Patent Description:
Since the first pseudouridine was discovered more than <NUM> years ago, to date, more than <NUM> different RNA modifications have been identified in the biological world. These modifications mainly occur in the four nucleotide bases of the newly generated precursor RNAs, and widely exist in various RNA molecules, including transfer RNA, ribosomal RNA, messenger RNA, and various microRNAs. A large number of studies have shown that plant microRNAs (miRNAs) also have various modifications such as uridylation and methylation, etc., and they participate in the regulation of miRNA production and stability, etc. Recent studies have shown that a variety of small molecular RNAs in insects and mammals also have <NUM>' terminal methylation, such as siRNA, hc-siRNA, ta-siRNA, nat-siRNA and piRNA. The <NUM>' terminal methylation of these small molecular RNAs can protect them from enzymatic attack of a variety of exonucleases, ligases, terminal transferases, polymerases, etc., that can act on the <NUM>' terminal hydroxyl groups of nucleic acids, thereby maintaining the stability of small molecular RNAs.

Studies have shown that miRNAs of many plants undergo the regulation of methylation by HEN1 enzyme during the formation process, which is a mechanism of protecting them from uridylation. In vitro activity tests showed that the reconstructed Arabidopsis Hen1 protein can act on miRNA/miRNA dimers ranging from <NUM> to <NUM> nt, adding a methyl group to the <NUM>'-OH site of the <NUM>' terminal nucleotide of each chain (<NUM>'-O-methylated group). In addition, it was also found that the protruding of two bases at the <NUM>' end of the dsRNA substrate and the <NUM>'-OH and <NUM>'-OH on the <NUM>' terminal nucleotides are two very important features of the Arabidopsis Hen1 protein acting substrate. The <NUM>'-OH and <NUM>'-OH on the <NUM>' terminal nucleotides of the substrate RNA are very important for the methylation activity of Hen1 protein, and these two sites are likely to play an important role in the process of substrate recognition. Studies on the crystal structure of Arabidopsis Hen1 protein showed that Hen1 protein binds to miRNA/miRNA dimer substrates in the form of monomers. The substrate forms an A-fold conformation on its tertiary structure, and the end of each chain can be specifically recognized by the Hen1 protein. Further studies on Henmt1 showed that in test-tube experiments, the Hen1 homologous protein Henmt1 in mammals can use <NUM> to <NUM> nt single-stranded small RNA (ssRNA) as the substrate and adenosylmethionine (AdoMet) as the methyl donor for the methylation modification of the small RNA.

Studies have shown that there are multiple modifications of miRNA, and the abundance of multiple modifications is increased in tumor samples. LC-MS/MS analysis results have found that a total of <NUM> methylation modifications were found in miRNA samples, including <NUM>'-end <NUM>'-O-methyladenosine (Am), <NUM>'-O-methyluridine (Um), <NUM>'-O-methylguanosine (Gm), <NUM>'-O-methylcytidine (Cm), and more interestingly, the abundance of <NUM>'-end <NUM>'-O-methylation modification in tumor samples is significantly increased. These results suggested that there are a variety of modifications in mammalian miRNAs, and these modifications are likely to be involved in the occurrence and the development of diseases such as tumors, etc. Moreover, these modifications may have the potential to become markers for tumor diagnosis.

At present, a variety of methods have been applied to the detection of <NUM>'-O-methylation. CHEN Xuemei's research team used miRNA terminal <NUM>'-O-methylation to protect the hydroxyl group, and a series of operations: sodium periodate oxidation, β elimination, RNA purification, etc., to detect methylated and unmethylated miRNAs. DONG Zhiwei et al. used specially designed reverse transcription primers to recognize the methylation sites. In the case of low concentrations of dNTPs and in the presence of <NUM>'-O-methylation modification, reverse transcription of RNAs will not be performed; however, the unmethylated site can be reverse transcribed normally. This method has been verified in the ribosomal RNA of human and yeast, and the piRNA of mice. It has been reported that due to the presence of plant miRNA methylation, the stem-loop method is more efficient than the tailing method in detecting plant miRNAs. At the same time, Andreas Marx et al. designed a reverse transcriptase that does not differentially amplify methylation sites which iscompared with enzymes that are not sensitive to methylation, wherein the difference in different detection methods is used to detect methylation. WANG Xiangdong designed three isothermal primer reaction methods to make it more sensitive to detect the <NUM>' terminal <NUM>'-O-methylated plant miRNAs. STEITZ et al. developed a more sensitive method to detect methylation sites. They designed a mononucleotide of an RNA-DNA-RNA sequence to recognize the methylation sites and guide RNAse H to degrade the recognized sites, wherein if the site is protected by methylation, it will not be degraded by RNAse H, thereby sensitively distinguishing between methylation and non-methylation. At the same time, some studies have also found that different sources of RNAseH have different recognition on the cleavage sites. However, the various methods for detecting miRNA methylation above still have the disadvantages of low sensitivity and large sample amount.

Lalonde et al. disclose that exoribonuclease R inMycoplasma genitalium can carry out both RNA processing and degradative functions, and is sensitive to RNA ribose methylation. The sensitivity of MgR to ribose methylation was confirmed by treating a synthetic methylated RNA oligonucleotide, where MgR stopped at the methylated position while EcR and EcII degraded through it. This demonstrates MgR's distinctive properties in RNA processing and degradation. However, the study does not disclose the method for identifying whether an RNA molecule has a <NUM>'-O-methylation modification on the <NUM>' terminal nucleotide.

<CIT> is one example of the use of miRNA for the diagnosis of lung cancer, including lung adenocarcinoma. This document does not disclose <NUM>' methylation as a marker for the disease. <CIT> discloses that short non-coding protein regulatory RNAs (sprRNAs) <NUM>'-end <NUM>'-O-methylation modification shows differential patterns in lung cancer.

Dai Qing et al. and <CIT> are other examples of methods for detecting <NUM>'-O-methylation sites of RNA.

In one aspect, the present invention provides a method for identifying whether an RNA molecule has a <NUM>'-O-methylation modification on the <NUM>' terminal nucleotide, comprising:.

wherein, if the RNA molecule is degraded, this indicates that the RNA molecule does not have the <NUM>'-O-methylation modification on the <NUM>' terminal nucleotide.

In some embodiments, the ribonuclease Rnase R has an amino acid sequence as shown in SEQ ID NO: <NUM> or <NUM>, or, compared with the amino acid sequence as shown in SEQ ID NO: <NUM> or <NUM>, the ribonuclease Rnase R has at least <NUM>% sequence identity, and has exonuclease activity.

In some embodiments, the step (<NUM>) comprises performing the detection using gel electrophoresis or RT-PCR.

In some embodiments, the RNA molecule is a miRNA.

In another aspect, the present invention provides a method for screening a miRNA that can be used as a disease diagnosis target, comprising:.

wherein, the miRNA with the <NUM>'-O-methylation modification on the <NUM>' terminal nucleotide in the patient while without the <NUM>'-O-methylation modification on the <NUM>' terminal nucleotide in the normal person is used as the disease diagnosis target.

In some embodiments, the disease is a cancer, a heritable disease with a genetic disorder, or a developmental error. Preferably, the disease is lung adenocarcinoma.

In another aspect, the present invention provides a method for identifying diseases associated with a <NUM>'-O-methylation modification on the <NUM>' terminal nucleotide of a miRNA in a subject, comprising:.

wherein, if the miRNA has a <NUM>'-O-methylation modification on the <NUM>' terminal nucleotide, while the miRNA in a normal person does not have a <NUM>'-O-methylation modification on the <NUM>' terminal nucleotide, it is considered that the subject has a disease associated with the <NUM>'-O-methylation modification on the <NUM>' terminal nucleotide of miRNA.

In some embodiments, the miRNA is extracted from the serum of the subject.

In another aspect, the present invention provides use of a miRNA listed in Table <NUM> as a disease diagnosis target.

In some embodiments, the disease is lung adenocarcinoma.

In another aspect, the present invention provides a method for determining a <NUM>'-O-methylation modification site in an RNA molecule, comprising:.

In some embodiments, the small molecular RNA produced in step (<NUM>) is <NUM> to <NUM> nucleotides in length.

In some embodiments, the RNA molecule is rRNA or mRNA molecule.

The methods provided by the invention do not depend on a specific RNA sequence and have universal applicability, with high sensitivity, mild reaction conditions and simple operation.

Unless otherwise stated, all technical and scientific terms as used herein have the meanings commonly understood by those of ordinary skill in the art.

"Ribonuclease (Rnase)" refers to a nuclease that can catalyze the hydrolysis of RNA into single nucleotides or small fragments, and is mainly divided into two categories: endonuclease and exonuclease.

Ribonuclease Rnase R has a well-known meaning in the art, and refers to a class of exoribonucleases, including many family members. The members of the RNase R family are widely distributed in species and have wide varieties. They have the highest similarity with the RNase II family, and usually are exonucleases that degrade RNA molecules from the <NUM>' end to the <NUM>' end. Previous studies have suggested that members of the RNase R family may be involved in the degradation of foreign RNA fragments or involved in the post-transcriptional modification of endogenous RNAs under specific conditions.

In a particularly preferred embodiment, the Rnase R is an exoribonuclease isolated from Mycoplasma genitalium (may be referred to as "MgR" for short). Mycoplasma genitalium has a very small genome, and this ribonuclease Rnase R is also the only exoribonuclease that has been identified therein currently. The inventors found that the activity of this ribonuclease MgR is different from many other RNase R family members. If the <NUM>' terminal nucleotide of the substrate single-stranded RNA has a <NUM>'-O-methylation modification, it will not exert the RNase hydrolytic activity; and if the <NUM>' terminal nucleotide of the substrate single-stranded RNA has no modification, it can degrade the substrate like many other RNase R family members (see <FIG>). The RNA exonuclease activity of the Rnase R is not limited by the sequence and secondary structure of the RNA itself and has universal applicability, and the reaction conditions are mild and simple, and the reaction speed is also very rapid.

The ribonuclease Rnase R herein may also include its natural mutants or homologs in other species. In addition, those skilled in the art know that some conservative changes or modifications can be made to the amino acid sequence of the enzyme, such as the substitutions, deletions, or additions of one or more amino acid residues while basically retaining the enzymatic activity, and such changes or modifications are also included in the scope of the present invention.

For example, the inventors found that removing up to <NUM> amino acids from the amino terminus of ribonuclease MgR does not affect the exonuclease activity of the enzyme. Therefore, reference to the ribonuclease RNase R (or MgR) herein also includes these truncated forms of the enzyme (also referred as truncated enzymes).

The inventors also found that, in the case of the <NUM>'-O-methylation modification in a RNA molecule, the degradation of the RNA molecule by the ribonuclease Rnase R will terminate at the nucleotide behind the <NUM>'-O-methylation modification site (i.e. the adjacent nucleotide in the <NUM>' direction) (degradation termination site). When the RNA molecules are numerous (for example, <NUM>, <NUM>, or <NUM> copies or more), as they are degraded by the ribonuclease Rnase R, the degradation will be found to terminate at the nucleotide next to the <NUM>'-O-methylation modification site in a significant amount of fragments. Therefore, this site is also referred to herein as the "concentrated degradation termination site". If the RNA molecule has multiple <NUM>'-O-methylation modifications, the RNA molecule can firstly be fragmented into, for example, <NUM> to <NUM> nucleotide fragments, and then degraded by the ribonuclease Rnase R. Accordingly, multiple concentrated degradation termination sites are generated.

"MiRNA (microRNA)" refers to a small non-coding RNA, usually a single-stranded RNA molecule of about <NUM> to <NUM> nucleotides. The 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 target mRNA through base pairing to prevent the translation of mRNA, thereby participating in the regulation of gene expression. Studies have shown that the <NUM>' terminal <NUM>'-O-methylation modification of miRNA is related to the occurrence and development of diseases such as tumors, etc..

When referring to a disease, a "subject" as used herein refers to an individual (preferably a human) suffering from or suspected of suffering from a certain disease, and the individual may also be a healthy individual. This term can often be used interchangeably with "patient", "test subject", and "treatment subject", etc..

Hereinafter, the present invention will be further described in detail through specific embodiments. It should be understood that the specific embodiments are only used to explain the present invention and are not used to limit the protection scope of the present invention. The instruments, equipments, reagents, and methods, etc., used in the examples are all commonly used instruments, equipments, reagents, and methods in the art unless otherwise specified.

Invitrogen was commissioned to synthesize the Rnase R gene sequence and the truncated gene sequence (SEQ ID NO: <NUM> and <NUM>) with the first <NUM> amino acids removed, and the Nco I endonuclease site was introduced at the <NUM>' end of the gene fragments and the Hind III endonuclease site was introduced at the <NUM>' end of the gene fragments. The synthesized gene fragments and pET28a(+) vector were double digested with Nco I and Hind III, and the gene fragments and vector fragments were ligated with T4 DNA ligase, and DH5α competent cells were routinely transformed (Tiangen Biotech (Beijing) Co. Positive clones were screened based on kanamycin resistance and plasmids were extracted. The recombinant plasmids were identified by double digestion with Nco I and Hind III and agarose gel electrophoresis. Invitrogen was commissioned to sequence the recombinant plasmids, and the sequencing results were analyzed using BioEdit software. The results were the same as the designed sequences, indicating the successful construction of recombinant bacteria.

The obtained positive clone plasmids were transformed into E. Coli BL21 (DE3) competent cells (Tiangen Biotech (Beijing) Co. ), and cultured overnight at <NUM> in LB medium containing <NUM>µg/mL kanamycin, then transferred to <NUM> of the same LB medium and cultured at <NUM> until OD = about <NUM>. The medium was then cooled to <NUM>, and <NUM> IPTG was added to induce expression for about <NUM> hours. The cells were collected through <NUM> centrifugation and resuspended in lysis buffer (<NUM> Tris pH=<NUM>, <NUM> NaCl, <NUM>% glycerol, <NUM> TCEP). The cells were lysed by an ultrasonic method (6W output for <NUM> minutes, <NUM> seconds on and <NUM> seconds off), and the supernatant was separated by <NUM> centrifugation. The supernatant was placed together with Nickel resin (ThermoFisher) at <NUM> for <NUM> hour, and then passed through a gravity column and washed with <NUM> of lysis buffer solution. The recombinant protein was eluted with a lysis buffer solution containing <NUM> imidazole, diluted to <NUM> NaCl and concentrated to <NUM>/ml with a centrifuge tube. The quality and concentration of the recombinant protein were determined by SDS-PAGE.

The sequences of the recombinant proteins are as shown in SEQ ID NO: <NUM> and <NUM>, respectively (with 6his tag not shown). Unless otherwise specified, the following examples are all performed using the full-length Rnase R enzyme with a 6his tag.

Invitrogen was commissioned to synthesize single-stranded small RNAs with the same sequences (miR-<NUM>) (SEQ ID NO: <NUM>): one without any additional modification (miR-<NUM>), one with <NUM>' terminal <NUM>'-O-methylation modification (miR-<NUM>-ch3). They were diluted to <NUM> nmol and store at -<NUM> protected from light.

<NUM>µg, <NUM>µg, or <NUM>µg of the recombinant fusion proteins obtained in Example <NUM> were mixed with <NUM>µL of the diluted RNA substrate obtained in Example <NUM>, an appropriate amount of buffer solution and water were added to a final volume of <NUM>µL (<NUM> Tris pH <NUM>, <NUM> KCl, <NUM> ZnCl<NUM>), reacted at <NUM> for <NUM> hour, and then treated at <NUM> for <NUM> minutes to inactivate the enzyme. Afterwards, the product was separated on <NUM>% TBE-urea gel.

The results are shown in <FIG>. In the control group using enzyme buffer, the RNA substrates were clearly displayed on the gel regardless of whether the <NUM>' end was modified or not. After reacting with three different concentrations of Rnase R enzymes, the RNA substrates with <NUM>' terminal <NUM>'-O-methylation modification were also clearly displayed on the gel. After reacting with three different concentrations of Rnase R enzymes, the RNA substrates without <NUM>' terminal modification were completely degraded.

Similar to the steps of the enzymatic reaction in Example <NUM>, <NUM>µg, <NUM>µg, or <NUM>µg of the recombinant fusion proteins obtained in Example <NUM> were mixed with <NUM>µL of the <NUM>-fold dilution (<NUM> nmol) of the RNA substrate obtained in Example <NUM>, an appropriate amount of buffer solution and water were added to a final volume of <NUM>µL (<NUM> Tris pH <NUM>, <NUM> KCl, <NUM> ZnCl<NUM>), reacted at <NUM> for <NUM> hour, and then treated at <NUM> for <NUM> minutes to inactivate the enzyme.

The qRT-PCR TaqMan ™ probe kit for miR-<NUM> sequence was ordered from ThermoFisher, and the reverse transcription and PCR procedure were completed according to the kit's operation manual. As can be seen from the results in Table <NUM>, after the Rnase R enzyme treatment, the CT values of the amplification of RNA substrates (miR-<NUM>) without <NUM>' terminal <NUM>'-O-methylation modification were all about <NUM> at three different enzyme concentrations, while the reaction CT value of the control group using Rnase R enzyme buffer solution instead of the enzyme was about <NUM>, with a very obvious difference between the two groups; and after the Rnase R enzyme treatment, the CT values of the amplification of RNA substrates (miR-<NUM>-ch3) with <NUM>' terminal <NUM>'-O-methylation modification were all about <NUM> at three different enzyme concentrations, while the reaction CT value of the control group using Rnase R enzyme buffer solution instead of enzyme was also about <NUM>, with no obvious difference between the two groups. It can be seen that Rnase R enzyme can selectively hydrolyze the RNA without <NUM>' terminal <NUM>'-O-methylation modification, but does not hydrolyze the RNA with <NUM>' terminal <NUM>'-O-methylation modification. Using qRT-PCR, it is possible to identify whether the target RNA has a <NUM>'terminal <NUM>'-O-methylation modification at nanomole level, at a concentration of <NUM> times lower than that of gel electrophoresis.

<NUM> of blood from each of <NUM> patients with stage I lung adenocarcinoma and <NUM> normal persons were collected, mixed into <NUM> blood samples of lung cancer group and normal person group separately, and sodium citrate was added for anticoagulation. It was centrifuged at <NUM> for <NUM> minutes at room temperature, the supernatant was aspirated; which was continued to be centrifuged at <NUM>,<NUM> at room temperature for <NUM> minutes, and about <NUM> of supernatant plasma was aspirated. <NUM> of Trizol ™ was added to <NUM> plasma and shaken well. <NUM> of chloroform was further added and shaken well. It was centrifuged at <NUM> for <NUM> minutes at room temperature. After centrifugation, it could be seen that the solution was divided into three layers: the lower layer was organic solvent, the middle layer was protein, and the upper layer was water-soluble materials. The upper layer of the water-soluble material was transferred to a new centrifuge tube, an equal volume of isopropanol was added, and mixed well, afterwards it was kept still overnight at -<NUM>. On the next day, it was centrifuged at <NUM> at <NUM> for <NUM> minutes, and the supernatant was removed after the centrifugation. <NUM> of <NUM>% ethanol was added to wash precipitates at the bottom and wall of the tube, which was then transferred to a new <NUM> centrifuge tube. It was centrifuged at <NUM> at <NUM> for <NUM> minutes, and the supernatant was removed after the centrifugation. The RNA precipitate was dissolved with <NUM>µL of DEPC-treated water and it was kept still for <NUM> minutes.

After measuring the total amount of RNA, <NUM> times the amount of Rnase R enzyme obtained in Example <NUM> (the buffer solution of the enzyme itself was <NUM> Tris-HCl, pH <NUM>, <NUM> NaCl, <NUM> TCEP), <NUM>µL 10XRnase R buffer (<NUM> Tris-Cl (pH <NUM>), <NUM> KCl, <NUM> ZnCl<NUM>) was added, and the final volume was brought up to <NUM> with DEPC-treated water. The reaction was carried out at <NUM> for <NUM> minutes, and then treated at <NUM> for <NUM> minutes to inactivate the Rnase R enzyme.

<NUM> of Trizol ™ was added and shaken well. <NUM>µL of chloroform was added and shaken well. It was centrifuged at <NUM> for <NUM> minutes at room temperature. After centrifugation, it can be seen that the solution was divided into three layers: the lower layer was organic solvent, the middle layer was protein, and the upper layer was water-soluble materials. The upper layer of the water-soluble material was transferred to a new centrifuge tube, an equal volume of isopropanol was added, and mixed well, afterwards it was kept still for <NUM> hour at -<NUM>. Subsequently, it was centrifuged at <NUM> at <NUM> for <NUM> minutes, and the supernatant was removed after centrifugation. <NUM> of <NUM>% ethanol was added to wash precipitates at the bottom and wall of the tube, which was then transferred to a new <NUM> centrifuge tube. It was centrifuged at <NUM> at <NUM> for <NUM> minutes, and the supernatant was removed after centrifugation. The RNA precipitate was dissolved with <NUM>µL of DEPC-treated water and it was kept still for <NUM> minutes. This sample can be used directly for sequencing.

The samples of lung adenocarcinoma and normal persons were sent to BGI for miRNA low-density chip sequencing (Applied Biosystems). The chip will detect <NUM> of the more common and less common miRNAs in the samples, totaling <NUM>. The principle is equivalent to performing <NUM> sets of qRT-PCR at the same time. The analysis of the results was similar to that of general qRT-PCR. The smaller the CT value, the higher the content, and a CT value greater than or equal to <NUM> could be considered as basically non-existent. Therefore, we were interested in miRNAs with a CT value less than <NUM> as much as possible in samples of lung adenocarcinoma and with a CT value greater than or equal to <NUM> in normal persons, that is, the miRNAs with <NUM>' terminal <NUM>'-O-methylation modification in blood samples of patients having lung adenocarcinoma and without the same modification in normal persons. Some of the results were shown in Table <NUM> below.

As shown in Table <NUM>, these miRNAs all showed <NUM> 'terminal <NUM>'-O-methylation modification (CT <<NUM>) in blood samples of patients having lung adenocarcinoma, while they were completely hydrolyzed by Rnase R enzyme in blood samples of normal persons (CT > <NUM>). The <NUM>' terminal <NUM>'-O-methylation modification of these miRNAs could all become potential diagnosis targets for lung adenocarcinoma.

Principle: The overall procedure was as shown in <FIG>, which was roughly divided into several processes: RNA acquisition, RNA fragmentation, Rnase R enzyme treatment, fragment tailing and high-throughput sequencing, sequencing result comparison, and modification site recovery. Specifically, the RNAs to be tested can be fragmented into fragments with a length of about <NUM> to <NUM> nt, and then the product can be processed with the exonuclease Rnase R. The inventors found that RNA fragments without <NUM>'-O-methylation modifications will be degraded, while sites with <NUM>'-O-methylation modifications (Nm) will be recognized by the Rnase R enzyme, and the degradation terminates at Nm+<NUM> site. Subsequently, the degraded RNA samples were purified and a library was prepared and sequenced. Since the Rnase R enzyme generates a concentrated degradation termination site at the nucleotide downstream of each <NUM>'-O-methylation modification site (Nm+<NUM> site), the <NUM>'-O-methylation sites can be identified by comparing the sequencing sequence with the genome.

Operation: Total RNA purification kit (Qiagen) was used to extract total RNA from Hela cell line. <NUM>µL of DEPC-treated water was used to dissolve the total RNA obtained. <NUM>% agarose gel was made, and 1XTAE electrophoresis buffer was formulated with DEPC-treated water. The obtained total RNA was subjected to electrophoresis detection, the <NUM> rRNAband was cut, and recovered with the RNA agarose recovery kit (zymo research). RNAs were dissolved with <NUM>µL of DEPC-treated water to obtain <NUM>µL of <NUM> RNAs. It was determined that A260:<NUM> = <NUM>, suggesting a relatively pure <NUM> product. A portion of the sample can be used for <NUM>% agarose gel electrophoresis to test the integrity of <NUM>.

<NUM>µg of the <NUM> rRNA (<NUM>µL) obtained in the previous step was mixed well with <NUM>µL of <NUM> Na<NUM>CO<NUM> (pH <NUM>), and treated in a water bath at <NUM> for <NUM>. Then <NUM>µL <NUM> mol/L Na-oAC (pH <NUM>) was added to terminate the reaction. <NUM>µL of <NUM>µg/µL glycogen was added to the system and mixed well. <NUM>µL of <NUM>% ethanol was added to the system, mixed well, and placed in liquid nitrogen. It was centrifuged for <NUM> minutes at a condition of <NUM> and <NUM>. The supernatant was discarded from the obtained centrifugal product, and <NUM> of <NUM>% ethanol was added to wash the precipitated RNA, and centrifuged again at a condition of <NUM> and <NUM> for <NUM> minutes. The supernatant of the obtained centrifugal product was discarded, the residual ethanol was completely volatilized in an ultra-clean workbench, and <NUM>µL of DEPC-treated water was added to dissolve the remaining RNAs.

<NUM>µg of the Rnase R enzyme obtained in Example <NUM> was added to <NUM>µL of the fragmented <NUM> rRNAs obtained in the previous step (the buffer solution of the enzyme itself was <NUM> Tris-HCl, pH <NUM>, <NUM> NaCl, <NUM> TCEP), <NUM>µL 10X Rnase R buffer (<NUM> Tris-Cl (pH <NUM>), <NUM> KCI, <NUM> ZnCl<NUM>). The reaction was carried out at <NUM> for <NUM> minutes, and then treated at <NUM> for <NUM> minutes to inactivate the Rnase R enzyme. The total RNA purification kit (Qiagen) was used again to purify the RNA fragments after the reaction.

The tailing/reverse transcription kit (Sangon Biotech) was used to generate a cDNA library from <NUM>µg of the treated product, and the high-throughput sequencing was used to obtain the sequence information of the fragments. By comparing the sequence information of the fragments obtained by high-throughput sequencing with the <NUM> rRNAs, it could be found that most of the fragments terminated in certain positions (<FIG>), and <NUM>'-O-methylation occurs on the nucleotide at the position immediately preceding these positions. In <FIG>, the abscissa represents a nucleotide site, and the ordinate represents a relative content of the fragments whose <NUM>' end is located at the corresponding amino acid sites. For example, the statistics of the termination point near the nucleotide at the 850th position showed that about <NUM>% of the fragments terminated on nucleotide at the 868th position, indicating that most of the <NUM> RNA molecules had a <NUM>'-O-methylation modification on nucleotide at the 867th position. The other fragment termination sites <NUM>, <NUM>, <NUM>, and <NUM> might be products of incomplete enzymatic hydrolysis after random interruption or a small amount of products after hydrolysis of <NUM>'-O-methylation sites with low ratio of modifications.

The method of this example can be used to identify all <NUM>'-O-methylation sites on single-stranded RNA, and these sites may be related to a disease such as a cancer, a heritable disease with a genetic disorder, or a developmental error, etc., in animals. Therefore, the method can be used for in vitro diagnosis of these diseases.

By way of the Hela cell line as an example, the principle and operation are the same as those in Example <NUM>. After obtaining the sequencing results, by using the ratio of the number of fragments terminating at each site to the number of fragments terminating at the previous site, based on the principle as described in Example <NUM>, we can determine whether it may be a <NUM>'-O-methylation site. <FIG> showed the ratio of each site by way of <NUM> rRNA as an example.

If ranked from high to low ratios, <NUM> of the top <NUM> sites (><NUM>) with the highest ratio have been reported in other literatures to have <NUM>'-O-methylation modification, while the site that hasn't been reported (that is, site <NUM>) was further verified by us with mass spectrometry that <NUM>'-O-methylation did exist, which fully demonstrated the accuracy of our methods.

Table <NUM> and Table <NUM> listed the distribution of some sites with higher ratios in <NUM> rRNA and <NUM> rRNA.

Similarly, we can also obtain the distribution of the sites with higher ratios in the mRNA, which correspond to the corresponding positions on the chromosome. Table <NUM> below shows some of the results.

Claim 1:
An ex vivo method for identifying whether an RNA molecule has a <NUM>'-O-methylation modification on the <NUM>' terminal nucleotide, comprising:
(<NUM>) contacting the RNA molecule with a ribonuclease Rnase R; and
(<NUM>) detecting whether the RNA molecule is degraded,
wherein, if the RNA molecule is degraded, this indicates that the RNA molecule does not have the <NUM>'-O-methylation modification on the <NUM>' terminal nucleotide;
the ribonuclease Rnase R has an amino acid sequence as shown in SEQ ID NO: <NUM> or <NUM>, or, compared with the amino acid sequence as shown in SEQ ID NO: <NUM> or <NUM>, the ribonuclease Rnase R has at least <NUM>% sequence identity, and has exonuclease activity.