7 Rnase R酶处理在鉴定18s rRNA、28s rRNA及mRNA中2’-O-甲基化位点中的应用
同样以Hela细胞系为例，原理与操作与实施例6相同。获得测序结果后，通过终止于每一个位点的片段数与终止于前一位点的片段数比值，基于实施例6阐述的原理，我们可以判断其是否可能是2’-O-甲基化位点。图5以18s rRNA为例展示了每个位点的比值情况。
如果以比值由高到低来排列，比值最高的前19个点(>20)有18个点都曾被其它文献报道过具有2’-O-甲基化修饰，而未被报道过的那个修饰位点(即位点889)也被我们用质谱进一步验证了2’-O-甲基化修确实存在，充分说明了我们方法的准确性。
表3和表4列出了部分比值较高的位点在18s rRNA和28s rRNA中的分布情况。
表3高通量测序结果中18s rRNA检测到位点的比值(修饰位点为检测位点前一位)
Figure PCTCN2019119168-appb-000008
Figure PCTCN2019119168-appb-000009
表4高通量测序结果中28s rRNA检测到位点的比值(修饰位点为检测位点前一位)
Figure PCTCN2019119168-appb-000010
Figure PCTCN2019119168-appb-000011
类似地，我们也可以得到mRNA中比值较高的位点在mRNA中的分布情况，此处已对应到染色体上的相应位置，下表5为部分结果。
表5高通量测序数据中mRNA比值排名靠前的修饰位点检测结果(修饰位点为检测位点前一位)
Figure PCTCN2019119168-appb-000012
Figure PCTCN2019119168-appb-000013
采用截短体酶替代完整Rnase R酶进行以上实施例的操作，可以得到类似的结果。例如，采用类似于实施例3的操作，用截短体酶对miR-21和miR-21-ch3进行处理，结果如图6所示：miR-21被降解，miR-21-ch3未被降解。这说明去除Rnase R酶的前(氨基端)81个氨基酸，不影响其RNA外切酶活性，并且该外切酶活性被2’-O-甲基化所抑制。
以上实施例仅用以说明本发明的技术方案，而非对其限制；本领域的普通技术人员应当理解：可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围，其均应涵盖在本发明说明书的范围中。
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Claims (20)

  1. 一种鉴定RNA分子是否在3’末端核苷酸上具有2’-O-甲基化修饰的方法，包括：
    (1)让所述RNA分子与核糖核酸酶Rnase R接触；以及
    (2)检测所述RNA分子是否被降解，
    其中，如果所述RNA分子被降解，则表明所述RNA分子在3’末端核苷酸上不具有2’-O-甲基化修饰。
  2. 如权利要求1所述的方法，其中所述核糖核酸酶Rnase R具有SEQ ID NO：3或4所示的氨基酸序列，或者，与SEQ ID NO：3或4所示的氨基酸序列相比，所述核糖核酸酶Rnase R具有一个或几个氨基酸残基的取代、缺失、添加，或者具有至少95％序列一致性，并具有外切核糖核酸酶活性。
  3. 如权利要求1或2所述的方法，其中步骤(2)包括使用凝胶电泳或逆转录PCR进行所述检测。
  4. 如权利要求1或2所述的方法，其中所述RNA分子为miRNA。
  5. 一种筛选能够作为疾病的诊断靶点的miRNA的方法，包括：
    (1)分别从患有所述疾病的患者和正常人获取所述miRNA；以及
    (2)以权利要求1至4中任一项的方法鉴定所述miRNA是否具有3’末端核苷酸2’-O-甲基化修饰，
    其中，将所述患者中具有3’末端核苷酸2’-O-甲基化修饰而正常人不具有3’末端核苷酸2’-O-甲基化修饰的miRNA作为所述疾病的诊断靶点。
  6. 如权利要求5所述的方法，其中所述疾病为癌症。
  7. 如权利要求5所述的方法，其中步骤(1)包括从所述患者和正常人的血清中提取所述miRNA。
  8. 一种在受试者中确定与miRNA的3’末端核苷酸2’-O-甲基化修饰相关的疾病的方法，包括：
    (1)从所述受试者获取miRNA；以及
    (2)以权利要求1至4中任一项的方法鉴定所述miRNA是否具有3’末端核苷酸2’-O-甲基化修饰，
    其中，如果所述miRNA具有3’末端核苷酸2’-O-甲基化修饰，而正常人所述miRNA不具有3’末端核苷酸2’-O-甲基化修饰，则认为所述受试者具有所述与miRNA的3’末端核苷酸2’-O-甲基化修饰相关的疾病。
  9. 如权利要求8所述的方法，其中所述疾病为癌症。
  10. 如权利要求9所述的方法，其中所述疾病为肺腺癌。
  11. 如权利要求8所述的方法，其中步骤(1)包括从所述受试者的血清中提取所述 miRNA。
  12. 表2中列出的miRNA作为疾病诊断靶点的应用。
  13. 如权利要求12所述的应用，其中所述疾病为肺腺癌。
  14. 一种用于在受试者中检测与miRNA的3’末端核苷酸2’-O-甲基化修饰相关的疾病的试剂盒，所述试剂盒包括核糖核酸酶Rnase R。
  15. 如权利要求14所述的试剂盒，其中所述核糖核酸酶Rnase R具有SEQ ID NO：3或4所示的氨基酸序列，或者，与SEQ ID NO：3或4所示的氨基酸序列相比，所述核糖核酸酶Rnase R具有一个或几个氨基酸残基的取代、缺失、添加，或者具有至少95％序列一致性，并具有外切核糖核酸酶活性。
  16. 如权利要求14所述的试剂盒，其中还包括作为标准品的不具有3’末端核苷酸2’-O-甲基化修饰的所述miRNA。
  17. 一种在RNA分子中确定2’-O-甲基化修饰位点的方法，包括：
    (1)将所述RNA分子片段化为多个小分子RNA；
    (2)用核糖核酸酶Rnase R降解所述小分子RNA，当所述小分子RNA内具有2’-O-甲基化修饰位点时，会形成集中的降解终止位点；
    (3)将经步骤(2)处理得到的消化产物进行高通量测序；以及
    (4)比对测序结果，并将所述集中的消化终止位点的前一个核苷酸确定为2’-O-甲基化修饰位点。
  18. 如权利要求17所述的方法，其中步骤(1)产生的所述小分子RNA的长度为60至200个核苷酸。
  19. 如权利要求17所述的方法，其中所述核糖核酸酶Rnase R具有SEQ ID NO：3或4所示的氨基酸序列，或者，与SEQ ID NO：3或4所示的氨基酸序列相比，所述核糖核酸酶Rnase R具有一个或几个氨基酸残基的取代、缺失、添加，或者具有至少95％序列一致性，并具有外切核糖核酸酶活性。
  20. 如权利要求17所述的方法，其中所述RNA分子为rRNA或mRNA分子。
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