WO2022257383A1 - 一种修饰核酸的捕捉方法及其应用 - Google Patents

一种修饰核酸的捕捉方法及其应用 Download PDF

Info

Publication number
WO2022257383A1
WO2022257383A1 PCT/CN2021/135412 CN2021135412W WO2022257383A1 WO 2022257383 A1 WO2022257383 A1 WO 2022257383A1 CN 2021135412 W CN2021135412 W CN 2021135412W WO 2022257383 A1 WO2022257383 A1 WO 2022257383A1
Authority
WO
WIPO (PCT)
Prior art keywords
rna
nad
formula
cap
present application
Prior art date
Application number
PCT/CN2021/135412
Other languages
English (en)
French (fr)
Inventor
刘南
牛孔艳
Original Assignee
中国科学院上海有机化学研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN202110646514.4A external-priority patent/CN115466762A/zh
Priority claimed from CN202111060086.3A external-priority patent/CN115786452A/zh
Application filed by 中国科学院上海有机化学研究所 filed Critical 中国科学院上海有机化学研究所
Publication of WO2022257383A1 publication Critical patent/WO2022257383A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • the present application relates to the field of biomedicine, in particular to a method for capturing modified nucleic acids and its application.
  • mRNA Messenger ribonucleic acid
  • m7G 7-methylguanylic acid
  • NAD Nicotinamide adenine dinucleotide
  • NAD-RNA is an important coenzyme factor in cells.
  • NAD-RNA is ubiquitous in prokaryotic and eukaryotic organisms, suggesting its possible involvement in gene regulation.
  • NAD is the core coenzyme of cellular oxidation and various metabolic pathways, and NAD decreases significantly during natural aging and aging-related diseases.
  • Systematic characterization of the type and abundance of NAD-RNA provides important clues for understanding cell physiology and pathology. Therefore, there is an urgent need in the art for a method capable of capturing NAD-RNA.
  • the application provides a method for enriching NAD cap-modified RNA.
  • the compound shown in formula (I) is contacted with the RNA to be tested in the presence of adenosine diphosphoribosyl cyclase, so that the formula (I) ) is combined with the RNA containing NAD cap modification in the RNA to be tested, and through affinity treatment, so as to obtain the RNA modified by NAD cap, X-L-B (I), wherein, X represents a nucleophilic group, and L represents a connection Group, B represents biotin or desthiobiotin, X and B are connected through L.
  • the present application provides a method for enriching RNA, according to the method described in the present application, the NAD cap modified RNA is enriched.
  • the application provides the use of the compound represented by formula (I) in enriching RNA modified by NAD cap, X-L-B (I), wherein, X represents a nucleophilic group, L represents a linking group, and B represents biotin or desulfurization biological Element, X and B are connected through L.
  • the present application provides a method for selectively enriching NAD cap-modified RNA, characterized in that it comprises:
  • RNA containing the NAD cap modification is combined, and the RNA modified by the NAD cap is obtained by affinity treatment, X-L-B (I), wherein, X represents a nucleophilic group, L represents a linking group, and B represents biotin or desthiobiotin , X and B are connected through L.
  • the present application provides a method for enriching RNA, according to the method described in the present application, the NAD cap modified RNA is enriched.
  • the present application provides a method for constructing a sequencing library, and RNA is enriched according to the method of the present application.
  • the application provides a method for capturing NAD cap-modified RNA or enriching NAD cap-modified RNA in a sample, the method comprising contacting the compound represented by formula (I) with the sample to be tested, and the contact is Carry out under the condition that phosphoribosyl cyclase exists, described test sample comprises the RNA of described NAD cap modification, X-L-B (I), wherein, X represents nucleophilic group, L represents linking group, B represents biotin Or desthiobiotin, X and B are connected through L.
  • the present application provides a set of oligonucleotides, comprising RNA captured or enriched according to any method of the present application or its complementary sequence, or the above-mentioned reverse transcription sequence.
  • the present application provides a storage medium, which records a program capable of running any one of the methods described in the present application.
  • the present application provides a device comprising the storage medium described in the present application.
  • the application provides a method as described in any one of the applications for use in assessing the risk of developing a disease or condition.
  • the present application provides an application of any one of the methods described in the present application in screening substances capable of improving diseases or conditions.
  • the present application provides a method according to any one of the present application for use in diagnosing or monitoring a disease or condition.
  • the effect that the method of the present application can have is selected from the following groups: 1. Avoid the 3-terminal fragment of RNA from being interrupted by Cu, and avoid the click chemical group reacting with impurities; 2. No additional steps are required to prevent non-specific reactions, such as separating first Obtain mRNA, fill it up with tRNA, and then remove m7G with IgG. This application can avoid the influence of m7G impurities through pre-treatment or post-treatment, that is, this application can make non-specific reactions not affect the results; and 3. The reaction steps are simple, and the yield of the obtained target RNA is high.
  • Fig. 1 is the test figure of the reaction efficiency of different nucleophilic groups according to the embodiment of the application;
  • Fig. 2 is the test chart of the reaction efficiency of different PEG lengths according to the embodiment of the application;
  • Fig. 3 is the electrophoresis figure of 8% polyacrylamide urea TBE gel according to the embodiment of the present application;
  • Fig. 4 is the technical flowchart of the present application according to the embodiment of the present application.
  • Fig. 5 is a schematic diagram of the specific effect of yDcpS according to the embodiment of the present application.
  • FIG. 6 is a schematic diagram of simultaneous hydrolysis of RppH according to an embodiment of the present application.
  • Fig. 7 is a diagram of the reaction efficiency of different nucleophilic groups according to an embodiment of the present application.
  • NAD nicotinamide adenine dinucleotide
  • NAD in the present application may refer to NAD+ and/or NADH, and derivatives of NAD.
  • the compounds of this application can be optionally substituted by one or more substituents, such as the above general formula compounds, or as specific examples in the examples, subclasses, and contained in this application A class of compounds.
  • substituents such as the above general formula compounds, or as specific examples in the examples, subclasses, and contained in this application A class of compounds.
  • substituents such as the above general formula compounds, or as specific examples in the examples, subclasses, and contained in this application A class of compounds.
  • substituents such as the above general formula compounds, or as specific examples in the examples, subclasses, and contained in this application A class of compounds.
  • alkyl groups include, but are not limited to, methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), n-propyl (n-Pr, -CH 2 CH 2 CH 3 ), Isopropyl (i-Pr, -CH(CH 3 ) 2 ), n-butyl (n-Bu, -CH 2 CH 2 CH 2 CH 3 ), isobutyl (i-Bu, -CH 2 CH(CH 3 ) 2 ), sec-butyl (s-Bu, -CH(CH 3 )CH 2 CH 3 ), tert-butyl (t-Bu, -C(CH 3 ) 3 ), n-pentyl (-CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3 )CH 2 CH 2 CH 3 ), 3-pentyl (-CH(CH 2 CH 3 ) 2 ), 2-methyl-2 -Butyl (-C(CH 3 ), 2-
  • alkyl and its prefix “alk” are used herein to include straight and branched saturated carbon chains.
  • alkylene is used herein to denote a saturated divalent hydrocarbon radical obtained by elimination of two hydrogen atoms from a linear or branched saturated hydrocarbon, examples of which include, but are not limited to, methylene, ethylene , Hypoisopropyl and so on.
  • heteroalkyl means that one or more heteroatoms may be inserted into the alkyl chain, wherein the alkyl group and heteroatom have the meanings described herein. Unless otherwise specified, heteroalkyl groups contain 1-10 carbon atoms, in other embodiments, heteroalkyl groups contain 1-8 carbon atoms, in other embodiments, heteroalkyl groups contain 1 - 6 carbon atoms, still other embodiments, heteroalkyl groups contain 1-4 carbon atoms, still other embodiments, heteroalkyl groups contain 1-3 carbon atoms.
  • Such examples include, but are not limited to, CH3OCH2- , CH3CH2OCH2- , CH3SCH2- , ( CH3 ) 2NCH2- , ( CH3 ) 2CH2OCH2- , CH 3 OCH 2 CH 2 -, CH 3 CH 2 OCH 2 CH 2 -, etc.
  • heterocycle refers to monocyclic, bicyclic or tricyclic ring systems in which one or more The carbon atoms are independently and optionally substituted by heteroatoms having the meanings described herein, the rings may be fully saturated or contain one or more unsaturations, but are never aromatic, and There are one or more junctions to other parts of the molecule.
  • One or more ring hydrogen atoms are independently and optionally substituted with one or more substituents described herein.
  • a “heterocycle”, “heterocyclyl”, “heteroalicyclic” or “heterocyclic” group is a 3-7 membered monocyclic ring (1-6 carbon atoms and selected from 1-3 heteroatoms of N, O, P, S, where S or P is optionally substituted by one or more oxygen atoms to obtain groups such as SO, SO 2 , PO, PO 2 , when said When the ring is a three-membered ring, wherein there is only one heteroatom), or a 7-10 membered bicyclic ring (4-9 carbon atoms and 1-3 heteroatoms selected from N, O, P, S, where S Or P is optionally substituted by one or more oxygen atoms to give a group such as SO, SO2, PO, PO2 ) .
  • a heterocyclic group may be a carbon group or a heteroatom group.
  • Heterocyclyl also includes the combination of a heterocyclic group and a saturated or partially unsaturated ring or heterocycle.
  • heterocycles include, but are not limited to, pyrrolidinyl, tetrahydrofuryl, dihydrofuryl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, Morpholinyl, Thiomorpholinyl, Thioxanyl, Thiazolidinyl, Oxazolidinyl, Piperazinyl, Homopiperazinyl, Azetidinyl, Oxetanyl, Thietanyl , homopiperidinyl, epoxypropyl, azepanyl, oxepyl, thiepanyl, 4-methoxy-piperidin-1-yl, 1,2,
  • heterocyclic groups also include 1,1-dioxothiomorpholinyl and pyrimidinedione in which two ring carbon atoms are replaced by oxygen atoms.
  • RNA containing NAD cap modifications had technical problems to be overcome.
  • One of the methods is the NAD captureSeq technology, which has two main disadvantages: (1) the participation of metal copper ions will lead to severe degradation of RNA and the loss of full-length RNA information, thereby greatly reducing the detection efficiency; The RNA of m7G cap was enriched non-specifically, thus affecting the detection accuracy of NAD-RNA.
  • Another method, SPAAC-NAD-seq detection technology has two main disadvantages: (1) The steps are cumbersome, and the multi-step reaction reduces the overall detection efficiency.
  • RNA transfer RNA
  • the present application provides a one-step method for enriching RNA containing NAD cap modifications.
  • the method of the present application simplifies the reaction steps, improves detection efficiency and accuracy, and/or significantly reduces the amount of RNA used.
  • the present application proposes a method for enriching NAD cap-modified RNA.
  • the method comprises contacting the compound represented by formula (I) with the RNA to be tested in the presence of adenosine diphosphoribosyl cyclase, so that the compound represented by formula (I) and The RNA to be tested contains NAD cap modified RNA for binding, through affinity treatment, so as to obtain NAD cap modified RNA, X-L-B (I), wherein, X represents a nucleophilic group, L represents a linking group, and B represents Biotin or desthiobiotin, X and B are connected through L.
  • the affinity treatment here is a conventional operation method, which can be a specific reaction between biotin or desthiobiotin groups and streptavidin resin, so as to achieve the effect of affinity enrichment.
  • X of the compound shown in formula (I) is a nucleophilic group, which can undergo nucleophilic substitution with the nicotinamide group on the NAD cap in RNA to replace the nicotinamide group
  • the B group is an affinity group Group, which is convenient for affinity purification after nucleophilic substitution reaction, without going through cumbersome steps (such as click chemistry), simplifying the operation process and improving efficiency.
  • the above method may further include at least one of the following technical features:
  • X is selected from -OH, -SH, -NH 2 , -NHR, -NRR' and N-containing heteroaryl, and R and R' are independently selected from optionally substituted alkyl, hetero Alkyl, cycloalkyl, heterocycloalkyl.
  • R and R' are independently selected from optionally substituted alkyl, hetero Alkyl, cycloalkyl, heterocycloalkyl.
  • L is selected from n is selected from 1, 2 or 3.
  • L is selected from n is selected from 1, 2 or 3.
  • the compound shown in formula (I) is selected from the following structures:
  • the compound shown in formula (I) is selected from the following structures:
  • the compound shown in formula (I) is selected from the following structures:
  • the present application also proposes a method for enriching RNA.
  • the method includes enriching NAD cap-modified RNA according to the method described above. The applicant found that the method in the embodiment of the application is easy to operate and has high efficiency.
  • the above method may further include at least one of the following additional technical features:
  • the above method further includes decapping the enriched NAD-capped RNA.
  • the above decapping treatment is catalyzed by NudC pyrophosphatase.
  • the present application found that the treatment with NudC pyrophosphatase can specifically decap the RNA containing NAD cap modification, and the released RNA can be used for subsequent operations (such as constructing an RNA sequencing library).
  • the RNA containing the m7G cap will not be decapped by NudC pyrophosphatase because of the three phosphate groups in the cap structure.
  • the application of NudC pyrophosphatase is equivalent to "secondary purification" in the process, without the need for m7G antibody pretreatment, which simplifies the operation and reduces the amount of RNA used.
  • the method according to the embodiment of the present application can realize the enrichment of a small amount of cellular RNA.
  • the present application also proposes the use of the compound represented by formula (I) in enriching RNA modified by NAD cap, X-L-B (I), wherein, X represents a nucleophilic group, L represents a linking group, and B represents Biotin or desthiobiotin, X and B are connected through L.
  • the compound represented by formula (I) is used to enrich NAD cap-modified RNA, and the enrichment can be achieved by using the compound in only one step reaction, and the operation is simple and the cost is low.
  • the above use may further include at least one of the following additional technical features:
  • X is selected from -OH, -SH, -NH 2 , -NHR, -NRR' and N-containing heteroaryl, and R and R' are independently selected from optionally substituted alkyl, hetero Alkyl, cycloalkyl, heterocycloalkyl.
  • L is selected from n is selected from 1, 2 or 3.
  • L is selected from n is selected from 1, 2 or 3.
  • the compound shown in formula (I) is selected from the following structures:
  • the compound shown in formula (I) is selected from the following structures:
  • the present application proposes a method for selectively enriching NAD cap-modified RNA.
  • the method includes: 1) contacting the triphosphatase yDcpS with the RNA to be tested to obtain the quasi-tested RNA; Under the condition that glycoside diphosphoribosyl cyclase exists, contact, so that make the compound shown in formula (I) combine with the RNA that contains NAD cap modification in the quasi-test RNA, through affinity treatment, so that obtain NAD cap Modified RNA, X-L-B (I), wherein X represents a nucleophilic group, L represents a linking group, B represents biotin or desthiobiotin, and X and B are connected through L.
  • the triphosphatase yDcpS pre-treats the RNA to be tested and specifically removes the RNA containing the m7G cap, which can avoid subsequent non-specific reactions, thereby greatly simplifying the reaction steps, improving detection efficiency and accuracy, and significantly reducing the amount of RNA used.
  • the above method may further include at least one of the following technical features:
  • X is selected from -OH, -SH, -NH 2 , -NHR, -NRR' and N-containing heteroaryl, and R and R' are independently selected from optionally substituted alkyl, hetero Alkyl, cycloalkyl, heterocycloalkyl.
  • R and R' are independently selected from optionally substituted alkyl, hetero Alkyl, cycloalkyl, heterocycloalkyl.
  • L is selected from n is selected from 1, 2 or 3.
  • the compound shown in formula (I) is selected from the following structures:
  • the compound shown in formula (I) is selected from the following structures:
  • the present application also proposes a method for enriching RNA.
  • the method includes enriching NAD cap-modified RNA according to the method described above. The applicant found that the method in the embodiment of the application is easy to operate and has high efficiency.
  • the present application also proposes a method for constructing a sequencing library.
  • the method includes enriching RNA according to the method described above.
  • the application provides a method for capturing NAD cap-modified RNA or enriching NAD cap-modified RNA in a sample
  • the method may comprise contacting the compound represented by formula (I) with the sample to be tested, and the contact is Carried out under the condition that diphosphoribosyl cyclase exists
  • the sample to be tested can comprise the RNA modified by the NAD cap, X-L-B (I), wherein, X can represent a nucleophilic group, and L can represent a linking group, B can represent biotin or desthiobiotin, and X and B can be linked by L.
  • X in the compound represented by the formula (I), can be selected from the following groups: -OH, -SH, -NH 2 , -NHR, -NRR' and N-containing heteroaryl groups, and R and R' can be independently optionally substituted groups selected from the group consisting of alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl.
  • the X may comprise -OH.
  • L can be selected from n can be selected from 1, 2 or 3.
  • n can be 2.
  • n can be 1, 2, 3, 4, 5, or 10.
  • the compound shown in formula (I) can be selected from the following groups:
  • the compound shown in formula (I) can be selected from the following groups:
  • the labeled RNA can be obtained; contacting the labeled RNA with a substance that may contain streptavidin, the labeled RNA bound to the streptavidin can be obtained.
  • the substance that may comprise streptavidin may comprise streptavidin magnetic beads.
  • the method may further comprise, after obtaining the labeled RNA bound to the streptavidin, contacting the obtained labeled RNA with NudC pyrophosphatase to obtain target RNA.
  • the method may further comprise, after contacting the test sample with triphosphatase yDcpS, obtaining a test sample treated with yDcpS, and the compound represented by formula (I) may be combined with the test sample treated with yDcpS touch.
  • the application may comprise only post-exposure treatment with NudC pyrophosphatase.
  • the application may comprise only a pretreatment with contact with the triphosphatase yDcpS.
  • the method may further comprise sequencing the target RNA.
  • the sequencing may comprise fluorescent PCR and/or next generation sequencing.
  • kits for enriching NAD cap-modified RNA may include the compound represented by formula (I) in the use described in the present application.
  • the present application provides the use of a kit in assessing the risk of developing a disease or condition, and the kit may include the compound represented by formula (I) in the use described in the present application.
  • kits for screening substances capable of improving diseases or conditions may include the compound represented by formula (I) in the use described in the present application.
  • kits in diagnosing or monitoring a disease or condition may include the compound represented by formula (I) in the use described in the present application.
  • the kit of the present application can also include NudC pyrophosphatase (NAD-capped RNA hydrolase NudC, for example, the UniPort number can be P32664).
  • the kit of the present application may also comprise a triphosphatase yDcpS (eg, encoded by the Saccharomyces cerevisiae DCS1 gene, eg, the UniPort number may be Q06151).
  • the present application provides a set of oligonucleotides, which may comprise RNA captured or enriched according to any method of the present application or its complementary sequence, or the above-mentioned reverse transcription sequence.
  • the present application provides a storage medium, which records a program capable of running any one of the methods described in the present application.
  • the present application provides a device, which may include the storage medium described in the present application.
  • the device may further include a processor coupled to the storage medium, and the processor is configured to execute based on a program stored in the storage medium to implement the method described in the present application.
  • the present application provides the use of the methods described herein for assessing the risk of developing a disease or condition.
  • the present application provides the application of the method described in the present application in screening substances capable of improving diseases or conditions.
  • the present application provides the methods described herein for use in diagnosing or monitoring a disease or condition.
  • the disease or condition can be associated with an increase or decrease in the subject's NAD+ levels, eg, NAD levels and/or NADH levels.
  • the disease or condition may be selected from the group consisting of diseases or symptoms associated with aging, diseases or symptoms associated with oxidative damage, metabolic syndrome, neurodegenerative diseases, cardiovascular diseases, tumors, fertility or reproductive diseases, Kidney disorders, chronic inflammatory disease, stem cell or tissue degeneration, and nonalcoholic fatty liver disease.
  • the disease or condition may be selected from the group consisting of Alzheimer's disease and Parkinson's disease.
  • the present application provides a method for capturing NAD cap-modified RNA or enriching NAD cap-modified RNA in a sample, the method comprising contacting the compound represented by formula (I) with the sample to be tested, and the contact is Carried out under the condition that diphosphoribosyl cyclase exists, the sample to be tested comprises the RNA modified by the NAD cap, X-L-B (I), wherein, X represents a nucleophilic group, L represents a linking group, and B represents a biological X and B are connected through L.
  • the method of the present application may comprise carrying a biotin group on the NAD-modified RNA through a one-step method.
  • the method of the present application is used to detect target RNA, and the target RNA is RNA with NAD modification.
  • target RNA with a biotin group can be bound and enriched with avidin.
  • avidin For example, N-[2-(2-hydroxyethoxy)ethyl]-biotinamide can be used as an avidin label.
  • the hydroxyl group of the avidin tag of the present application can be used as a nucleophilic group.
  • the nicotinamide part of NAD can be replaced, so that the target RNA is biotin and thus can be bound and enriched by streptavidin.
  • the present application provides NudC, which has the ability to specifically hydrolyze diphosphate (but not triphosphate) bonds, so NudC of the present application can be specifically isolated from RNA bound on streptavidin beads.
  • Target RNA for NAD RNA is used for transcriptome analysis and gene-specific qRT-PCR analysis.
  • NudC-based post-processing can eliminate the influence of m7G RNA impurities.
  • the RNA derived from m7G RNA can be labeled with the avidin label of the present application, but the RNA derived from m7G RNA is connected to the avidin label of the present application through a triphosphate bond, so Can not be enzymatically digested by NudC, RNA derived from m7G RNA is still bound on streptavidin beads; RNA derived from NAD RNA can be labeled with the avidin marker of the application, and derived from NAD RNA The RNA is linked to the avidin label of the application through a diphosphate bond, so it can be digested by NudC, and the RNA derived from NAD RNA can be released and isolated from the streptavidin beads.
  • NudC can be assessed. NudC can cleave the NAD cap of NAD-RNA (38nt), but not the m7G cap of m7GpppA RNA (38nt). For example, NudC treatment efficiently eluted NAD RNA (38nt) but not m7GpppA RNA (38nt) from streptavidin beads.
  • the effect of NudC can be verified by three sets of 106nt synthetic RNAs (ie, uncapped pppA RNA, NAD capped RNA, and m7G capped RNA (m7GpppA RNA). N-[2-(2-hydroxyethoxy)ethyl]-biotinamide can react with NAD-RNA(106nt) and m7GpppA RNA(106nt), but not with pppA RNA(106nt).
  • the total RNA extract is mixed with three sets of 106nt synthetic RNA, subjected to the capture method of the present application, and can optionally be sequenced, such as qRT PCR.
  • NudC treatment could elute RNA derived from NAD-RNA (106 nt), but not m7GpppA RNA (106 nt).
  • the elimination effect of NudC on impurities can be determined by comparative analysis of RNA-seq datasets.
  • polyA-selective RNA sequencing can confirm that the use of NudC will remove RNA impurities derived from m7GpppA RNA (106nt), which will help reduce false positive results.
  • the present application also confirms the practical effect of the capture method of the present application.
  • the method of the present application can capture NAD RNA from the liver tissues of young mice (8 weeks) and aged mice (72 weeks).
  • the method of the present application can capture nearly 2000 previously unknown NAD RNAs and can display the transcriptome dynamics of NAD RNA changes with age.
  • NAD modification can be on protein coding genes, but also on non-coding RNA and pseudogenes.
  • NAD modification can be related to age. For example, compared with young animals, the amount of RNA containing NAD and the proportion of RNA containing NAD are lower in older animals.
  • NAD RNA is involved in various cellular events, including nucleus-related, mitochondria-related and metabolic processes.
  • NAD-RNA 106nt
  • pppA RNA 62nt
  • the method of the present application can be used as an effective method for analyzing NAD RNA.
  • NAD is a central metabolite and redox agent in cells, and the combination of NAD and RNA can link basic metabolic regulation with gene expression, which can be used as a key content of epitranscriptome regulation.
  • the method of this application is easy to use and has strong specificity, which can not only accelerate the discovery of NAD RNA, but also accelerate the study of the dynamic changes of NAD RNA in the life process and even in the disease environment, providing a basis for functional research.
  • the present application found that although NAD declines with increasing age, unique NAD RNAs can be found in aged animals, suggesting that NAD modifications on RNA transcripts may be modulated by various physiological states of cells.
  • qRT PCR analysis can be performed on specific NAD RNA, and specific NAD RNA that can become a biomarker of health and disease can be found.
  • the compound represented by formula I) reacted with NAD-RNA at 37° C. for 1 hour under the catalysis of ADPRC enzyme, and then purified the reacted RNA with Zymo Column.
  • the purified RNA was verified by 8% polyacrylamide urea TBE gel electrophoresis, and the reaction efficiency was quantitatively calculated. As shown in Figure 1, the nucleophilic group -OH achieved the highest reaction efficiency of 76%.
  • the substrate molecule reacted with NAD-RNA at 37°C for 1 hour under the catalysis of ADPRC enzyme.
  • the reacted RNA was purified with Zymo Column, and then incubated with streptavidin resin magnetic beads at 25°C for 30 minutes.
  • the RNA bound to the magnetic beads was extracted by Trizol, verified by 8% polyacrylamide urea gel electrophoresis (as shown in Figure 2) and the enrichment efficiency was calculated. Under the condition of 60mM substrate molecules, the initial 1 ⁇ g NAD-RNA can be enriched to 0.16 ⁇ g RNA, and the enrichment rate is 16%.
  • the NAD-RNA standard was reacted with ADPRC enzyme at 37°C for 1 hour.
  • the reacted RNA was purified with Zymo Column, and incubated with streptavidin resin magnetic beads at 25°C for 30 minutes.
  • NudC pyrophosphatase catalyzed the reaction at 37°C for 1 hour, and collected the supernatant.
  • 8% polyacrylamide urea TBE gel electrophoresis (as shown in FIG. 3 )
  • NudC pyrophosphatase released NAD-RNA through decapping reaction.
  • the present invention utilizes yDcpS for preprocessing (as shown in FIG. 4 ).
  • yDcpS can hydrolyze the phosphodiester bond between the gamma and beta phosphate groups in the m7G cap structure, and the products are diphosphorylated 5' terminal RNA and free m7GMP.
  • yDcpS pretreatment can decap m7G-RNA, but does not react with NAD-RNA.
  • the yDcpS enzyme digestion is simple and feasible, without the cumbersome steps of affinity removal of m7G antibody.
  • the present invention is based on the principle that ADPRC enzyme catalyzes the exchange reaction between nicotinamide and nucleophile, and adds a biotin label on the basis of the original simple nucleophile (reaction substrate in step 1 of Figure 4: nucleophilic polyethylene glycol biotin) , so that it has the characteristics of affinity purification and becomes a bifunctional molecule (nucleophilic and affinity).
  • the RNA generated by the exchange reaction can be directly affinity-purified without going through cumbersome steps (such as click chemistry), which simplifies the operation process and improves efficiency.
  • m7G-RNA standard and the NAD-RNA standard were respectively reacted with yDcpS triphosphatase at 37°C for 1 hour, and the supernatant was collected.
  • yDcpS triphosphatase releases m7G-RNA through decapping reaction, but does not participate in NAD-RNA decapping reaction.
  • the m7G-RNA standard and the NAD-RNA standard were respectively reacted with RppH triphosphatase at 37°C for 1 hour, and the supernatant was collected. It was shown by 8% polyacrylamide urea TBE gel electrophoresis (as shown in FIG. 6 ), that RppH triphosphatase released m7G-RNA through uncapping reaction and partially hydrolyzed NAD-RNA at the same time.
  • yDcpS can specifically act on m7G-RNA, but does not react with NAD-RNA ( Figure 5); RppH can hydrolyze m7G-RNA and NAD-RNA simultaneously, without specificity ( Figure 6). It can be seen that yDcpS is more effective as the pre-treated m7G decapping enzyme.
  • the compound represented by formula I) reacted with NAD-RNA at 37° C. for 1 hour under the catalysis of ADPRC enzyme, and then purified the reacted RNA with Zymo Column.
  • the purified RNA was verified by 8% polyacrylamide urea TBE gel electrophoresis, and the reaction efficiency was quantitatively calculated. As shown in Figure 7, the nucleophilic group -OH achieved the highest reaction efficiency of 76%.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

涉及一种修饰核酸的捕捉方法及其应用。具体提供了一种捕获NAD帽修饰的RNA或富集样品中NAD帽修饰的RNA的方法，所述方法包含使式(I)所示化合物与待测样品接触，所述接触在腺苷二磷酸核糖基环化酶存在的条件下进行，所述待测样品包含所述NAD帽修饰的RNA，X-L-B(I)，其中，X代表亲核基团，L代表连接基团，B代表生物素或脱硫生物素，X与B通过L相连。

Description

一种修饰核酸的捕捉方法及其应用 技术领域
本申请涉及生物医学领域，具体的涉及一种修饰核酸的捕捉方法及其应用。
背景技术
真核细胞中的信使核糖核酸(以下简称：mRNA)通常具有5’端帽子修饰。长期以来，7-甲基鸟苷酸(以下简称：m7G)被认为是真核细胞mRNA唯一的帽子修饰。烟酰胺腺嘌呤二核苷酸(以下简称：NAD)是细胞中重要辅酶因子。最新研究发现，NAD也可作为RNA5’端帽子修饰(以下简称：NAD-RNA)。NAD-RNA在原核和真核生物体中广泛存在，表明其有可能参与基因调控。NAD是细胞氧化及多种代谢通路的核心辅酶，且NAD在自然衰老和衰老相关疾病过程中发生显著下降。系统表征NAD-RNA的类型及丰度，为理解细胞生理及病理提供重要线索。因此，本领域急需一种能够捕捉NAD-RNA的方法。
发明内容
本申请提供了一种富集NAD帽修饰的RNA的方法，将式(I)所示化合物与待测RNA在腺苷二磷酸核糖基环化酶存在的条件下进行接触，以便使得式(I)所示化合物与所述待测RNA中含有NAD帽修饰的RNA进行结合，通过亲和处理，以便获得NAD帽修饰的RNA，X-L-B(I)，其中，X代表亲核基团，L代表连接基团，B代表生物素或脱硫生物素，X与B通过L相连。
本申请提供了一种富集RNA的方法，根据本申请所述的方法富集NAD帽修饰的RNA。
本申请提供了式(I)所示化合物在富集NAD帽修饰的RNA中的用途，X-L-B(I)，其中，X代表亲核基团，L代表连接基团，B代表生物素或脱硫生物素，X与B通过L相连。
本申请提供了一种选择性富集NAD帽修饰RNA的方法，其特征在于，包括：
1)将三磷酸酶yDcpS与待测RNA进行接触，获得准待测RNA，
2)将式(I)所示化合物所述准待测RNA在腺苷二磷酸核糖基环化酶存在的条件下进行接触，以便使得式(I)所示化合物与所述准待测RNA中含有NAD帽修饰的RNA进行结合，通过亲和处理，以便获得NAD帽修饰的RNA，X-L-B(I)，其中，X代表亲核基团，L代表 连接基团，B代表生物素或脱硫生物素，X与B通过L相连。
本申请提供了一种富集RNA的方法，根据本申请所述的方法富集NAD帽修饰的RNA。
本申请提供了一种构建测序文库的方法，根据本申请的方法富集RNA。
本申请提供了一种捕获NAD帽修饰的RNA或富集样品中NAD帽修饰的RNA的方法，所述方法包含使式(I)所示化合物与待测样品接触，所述接触在腺苷二磷酸核糖基环化酶存在的条件下进行，所述待测样品包含所述NAD帽修饰的RNA，X-L-B(I)，其中，X代表亲核基团，L代表连接基团，B代表生物素或脱硫生物素，X与B通过L相连。
本申请提供了一种寡核苷酸集合，包含根据本申请任一项方法捕获或富集的RNA或其互补序列，或上述的逆转录序列。
本申请提供了一种储存介质，其记载可以运行本申请任一项所述的方法的程序。
本申请提供了一种设备，其包含本申请所述的储存介质。
本申请提供了一种本申请任一项所述的方法，在评估发展疾病或病症的风险中的应用。
本申请提供了一种本申请任一项所述的方法，在筛选能够改善疾病或病症的物质中的应用。
本申请提供了一种本申请任一项所述的方法，在诊断或监测疾病或病症中的应用。
本申请的方法可以具有的效果选自以下组：1.避免RNA的3端的片段被Cu打断，避免点击化学基团与杂质反应；2.不需要额外的步骤防止非特异性反应，例如先分离得到mRNA，再用tRNA补齐，然后用IgG去除m7G，本申请通过前处理或后处理任一种，即可以避免m7G的杂质影响，即本申请可以使得非特异性反应不影响结果；和3.反应步骤简单，所得目标RNA产率高。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1是根据本申请实施例的不同亲核基团反应效率的测试图；
图2是根据本申请实施例的不同PEG长度反应效率的测试图；
图3是根据本申请实施例的8％聚丙烯酰胺尿素TBE胶电泳图；
图4是根据本申请实施例的本申请技术流程图；
图5是根据本申请实施例的yDcpS可以特异性作用示意图；
图6是根据本申请实施例的RppH可以同时水解示意图；
图7是根据本申请实施例的不同亲核基团反应效率图。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
本申请将应用以下定义除非其他方面表明。根据本申请的目的，化学元素根据元素周期表，CAS版本和化学物理手册，75 th Ed.，1994来定义。另外，有机化学一般原理见“Organic Chemistry”,Thomas Sorrell,University Science Books,Sausalito,1999,和“March's Advanced Organic Chemistry”,Michael B.Smith和Jerry March,John Wiley&Sons,New York,2007，所有上述参考文献均通过引用并入本文中。本申请中术语"NAD"通常是指烟酰胺腺嚷呤二核苷酸。例如，本申请的NAD可以是指NAD+和/或NADH，以及NAD的衍生物。
如本申请所描述的，本申请的化合物可以任选地被一个或多个取代基所取代，如上面的通式化合物，或者如实施例里面特殊的例子，子类，和本申请所包含的一类化合物。应了解“任选取代的”这个术语与“取代或未取代的”这个术语可以交换使用。一般而言，术语“任选地”不论是否位于术语“取代的”之前，表示所给结构中的一个或多个氢原子被具体取代基所取代。除非其他方面表明，一个任选的取代基团可以有一个取代基在基团各个可取代的位置进行取代。当所给出的结构式中不只一个位置能被选自具体基团的一个或多个取代基所取代，那么取代基可以相同或不同地在各个位置取代。其中所述的取代基可以是，但并不限于，氘、羟基、氨基、卤素、氰基、芳基、杂芳基、烷氧基、烷氨基、烷硫基、烷基、烯基、炔基、杂环基、巯基、硝基、芳氧基、杂芳氧基、氧代(＝O)、羧基、羟基取代的烷氧基、羟基取代的烷基-C(＝O)-、烷基-C(＝O)-、烷基-S(＝O)-、烷基-S(＝O) 2-、羟基取代的烷基-S(＝O)-、羟基取代的烷基-S(＝O) 2-、羧基取代的烷氧基等等。
除非另有说明，术语“烷基”，表示1-20个碳原子，或1-10个碳原子，或1-8个碳原子， 或1-6个碳原子，或1-4个碳原子，或1-3个碳原子的饱和直链或支链的单价烃基，其中烷基可以独立且任选地被一个或多个本申请所描述的取代基所取代，取代基包括但不限于，氘、氨基、羟基、氰基、F、Cl、Br、I、巯基、硝基、氧代(＝O)等等。烷基的实例包括，但并不限于，甲基(Me，-CH 3)、乙基(Et，-CH 2CH 3)、正丙基(n-Pr，-CH 2CH 2CH 3)、异丙基(i-Pr，-CH(CH 3) 2)、正丁基(n-Bu，-CH 2CH 2CH 2CH 3)、异丁基(i-Bu，-CH 2CH(CH 3) 2)、仲丁基(s-Bu，-CH(CH 3)CH 2CH 3)、叔丁基(t-Bu，-C(CH 3) 3)、正戊基(-CH 2CH 2CH 2CH 2CH 3)、2-戊基(-CH(CH 3)CH 2CH 2CH 3)、3-戊基(-CH(CH 2CH 3) 2)、2-甲基-2-丁基(-C(CH 3) 2CH 2CH 3)、3-甲基-2-丁基(-CH(CH 3)CH(CH 3) 2)、3-甲基-1-丁基(-CH 2CH 2CH(CH 3) 2)、2-甲基-1-丁基(-CH 2CH(CH 3)CH 2CH 3)、正己基(-CH 2CH 2CH 2CH 2CH 2CH 3)、2-己基(-CH(CH 3)CH 2CH 2CH 2CH 3)、3-己基(-CH(CH 2CH 3)(CH 2CH 2CH 3))、2-甲基-2-戊基(-C(CH 3) 2CH 2CH 2CH 3)、3-甲基-2-戊基(-CH(CH 3)CH(CH 3)CH 2CH 3)、4-甲基-2-戊基(-CH(CH 3)CH 2CH(CH 3) 2)、3-甲基-3-戊基(-C(CH 3)(CH 2CH 3) 2)、2-甲基-3-戊基(-CH(CH 2CH 3)CH(CH 3) 2)、2,3-二甲基-2-丁基(-C(CH 3) 2CH(CH 3) 2)、3,3-二甲基-2-丁基(-CH(CH 3)C(CH 3) 3)、正庚基、正辛基等等。术语“烷基”和其前缀“烷”在此处使用，都包含直链和支链的饱和碳链。术语“烷撑”在此处使用，表示从直链或支链饱和碳氢化物消去两个氢原子得到的饱和二价烃基，这样的实例包括，但并不限于，亚甲基、次乙基、次异丙基等等。
术语“杂烷基”表示烷基链中可以插入一个或多个杂原子，其中烷基基团和杂原子具有如本申请所述的含义。除非另外详细说明，杂烷基基团含有1-10个碳原子，另外一些实施方案是，杂烷基基团含有1-8个碳原子，另外一些实施方案是，杂烷基基团含有1-6个碳原子，另外一些实施方案是，杂烷基基团含有1-4个碳原子，另外一些实施方案是，杂烷基基团含有1-3个碳原子。这样的实例包括，但并不限于，CH 3OCH 2-、CH 3CH 2OCH 2-、CH 3SCH 2-、(CH 3) 2NCH 2-、(CH 3) 2CH 2OCH 2-、CH 3OCH 2CH 2-、CH 3CH 2OCH 2CH 2-等。
术语“杂环”、“杂环基”、“杂脂环族”或“杂环的”在此处可交换使用，都是指单环、双环或三环体系，其中环上一个或多个碳原子独立且任选地被杂原子所取代，所述杂原子具有如本申请所述的含义，环可以是完全饱和的或包含一个或多个不饱和度，但绝不是芳香族类，且有一个或多个连接点连接到分子的其他部分。一个或多个环上的氢原子独立且任选地被一个或多个本申请所描述的取代基所取代。其中一些实施方案是，“杂环”、“杂环基”、“杂脂环族”或“杂环的”基团是3-7元环的单环(1-6个碳原子和选自N、O、P、S的1-3个杂原子，在此S或P任选地被一个或多个氧原子所取代得到例如SO、SO 2、PO、PO 2的基团，当所述的环为三元环时，其中只有一个杂原子)，或7-10元的双环(4-9个碳原子和选自N、O、P、S的1-3个杂原子，在此S或P任选地被一个或多个氧原子所取代得到例如SO、SO 2、PO、PO 2的基团)。
杂环基可以是碳基或杂原子基。“杂环基”同样也包括杂环基团与饱和或部分不饱和环或杂环并合所形成的基团。杂环的实例包括，但并不限于，吡咯烷基、四氢呋喃基、二氢呋喃基、四氢噻吩基、四氢吡喃基、二氢吡喃基、四氢噻喃基、哌啶基、吗啉基、硫代吗啉基、噻噁烷基、噻唑烷基、噁唑烷基、哌嗪基、高哌嗪基、氮杂环丁基、氧杂环丁基、硫杂环丁基、高哌啶基、环氧丙基、氮杂环庚基、氧杂环庚基、硫杂环庚基、4-甲氧基-哌啶-1-基、1,2,3,6-四氢吡啶-1-基、氧氮杂
Figure PCTCN2021135412-appb-000001
基、二氮杂
Figure PCTCN2021135412-appb-000002
基、硫氮杂
Figure PCTCN2021135412-appb-000003
基、吡咯啉-1-基、2-吡咯啉基、3-吡咯啉基、二氢吲哚基、2H-吡喃基、4H-吡喃基、二氧杂环己基、1,3-二氧戊基、吡唑啉基、二噻烷基、二噻茂烷基、二氢噻吩基、吡唑烷基、咪唑啉基、咪唑烷基、1,2,3,4-四氢异喹啉基、1,2,6-噻二嗪烷1,1-二氧代-2-基、4-羟基-1,4-氮杂磷烷4-氧化物-1-基、2-羟基-1-(哌嗪-1-基)乙酮-4-基、2-羟基-1-(5,6-二氢-1,2,4-三嗪-1(4H)-基)乙酮-4-基、5,6-二氢-4H-1,2,4-噁二嗪-4-基、2-羟基-1-(5,6-二氢吡啶-1(2H)-基)乙酮-4-基、3-氮杂双环[3.1.0]己基、3-氮杂双环[4.1.0]庚基、氮杂双环[2.2.2]己基、2-甲基-5,6,7,8-四氢-[1.2.4]三唑[1,5-c]嘧啶-6-基、4,5,6,7-四氢异噁唑[4,3-c]吡啶-5-基、3H-吲哚基2-氧-5-氮杂双环[2.2.1]庚烷-5-基、2-氧-5-氮杂双环[2.2.2]辛烷-5-基、喹嗪基和N-吡啶基尿素。杂环基团的实例还包括，1,1-二氧代硫代吗啉基和其中环上两个碳原子被氧原子所取代如嘧啶二酮基。并且所述杂环基可以是取代或未取代的，其中取代基可以是，但并不限于，氘、氧代(＝O)、羟基、氨基、卤素、氰基、杂芳基、烷氧基、烷氨基、烷基、烯基、炔基、杂环基、巯基、硝基、芳氧基、羟基取代的烷氧基、羟基取代的烷基-C(＝O)-、烷基-C(＝O)-、烷基-S(＝O)-、烷基-S(＝O)2-、羟基取代的烷基-S(＝O)-、羟基取代的烷基-S(＝O)2-、羧基取代的烷氧基等等。
发明详述
本申请发现，两种用来富集含有NAD帽修饰的RNA的方法存在需要克服的技术问题。其中一种方法是NAD captureSeq技术，该技术的缺点主要有两个：(1)金属铜离子的参与会导致RNA的严重降解导致RNA全长信息丢失，从而大大降低检测效率；(2)对于含有m7G帽子的RNA发生非特异性富集，从而影响NAD-RNA检测准确性。另一种方法SPAAC-NAD-seq检测技术，该技术的缺点也主要有两个：(1)步骤繁琐，多步反应降低总体检测效率，去除m7G帽子RNA后，需额外的步骤添加转运RNA(tRNA)以确保反应过程RNA总量不变；(2)多步反应造成RNA消耗，对RNA起始量要求高(不少于400微克总RNA)。
本申请基于上述发现，提供了通过一步法富集含有NAD帽修饰的RNA。本申请的方法简化反应步骤，提高检测效率和准确性，和/或显著降低RNA用量。
第一方面，本申请提出了一种富集NAD帽修饰的RNA的方法。根据本申请的实施例，所述方法包括将式(I)所示化合物与待测RNA在腺苷二磷酸核糖基环化酶存在的条件下进 行接触，以便使得式(I)所示化合物与所述待测RNA中含有NAD帽修饰的RNA进行结合，通过亲和处理，以便获得NAD帽修饰的RNA，X-L-B(I)，其中，X代表亲核基团，L代表连接基团，B代表生物素或脱硫生物素，X与B通过L相连。需要说明的是，这里的亲和处理是常规的操作方法，可以为生物素或脱硫生物素基团与链霉亲和素树脂发生的特异性反应，从而达到亲和富集的效果。本申请发现，式(I)所示化合物的X为亲核基团，可以与RNA中的NAD帽上的烟酰胺基团发生亲核取代，替换烟酰胺基团，B基团为亲和基团，便于亲核取代反应后进行亲和纯化，不必再通过繁琐的步骤(比如点击化学)，简化操作过程并提高效率。
根据本申请的实施例，上述方法还可以进一步包括如下技术特征至少之一：
根据本申请的实施例，X选自-OH、-SH、-NH 2、-NHR、-NRR’和含N杂芳基，R、R’分别独立地选自任选取代的烷基、杂烷基、环烷基、杂环烷基。本申请发现，上述基团都可以和NAD帽上的烟酰胺基团发生亲核取代反应，且当X为OH时，亲核取代的效率能达到76％。
根据本申请的实施例，L选自
Figure PCTCN2021135412-appb-000004
n选自1、2或3。本申请发现，在生物素与亲核基团之间加入具有亲水性质的连接片段(L)，使最终的工具分子能以较高浓度在水溶性样品中稳定存在。通过比较PEG(带有氧原子)和全碳的烷烃链，带有PEG的工具分子在水溶性样品中具有更好的溶解性，因此用PEG作为底物分子的L部分。
根据本申请的实施例，式(I)所示化合物选自如下所示结构：
Figure PCTCN2021135412-appb-000005
根据本申请的实施例，式(I)所示化合物选自如下所示结构：
Figure PCTCN2021135412-appb-000006
Figure PCTCN2021135412-appb-000007
根据本申请的实施例，式(I)所示化合物选自如下所示结构：
Figure PCTCN2021135412-appb-000008
另一方面，本申请还提出了一种富集RNA的方法。根据本申请的实施例，所述方法包括根据前面所述的方法富集NAD帽修饰的RNA。本申请发现，本申请实施例的方法操作简单并且效率高。
根据本申请的实施例，上述方法还可以进一步包括如下附加技术特征至少之一：
根据本申请的实施例，上述方法进一步包括对富集的NAD帽修饰的RNA进行脱帽处理。
根据本申请的实施例，上述脱帽处理是通过NudC焦磷酸酶催化进行。本申请发现，采用NudC焦磷酸酶处理，可特异性的对含有NAD帽修饰的RNA进行脱帽，释放的RNA可用于后续操作(如构建RNA测序文库)。含有m7G帽子的RNA，因其帽子结构中含有三个磷酸基团，不会被NudC焦磷酸酶催化发生脱帽反应。NudC焦磷酸酶的应用，在流程上相当于“二次纯化”，无需m7G抗体预处理，简化操作，减少RNA用量。根据本申请实施例的方法可以实现少量细胞RNA的富集。
另一方面，本申请还提出了式(I)所示化合物在富集NAD帽修饰的RNA中的用途，X-L-B(I)，其中，X代表亲核基团，L代表连接基团，B代表生物素或脱硫生物素，X与B通过L相连。本申请，首次将式(I)所示化合物用于富集NAD帽修饰的RNA，且利用该化合物只需要一步反应就能实现富集，操作简单，成本低廉。
根据本申请的实施例，上述用途还可以进一步包括如下附加技术特征至少之一：
根据本申请的实施例，X选自-OH、-SH、-NH 2、-NHR、-NRR’和含N杂芳基，R、R’分别独立地选自任选取代的烷基、杂烷基、环烷基、杂环烷基。本申请发现，上述基团都可以和NAD基团发生亲核取代反应，且当X为OH时，亲核取代的效率能达到76％。
根据本申请的实施例，L选自
Figure PCTCN2021135412-appb-000009
n选自1、2或3。本申请发现，在生物素与亲核基团之间加入具有亲水性质的连接片段(L)，使最终的工具分子能以较高浓度在水溶性样品中稳定存在。通过比较PEG(带有氧原子)和全碳的烷烃链，带有PEG的工具分子在水溶性样品中具有更好的溶解性，因此用PEG作为底物分子的L部分。
根据本申请的实施例，式(I)所示化合物选自如下所示结构：
Figure PCTCN2021135412-appb-000010
根据本申请的实施例，式(I)所示化合物选自如下所示结构：
Figure PCTCN2021135412-appb-000011
第一方面，本申请提出了一种选择性富集NAD帽修饰RNA的方法。根据本申请的实施例，所述方法包括：1)将三磷酸酶yDcpS与待测RNA进行接触，获得准待测RNA，2)将式(I)所示化合物所述准待测RNA在腺苷二磷酸核糖基环化酶存在的条件下进行接触，以便使得式(I)所示化合物与所述准待测RNA中含有NAD帽修饰的RNA进行结合，通过亲 和处理，以便获得NAD帽修饰的RNA，X-L-B(I)，其中，X代表亲核基团，L代表连接基团，B代表生物素或脱硫生物素，X与B通过L相连。本申请发现，三磷酸酶yDcpS前处理待测RNA，特异性去除含有m7G帽的RNA，可规避后续非特异性反应，进而极大简化反应步骤，提高检测效率和准确性，且显著降低RNA用量。
根据本申请的实施例，上述方法还可以进一步包括如下技术特征至少之一：
根据本申请的实施例，X选自-OH、-SH、-NH 2、-NHR、-NRR’和含N杂芳基，R、R’分别独立地选自任选取代的烷基、杂烷基、环烷基、杂环烷基。本申请发现，上述基团都可以和NAD帽上的烟酰胺基团发生亲核取代反应，且当X为OH时，亲核取代的效率能达到76％。
根据本申请的实施例，L选自
Figure PCTCN2021135412-appb-000012
n选自1、2或3。本申请发现，由于生物素或脱硫生物素疏水性很强，因此需要在生物素与亲核基团之间加入具有亲水性质的连接片段(L)，使最终的工具分子能以较高浓度在水溶性样品中稳定存在。通过比较PEG(带有氧原子)和全碳的烷烃链，带有PEG的工具分子在水溶性样品中具有更好的溶解性，因此用PEG作为底物分子的L部分。
根据本申请的实施例，式(I)所示化合物选自如下所示结构：
Figure PCTCN2021135412-appb-000013
根据本申请的实施例，式(I)所示化合物选自如下所示结构：
Figure PCTCN2021135412-appb-000014
Figure PCTCN2021135412-appb-000015
在本申请的再一方面，本申请还提出了一种富集RNA的方法。根据本申请的实施例，所述方法包括根据前面所述的方法富集NAD帽修饰的RNA。本申请发现，本申请实施例的方法操作简单并且效率高。
在本申请的再一方面，本申请还提出了一种构建测序文库的方法。根据本申请的实施例，所述方法包括根据前面所述的方法富集RNA。
本申请提供了一种捕获NAD帽修饰的RNA或富集样品中NAD帽修饰的RNA的方法，所述方法可以包含使式(I)所示化合物与待测样品接触，所述接触在腺苷二磷酸核糖基环化酶存在的条件下进行，所述待测样品可以包含所述NAD帽修饰的RNA，X-L-B(I)，其中，X可以代表亲核基团，L可以代表连接基团，B可以代表生物素或脱硫生物素，X与B通过可以L相连。
例如，所述式(I)所示化合物中，X可以选自以下组：-OH、-SH、-NH 2、-NHR、-NRR’和含N杂芳基，R、R’可以分别独立地选自以下组任选取代的基团：烷基、杂烷基、环烷基、和杂环烷基。例如，所述X可以包含-OH。
例如，L可以选自
Figure PCTCN2021135412-appb-000016
n可以选自1、2或3。例如，n可以为2。例如，n可以为1、2、3、4、5、或10。
例如，式(I)所示化合物可以选自以下组：
Figure PCTCN2021135412-appb-000017
例如，式(I)所示化合物可以选自以下组：
Figure PCTCN2021135412-appb-000018
例如，式(I)所示化合物与待测样品中NAD帽修饰的RNA接触之后，可以获得经标记的RNA；所述方法还可以包含在使式(I)所示化合物与待测样品接触之后，使所述经标记的RNA与可以包含链霉亲和素的物质进行接触，可以获得与所述链霉亲和素结合的所述经标记的RNA。
例如，所述可以包含链霉亲和素的物质可以包含链霉亲和素磁珠。
例如，所述方法还可以包含在获得与所述链霉亲和素结合的所述经标记的RNA后，可以使获得的所述经标记的RNA与NudC焦磷酸酶接触，获得目标RNA。例如，所述方法还可以包含在使所述待测样品与三磷酸酶yDcpS接触之后，获得yDcpS处理的待测样品，可以使式(I)所示化合物与所述与yDcpS处理的待测样品接触。例如，本申请可以只包含与NudC焦磷酸酶接触后处理。例如，本申请可以只包含与三磷酸酶yDcpS接触的前处理。
例如，所述方法还可以包含对所述目标RNA进行测序。
例如，所述测序可以包含荧光PCR和/或二代测序。
本申请提供了试剂盒在富集NAD帽修饰的RNA中的用途，所述试剂盒可以包含如本申请所述用途中的式(I)所示化合物。
本申请提供了试剂盒在评估发展疾病或病症的风险中的用途，所述试剂盒可以包含如本申请所述用途中的式(I)所示化合物。
本申请提供了试剂盒在筛选能够改善疾病或病症的物质中的用途，所述试剂盒可以包含如本申请所述用途中的式(I)所示化合物。
本申请提供了试剂盒在诊断或监测疾病或病症中的用途，所述试剂盒可以包含如本申请所述用途中的式(I)所示化合物。例如，本申请的试剂盒还可以包含NudC焦磷酸酶(NAD-capped RNA hydrolase NudC，例如UniPort编号可以是P32664)。例如，本申请的试剂盒还可以包含三磷酸酶yDcpS(例如被Saccharomyces cerevisiae DCS1基因编码，例如UniPort编号可以是Q06151)。
本申请提供了一种寡核苷酸集合，可以包含根据本申请任一项方法捕获或富集的RNA或其互补序列，或上述的逆转录序列。
本申请提供了一种储存介质，其记载可以运行本申请任一项所述的方法的程序。
本申请提供了一种设备，其可以包含本申请所述的储存介质。
例如，该设备还可以包含耦接至所述储存介质的处理器，所述处理器被配置为基于存储在所述储存介质中的程序执行以实现本申请所述的方法。
本申请提供了本申请所述的方法，在评估发展疾病或病症的风险中的应用。
本申请提供了本申请所述的方法，在筛选能够改善疾病或病症的物质中的应用。
本申请提供了本申请所述的方法，在诊断或监测疾病或病症中的应用。
例如，所述疾病或病症可以与受试者NAD+水平，例如NAD水平和/或NADH水平升高或降低有关。
例如，所述疾病或病症可以选自以下组：与衰老相关的疾病或症状、与氧化损伤相关的疾病或症状、代谢综合征、神经退行性疾病、心血管疾病、肿瘤、生育或生殖疾病、肾脏病症、慢性炎性疾病、干细胞或组织退化、和非酒精性脂肪性肝病。
例如，所述疾病或病症可以选自以下组：阿尔茨海默病和帕金森病。
例如，本申请提供一种捕获NAD帽修饰的RNA或富集样品中NAD帽修饰的RNA的方法，所述方法包含使式(I)所示化合物与待测样品接触，所述接触在腺苷二磷酸核糖基环化酶存在的条件下进行，所述待测样品包含所述NAD帽修饰的RNA，X-L-B(I)，其中，X代表亲核基团，L代表连接基团，B代表生物素或脱硫生物素，X与B通过L相连。
例如，本申请的方法可以包含通过一步法，将带有NAD修饰的RNA带有生物素基团。例如，本申请的方法用于检测目的RNA，所述目的RNA是带有NAD修饰的RNA。例如， 带有生物素基团的目的RNA，可以利用亲和素进行结合和富集。例如，可以将N-[2-(2-羟基乙氧基)乙基]-生物素酰胺作为亲和素标记物。首先，本申请的亲和素标记物具有的羟基可以作为亲核基团，在ADPRC(腺苷二磷酸核糖环化酶)催化下，可以将NAD的烟酰胺部分取代，使得目的RNA被生物素化，因此可以通过链霉亲和素进行结合和富集。其次，本申请提供了NudC，NudC具有特异性水解二磷酸(而非三磷酸)键的能力，因此本申请的NudC可以从链霉亲和素珠上结合的RNA中，特异性地分离源自于NAD RNA的目标RNA。最后，洗脱的目的RNA用于转录组的分析以及基因特异性qRT PCR分析。
可以验证一步化学酶反应的可行性。例如，可以通过HPLC和LC-MS确认与生物素化NAD衍生结构相对应的产物，该结构是由ADPRC催化的NAD分子和N-[2-(2-羟基乙氧基)乙基]-生物素酰胺之间的反应。其次，可以将合成的38核苷酸(nt)带有NAD的RNA置于N-[2-(2-羟基乙氧基)乙基]-生物素酰胺反应中，产生剂量依赖性的产量，例如可以通过凝胶中上层带的积累验证。基于荧光团的斑点杂交分析证实RNA产物带有生物素标记。链霉亲和素珠可以保留反应产物的上层带组分，而未反应形式的下层带组分则会被流动以丢弃。
例如，基于NudC的后处理，可以消除m7G RNA杂质带来的影响。在NudC酶解步骤中，源自于m7G RNA的RNA可以被本申请的亲和素标记物标记，但是源自于m7G RNA的RNA通过三磷酸键与本申请的亲和素标记物连接，因此可以不被NudC酶解，源自于m7G RNA的RNA依然在链霉亲和素珠上结合；源自于NAD RNA的RNA可以被本申请的亲和素标记物标记，且源自于NAD RNA的RNA通过二磷酸键与本申请的亲和素标记物连接，因此可以被NudC酶解，源自于NAD RNA的RNA可以从链霉亲和素珠释放和分离。
可以评估NudC的效果。NudC能够剪切NAD-RNA(38nt)的NAD帽，但不能剪切m7GpppA RNA(38nt)的m7G帽。例如，NudC处理可有效洗脱链霉亲和素珠中的NAD RNA(38nt)，但不洗脱m7GpppA RNA(38nt)。可以通过三组106nt合成RNA(即，不含帽的pppA RNA、含NAD帽RNA和含m7G帽RNA(m7GpppA RNA))验证NudC的效果。N-[2-(2-羟基乙氧基)乙基]-生物素酰胺可以与NAD-RNA(106nt)和m7GpppA RNA(106nt)反应，但不与pppA RNA(106nt)反应。
将总RNA提取物与三组106nt合成RNA混合，进行本申请的捕获方法，并可以任选地测序，例如qRT PCR。例如，NudC的处理可以洗脱源自于NAD-RNA(106nt)的RNA，但是不会洗脱源自于m7GpppA RNA(106nt)的RNA。可以通过RNA序列数据集的比较分析确定了NudC对于杂质的消除效果。例如，进行polyA选择性RNA测序，可以确定NudC的使用将源自于m7GpppA RNA(106nt)的RNA杂质去除，有利于减少假阳性结果。
本申请还确认了本申请的捕获方法的实用效果。例如，本申请的方法可以从年轻小鼠(8周)和老年小鼠(72周)肝组织中捕获NAD RNA。本申请的方法可以捕获近2000个以前未知的NAD RNA，并可以显示NAD RNA随年龄变化的转录组动力学。检测结果可以发现，NAD修饰可以在蛋白质编码基因上，但也可以在非编码RNA和假基因上。检测结果可以发现，NAD修饰可以与年龄相关，例如与年轻动物相比，老年动物中含有NAD的RNA数量以及含有NAD的比例更少。基因本体分析结果可以表明，NAD RNA参与多种细胞事件，包括细胞核相关、线粒体相关和代谢过程。例如，将人工NAD-RNA(106nt)和pppA RNA(62nt)分别作为阳性和阴性对照，可以对特定基因上的NAD修饰情况进行比较和/或定量评估。
本申请的方法可以作为分析NAD RNA的有效方法。例如，NAD作为细胞的中枢代谢物和氧化还原剂，NAD与RNA的结合可以将基本代谢调节与基因表达联系起来，可以作为表观转录组调控的关键内容。本申请的方法使用简单，特异性强，不仅可以加速NAD RNA的发现，而且加速研究NAD RNA在生命过程中甚至在疾病环境中的动态变化，为功能研究提供了基础。例如，本申请发现尽管年龄增加使得NAD下降，但在老年动物中可以发现独特的NAD RNA，这表明RNA转录物上的NAD修饰可能受到细胞生理状态的多种调整。基于本申请的方法，可以对特定NAD RNA进行例如qRT PCR分析，可以发现能够成为健康和疾病的生物标志物的特定NAD RNA。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1
底物分子设计及测试
1)亲核基团的选择：
通过比较亲核基团-OH、-SH、-NH 2、-NHCH 3、-N(CH 3) 2、或吡啶的反应活性，-OH的反应效率最好(见图1)，因此以-OH作为底物分子的亲核基团。
式I)所示化合物分别与NAD-RNA在ADPRC酶催化下37℃反应1小时，然后用Zymo Column纯化反应后的RNA。将纯化好的RNA进行8％聚丙烯酰胺尿素TBE胶电泳验证，并定量计算反应效率。如图1所示，亲核基团-OH取得76％的最高反应效率。
2)PEG基团的选择：
将带有不同PEG长度(n＝1，2，3)的式(I)所示化合物与NAD-RNA在ADPRC酶催化下37℃反应1小时，用Zymo Column将反应后的RNA进行纯化，然后进行8％聚丙烯酰 胺尿素TBE胶电泳验证。如图2所示，PEG(n＝2)的产物生物素化RNA最多，说明PEG(n＝2)具有最高的反应活性。
底物分子与NAD-RNA在ADPRC酶催化下37℃反应1小时。反应后的RNA用Zymo Column进行纯化，再与链霉亲和素树脂磁珠在25℃孵育30分钟。通过Trizol提取与磁珠结合的RNA，进行8％聚丙烯酰胺尿素胶电泳验证(如图2所示)并计算富集效率。在60mM底物分子条件下，起始1μg NAD-RNA最终能够富集到0.16μg RNA，富集率为16％。
将NAD-RNA标准品与ADPRC酶催化37℃反应1小时。将反应后的RNA用Zymo Column进行纯化，与链霉亲和素树脂磁珠在25℃孵育30分钟。NudC焦磷酸酶催化37℃反应1小时，收集上清液。通过8％聚丙烯酰胺尿素TBE胶电泳显示(如图3所示)，NudC焦磷酸酶通过脱帽反应释放NAD-RNA。
实施例2
本发明利用yDcpS进行前处理(如图4所示)。yDcpS可水解m7G帽结构中gamma和beta位磷酸集团之间的磷酸二脂键，产物为二磷酸化的5‘末端RNA和游离的m7GMP。以总RNA为底物，yDcpS前处理可对m7G-RNA脱帽，而与NAD-RNA不反应。yDcpS酶消化简单可行，无需繁琐的m7G抗体亲和去除的步骤。
本发明以ADPRC酶催化烟酰胺与亲核物发生交换反应为原理，在原先简单的亲核物基础上加上生物素标签(图4步骤1反应底物：亲核聚乙二醇生物素)，使其带有亲和纯化的特性，成为双功能分子(亲核和亲和)。通过交换反应生成的RNA即可直接进行亲和纯化，不必再通过繁琐的步骤(比如点击化学)，简化操作过程并提高效率。
将m7G-RNA标准品和NAD-RNA标准品分别与yDcpS三磷酸酶37℃反应1小时，收集上清液。通过8％聚丙烯酰胺尿素TBE胶电泳显示(如图5所示)，yDcpS三磷酸酶通过脱帽反应释放m7G-RNA，但是不参与NAD-RNA脱帽反应。
将m7G-RNA标准品和NAD-RNA标准品分别与RppH三磷酸酶37℃反应1小时，收集上清液。通过8％聚丙烯酰胺尿素TBE胶电泳显示(如图6所示)，RppH三磷酸酶通过脱帽反应释放m7G-RNA，同时部分水解NAD-RNA。
通过比较不同的三磷酸酶，yDcpS可以特异性作用m7G-RNA，而与NAD-RNA不反应(图5)；RppH可以同时水解m7G-RNA和NAD-RNA，不具有特异性(图6)。可见yDcpS作为前处理的m7G脱帽酶效果更优。
1)亲核基团的选择：
通过比较亲核基团-OH、-SH、-NH 2、-NHCH 3、-N(CH 3) 2、或吡啶的反应活性，-OH的 反应效率最好(见图1)，因此以-OH作为底物分子的亲核基团。
式I)所示化合物分别与NAD-RNA在ADPRC酶催化下37℃反应1小时，然后用Zymo Column纯化反应后的RNA。将纯化好的RNA进行8％聚丙烯酰胺尿素TBE胶电泳验证，并定量计算反应效率。如图7所示，亲核基团-OH取得76％的最高反应效率。
2)PEG基团的选择：
将带有不同PEG长度(n＝1，2，3)的式(I)所示化合物与NAD-RNA在ADPRC酶催化下37℃反应1小时，用Zymo Column将反应后的RNA进行纯化，然后进行8％聚丙烯酰胺尿素TBE胶电泳验证。如图2所示，PEG(n＝2)的产物生物素化RNA最多，说明PEG(n＝2)具有最高的反应活性。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (39)

  1. 一种富集NAD帽修饰的RNA的方法，其特征在于，将式(I)所示化合物与待测RNA在腺苷二磷酸核糖基环化酶存在的条件下进行接触，以便使得式(I)所示化合物与所述待测RNA中含有NAD帽修饰的RNA进行结合，通过亲和处理，以便获得NAD帽修饰的RNA，
    X-L-B(I)，
    其中，X代表亲核基团，L代表连接基团，B代表生物素或脱硫生物素，X与B通过L相连。
  2. 根据权利要求1所述的方法，其特征在于，X选自-OH、-SH、-NH 2、-NHR、-NRR’和含N杂芳基，
    R、R’分别独立地选自任选取代的烷基、杂烷基、环烷基、杂环烷基；
    任选地，L选自
    Figure PCTCN2021135412-appb-100001
    n选自1、2或3。
  3. 根据权利要求1-2中任一项所述的方法，其特征在于，式(I)所示化合物选自如下所示结构：
    Figure PCTCN2021135412-appb-100002
  4. 根据权利要求1-3中任一项所述的方法，其特征在于，式(I)所示化合物选自如下所示结构：
    Figure PCTCN2021135412-appb-100003
    Figure PCTCN2021135412-appb-100004
  5. 一种富集RNA的方法，其特征在于，根据权利要求1-4中任一项所述的方法富集NAD帽修饰的RNA。
  6. 根据权利要求5所述的方法，其特征在于，进一步包括对富集的NAD帽修饰的RNA进行脱帽处理；
    任选地，所述脱帽处理是通过NudC焦磷酸酶催化进行。
  7. 式(I)所示化合物在富集NAD帽修饰的RNA中的用途，
    X-L-B(I)，
    其中，X代表亲核基团，L代表连接基团，B代表生物素或脱硫生物素，X与B通过L相连。
  8. 根据权利要求7所述的用途，其特征在于，X选自-OH、-SH、-NH 2、-NHR、-NRR’和含N杂芳基，
    R、R’分别独立地选自任选取代的烷基、杂烷基、环烷基、杂环烷基；
    任选地，L选自
    Figure PCTCN2021135412-appb-100005
  9. 根据权利要求7-8中任一项所述的用途，其特征在于，式(I)所示化合物选自如下所示结构：
    Figure PCTCN2021135412-appb-100006
  10. 根据权利要求7-9中任一项所述的用途，其特征在于，式(I)所示化合物选自如下所示结构：
    Figure PCTCN2021135412-appb-100007
  11. 一种选择性富集NAD帽修饰RNA的方法，其特征在于，包括：
    1)将yDcpS三磷酸酶与待测RNA进行接触，获得准待测RNA，
    2)将式(I)所示化合物所述准待测RNA在腺苷二磷酸核糖基环化酶存在的条件下进行接触，以便使得式(I)所示化合物与所述准待测RNA中含有NAD帽修饰的RNA进行结合，通过亲和处理，以便获得NAD帽修饰的RNA，
    X-L-B(I)，
    其中，X代表亲核基团，L代表连接基团，B代表生物素或脱硫生物素，X与B通过L相连。
  12. 根据权利要求11所述的方法，其特征在于，X选自-OH、-SH、-NH 2、-NHR、-NRR’和含N杂芳基，
    R、R’分别独立地选自任选取代的烷基、杂烷基、环烷基、杂环烷基。
  13. 根据权利要求11-12中任一项所述的方法，其特征在于，L选自
    Figure PCTCN2021135412-appb-100008
    n选自1、2或3。
  14. 根据权利要求11-13中任一项所述的方法，其特征在于，式(I)所示化合物选自如下所示结构：
    Figure PCTCN2021135412-appb-100009
  15. 根据权利要求11-14中任一项所述的方法，其特征在于，式(I)所示化合物选自如下所示结构：
    Figure PCTCN2021135412-appb-100010
  16. 一种富集RNA的方法，其特征在于，根据权利要求11-15任一项所述的方法富集NAD帽修饰的RNA。
  17. 一种构建测序文库的方法，其特征在于，根据权利要求16中的方法富集RNA。
  18. 一种捕获NAD帽修饰的RNA或富集样品中NAD帽修饰的RNA的方法，所述方法包含使式(I)所示化合物与待测样品接触，所述接触在腺苷二磷酸核糖基环化酶存在的条件下进行，所述待测样品包含所述NAD帽修饰的RNA，
    X-L-B(I)，
    其中，X代表亲核基团，L代表连接基团，B代表生物素或脱硫生物素，X与B通过L相连。
  19. 根据权利要求18所述的方法，所述式(I)所示化合物中，X选自以下组：-OH、-SH、-NH 2、-NHR、-NRR’和含N杂芳基，R、R’分别独立地选自以下组任选取代的基团：烷基、杂烷基、环烷基、和杂环烷基。
  20. 根据权利要求18-19中任一项所述的方法，L选自
    Figure PCTCN2021135412-appb-100011
    n选自1、2或3。
  21. 根据权利要求18-20中任一项所述的方法，式(I)所示化合物选自以下组：
    Figure PCTCN2021135412-appb-100012
  22. 根据权利要求18-21中任一项所述的方法，式(I)所示化合物选自以下组：
    Figure PCTCN2021135412-appb-100013
  23. 根据权利要求18-22中任一项所述的方法，式(I)所示化合物与待测样品中NAD帽修饰的RNA接触之后，获得经标记的RNA；所述方法还包含在使式(I)所示化合物与待测样品接触之后，使所述经标记的RNA与包含链霉亲和素的物质进行接触，获得与所述链霉亲和素结合的所述经标记的RNA。
  24. 根据权利要求23所述的方法，所述包含链霉亲和素的物质包含链霉亲和素磁珠。
  25. 根据权利要求18-24中任一项所述的方法，所述方法还包含在获得与所述链霉亲和素结合的所述经标记的RNA后，使获得的所述经标记的RNA与NudC焦磷酸酶接触，获得目标RNA。
  26. 根据权利要求18-25中任一项所述的方法，所述方法还包含在使所述待测样品与yDcpS三磷酸酶接触之后，获得yDcpS处理的待测样品，使式(I)所示化合物与所述与yDcpS处理的待测样品接触。
  27. 根据权利要求18-26中任一项所述的方法，所述方法还包含对所述目标RNA进行测序。
  28. 根据权利要求27所述的方法，所述测序包含荧光PCR和/或二代测序。
  29. 试剂盒在富集NAD帽修饰的RNA中的用途，所述试剂盒包含如权利要求7-10中任一项所述用途中的式(I)所示化合物。
  30. 一种寡核苷酸集合，包含根据权利要求1-6、11-16和18-28中任一项方法捕获或富集的RNA或其互补序列，或上述的逆转录序列。
  31. 一种储存介质，其记载可以运行权利要求1-6、11-16和18-28中任一项所述的方法的程序。
  32. 一种设备，其包含权利要求31所述的储存介质。
  33. 如权利要求21所述的设备，还包含耦接至所述储存介质的处理器，所述处理器被配置为基于存储在所述储存介质中的程序执行以实现权利要求1-6、11-16和18-28中任一项所述的方法。
  34. 权利要求1-6、11-16和18-28中任一项所述的方法，在评估发展疾病或病症的风险中的应用。
  35. 权利要求1-6、11-16和18-28中任一项所述的方法，在筛选能够改善疾病或病症的物质中的应用。
  36. 权利要求1-6、11-16和18-28中任一项所述的方法，在诊断或监测疾病或病症中的应用。
  37. 如权利要求34-36中任一项所述的应用，所述疾病或病症与受试者NAD+水平升高或降低有关。
  38. 如权利要求34-37中任一项所述的应用，所述疾病或病症选自以下组：与衰老相关的疾病或症状、与氧化损伤相关的疾病或症状、代谢综合征、神经退行性疾病、心血管疾 病、肿瘤、生育或生殖疾病、肾脏病症、慢性炎性疾病、干细胞或组织退化、和非酒精性脂肪性肝病。
  39. 如权利要求34-37中任一项所述的应用，所述疾病或病症选自以下组：阿尔茨海默病和帕金森病。
PCT/CN2021/135412 2021-06-10 2021-12-03 一种修饰核酸的捕捉方法及其应用 WO2022257383A1 (zh)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202110646514.4A CN115466762A (zh) 2021-06-10 2021-06-10 一步法富集含有nad帽修饰的rna
CN202110646514.4 2021-06-10
CN202111060086.3A CN115786452A (zh) 2021-09-10 2021-09-10 一种选择性富集nad帽修饰rna的方法
CN202111060086.3 2021-09-10

Publications (1)

Publication Number Publication Date
WO2022257383A1 true WO2022257383A1 (zh) 2022-12-15

Family

ID=84424726

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/135412 WO2022257383A1 (zh) 2021-06-10 2021-12-03 一种修饰核酸的捕捉方法及其应用

Country Status (1)

Country Link
WO (1) WO2022257383A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110262917A1 (en) * 2010-04-21 2011-10-27 Pierce Biotechnology, Inc. Modified nucleotides
CN103298949A (zh) * 2010-10-29 2013-09-11 生命科技公司 生物素衍生物
US20170298402A1 (en) * 2014-10-03 2017-10-19 President And Fellows Of Harvard College Self-labeling nucleic acids and methods of use
WO2019226822A1 (en) * 2018-05-23 2019-11-28 The Regents Of The University Of California Methods of analyzing capped ribonucleic acids

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110262917A1 (en) * 2010-04-21 2011-10-27 Pierce Biotechnology, Inc. Modified nucleotides
CN103298949A (zh) * 2010-10-29 2013-09-11 生命科技公司 生物素衍生物
US20170298402A1 (en) * 2014-10-03 2017-10-19 President And Fellows Of Harvard College Self-labeling nucleic acids and methods of use
WO2019226822A1 (en) * 2018-05-23 2019-11-28 The Regents Of The University Of California Methods of analyzing capped ribonucleic acids

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ABELE FLORIAN, HÖFER KATHARINA, BERNHARD PATRICK, GRAWENHOFF JULIA, SEIDEL MAXIMILIAN, KRAUSE ANDRÉ, KOPF SARA, SCHRÖTER MARTIN, J: "A Novel NAD-RNA Decapping Pathway Discovered by Synthetic Light-Up NAD-RNAs", BIOMOLECULES, vol. 10, no. 4, 28 March 2020 (2020-03-28), pages 513, XP093014139, DOI: 10.3390/biom10040513 *
CAHOVÁ HANA, WINZ MARIE-LUISE, HÖFER KATHARINA, NÜBEL GABRIELE, JÄSCHKE ANDRES: "NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs", NATURE, NATURE PUBLISHING GROUP UK, LONDON, vol. 519, no. 7543, 19 March 2015 (2015-03-19), London, pages 374 - 377, XP093014138, ISSN: 0028-0836, DOI: 10.1038/nature14020 *
HU HAO, FLYNN NORA, ZHANG HAILEI, YOU CHENJIANG, HANG RUNLAI, WANG XUFENG, ZHONG HUAN, CHAN ZHULONG, XIA YIJI, CHEN XUEMEI: "SPAAC-NAD-seq, a sensitive and accurate method to profile NAD+-capped transcripts", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 118, no. 13, 30 March 2021 (2021-03-30), XP009541830, ISSN: 0027-8424, DOI: 10.1073/pnas.2025595118 *

Similar Documents

Publication Publication Date Title
US11591653B2 (en) Methods and systems for genetic analysis
EP3105349B1 (en) Targeted sequencing and uid filtering
Gahan et al. Circulating nucleic acids in plasma and serum: recent developments
CA2929471C (en) Methods for predicting age and identifying agents that induce or inhibit premature aging
AU2011215753B2 (en) Compositions and methods for the detection of small RNAs
JP2021511309A (ja) 核酸を解析するための方法および組成物
EP3965111A1 (en) Methods and systems for genomic analysis
JP2017529104A (ja) Ｒｎａスティッチシーケンシング：細胞におけるｒｎａ：ｒｎａ相互作用の直接マッピングのための解析
WO2020146312A1 (en) Compositions and methods for genomic dna and gene expression analysis in single cells
WO2022257383A1 (zh) 一种修饰核酸的捕捉方法及其应用
JP2023531720A (ja) 核酸を解析するための方法および組成物
CN115786452A (zh) 一种选择性富集nad帽修饰rna的方法
US11667955B2 (en) Methods for isolation of cell-free DNA using an anti-double-stranded DNA antibody
CN115466762A (zh) 一步法富集含有nad帽修饰的rna
WO2024015800A2 (en) Methods and compositions for modification and detection of 5-methylcytosine
Wu Phthalates, Embryo Development, and Sperm DNA Methylation
SA MERLI SAARE

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21944879

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21944879

Country of ref document: EP

Kind code of ref document: A1