WO2023116575A1 - Adapter for characterizing target polynucleotide, method, and use thereof - Google Patents

Adapter for characterizing target polynucleotide, method, and use thereof Download PDF

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WO2023116575A1
WO2023116575A1 PCT/CN2022/139679 CN2022139679W WO2023116575A1 WO 2023116575 A1 WO2023116575 A1 WO 2023116575A1 CN 2022139679 W CN2022139679 W CN 2022139679W WO 2023116575 A1 WO2023116575 A1 WO 2023116575A1
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polynucleotide
helicase
target
rna
adapter
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PCT/CN2022/139679
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French (fr)
Chinese (zh)
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刘先宇
王慕旸
常馨
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成都齐碳科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/04—Hydrolases acting on acid anhydrides (3.6) acting on acid anhydrides; involved in cellular and subcellular movement (3.6.4)
    • C12Y306/04012—DNA helicase (3.6.4.12)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis

Definitions

  • the application belongs to the field of gene sequencing, and relates to an adapter used in characterizing polynucleotides, and also relates to a method for characterizing polynucleotides using the adapter.
  • Nanopore sequencing technology has the characteristics of long read length, direct reading of modification information and parallel analysis of real-time data production.
  • Nucleic acid-related variations such as shearing and RNA editing
  • modification information including but not limited to methylation, acetylation, etc.
  • the platform supports parallel data production and analysis to realize real-time mutation/modification detection and diagnosis, and the portable design makes it have a wide range of application prospects.
  • Nanopores detect nucleotides that give current changes of known character and duration.
  • Messenger RNA provides insight into the dynamics of an organism, and the benefits and applications of direct RNA sequencing are enormous, including for health screening; eg the metastatic process of some cancers and heart disease.
  • Direct RNA sequencing has applications in investigating disease resistance in crop plants, determining crop responses to stress factors such as drought, UV light, and salinity, and in cell differentiation and determination during embryonic development.
  • RNA of 500 nucleotides or more A problem in the direct sequencing of RNA, especially RNA of 500 nucleotides or more, is to find suitable molecular motors capable of controlling the translocation of RNA across transmembrane pores. So far, molecular motors that work with RNA and provide sustained locomotion have not emerged. For characterizing or sequencing polynucleotides, the sustained movement of RNA polymers and the ability to read long polymers is required.
  • RNA ribonucleic acid
  • the purpose of this application is to provide a new adapter, and this application also provides a preparation method of the adapter and its use for nanopore sequencing.
  • the adapter of this application directly uses the modified RNA-binding helicase, which greatly enriches the diversity of RNA sequencing and provides a good basis for the further development of nanopore RNA sequencing.
  • the first aspect of the present application provides an adapter for characterizing a target polynucleotide, the adapter comprising a helicase binding region, the helicase binding region comprising a modified RNA polynucleotide, with for binding or loading the helicase.
  • the helicase comprises a DNA helicase
  • the modified RNA polynucleotide is selected from sugar ring 2'-F modified RNA; and/or
  • the binding region of the helicase does not contain DNA.
  • the adapter comprises a leader sequence that preferentially penetrates into a nanopore
  • the binding region of the helicase is located in the leader sequence.
  • the target polynucleotide is a target RNA polynucleotide and/or a target DNA polynucleotide, optionally a target RNA polynucleotide;
  • the target polynucleotide is single-stranded or double-stranded
  • the adapter is linked to the target polynucleotide by a covalent bond formed between the RNA polynucleotide and at least one reactive group each of the non-nucleotides ;and / or
  • the adapter is ligated to the RNA polynucleotide by chemical or enzymatic ligation.
  • the DNA helicase is:
  • a second aspect of the present application provides a method of characterizing a target polynucleotide, said method using said adapter.
  • the target polynucleotide is a target RNA polynucleotide and/or a target DNA polynucleotide, optionally a target RNA polynucleotide;
  • the target polynucleotide is single-stranded or double-stranded
  • the method includes:
  • the one or more characteristics are selected from (i) the length of the target polynucleotide, (ii) the identity of the target polynucleotide, (iii) the target polynucleotide The sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide of interest and (v) whether the polynucleotide of interest is modified.
  • step c) comprises measuring the current flowing through the transmembrane pore as the target polynucleotide moves relative to the transmembrane pore, wherein the current represents the target polynucleotide One or more characteristics of the nucleotide and thereby characterize the target polynucleotide.
  • the target RNA polynucleotide is additionally or further through methylation, oxidation, damage, with one or more proteins, or with one or more markers, labels or blocking chain modification.
  • the target polynucleotide may be coupled to the membrane using one or more anchors.
  • the helicase comprises a modification to reduce the size of an opening in the polynucleotide binding domain through which the target polynucleotide can pass in at least one conformational state from Unbound on the helicase.
  • the one or more helicases are as described above.
  • the method further comprises using one or more molecular brakes derived from a helicase, the molecular brakes being modified such that they bind polynucleotides but do not function as a helicase.
  • the transmembrane pore may be a protein pore or a solid pore.
  • the transmembrane protein pore is a protein pore, and is derived from any one or more of the following: hemolysin, leukocidin, Mycobacterium smegmatis (Mycobacterium smegmatis) porin A (MspA), MspB, MspC, MspD, lysenin, CsgG, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria Autotransport lipoprotein (NalP) and WZA.
  • the third aspect of the present application also provides a method for moving a target polynucleotide relative to a transmembrane pore, the movement being controlled by a helicase, the method comprising:
  • the modified RNA polynucleotide region of the helicase acts as a binding region for the DNA helicase;
  • the modified RNA polynucleotide comprises 2'-F modified RNA
  • the helicase includes the DNA helicase
  • the fourth aspect of the present application provides a complex, the complex comprising the adapter and helicase;
  • the helicase includes the DNA helicase
  • the DNA helicase is selected from:
  • the fifth aspect of the present application provides a kit for characterizing a target polynucleotide, the kit comprising the adapter and the helicase or the complex;
  • the target polynucleotide is a target RNA polynucleotide or a target DNA polynucleotide.
  • the sixth aspect of the present application provides an isolated polynucleotide, the polynucleotide comprising RNA polynucleotide or DNA polynucleotide, and a modified RNA polynucleotide region, the modified RNA polynucleotide and / or a non-nucleotide region for binding a helicase;
  • the modified RNA polynucleotide comprises 2'-F modified RNA
  • the helicase includes the DNA helicase.
  • the present application provides a modified RNA that can be combined with DNA helicase.
  • the modified RNA of the present application is less prone to degradation, and it can be used in nanopore polynucleotides including RNA and Adapter preparation for DNA sequencing, the use of this adapter greatly enriches the diversity of RNA sequencing, and provides a good foundation for the further development of nanopore RNA sequencing.
  • Fig. 1 shows that DNA helicase T4 Dda binds to ssDNA of the same length and 2'-F-RNA;
  • Figure 2 shows the binding of DNA helicase Hel308 to ssDNA of the same length and 2'-F-RNA;
  • Fig. 3 shows the electrophoretic detection diagram of the complex formed after the DNA helicase Hel308 binds to the Y-shaped adapter
  • Figure 4 shows the purified electrophoresis of a complex formed after DNA helicase Hel308 binds to a Y-shaped adapter
  • Figure 5 is a graph showing the signal that the complex can be used for nanopore sequencing.
  • the present application first provides an adapter for characterizing a target polynucleotide, the adapter comprises a DNA helicase binding region, and the binding region comprises a modified RNA polynucleotide for binding the DNA helicase.
  • the helicase comprises a DNA helicase.
  • the aforementioned helicase may be a polymeric or oligomeric helicase. It will be appreciated that helicases may need to form polymers or oligomers such as dimers in order to function. In such embodiments, the two or more moieties cannot be on different monomers.
  • Helicases are optionally monomeric. It will be appreciated that the helicase optionally does not need to form multimers or oligomers such as dimers to function. For example, Hel308, RecD, TraI and XPD helicases are all monomeric helicases.
  • a monomeric helicase may comprise several domains attached together.
  • TraI helicases and TraI subgroup helicases may contain two RecD helicase domains, a releasease domain and a C-terminal domain. These domains generally form monomeric helicases capable of functioning without forming oligomers.
  • the modified RNA polynucleotide is selected from sugar ring 2'-F modified RNA.
  • the binding region of the helicase does not comprise DNA.
  • the adapter comprises a leader sequence that preferentially penetrates the nanopore
  • the DNA helicase binding region is located at the leader sequence.
  • the adapters of the present application are more suitable for the characterization of target RNA polynucleotides.
  • the adapter may be attached to the target RNA polynucleotide via a covalent bond formed in at least one reaction of each of the RNA polynucleotide and the adapter. groups; and/or attaching the adapter to the RNA polynucleotide by chemical or enzymatic ligation.
  • the target RNA polynucleotide is optionally modified by ligating an adapter of the present application to the RNA.
  • the adapters of the present application are described to facilitate the characterization methods of the present application.
  • the adapters of the present application are designed to preferentially penetrate the pore and thus facilitate the movement of the polynucleotide through the pore.
  • the adapters of the present application can also be used to join the target RNA polynucleotide to one or more anchors as described below.
  • the adapter of the present application can be ligated to the target RNA polynucleotide.
  • Adapters of the present application typically comprise polymeric domains.
  • the polymer domains are optionally negatively charged.
  • the polymer can be optionally a polynucleotide, such as DNA, a modified polynucleotide (eg, abasic DNA), PNA, LNA, polyethylene glycol (PEG), or a polypeptide.
  • a polynucleotide such as DNA, a modified polynucleotide (eg, abasic DNA), PNA, LNA, polyethylene glycol (PEG), or a polypeptide.
  • Adapters of the present application optionally comprise one or more blocking strands.
  • One or more blocker strands are included in the target polynucleotide.
  • One or more blocker strands are included in the target RNA polynucleotide and/or the target DNA polynucleotide.
  • the one or more blocking strands are optionally part of the target polynucleotide, eg it/they interrupt the polynucleotide sequence.
  • the one or more blocker strands are optionally not part of one or more block molecules such as speed bumps for hybridization to the polynucleotide of interest.
  • blocker strands in the target polynucleotide such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more blocks chain.
  • blocker strands in the polynucleotide of interest there are 2, 4 or 6 blocker strands in the polynucleotide of interest.
  • blocker strands in different regions of the polynucleotide of interest for example a blocker strand in the leader sequence and a blocker strand in the hairpin loop.
  • the one or more blocking strands each provide an energy barrier that the one or more helicases cannot overcome even in active mode.
  • One or more blocking strands can be achieved by reducing the pull of the helicase (e.g. by removing bases of nucleotides in the target polynucleotide) or physically blocking the movement of the one or more helicases (e.g. Utilize bulky chemical groups) to stall one or more helicases.
  • the one or more blocking strands may comprise any molecule or combination of any molecules that stall one or more helicases.
  • the one or more blocker strands may comprise any molecule or combination of any molecules that prevent movement of the one or more helicases along the target polynucleotide. It directly determines whether, in the absence of a transmembrane pore and an applied potential, one or more helicases lodges at one or more blocking strands. For example, tests are performed as shown in the Examples, eg the ability of the helicase to pass through the blocking strand and displace the complementary strand of DNA can be measured by PAGE.
  • the one or more blocking chains typically comprise linear molecules such as polymers.
  • the one or more blocking strands typically have a different structure than the target polynucleotide.
  • the target polynucleotide is DNA
  • the one or more blocking strands are typically not deoxyribonucleic acid.
  • the polynucleotide of interest is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)
  • the one or more blocking strands optionally include peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid ( TNA), locked nucleic acid (LNA) or synthetic polymers with nucleotide side chains.
  • One or more blocker strands optionally include one or more nitroindole, for example one or more 5-nitroindole, one or more inosine, one or more acridine, one or more 2 - aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more reverse thymidines (reverse dTs), one or more reverse deoxythymidines glycosides (ddTs), one or more dideoxycytidines (ddCs), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2' alkoxy modifications Ribonucleotides (optionally 2'methoxy-modified ribonucleotides), one or more isodeoxycytidines (iso-dCs), one or more isodeoxyguanosines (iso-dGs), one or Multiple iSpC3 groups (i.e.
  • One or more of the blocking chains may contain any number of these groups.
  • One or more blocker strands optionally comprise 2, 3, 4, 5, 6, 7, 8 or more iSp9 groups.
  • One or more blocker strands optionally comprise 2, 3, 4, 5 or 6 or more iSpl8 groups.
  • the most optional chain-blocking groups are 4 iSp18 groups.
  • the polymer can be optionally a polypeptide or polyethylene glycol (PEG).
  • the polypeptide optionally comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acids.
  • the PEG optionally comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more monomeric units.
  • One or more blocking strands optionally include one or more abasic nucleotides (i.e., nucleotides lacking nucleobases), e.g., 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12 or more abasic nucleotides.
  • a nucleobase can be replaced by -H(idSp) or -OH in an abasic nucleotide.
  • An abasic blocker strand can be inserted into a polynucleotide of interest by removing a nucleobase from one or more adjacent nucleotides.
  • the one or more blocking strands optionally contain one or more chemical groups that physically cause the one or more helicases to stall.
  • the one or more chemical groups may be one or more pendant chemical groups.
  • the one or more chemical groups may be linked to one or more nucleobases in the target polynucleotide.
  • the one or more chemical groups may be attached to the backbone of the polynucleotide of interest. There may be any number, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more of these chemical groups.
  • Suitable groups include, but are not limited to, fluorophores, streptavidin and/or biotin, cholesterol, methylene blue, dinitrophenols (DNPs), digoxigenin and/or anti-digoxigenin and diphenyl Cyclooctyne group.
  • one blocker strand can include a linear molecule as discussed above, and the other blocker strand can include one or more chemical groups that physically cause one or more helicases to stall.
  • a blocking strand may comprise any linear molecule as discussed above and one or more chemical groups that physically cause one or more helicases to stall, such as one or more abasic groups and fluorophores.
  • the problem addressed by the method of the present application is how to characterize the target polynucleotide.
  • Methods for characterizing target polynucleotides including:
  • the helicase comprises a DNA helicase;
  • the adapter of the present application is used, and the adapter binds DNA helicase and ligates to the target polynucleotide, so that the target polynucleotide is transported to the transmembrane pore, and the pore is used to characterize the target polynucleotide. the target polynucleotide.
  • the present application provides for characterizing target ribonucleic acid (RNA and/or DNA) polynucleotides by taking one or more measurements as the target polynucleotide moves relative to the transmembrane pore under the control of a DNA helicase sour method.
  • target polynucleotides include target RNA polynucleotides and target DNA polynucleotides.
  • the transmembrane pore is capable of detecting a single molecule of the target polynucleotide, there is no need to amplify (amplify) the target polynucleotide.
  • the methods typically do not involve polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR). This greatly reduces the effort required to characterize the target polynucleotide. This also avoids any bias and artefacts caused by PCR.
  • the methods of the present application may involve determining or measuring one or more characteristics of an RNA polynucleotide or a DNA polynucleotide.
  • the method may comprise determining or measuring one, two, three, four or five or more characteristics of the RNA polynucleotide or DNA polynucleotide.
  • the one or more features are optionally selected from (i) the length of the RNA polynucleotide, (ii) the identity of the RNA polynucleotide, (iii) the RNA polynucleotide sequence, (iv) the secondary structure of the RNA polynucleotide and (v) whether the RNA polynucleotide is modified.
  • any combination of (i) to (v) can be measured according to this application, such as ⁇ i ⁇ , ⁇ ii ⁇ , ⁇ iii ⁇ , ⁇ iv ⁇ , ⁇ v ⁇ , ⁇ i, ii ⁇ , ⁇ i, iii ⁇ , ⁇ i, iv ⁇ , ⁇ i, v ⁇ , ⁇ ii, iii ⁇ , ⁇ iii, iv ⁇ , ⁇ iii, v ⁇ , ⁇ iii, v ⁇ , ⁇ iv, v ⁇ , ⁇ i, ii, ii ⁇ , ⁇ i, iii, v ⁇ , ⁇ i, iii, v ⁇ , ⁇ i, iii, v ⁇ , ⁇ i, iii, v ⁇ , ⁇ i, iii, v ⁇ , ⁇ i, iii, v ⁇ , ⁇ i, iii, v ⁇ , ⁇ i, iii, v ⁇ ,
  • the length of the target RNA polynucleotide can be determined, for example, by determining the number of interactions between the target RNA polynucleotide and the pore, or the number of interactions between the target RNA polynucleotide and the pore. The duration of the interaction is measured.
  • the identity of the target RNA polynucleotide can be determined in a number of ways.
  • the identity of the target RNA polynucleotide may or may not be determined in conjunction with sequence determination of the target RNA polynucleotide.
  • the former is straightforward; the polynucleotide is sequenced and the identity of the target RNA polynucleotide is thereby identified.
  • the latter can be done in several ways. For example, the presence of a particular motif in the target RNA polynucleotide can be determined (without determining the remaining sequence of the RNA polynucleotide).
  • measurements of specific electrical and/or optical signals determined in the methods can identify RNA polynucleotides from a specific source.
  • the sequence of the target RNA polynucleotide can be determined as previously described. Suitable sequencing methods, especially those using electrical measurements, are described in Stoddart D et al., Proc Natl Acad Sci, 12; 106(19):7702-7, Lieberman KR et al, J Am Chem Soc. 2010; 132(50):17961-72, and described in International Application WO 2000/28312.
  • the secondary structure can be measured in a variety of ways. For example, if the method includes electrical measurements, the secondary structure may be measured using changes in residence time or changes in current through the pores. This allows regions of single- and double-stranded RNA polynucleotides to be distinguished.
  • the method optionally includes determining whether the polynucleotide is modified by methylation, oxidation, damage, with one or more proteins, or with one or more markers, tags, or blocker strands. Specific modifications will result in specific interactions with the pore, which can be determined using the methods described below.
  • cytosine can be distinguished from methylcytosine based on the current flow through the pore during the pore's interaction with each nucleotide.
  • the method of the present application can be used to discriminate between RNA and DNA, even in a single sample: RNA and DNA can be distinguished from each other even when the RNA and DNA sequences are identical, as a function of the mean amplitude and range.
  • the method can be carried out using any device suitable for studying membrane/pore systems in which the pores are present in the membrane.
  • the method can be performed using any device suitable for transmembrane pore sensing.
  • the device comprises a chamber comprising an aqueous solution and a barrier dividing the chamber into two parts.
  • the barrier typically has slits in which a membrane comprising pores is formed.
  • the barrier forms a membrane in which pores are present.
  • the method can be carried out using the apparatus described in International Application No. PCT/GB08/000562 (WO 2008/102120).
  • the method can include measuring a current through the pore as the RNA polynucleotide moves relative to the pore.
  • the device may therefore also include circuitry capable of applying a potential across the membrane and pore and measuring the current signal.
  • the method can be performed using patch clamp or voltage clamp.
  • the method optionally includes the use of a voltage clamp.
  • the methods of the present application may comprise measuring the current flowing through the pore as the RNA polynucleotide moves relative to the pore.
  • the current flowing through the pore as the polynucleotide moves relative to the pore is used to determine the sequence of the target RNA polynucleotide. This is strand sequencing.
  • Suitable conditions for measuring ionic currents through transmembrane protein pores are known in the art and disclosed in the Examples.
  • the method is carried out by applying a voltage across the membrane and pore.
  • the voltage used is usually +5V to -5V, for example from +4V to -4V, +3V to -3V or +2V to -2V.
  • Commonly used voltages are typically -600mV to +600mV, or -400mV to +400mV.
  • the voltage used is optionally within a range having a lower limit selected from -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV and 0mV, and an upper limit independently Select from +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300mV and +400mV.
  • the voltage used is more preferably in the range of 100 mV to 240 mV and most preferably in the range of 120 mV to 220 mV.
  • the resolution of different ribonucleotides can be increased by applying an increased potential to the well.
  • the method is generally carried out in the presence of any charge carrier, such as a metal salt, eg an alkali metal salt, a halide salt, eg a chloride salt, eg an alkali metal chloride salt.
  • Chargers may include ionic liquids or organic salts such as tetramethylammonium chloride, tricresyl ammonium chloride, phenyltrimethylbenzene chloride, or 1-ethyl-3-methylimidazolium chloride.
  • the salt is present in an aqueous solution in the chamber.
  • KCl potassium chloride
  • NaCl sodium chloride
  • CsCl cesium chloride
  • a mixture of potassium ferrocyanide and potassium ferricyanide is used.
  • Potassium chloride, sodium chloride and mixtures of potassium ferrocyanide and potassium ferricyanide are optional.
  • the charge carriers may pass through the membrane asymmetrically. For example, the type and/or concentration of charge carriers may be different on each side of the membrane.
  • the salt concentration can be saturated.
  • the salt concentration may be 3M or less, typically 0.1M to 2.5M, 0.3M to 1.9M, 0.5M to 1.8M, 0.7M to 1.7M, 0.9M to 1.6M or 1M to 1.4M.
  • the optional salt concentration is 150mM to 1M.
  • the method optionally uses at least 0.3M, such as at least 0.4M, at least 0.5M, at least 0.6M, at least 0.8M, at least 1.0M, at least 1.5M, at least 2.0M, at least 2.5 M, or at least a salt concentration of 3.0M for implementation.
  • the high salt concentration provides a high signal-to-noise ratio and allows currents representing the presence of ribonucleotides to be identified against the background of normal current fluctuations.
  • the methods are generally performed in the presence of a buffer.
  • the buffer is present in the aqueous solution in the chamber. Any buffer may be used in the methods of the present application.
  • the buffer is phosphate buffered saline.
  • Other suitable buffers are HEPES and Tris-HCl buffers.
  • the method is typically carried out at a pH of 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7, 7.0 to 8.8, or 7.5 to 8.5.
  • the pH used is optionally about 7.5.
  • the method can be performed at 0°C to 100°C, 15°C to 95°C, 16°C to 90°C, 17°C to 85°C, 18°C to 80°C, 19°C to 70°C, or 20°C to 60°C.
  • the method is generally carried out at room temperature.
  • the method is optionally performed at a temperature that supports enzyme function, such as about 37°C.
  • the method may be performed in the presence of free nucleotides or free nucleotide analogs and/or enzyme cofactors that facilitate the function of the helicase or construct.
  • the method can also be performed in the absence of free nucleotides or free nucleotide analogs and in the absence of enzyme cofactors.
  • the free nucleotides may be one or more of any single nucleotide.
  • the free nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), triphosphate Guanosine phosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate Cytidine monophosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxygenate monophosphate Adenosine (dAMP), deoxyadenosine diphosphat
  • the free nucleotides may be selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP.
  • the free nucleotide may be adenosine triphosphate (ATP).
  • the enzyme cofactor is the factor that enables the function of the helicase or construct.
  • the enzyme cofactor is optionally a divalent metal cation.
  • the divalent metal cation may be Mg 2+ , Mn 2+ , Ca 2+ or Co 2+ .
  • the enzyme cofactor is most preferably Mg 2+ .
  • RNA is a macromolecule comprising two or more ribonucleotides.
  • the target RNA polynucleotide can be eukaryotic or prokaryotic RNA.
  • the target RNA polynucleotide may comprise any combination of ribonucleotides.
  • the ribonucleotides may be naturally occurring or man-made.
  • One or more ribonucleotides in the target RNA polynucleotide may be oxidized or methylated.
  • One or more ribonucleotides in the target RNA may be damaged.
  • the target RNA may comprise a pyrimidine dimer, such as a uracil dimer.
  • RNA polynucleotide may be modified, eg, with a label or tag. Suitable markers are described below.
  • the target RNA may comprise one or more blocker strands.
  • Ribonucleotides contain a base, ribose sugar and at least one phosphate group.
  • the bases are typically heterocyclic.
  • Bases include, but are not limited to, purines and pyrimidines, more specifically, adenine, guanine, thymine, uracil, and cytosine.
  • the nucleotides generally contain monophosphates, diphosphates or triphosphates. Phosphate can be attached to the 5' or 3' side of the nucleotide.
  • Ribonucleotides include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), 5-Methylcytidine Phosphate, 5-Methylcytidine Diphosphate, 5-Methylcytidine Triphosphate, 5-Hydroxymethylcytidine Monophosphate, 5-Hydroxymethylcytidine Diphosphate, and 5-Hydroxymethylcytidine Triphosphate Hydroxymethylcytidine.
  • the nucleotides may optionally be selected from AMP, TMP, GMP, CMP and UMP.
  • Ribonucleotides may be abasic (ie, lack a base). Ribonucleotides can also lack bases and sugars (i.e. C3 blocks the chain).
  • the ribonucleotides of the target RNA polynucleotide may be linked to each other in any manner.
  • the ribonucleotides are usually linked through their sugar and phosphate groups.
  • the ribonucleotides may be linked via their bases.
  • RNA is a very diverse molecule.
  • the target RNA polynucleotide can be any naturally occurring or synthetic ribonucleotide molecule, for example, RNA, messenger RNA (mRNA), ribosomal RNA (rRNA), nuclear heterogeneous RNA (hnRNA), transfer RNA ( tRNA), transfer messenger RNA (tmRNA), microRNA (miRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), signal recognition particle (SRP RNA), SmY RNA, small Cajal body-speicifc) RNA (scaRNA), guide RNA (gRNA), splicing leader RNA (SL RNA), antisense RNA (asRNA), long non-coding RNA (lncRNA), Piwi-interacting (Piwi-interacting) RNA (piRNA ), small interfering RNA (siRNA), trans-acting siRNA (tasiRNA), repeat linkage siRNA (rasiRNA), Y
  • the target RNA polynucleotide is optionally messenger RNA (mRNA).
  • mRNA messenger RNA
  • the target mRNA may be an alternate splice variant.
  • the amount (or grade) of variation in mRNA and/or alternate mRNA splice variants may be associated with a disease or health condition.
  • the target RNA polynucleotide is a microRNA (or miRNA).
  • a group of RNAs that are difficult to detect at low concentrations are microRNAs (micro-RNAs or miRNAs).
  • miRNAs are highly stable RNA oligomers that post-transcriptionally regulate protein production. They work by one of two mechanisms.
  • miRNAs In plants, miRNAs have been shown to function primarily by directing the cleavage of messenger RNAs, whereas in animals, gene regulation by miRNAs often involves hybridization of miRNAs to the 3'UTRs of messenger RNAs, which hinders translation (Lee et al ., Cell 75, 843-54 (1993); Wightman et al., Cell 75, 855-62 (1993); and Esquela-Kerscher et al., Cancer 6, 259-69 (2006)). miRNAs often bind their targets with defective complementarity. They have been predicted to individually bind as many as 200 or more gene targets and regulate more than one-third of all human genes (Lewis et al., Cell 120, 15-20 (2005)).
  • Suitable miRNAs for use in this application are known in the art.
  • suitable miRNAs deposited on publicly available databases (Jiang Q., Wang Y., Hao Y., Juan L., Teng M., Zhang X., Li M., Wang G., Liu Y. , (2009) miR2Disease: a manually curated database for microRNA deregulation in human disease. Nucleics Acides Res.).
  • the expression levels of certain microRNAs are known to be altered in tumors, resulting in patterns of microRNA expression characteristic of different tumor types (Rosenfeld, N. et al., Nature Biotechnology 26, 462-9 (2008)).
  • miRNA expression profiling is known to reveal the stage of tumor development more precisely than messenger RNA expression profiling (Lu et al., Nature 435, 834-8 (2005) and Barshack et al., The International Journal of Biochemistry & Cell Biology 42, 1355-62 (2010) ).
  • These findings combined with the high stability of miRNAs, and the ability to detect circulating miRNAs in serum and plasma (Wang et al., Biochemistry and Biophysical Research Communications 394, 184-8 (2010); Gilad et al., PloS One 3, e3148 (2008 ); and Keller et al., Nature Methods 8,841-3 (2011)), have aroused a great deal of interest in the potential application of microRNAs as cancer biomarkers.
  • cancers need to be accurately classified and treated differently, but the power of tumor morphology assessment as a method of classification is diminished by the fact that many different types of cancer share morphological features.
  • miRNAs offer a potentially more reliable and less invasive solution.
  • mRNAs and miRNAs are used to diagnose or predict a disease or condition.
  • RNA can be studied.
  • the methods of the present application may focus on determining the presence, absence or one or more characteristics of 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more RNA molecules.
  • the polynucleotides may be naturally occurring or synthetic.
  • the method can be used to verify the sequence of two or more artificially produced oligonucleotides.
  • the methods are typically performed in vitro.
  • the target RNA polynucleotide can be of any length.
  • the RNA polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 ribonucleotides in length.
  • the target RNA can be 1000 or more ribonucleotides, 5000 or more nucleotides or at 100000 or more ribonucleotides in length. All or only a portion of the target RNA can be characterized using this method.
  • the portion of RNA to be sequenced optionally comprises the entire target molecule, but can eg be less than the entire molecule, eg 4 bases to 1 kb, eg 4 to 100 bases.
  • the target RNA polynucleotide is typically present in or derived from any suitable sample.
  • the application is generally practiced on samples known to contain or suspected to contain the RNA polynucleotide of interest.
  • the present application can be performed on a sample to confirm the presence in the sample of one or more target RNAs whose identity is known or expected.
  • the sample can be a biological sample.
  • the present application may be practiced in vitro on samples obtained or extracted from any organism or microorganism.
  • the organisms or microorganisms are generally archaeal, prokaryotic or eukaryotic, and generally belong to one of the following five kingdoms: Plantae, Animalia, Fungi, Prokaryotes and Protista.
  • the RNA polynucleotide of interest may be derived from a eukaryotic cell or may be derived from a virus that uses the transcriptional machinery of a eukaryotic cell.
  • the present application can be practiced in vitro on samples obtained or extracted from any virus.
  • the sample is optionally a liquid sample.
  • Samples typically include bodily fluids of patients.
  • the sample may be urine, lymph, saliva, mucus or amniotic fluid, but may alternatively be blood, plasma or serum.
  • the sample will be of human origin, but may alternatively be from other mammals, such as from a commercially bred animal such as a horse, cow, sheep or pig or alternatively may be a pet such as a cat or dog.
  • samples of plant origin are usually obtained from commercial crops such as cereals, legumes, fruits or vegetables such as wheat, barley, oats, rapeseed oil (canola), corn, soybeans, rice, bananas, apples, tomatoes, potatoes, Grapes, tobacco, beans, lentils, sugar cane, cocoa or cotton.
  • commercial crops such as cereals, legumes, fruits or vegetables such as wheat, barley, oats, rapeseed oil (canola), corn, soybeans, rice, bananas, apples, tomatoes, potatoes, Grapes, tobacco, beans, lentils, sugar cane, cocoa or cotton.
  • the sample can be a non-biological sample.
  • the non-biological sample can optionally be a fluid sample.
  • Examples of non-biological samples include surgical fluids, water such as drinking water, sea water or river water, and reagents used in laboratory tests.
  • the sample is typically processed before being analyzed, for example by centrifugation, or by membrane filtration to remove unwanted molecules or cells, such as red blood cells.
  • the samples can be measured immediately after collection. Samples can also usually be stored prior to analysis, optionally below -70°C.
  • the target RNA polynucleotides are typically extracted from the sample prior to use in the methods of the present application. RNA extraction kits are available, eg, from New England and commercially.
  • the adapter is ligated to the target RNA polynucleotide to form a modified RNA polynucleotide.
  • the adapter is linked to the target RNA polynucleotide by a covalent bond formed at least one reactive group on each of the RNA polynucleotide and the adapter. between groups; and/or
  • the adapter is ligated to the RNA polynucleotide by chemical or enzymatic ligation.
  • the target RNA polynucleotide can be chemically linked to the adapter, for example by a covalent bond.
  • the target RNA polynucleotide can be ligated to the adapter by chemical or enzymatic conjugation.
  • the target RNA polynucleotide can be ligated to the adapter by hybridization and/or synthetic methods.
  • the RNA polynucleotide can be ligated to the adapter using a topoisomerase.
  • the RNA polynucleotide may be linked to the adapter at more than one, eg two or three positions. Connection methods may include one, two, three, four, five or more different connection methods. Combinations of any of the attachment methods described below may be used in accordance with the present application.
  • RNA polynucleotide and the adapter can be prepared separately and then ligated together. These two components can be linked in any configuration. For example, they can be linked via their ends (ie 5' or 3'). Suitable configurations include, but are not limited to, ligation of the 5' end of the RNA polynucleotide to the 3' end of the adapter and vice versa. Alternatively, the two components can be linked via nucleotides within their sequences.
  • RNA polynucleotide can be linked to the adapter using one or more chemical cross-linkers or one or more peptide linkers.
  • Suitable chemical crosslinkers are well known in the art.
  • Suitable chemical cross-linking agents include, but are not limited to, chemical cross-linking agents comprising the following functional groups: maleimides, active esters, succinimides, azides, alkynes (e.g.
  • dibenzocyclooctyl alcohols DIBO or DBCO
  • difluoroalicyclic hydrocarbons and linear alkynes difluoroalicyclic hydrocarbons and linear alkynes
  • phosphines such as those used in traceless and non-traceless Staudinger linkages
  • haloacetyls such as iodoethyl Amides
  • phosgene reagents sulfonyl chloride reagents
  • isothiocyanates acid halides, hydrazines, disulfides, vinyl sulfones, aziridines and photosensitive reagents (such as aromatic azides, diazacyclones propane).
  • the reaction between the RNA polynucleotide and the adapter may be spontaneous, such as cysteine/maleimide, or may require external reagents, such as for ligation of azide and linear alkyne Cu(I).
  • Optional crosslinkers include 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanyl)propionate, 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butyrate and 2,5-dioxopyrrolidin-1-yl 8-(pyridin-2-yldisulfanyl)octanoate, dimale Imide PEG 1k, bismaleimide PEG 3.4k, bismaleimide PEG 5k, bismaleimide PEG 10k, bis(maleimide) ethane (BMOE), bis Maleimide hexane (BMH), 1,4-bismaleimide butane (BMB), 1,4-bismaleimide-2,3-dihydroxybutane (BMDB) , BM[PEO]2 (1,8-bismaleimide diethylene glycol), BM[PEO]3 (1,11-bismaleimide
  • the linker can be labeled. Suitable labels include, but are not limited to, fluorescent molecules (eg Cy3 or 555), radioisotopes such as125I,35S, enzymes, antibodies, antigens, polynucleotides and ligands such as biotin.
  • This tag allows the amount of the linker to be determined.
  • the tag may also be a cleavable purification tag, such as biotin, or a specific sequence present in the identification method.
  • RNA polynucleotide or the adapter itself can be prevented by maintaining the linker in a large excess in solubility over the RNA polynucleotide and/or the adapter.
  • a "lock and key" arrangement may be used. Only one end of each linker can be reacted together to form a longer linker, the other end of each linker reacting with a different part of the construct (ie, the RNA polynucleotide or the adapter).
  • the target RNA polynucleotide can be covalently linked to the adapter.
  • the adapter may or may not contain a pre-bound DNA helicase.
  • free copper click chemistry or copper catalyzed click chemistry can be used to make a covalent bond between the RNA polynucleotide and the adapter.
  • Click chemistry is used in these applications because of its desirable properties and its scope for generating covalent linkages between a variety of building blocks. For example, it is fast, clean and non-toxic, producing only harmless by-products. Click chemistry is a term first introduced by Kolb et al.
  • Required process features include simple reaction conditions (ideally the process should be insensitive to oxygen and water), readily available starting materials and reagents, solvent-free or use, the solvent is mild (e.g. water) or easily removed, and simple product isolation. Purification must be by non-chromatographic methods if necessary, such as crystallization or distillation, and the product must be stable under physiological conditions of".
  • Example 1 2'-F-RNA can bind to DNA helicase
  • GCCAGAAACG-3' sequence length: greater than 6nt, no sequence preference
  • T4 Dda-M1G/E94C/C109A/C136A/A360C (3 ⁇ M) and DNA helicase Hel308 were mixed in buffer (20 mM HEPES (pH 7.0); 50 mM NaCl; 0.5 mM TMAD) and incubated at room temperature for 60 min. Then use TBE (native) PAGE gel to analyze its binding efficiency, TBE (native) PAGE is 4-20% gel, run at 160V for 40 minutes, and then use SYBR gold dye to stain the nucleic acid.
  • DNA helicase T4 Dda-M1G/E94C/C109A/C136A/A360C and DNA helicase Hel308 can be well with 2'-F-RNA , and the binding effect is not inferior to the binding of the enzyme to DNA. Therefore, 2'-F-RNA sequences can be used for adapter preparation for nanopore RNA sequencing.
  • Example 2 Incubation and preparation of sequencing adapter complexes containing 2'-F-RNA leading strand
  • RNA-Y1; RNA-YB and RNA-Y2 strands respectively.
  • the annealing process is to slowly cool down from 95°C to 25°C , the cooling range does not exceed 0.1°C/s.
  • Annealing buffer includes 160mM HEPES 7.0, 200mM NaCl.
  • the 2'-F modification of the sugar ring is a relatively common technique.
  • the bases of the 2'-F modification are all U, and there is no need to consider the formation of secondary structures too much.
  • the length of 15 U is determined according to the size of the space occupied by the specific enzyme. It has been verified that at least one enzyme can be combined, not two enzymes.
  • i2OMe represents a kind of sugar ring modification, that is, 2'-methoxy modification.
  • iXNA which is a kind of LNA, is used together with iSp18 in Y1 to block enzymes.
  • the sequencing adapter complex was then added to a DNAPac PA200 column and purified with elution buffer to elute the enzymes not bound to the sequencing adapter complex from the column.
  • the sequencing adapter complex was then eluted with 10 column volumes of a mixture of buffer A and buffer B. Then the main elution peaks were pooled, their concentrations were measured, and the RNA sequencing adapters were obtained and run for 40 minutes with a TBE PAGE gel at 160V.
  • buffer A 20mMNa-CHES, 250mM NaCl, 4% (W/V) glycerol, pH 8.6
  • buffer B 20mM Na-CHES, 1MNaCl, 4% (W/V) glycerol, pH 8.6, the final result As shown in Figure 4.
  • Example 3 On-machine testing of the sequencing adapter complex of the 2'-F-RNA leading strand
  • B corresponding to A means that B is associated with A, and B can be determined according to A.
  • determining B according to A does not mean determining B only according to A, and B may also be determined according to A and/or other information.

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Abstract

Provided are an adapter for characterizing a target polynucleotide, a method, and a use thereof. Provided is an adapter for characterizing a target polynucleotide. The adapter comprises a binding region of a DNA helicase. The binding region comprises a modified RNA polynucleotide for binding the DNA helicase. Further provided is a method for characterizing the target polynucleotide. The method uses the adapter.

Description

用于表征目标多核苷酸的衔接体、方法及其用途Adapters, methods and uses thereof for characterizing polynucleotides of interest
相关申请的交叉引用Cross References to Related Applications
本申请要求享有于2021年12月21日提交的名称为“用于表征目标多核苷酸的衔接体、方法及其用途”的中国专利申请202111573545.8的优先权，该申请的全部内容通过引用并入本文中。This application claims the priority of Chinese patent application 202111573545.8 entitled "Adapters, methods and uses thereof for characterizing target polynucleotides" filed on December 21, 2021, the entire contents of which are incorporated by reference In this article.
技术领域technical field
本申请属于基因测序领域，涉及一种表征多核苷酸中使用的衔接体，本申请还涉及使用所述衔接体表征多核苷酸的方法。The application belongs to the field of gene sequencing, and relates to an adapter used in characterizing polynucleotides, and also relates to a method for characterizing polynucleotides using the adapter.
背景技术Background technique
纳米孔测序技术具有长读长、直接读取修饰信息和实时数据生产并行分析的特点，在长片段核酸检测变异(包括但不仅限于点突变、***缺失、倒位易位、基因融合、RNA异常剪切、RNA编辑等多种核酸相关变异)和修饰信息(包括但不仅限于甲基化、乙酰化等)检测方面比二代测序或其他测序平台有更明显优势。该平台支持数据生产和分析并行的特点实现了实时变异/修饰检出和诊断，加上便携式的设计，使其具有广泛的应用前景。Nanopore sequencing technology has the characteristics of long read length, direct reading of modification information and parallel analysis of real-time data production. Nucleic acid-related variations such as shearing and RNA editing) and modification information (including but not limited to methylation, acetylation, etc.) have obvious advantages over next-generation sequencing or other sequencing platforms. The platform supports parallel data production and analysis to realize real-time mutation/modification detection and diagnosis, and the portable design makes it have a wide range of application prospects.
对纳米孔两侧施加电压后，当分析物(例如多核苷酸、多肽)通过纳米孔时造成电流下降，不同结构的分析物所引起的电流阻断程度不同。当分析物在纳米孔的桶(barrel)中暂时停留一段时间时，电流会发生变化。纳米孔检测核苷酸给出已知特征和持续时间的电流变化。After applying a voltage to both sides of the nanopore, when the analyte (such as polynucleotide, polypeptide) passes through the nanopore, the current will drop, and the degree of current blockage caused by the analyte with different structures is different. When the analyte temporarily stays in the barrel of the nanopore for a period of time, the current changes. Nanopores detect nucleotides that give current changes of known character and duration.
目前需要一种具有广泛的应用范围的快速且廉价的多核苷酸(如DNA或RNA)测序和鉴定技术。相关技术是缓慢的且昂贵的，这主要由于它们依赖于扩增技术来产生大量的多核苷酸，且需要大量特定的用于信号检测的荧光化学物质。There is a need for a rapid and inexpensive polynucleotide (eg DNA or RNA) sequencing and identification technique with a broad range of applications. Related techniques are slow and expensive, mainly because they rely on amplification techniques to generate large quantities of polynucleotides and require large quantities of specific fluorescent chemicals for signal detection.
信使RNA提供了对生物体动态的观察，并且直接RNA测序的益处和应用是巨大的，包括用于健康筛查；例如某些癌症的转移过程和心脏病。直接RNA测序能在调查农作物的抗病性中应用，确定农作物对应激因素，例如干旱、紫外线和盐度的反应，以及在胚胎发育过程的细胞分化和决定中应用。Messenger RNA provides insight into the dynamics of an organism, and the benefits and applications of direct RNA sequencing are enormous, including for health screening; eg the metastatic process of some cancers and heart disease. Direct RNA sequencing has applications in investigating disease resistance in crop plants, determining crop responses to stress factors such as drought, UV light, and salinity, and in cell differentiation and determination during embryonic development.
存在于RNA，特别是500个或更多的核苷酸的RNA的直接测序中的问题是寻找合适的能够控制RNA穿过跨膜孔的移位的分子马达。至今，用于RNA并提供持续移动的分子马达还没有出现。对于表征或测序多核苷酸，需要RNA聚合物的持续移动和读取长片段聚合物的能力。A problem in the direct sequencing of RNA, especially RNA of 500 nucleotides or more, is to find suitable molecular motors capable of controlling the translocation of RNA across transmembrane pores. So far, molecular motors that work with RNA and provide sustained locomotion have not emerged. For characterizing or sequencing polynucleotides, the sustained movement of RNA polymers and the ability to read long polymers is required.
国际专利申请No.PCT/GB2014/053121(WO 2015/056028)公开了表征目标核糖核酸(RNA)的方法，包括形成互补多核苷酸，然后使用跨膜孔表征所述互补多核苷酸。这种间接RNA表征易于出错并可能导致RNA的甲基化状态的重要信息的丢失。RNA到cDNA的转换过程中其他重要的修饰也可能被隐藏。International Patent Application No. PCT/GB2014/053121 (WO 2015/056028) discloses a method of characterizing a target ribonucleic acid (RNA) comprising forming a complementary polynucleotide and then characterizing the complementary polynucleotide using a transmembrane pore. This indirect RNA characterization is error-prone and can result in the loss of important information about the methylation status of the RNA. Other important modifications during the RNA-to-cDNA conversion may also be hidden.
国际专利申请WO2016059436A1公开了一种纳米孔RNA表征方法。其使用DNA解旋酶表征RNA，所述DNA解旋酶通过借助非-RNA前导序列的存在本质上“被欺骗地”("tricked")读取所述目标RNA序列。一旦通过所述非-RNA多核苷酸(其可以包含DNA或DNA类似物)引发所述DNA解旋酶的移动，它可以继续沿着所述RNA移动。International patent application WO2016059436A1 discloses a nanopore RNA characterization method. It characterizes RNA using a DNA helicase that is essentially "tricked" into reading the target RNA sequence by virtue of the presence of a non-RNA leader sequence. Once movement of the DNA helicase has been initiated by the non-RNA polynucleotide (which may comprise DNA or a DNA analog), it can continue to move along the RNA.
显然地，这两种方法都限定了目前的纳米孔RNA测序必须提供包含DNA修饰前导序列的RNA多核苷酸，这极大限制了现有RNA测序衔接体的序列多样性。Obviously, both methods limit the current nanopore RNA sequencing to provide RNA polynucleotides containing DNA-modified leader sequences, which greatly limits the sequence diversity of existing RNA sequencing adapters.
发明内容Contents of the invention
针对相关技术的不足，本申请的目的在于提供一种新的衔接体，本申请还提供了所述衔接体的制备方法，及其用于纳米孔测序的用途。本申请的衔接体直接使用修饰的RNA结合解旋酶，极大地丰富了RNA测序的多样性，并为纳米孔RNA测序的进一步发展提供了很好的基础。In view of the deficiencies in related technologies, the purpose of this application is to provide a new adapter, and this application also provides a preparation method of the adapter and its use for nanopore sequencing. The adapter of this application directly uses the modified RNA-binding helicase, which greatly enriches the diversity of RNA sequencing and provides a good basis for the further development of nanopore RNA sequencing.
本申请的目的是通过如下的实施例实现的：The purpose of this application is achieved through the following embodiments:
本申请的第一方面提供了一种用于表征目标多核苷酸的衔接体，所述衔接体包含解旋酶的结合区域，所述解旋酶的结合区域包含修饰的RNA多核苷酸，用于结合或装载所述解旋酶。The first aspect of the present application provides an adapter for characterizing a target polynucleotide, the adapter comprising a helicase binding region, the helicase binding region comprising a modified RNA polynucleotide, with for binding or loading the helicase.
根据本申请所述的衔接体，其中，According to the adapter described in the present application, wherein,
所述解旋酶包括DNA解旋酶；和/或The helicase comprises a DNA helicase; and/or
所述修饰的RNA多核苷酸选自糖环2’-F修饰的RNA；和/或The modified RNA polynucleotide is selected from sugar ring 2'-F modified RNA; and/or
所述解旋酶的结合区域不包含DNA。The binding region of the helicase does not contain DNA.
根据本申请所述的衔接体，其中，所述衔接体包含优先地穿入纳米孔的前导序列；The adapter according to the present application, wherein the adapter comprises a leader sequence that preferentially penetrates into a nanopore;
可选地，所述解旋酶的结合区域位于所述前导序列。Optionally, the binding region of the helicase is located in the leader sequence.
根据本申请所述的衔接体，其中，所述目标多核苷酸为目标RNA多核苷酸和/或目标DNA多核苷酸，可选为目标RNA多核苷酸；According to the adapter described in the present application, wherein the target polynucleotide is a target RNA polynucleotide and/or a target DNA polynucleotide, optionally a target RNA polynucleotide;
所述目标多核苷酸为单链或双链；The target polynucleotide is single-stranded or double-stranded;
可选地，通过共价键将所述衔接体连接到所述目标多核苷酸，所述共价键形成在所述RNA多核苷酸和所述非核苷酸的各自至少一个反应基团之间；和/或Optionally, the adapter is linked to the target polynucleotide by a covalent bond formed between the RNA polynucleotide and at least one reactive group each of the non-nucleotides ;and / or
通过化学或酶促连接将所述衔接体连接到所述RNA多核苷酸。根据本申请所述的衔接体，其中，所述DNA解旋酶为：The adapter is ligated to the RNA polynucleotide by chemical or enzymatic ligation. According to the adapter described in the present application, wherein the DNA helicase is:
a)Hel308解旋酶、RecD解旋酶、XPD解旋酶、Dda解旋酶、Tral解旋酶、或TrwC解旋酶；a) Hel308 helicase, RecD helicase, XPD helicase, Dda helicase, Tral helicase, or TrwC helicase;
b)衍生自a)中所述任何解旋酶的解旋酶；或b) a helicase derived from any of the helicases described in a); or
c)a)和/或b)中所述解旋酶的任意组合。c) Any combination of the helicases described in a) and/or b).
本申请的第二方面提供了一种表征目标多核苷酸的方法，所述方法使用所述的衔接体。A second aspect of the present application provides a method of characterizing a target polynucleotide, said method using said adapter.
根据本申请所述的方法，其中所述目标多核苷酸为目标RNA多核苷酸和/或目标DNA多核苷酸，可选为目标RNA多核苷酸；According to the method described in the present application, wherein the target polynucleotide is a target RNA polynucleotide and/or a target DNA polynucleotide, optionally a target RNA polynucleotide;
所述目标多核苷酸为单链或双链；The target polynucleotide is single-stranded or double-stranded;
可选地，所述方法包括：Optionally, the method includes:
a)提供(i)多核苷酸构建体和(ii)解旋酶，所述多核苷酸构建体包 含所述目标多核苷酸和所述的衔接体；所述解旋酶包括所述的DNA解旋酶；a) providing (i) a polynucleotide construct and (ii) a helicase, the polynucleotide construct comprising the target polynucleotide and the adapter; the helicase comprising the DNA Helicase;
b)将a)中提供的所述多核苷酸构建体和所述解旋酶与跨膜孔接触，使得所述解旋酶控制所述目标多核苷酸相对于所述跨膜孔的移动；b) contacting the polynucleotide construct provided in a) and the helicase with a transmembrane pore such that the helicase controls movement of the polynucleotide of interest relative to the transmembrane pore;
c)随着所述目标多核苷酸相对于所述跨膜孔移动，获取一个或多个测量值，其中所述测量值代表所述目标多核苷酸的一个或多个特征，并由此表征所述目标多核苷酸。c) taking one or more measurements as the target polynucleotide moves relative to the transmembrane pore, wherein the measurements represent one or more characteristics of the target polynucleotide and thereby characterize The target polynucleotide.
根据本申请所述的方法，其中，所述一个或多个特征选自(i)所述目标多核苷酸的长度，(ii)所述目标多核苷酸的同一性，(iii)所述目标多核苷酸的序列，(iv)所述目标多核苷酸的二级结构和(v)所述目标多核苷酸是否是修饰的。According to the method described in the present application, wherein, the one or more characteristics are selected from (i) the length of the target polynucleotide, (ii) the identity of the target polynucleotide, (iii) the target polynucleotide The sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide of interest and (v) whether the polynucleotide of interest is modified.
根据本申请所述的方法，其中所述目标多核苷酸的一个或多个特征可以通过电测量和/或光测量来测量。The method according to the present application, wherein one or more characteristics of the target polynucleotide can be measured by electrical measurement and/or optical measurement.
根据本申请所述的方法，其中步骤c)包括随着所述目标多核苷酸相对于所述跨膜孔移动，测量流过所述跨膜孔的电流，其中所述电流代表所述目标多核苷酸的一个或多个特征，并由此表征所述目标多核苷酸。According to the method described in the present application, wherein step c) comprises measuring the current flowing through the transmembrane pore as the target polynucleotide moves relative to the transmembrane pore, wherein the current represents the target polynucleotide One or more characteristics of the nucleotide and thereby characterize the target polynucleotide.
根据本申请所述的方法，其中，所述目标RNA多核苷酸额外地或进一步通过甲基化、氧化、损伤、用一个或多个蛋白，或用一个或多个标记物、标签或阻断链进行修饰。According to the method described in the present application, wherein, the target RNA polynucleotide is additionally or further through methylation, oxidation, damage, with one or more proteins, or with one or more markers, labels or blocking chain modification.
根据本申请所述的方法，其中，所述目标多核苷酸可以使用一个或多个锚耦合到所述膜。The method according to the present application, wherein the target polynucleotide may be coupled to the membrane using one or more anchors.
根据本申请所述的方法，其中，所述解旋酶包含修饰，以减小多核苷酸结合域中开口的大小，所述目标多核苷酸可以在至少一个构象状态下穿过所述开口从所述解旋酶上解绑。A method according to the present application, wherein the helicase comprises a modification to reduce the size of an opening in the polynucleotide binding domain through which the target polynucleotide can pass in at least one conformational state from Unbound on the helicase.
根据本申请所述的方法，其中，所述一个或多个解旋酶如前所述。According to the method described in the present application, wherein, the one or more helicases are as described above.
根据本申请所述的方法，其中，所述方法进一步包含使用一个或多个衍生自解旋酶的分子制动器，所述分子制动器被修饰使得其结合多核苷酸但不发挥解旋酶的功能。The method according to the present application, wherein the method further comprises using one or more molecular brakes derived from a helicase, the molecular brakes being modified such that they bind polynucleotides but do not function as a helicase.
根据本申请所述的方法，其中，所述跨膜孔可以是蛋白孔或固态孔。According to the method described in the present application, wherein the transmembrane pore may be a protein pore or a solid pore.
根据本申请所述的方法，其中，所述跨膜蛋白质孔是蛋白孔，并衍生自如下任一种或多种：溶血素、杀白细胞素，耻垢分枝杆菌(Mycobacterium smegmatis)孔蛋白A(MspA)、MspB、MspC、MspD、胞溶素(lysenin)、CsgG、外膜孔蛋白F(OmpF)、外膜孔蛋白G(OmpG)，外膜磷脂酶A、奈瑟球菌属(Neisseria)自转运脂蛋白(NalP)和WZA。According to the method described in the present application, wherein, the transmembrane protein pore is a protein pore, and is derived from any one or more of the following: hemolysin, leukocidin, Mycobacterium smegmatis (Mycobacterium smegmatis) porin A (MspA), MspB, MspC, MspD, lysenin, CsgG, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria Autotransport lipoprotein (NalP) and WZA.
本申请的第三方面还提供了一种目标多核苷酸相对于跨膜孔移动的方法，所述移动被解旋酶控制，所述方法包括：The third aspect of the present application also provides a method for moving a target polynucleotide relative to a transmembrane pore, the movement being controlled by a helicase, the method comprising:
a)提供(i)目标RNA多核苷酸或目标DNA多核苷酸，和(ii)解旋酶，所述目标RNA多核苷酸或目标DNA多核苷酸被修饰以包含用于结合或装载所述解旋酶的修饰的RNA多核苷酸区域充当DNA解旋酶的结合区域；a) providing (i) a target RNA polynucleotide or a target DNA polynucleotide, and (ii) a helicase, the target RNA polynucleotide or target DNA polynucleotide being modified to comprise The modified RNA polynucleotide region of the helicase acts as a binding region for the DNA helicase;
其中，所述修饰的RNA多核苷酸包含2’-F修饰的RNA；Wherein, the modified RNA polynucleotide comprises 2'-F modified RNA;
所述解旋酶包括所述的DNA解旋酶；The helicase includes the DNA helicase;
b)将a)中提供的所述目标RNA多核苷酸或目标DNA多核苷酸、和所述解旋酶与跨膜孔接触，使得所述解旋酶控制所述RNA多核苷酸相对于所述跨膜孔的移动。b) contacting the target RNA polynucleotide or the target DNA polynucleotide provided in a), and the helicase with a transmembrane pore such that the helicase controls the relative relation of the RNA polynucleotide to the Movement of the transmembrane pore.
本申请的第四方面提供了一种复合物，所述复合物包含所述的衔接体和解旋酶；The fourth aspect of the present application provides a complex, the complex comprising the adapter and helicase;
所述解旋酶包括所述的DNA解旋酶；The helicase includes the DNA helicase;
可选地，所述DNA解旋酶选自：Optionally, the DNA helicase is selected from:
a)Hel308解旋酶、RecD解旋酶、XPD解旋酶、Dda解旋酶、Tral解旋酶、或TrwC解旋酶；a) Hel308 helicase, RecD helicase, XPD helicase, Dda helicase, Tral helicase, or TrwC helicase;
b)衍生自a)中所述任何解旋酶的解旋酶；或b) a helicase derived from any of the helicases described in a); or
c)a)和/或b)中所述解旋酶的任意组合。c) Any combination of the helicases described in a) and/or b).
本申请的第五方面提供了一种用于表征目标多核苷酸的试剂盒，所述试剂盒包含所述的衔接体和所述解旋酶或所述的复合物；The fifth aspect of the present application provides a kit for characterizing a target polynucleotide, the kit comprising the adapter and the helicase or the complex;
所述目标多核苷酸为目标RNA多核苷酸或目标DNA多核苷酸。The target polynucleotide is a target RNA polynucleotide or a target DNA polynucleotide.
本申请的第六方面提供了一种分离的多核苷酸，所述多核苷酸包含RNA多核苷酸或DNA多核苷酸，和修饰的RNA多核苷酸区域，所述修 饰的RNA多核苷酸和/或非核苷酸区域用于结合解旋酶；The sixth aspect of the present application provides an isolated polynucleotide, the polynucleotide comprising RNA polynucleotide or DNA polynucleotide, and a modified RNA polynucleotide region, the modified RNA polynucleotide and / or a non-nucleotide region for binding a helicase;
其中，所述修饰的RNA多核苷酸包含2’-F修饰的RNA；Wherein, the modified RNA polynucleotide comprises 2'-F modified RNA;
所述解旋酶包括所述的DNA解旋酶。The helicase includes the DNA helicase.
与相关技术相比，本申请的技术方案具备以下优点：Compared with related technologies, the technical solution of the present application has the following advantages:
本申请提供了能够与DNA解旋酶结合的经修饰的RNA，与相关技术中的RNA相比，本申请的经修饰的RNA更不容易降解，其可以用于纳米孔多核苷酸包括RNA和DNA测序的衔接体制备，使用该衔接体极大丰富了RNA测序的多样性，并为纳米孔RNA测序的进一步发展提供了很好的基础。The present application provides a modified RNA that can be combined with DNA helicase. Compared with the RNA in the related art, the modified RNA of the present application is less prone to degradation, and it can be used in nanopore polynucleotides including RNA and Adapter preparation for DNA sequencing, the use of this adapter greatly enriches the diversity of RNA sequencing, and provides a good foundation for the further development of nanopore RNA sequencing.
附图说明Description of drawings
为了更清楚地说明本申请实施例的技术方案，下面将对本申请实施例中所需要使用的附图作简单地介绍，显而易见地，下面所描述的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the accompanying drawings that need to be used in the embodiments of the present application. Obviously, the accompanying drawings described below are only some embodiments of the present application. Those of ordinary skill in the art can also obtain other drawings based on these drawings without making creative efforts.
图1示出为DNA解旋酶T4 Dda与相同长度的ssDNA以及2’-F-RNA结合情况；Fig. 1 shows that DNA helicase T4 Dda binds to ssDNA of the same length and 2'-F-RNA;
图2示出为DNA解旋酶Hel308与相同长度的ssDNA以及2’-F-RNA结合情况；Figure 2 shows the binding of DNA helicase Hel308 to ssDNA of the same length and 2'-F-RNA;
图3示出为DNA解旋酶Hel308与Y型衔接体结合后形成复合物的电泳检测图；Fig. 3 shows the electrophoretic detection diagram of the complex formed after the DNA helicase Hel308 binds to the Y-shaped adapter;
图4示出为DNA解旋酶Hel308与Y型衔接体结合后形成复合物的纯化后的电泳图；Figure 4 shows the purified electrophoresis of a complex formed after DNA helicase Hel308 binds to a Y-shaped adapter;
图5示出为复合物可以用于纳米孔测序的信号图。Figure 5 is a graph showing the signal that the complex can be used for nanopore sequencing.
具体实施方式Detailed ways
应理解，公开的产品和方法的不同应用可以根据本领域的具体需求而调整。可以理解本文中使用的术语仅是为了描述本申请的具体实施方式的目的，而不意为对本申请的限制。It will be appreciated that various applications of the disclosed products and methods may be tailored to the specific needs of the art. It can be understood that the terms used herein are only for the purpose of describing specific embodiments of the application, and are not intended to limit the application.
另外，除非本文另有明确规定，否则本说明书和随附的权利要求中所使用的单数形式的“一”、“一个”和“所述”包括复数指代。因此，例如，涉及“多核苷酸”时包括两个或多核苷酸，涉及“多核苷酸结合蛋白质包括两个或多个这样的蛋白，涉及“解旋酶”时包括两个或多个解旋酶，涉及“单体”指的是两个或多个单体，涉及“孔”时包括两个或多个孔，等。Additionally, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "polynucleotide" includes two or more nucleotides, reference to "polynucleotide binding protein" includes two or more such proteins, and reference to "helicase" includes two or more helicases. For gyrase, reference to "monomer" means two or more monomers, reference to "pore" includes two or more pores, etc.
本文所引用的所有公开物、专利和专利申请，无论在前文或在后文，均以引用的方式全文引入。All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
衔接体Adapter
本申请首先提供了一种用于表征目标多核苷酸的衔接体，衔接体包含DNA解旋酶的结合区域，结合区域包含修饰的RNA多核苷酸，用于结合DNA解旋酶。The present application first provides an adapter for characterizing a target polynucleotide, the adapter comprises a DNA helicase binding region, and the binding region comprises a modified RNA polynucleotide for binding the DNA helicase.
在本申请的一个实施方案中，解旋酶包括DNA解旋酶。上述解旋酶可以是多聚或寡聚解旋酶。可以理解的是，解旋酶可能需要形成多聚体或寡聚体诸如二聚体以起作用。在这样的施例中，两个或更多个部分不能在不同的单体上。解旋酶可选地是单聚的。可以理解的是，解旋酶可选地不需要形成多聚体或寡聚体诸如二聚体即可起作用。例如，Hel308、RecD、TraI和XPD解旋酶都是单聚解旋酶。In one embodiment of the present application, the helicase comprises a DNA helicase. The aforementioned helicase may be a polymeric or oligomeric helicase. It will be appreciated that helicases may need to form polymers or oligomers such as dimers in order to function. In such embodiments, the two or more moieties cannot be on different monomers. Helicases are optionally monomeric. It will be appreciated that the helicase optionally does not need to form multimers or oligomers such as dimers to function. For example, Hel308, RecD, TraI and XPD helicases are all monomeric helicases.
单聚解旋酶可以包含附接在一起的若干结构域。例如，TraI解旋酶和TraI亚组解旋酶可以含有两个RecD解旋酶结构域、释放酶结构域和C末端结构域。这些结构域通常形成能够起作用而不会形成寡聚体的单聚解旋酶。A monomeric helicase may comprise several domains attached together. For example, TraI helicases and TraI subgroup helicases may contain two RecD helicase domains, a releasease domain and a C-terminal domain. These domains generally form monomeric helicases capable of functioning without forming oligomers.
在本申请的一个实施方案中，修饰的RNA多核苷酸选自糖环2’-F修饰的RNA。In one embodiment of the present application, the modified RNA polynucleotide is selected from sugar ring 2'-F modified RNA.
在本申请的一个实施方案中，解旋酶的结合区域不包含DNA。In one embodiment of the present application, the binding region of the helicase does not comprise DNA.
在本申请的一个实施方案中，衔接体包含优先地穿入纳米孔的前导序列；In one embodiment of the application, the adapter comprises a leader sequence that preferentially penetrates the nanopore;
在具体的实施方案中，DNA解旋酶的结合区域位于前导序列。In specific embodiments, the DNA helicase binding region is located at the leader sequence.
本申请的衔接体更适用于目标RNA多核苷酸的表征。在具体的实施 方案中，可以通过共价键将所述衔接体连接到所述目标RNA多核苷酸，所述共价键形成在所述RNA多核苷酸和所述衔接体的各自至少一个反应基团之间；和/或通过化学或酶促连接将所述衔接体连接到所述RNA多核苷酸。The adapters of the present application are more suitable for the characterization of target RNA polynucleotides. In specific embodiments, the adapter may be attached to the target RNA polynucleotide via a covalent bond formed in at least one reaction of each of the RNA polynucleotide and the adapter. groups; and/or attaching the adapter to the RNA polynucleotide by chemical or enzymatic ligation.
可选地通过将本申请的衔接体连接到RNA来修饰所述目标RNA多核苷酸。所述本申请的衔接体有助于本申请的表征方法。所述本申请的衔接体被设计为优先地穿入所述孔，并因此促进多核苷酸穿过孔的移动。所述本申请的衔接体也可以用于将所述目标RNA多核苷酸连接到如下所述的一个或多个锚。所述本申请的衔接体可连接到所述目标RNA多核苷酸。The target RNA polynucleotide is optionally modified by ligating an adapter of the present application to the RNA. The adapters of the present application are described to facilitate the characterization methods of the present application. The adapters of the present application are designed to preferentially penetrate the pore and thus facilitate the movement of the polynucleotide through the pore. The adapters of the present application can also be used to join the target RNA polynucleotide to one or more anchors as described below. The adapter of the present application can be ligated to the target RNA polynucleotide.
本申请的衔接体通常包含聚合物区域。所述聚合物区域可选带负电荷。所述聚合物可选为多核苷酸，例如DNA，修饰的多核苷酸(例如无碱基DNA)，PNA，LNA，聚乙二醇(PEG)或多肽。Adapters of the present application typically comprise polymeric domains. The polymer domains are optionally negatively charged. The polymer can be optionally a polynucleotide, such as DNA, a modified polynucleotide (eg, abasic DNA), PNA, LNA, polyethylene glycol (PEG), or a polypeptide.
本申请的衔接体可选包含一个或多个阻断链。Adapters of the present application optionally comprise one or more blocking strands.
b断链b broken chain
一个或多个阻断链包括在目标多核苷酸中。一个或多个阻断链包括在目标RNA多核苷酸和/或目标DNA多核苷酸中。一个或多个阻断链可选是目标多核苷酸的一部分，例如它/它们中断多核苷酸序列。一个或多个阻断链可选不为一个或多个嵌段分子的一部分，该嵌段分子如与目标多核苷酸杂交的减速带。One or more blocker strands are included in the target polynucleotide. One or more blocker strands are included in the target RNA polynucleotide and/or the target DNA polynucleotide. The one or more blocking strands are optionally part of the target polynucleotide, eg it/they interrupt the polynucleotide sequence. The one or more blocker strands are optionally not part of one or more block molecules such as speed bumps for hybridization to the polynucleotide of interest.
在目标多核苷酸中具有任意数量的阻断链，如1个，2个，3个，4个，5个，6个，7个，8个，9个，10个或更多个阻断链。可选在目标多核苷酸中具有2个，4个或6个阻断链。目标多核苷酸的不同区域中可具有阻断链，例如前导序列中的阻断链和发卡环中的阻断链。Have any number of blocker strands in the target polynucleotide, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more blocks chain. Optionally there are 2, 4 or 6 blocker strands in the polynucleotide of interest. There may be blocker strands in different regions of the polynucleotide of interest, for example a blocker strand in the leader sequence and a blocker strand in the hairpin loop.
一个或多个阻断链各提供了能量障碍，一个或多个解旋酶甚至在活动模式也不能克服该能量障碍。一个或多个阻断链可通过减少解旋酶的牵拉(例如通过除去目标多核苷酸中的核苷酸的碱基)或物理性地阻断一个或多个解旋酶的移动(例如利用庞大的化学基团)来停滞一个或多个解旋酶。The one or more blocking strands each provide an energy barrier that the one or more helicases cannot overcome even in active mode. One or more blocking strands can be achieved by reducing the pull of the helicase (e.g. by removing bases of nucleotides in the target polynucleotide) or physically blocking the movement of the one or more helicases (e.g. Utilize bulky chemical groups) to stall one or more helicases.
一个或多个阻断链可包括停滞一个或多个解旋酶的任意分子或任意分子的组合。所述一个或多个阻断链可以包括阻止所述一个或多个解旋酶沿目标多核苷酸移动的任意分子或任意分子的组合。其直接地确定在缺少跨膜孔和施加的电势的条件下，一个或多个解旋酶是否停留在一个或多个阻断链处。例如，实施例中所示进行测试，例如解旋酶穿过阻断链且置换DNA的互补链的能力可以通过PAGE进行测量。The one or more blocking strands may comprise any molecule or combination of any molecules that stall one or more helicases. The one or more blocker strands may comprise any molecule or combination of any molecules that prevent movement of the one or more helicases along the target polynucleotide. It directly determines whether, in the absence of a transmembrane pore and an applied potential, one or more helicases lodges at one or more blocking strands. For example, tests are performed as shown in the Examples, eg the ability of the helicase to pass through the blocking strand and displace the complementary strand of DNA can be measured by PAGE.
一个或多个阻断链通常包括直链分子如聚合物。所述一个或多个阻断链通常具有与目标多核苷酸不同的结构。例如，如果所述目标多核苷酸是DNA，一个或多个阻断链通常不是脱氧核糖核酸。特别是，如果目标多核苷酸是脱氧核糖核酸(DNA)或核糖核酸(RNA)，所述一个或多个阻断链可选包括肽核酸(PNA)，甘油核酸(GNA)，苏糖核酸(TNA)，锁核酸(LNA)或具有核苷酸侧链的合成聚合物。The one or more blocking chains typically comprise linear molecules such as polymers. The one or more blocking strands typically have a different structure than the target polynucleotide. For example, if the target polynucleotide is DNA, the one or more blocking strands are typically not deoxyribonucleic acid. In particular, if the polynucleotide of interest is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), the one or more blocking strands optionally include peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid ( TNA), locked nucleic acid (LNA) or synthetic polymers with nucleotide side chains.
一个或多个阻断链可选包括一个或多个硝基吲哚，例如一个或多个5-硝基吲哚，一个或多个肌苷，一个或多个吖啶，一个或多个2-氨基嘌呤，一个或多个2-6-二氨基嘌呤，一个或多个5-溴-脱氧尿嘧啶，一个或多个反向胸苷(反向dTs)，一个或多个反向脱氧胸苷(ddTs)，一个或多个二脱氧胞苷(ddCs)，一个或多个5-甲基胞苷，一个或多个5-羟甲基胞苷，一个或多个2’烷氧基修饰的核糖核苷酸(可选2’甲氧基修饰的核糖核苷酸)，一个或多个异脱氧胞苷(异-dCs)，一个或多个异脱氧鸟苷(异dGs)，一个或多个iSpC3基团(即缺少糖和碱基的核苷酸)，一个或多个光裂解(PC)基团，一个或多个己二醇基团，一个或多个阻断链9(iSp9)基团，一个或多个阻断链18(iSp18)基团，聚合物或一个或多个硫醇连接。所述一个或多个阻断链可包括这些基团的任意组合。许多这些基团可以购自(Integrated DNA)。One or more blocker strands optionally include one or more nitroindole, for example one or more 5-nitroindole, one or more inosine, one or more acridine, one or more 2 - aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more reverse thymidines (reverse dTs), one or more reverse deoxythymidines glycosides (ddTs), one or more dideoxycytidines (ddCs), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2' alkoxy modifications Ribonucleotides (optionally 2'methoxy-modified ribonucleotides), one or more isodeoxycytidines (iso-dCs), one or more isodeoxyguanosines (iso-dGs), one or Multiple iSpC3 groups (i.e., nucleotides lacking sugar and base), one or more photocleavage (PC) groups, one or more hexanediol groups, one or more blocker chain 9 (iSp9 ) group, one or more chain-blocking 18 (iSp18) groups, a polymer or one or more thiol linkages. The one or more blocking chains may comprise any combination of these groups. Many of these groups are commercially available from (Integrated DNA).
一个或多个阻断链可包含任何数量的这些基团。例如，对于2-氨基嘌呤，2-6-二氨基嘌呤，5-溴脱氧尿苷，反向dTs，ddTs，ddCs，5-甲基胞苷，5-羟甲基胞苷，2’烷氧基修饰的核糖核苷酸(可选2’甲氧基修饰的核糖核苷酸)，异dCs，异dGs，iSpC3基团，PC基团，己二醇基团和硫醇连接，一个或多个阻断链可选包含2个、3个、4个、5个、6个、7个、8 个、9个、10个、11个、12个或更多。一个或多个阻断链可选包含2个、3个、4个、5个、6个、7个、8个或更多iSp9基团。一个或多个阻断链可选包含2个、3个、4个、5个或6个或更多iSp18基团。最可选的阻断链基团是4个iSp18基团。One or more of the blocking chains may contain any number of these groups. For example, for 2-aminopurine, 2-6-diaminopurine, 5-bromodeoxyuridine, reverse dTs, ddTs, ddCs, 5-methylcytidine, 5-hydroxymethylcytidine, 2'alkoxy base-modified ribonucleotides (optionally 2'methoxy-modified ribonucleotides), iso-dCs, iso-dGs, iSpC3 groups, PC groups, hexanediol groups and thiol linkages, one or more Each blocking chain may optionally comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. One or more blocker strands optionally comprise 2, 3, 4, 5, 6, 7, 8 or more iSp9 groups. One or more blocker strands optionally comprise 2, 3, 4, 5 or 6 or more iSpl8 groups. The most optional chain-blocking groups are 4 iSp18 groups.
聚合物可选为多肽或聚乙二醇(PEG)。所述多肽可选地包含2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个或更多个氨基酸。所述PEG可选包含2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个或更多单体单元。The polymer can be optionally a polypeptide or polyethylene glycol (PEG). The polypeptide optionally comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acids. The PEG optionally comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more monomeric units.
一个或多个阻断链可选包括一个或多个无碱基核苷酸(即缺乏核碱基的核苷酸)，例如2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个或更多个无碱基的核苷酸。核碱基可以被无碱基核苷酸中的-H(idSp)或-OH置换。无碱基的阻断链可以通过从一个或多个相邻的核苷酸中除去核碱基而被***到目标多核苷酸中。One or more blocking strands optionally include one or more abasic nucleotides (i.e., nucleotides lacking nucleobases), e.g., 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12 or more abasic nucleotides. A nucleobase can be replaced by -H(idSp) or -OH in an abasic nucleotide. An abasic blocker strand can be inserted into a polynucleotide of interest by removing a nucleobase from one or more adjacent nucleotides.
一个或多个阻断链可选包含一个或多个物理上导致一个或多个解旋酶停滞的化学基团。所述一个或多个化学基团可选为一个或多个侧挂的化学基团。所述一个或多个化学基团可以连接到目标多核苷酸中的一个或更多个核碱基。所述一个或多个化学基团可以连接到目标多核苷酸的骨架。可存在任何数量，如2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个或更多个这些化学基团。合适的基团包括但不限于，荧光团，链霉亲和素和/或生物素，胆固醇，亚甲基蓝，二硝基苯酚(DNPs)，洋地黄毒苷和/或抗洋地黄毒苷和二苯基环辛炔基团。The one or more blocking strands optionally contain one or more chemical groups that physically cause the one or more helicases to stall. The one or more chemical groups may be one or more pendant chemical groups. The one or more chemical groups may be linked to one or more nucleobases in the target polynucleotide. The one or more chemical groups may be attached to the backbone of the polynucleotide of interest. There may be any number, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more of these chemical groups. Suitable groups include, but are not limited to, fluorophores, streptavidin and/or biotin, cholesterol, methylene blue, dinitrophenols (DNPs), digoxigenin and/or anti-digoxigenin and diphenyl Cyclooctyne group.
目标多核苷酸中的不同阻断链可以包含不同停滞分子。例如，一个阻断链可以包括如上讨论的一个线性分子，另一个阻断链可以包括一个或多个物理上导致一个或多个解旋酶停滞的化学基团。阻断链可包括如上讨论的任何线性分子和一个或多个物理上导致一个或多个解旋酶停滞的化学基团，例如一个或多个无碱基和荧光团。Different blocking strands in the target polynucleotide may contain different arresting molecules. For example, one blocker strand can include a linear molecule as discussed above, and the other blocker strand can include one or more chemical groups that physically cause one or more helicases to stall. A blocking strand may comprise any linear molecule as discussed above and one or more chemical groups that physically cause one or more helicases to stall, such as one or more abasic groups and fluorophores.
表征目标多核苷酸的方法Methods for Characterizing Polynucleotides of Interest
本申请的方法解决的问题是如何表征目标多核苷酸。The problem addressed by the method of the present application is how to characterize the target polynucleotide.
表征目标多核苷酸的方法，包括：Methods for characterizing target polynucleotides, including:
a)提供(i)多核苷酸构建体和(ii)解旋酶，所述多核苷酸构建体包含所述目标多核苷酸和如权利要求1至4中任一项所述的衔接体；所述解旋酶包括DNA解旋酶；A) providing (i) a polynucleotide construct and (ii) a helicase, the polynucleotide construct comprising the target polynucleotide and the adapter according to any one of claims 1 to 4; The helicase comprises a DNA helicase;
b)将a)中提供的所述多核苷酸构建体和所述解旋酶与跨膜孔接触，使得所述解旋酶控制所述目标多核苷酸相对于所述跨膜孔的移动；b) contacting the polynucleotide construct provided in a) and the helicase with a transmembrane pore such that the helicase controls movement of the polynucleotide of interest relative to the transmembrane pore;
c)随着所述目标多核苷酸相对于所述跨膜孔移动，获取一个或多个测量值，其中所述测量值代表所述目标多核苷酸的一个或多个特征，并由此表征所述目标多核苷酸。c) taking one or more measurements as the target polynucleotide moves relative to the transmembrane pore, wherein the measurements represent one or more characteristics of the target polynucleotide and thereby characterize The target polynucleotide.
上述方法中使用到本申请的衔接体，所述衔接体结合DNA解旋酶并与目标多核苷酸连接，从而使所述目标多核苷酸被运送到跨膜孔，并且用所述孔表征所述目标多核苷酸。本申请提供了通过在DNA解旋酶的控制下，随着目标多核苷酸相对于所述跨膜孔移动，获取一个或多个测量值来表征目标核糖核酸(RNA和/或DNA)多核苷酸的方法。In the above method, the adapter of the present application is used, and the adapter binds DNA helicase and ligates to the target polynucleotide, so that the target polynucleotide is transported to the transmembrane pore, and the pore is used to characterize the target polynucleotide. the target polynucleotide. The present application provides for characterizing target ribonucleic acid (RNA and/or DNA) polynucleotides by taking one or more measurements as the target polynucleotide moves relative to the transmembrane pore under the control of a DNA helicase sour method.
示例性，目标多核苷酸包括目标RNA多核苷酸和目标DNA多核苷酸。Exemplary, target polynucleotides include target RNA polynucleotides and target DNA polynucleotides.
由于跨膜孔能够检测目标多核苷酸的单个分子，从而不需要放大(扩增)所述目标多核苷酸。所述方法通常不包括聚合酶链反应(PCR)或反转录PCR(RT-PCR)。这极大地减少了表征目标多核苷酸需要的工作量。这也避免了由PCR引起的任何偏差和人为假象。Since the transmembrane pore is capable of detecting a single molecule of the target polynucleotide, there is no need to amplify (amplify) the target polynucleotide. The methods typically do not involve polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR). This greatly reduces the effort required to characterize the target polynucleotide. This also avoids any bias and artefacts caused by PCR.
本申请的方法可涉及确定或测量RNA多核苷酸或DNA多核苷酸的一个或多个特征。所述方法可包括确定或测量所述RNA多核苷酸或DNA多核苷酸的一个、两个、三个、四个或五个或更多个特征。The methods of the present application may involve determining or measuring one or more characteristics of an RNA polynucleotide or a DNA polynucleotide. The method may comprise determining or measuring one, two, three, four or five or more characteristics of the RNA polynucleotide or DNA polynucleotide.
示例性的，所述一个或多个特征可选地选自(i)所述RNA多核苷酸的长度，(ii)所述RNA多核苷酸的同一性，(iii)所述RNA多核苷酸的序列，(iv)所述RNA多核苷酸的二级结构和(v)所述RNA多核苷酸是否是修饰的。根据本申请可以测量(i)至(v)的任何组合，例如{i}，{ii}，{iii}，{iv}，{v}，{i，ii}，{i，iii}，{i，iv}，{i，v}，{ii，iii}，{ii，iv}，{ii，v}，{iii，iv}，{iii，v}，{iv，v}，{i，ii，iii}，{i，ii，iv}，{i，ii，v}，{i，iii，iv}，{i， iii，v}，{i，iv，v}，{ii，iii，iv}，{ii，iii，v}，{ii，iv，v}，{iii，iv，v}，{i，ii，iii，iv}，{i，ii，iii，v}，{i，ii，iv，v}，{i，iii，iv，v}，{ii，iii，iv，v}或{i，ii，iii，iv，v}。可以测量(i)至(v)的不同组合，包括以上列举的任何组合。本申请的方法可选地包含估计所述RNA多核苷酸的序列或对所述RNA多核苷酸测序。Exemplarily, the one or more features are optionally selected from (i) the length of the RNA polynucleotide, (ii) the identity of the RNA polynucleotide, (iii) the RNA polynucleotide sequence, (iv) the secondary structure of the RNA polynucleotide and (v) whether the RNA polynucleotide is modified. Any combination of (i) to (v) can be measured according to this application, such as {i}, {ii}, {iii}, {iv}, {v}, {i, ii}, {i, iii}, { i, iv}, {i, v}, {ii, iii}, {ii, iv}, {ii, v}, {iii, iv}, {iii, v}, {iv, v}, {i, ii, iii}, {i, ii, iv}, {i, ii, v}, {i, iii, iv}, {i, iii, v}, {i, iv, v}, {ii, iii, iv}, {ii, iii, v}, {ii, iv, v}, {iii, iv, v}, {i, ii, iii, iv}, {i, ii, iii, v}, {i, ii, iv, v}, {i, iii, iv, v}, {ii, iii, iv, v} or {i, ii, iii, iv, v}. Different combinations of (i) to (v) can be measured, including any combination listed above. The methods of the present application optionally comprise estimating the sequence of said RNA polynucleotide or sequencing said RNA polynucleotide.
对于(i)，目标RNA多核苷酸的长度例如可以通过确定所述目标RNA多核苷酸和所述孔之间的相互作用的次数，或所述目标RNA多核苷酸和所述孔之间的相互作用的持续时间来测定。For (i), the length of the target RNA polynucleotide can be determined, for example, by determining the number of interactions between the target RNA polynucleotide and the pore, or the number of interactions between the target RNA polynucleotide and the pore. The duration of the interaction is measured.
对于(ii)，目标RNA多核苷酸的同一性可以通过多种方式测定。所述目标RNA多核苷酸的同一性可联合所述目标RNA多核苷酸的序列测定来测定或不联合所述目标RNA多核苷酸的序列测定来测定。前者是直接的；对所述多核苷酸进行测序，并由此鉴定目标RNA多核苷酸的同一性。后者可以以几种方式来完成。例如，可以测定所述目标RNA多核苷酸中特定模序的存在(而无需测定所述RNA多核苷酸的其余序列)。或者，所述方法中测定的特定的电和/或光信号的测量值可鉴定来自特定来源的RNA多核苷酸。Regarding (ii), the identity of the target RNA polynucleotide can be determined in a number of ways. The identity of the target RNA polynucleotide may or may not be determined in conjunction with sequence determination of the target RNA polynucleotide. The former is straightforward; the polynucleotide is sequenced and the identity of the target RNA polynucleotide is thereby identified. The latter can be done in several ways. For example, the presence of a particular motif in the target RNA polynucleotide can be determined (without determining the remaining sequence of the RNA polynucleotide). Alternatively, measurements of specific electrical and/or optical signals determined in the methods can identify RNA polynucleotides from a specific source.
对于(iii)，所述目标RNA多核苷酸的序列可以如前所述确定。合适的测序方法，特别是那些使用电测量值的方法，在Stoddart D et al.,Proc Natl Acad Sci,12；106(19):7702-7,Lieberman KR et al,J Am Chem Soc.2010；132(50):17961-72，和国际申请WO 2000/28312中描述。For (iii), the sequence of the target RNA polynucleotide can be determined as previously described. Suitable sequencing methods, especially those using electrical measurements, are described in Stoddart D et al., Proc Natl Acad Sci, 12; 106(19):7702-7, Lieberman KR et al, J Am Chem Soc. 2010; 132(50):17961-72, and described in International Application WO 2000/28312.
对于(iv)，所述二级结构可以多种方法测量。例如，如果所述方法包括电测量，所述二级结构可以利用穿过孔的停留时间的变化或电流变化来测量。这使得单链和双链RNA多核苷酸的区域被区别。For (iv), the secondary structure can be measured in a variety of ways. For example, if the method includes electrical measurements, the secondary structure may be measured using changes in residence time or changes in current through the pores. This allows regions of single- and double-stranded RNA polynucleotides to be distinguished.
对于(v)，可以测定任何修饰的存在或不存在。所述方法可选地包括确定所述多核苷酸是否通过甲基化、氧化、损伤、用一个或多个蛋白，或用一个或多个标记物、标签或阻断链进行了修饰。特异性修饰将导致与孔的特异性相互作用，这可以使用下面描述的方法来测定。例如，可以基于孔与每个核苷酸的相互作用过程中穿过孔的电流，区别胞嘧啶与甲基胞嘧啶。本申请的方法可用于RNA和DNA之间的区别，甚至在单一样品中：作为 平均振幅和范围的函数，甚至RNA和DNA序列相同时，RNA和DNA可被相互区别。For (v), the presence or absence of any modification can be determined. The method optionally includes determining whether the polynucleotide is modified by methylation, oxidation, damage, with one or more proteins, or with one or more markers, tags, or blocker strands. Specific modifications will result in specific interactions with the pore, which can be determined using the methods described below. For example, cytosine can be distinguished from methylcytosine based on the current flow through the pore during the pore's interaction with each nucleotide. The method of the present application can be used to discriminate between RNA and DNA, even in a single sample: RNA and DNA can be distinguished from each other even when the RNA and DNA sequences are identical, as a function of the mean amplitude and range.
所述方法可使用任何适于研究膜/孔***的设备来实施，其中在所述膜/孔***中，孔存在于膜中。可使用任何适于跨膜孔传感的设备来实施所述方法。例如，所述设备包括一个室，所述室包括水溶液和将该室分割为两部分的屏障(barrier)。所述屏障通常具有缝隙，其中在缝隙中形成包括孔的膜。或者该屏障形成其中存在孔的膜。The method can be carried out using any device suitable for studying membrane/pore systems in which the pores are present in the membrane. The method can be performed using any device suitable for transmembrane pore sensing. For example, the device comprises a chamber comprising an aqueous solution and a barrier dividing the chamber into two parts. The barrier typically has slits in which a membrane comprising pores is formed. Or the barrier forms a membrane in which pores are present.
该方法可以使用在国际申请No.PCT/GB08/000562(WO 2008/102120)中描述的设备实施。The method can be carried out using the apparatus described in International Application No. PCT/GB08/000562 (WO 2008/102120).
该方法可以包括随着所述RNA多核苷酸相对于所述孔移动，测量通过所述孔的电流。因此该装置也可以包括能够跨膜和孔施加电势并测量电流信号的电路。该方法可以使用膜片钳或电压钳实施。所述方法可选包含使用电压钳。The method can include measuring a current through the pore as the RNA polynucleotide moves relative to the pore. The device may therefore also include circuitry capable of applying a potential across the membrane and pore and measuring the current signal. The method can be performed using patch clamp or voltage clamp. The method optionally includes the use of a voltage clamp.
本申请的方法可包括随着所述RNA多核苷酸相对于所述孔移动来测量流过所述孔的电流。随着所述多核苷酸相对于所述孔移动流经所述孔的电流被用于确定所述目标RNA多核苷酸的序列。这是链测序。用于测量通过跨膜蛋白质孔的离子电流的合适的条件是本领域已知的，并且在实施例中公开。所述方法通过跨膜和孔施加的电压进行实施。使用的电压通常为+5V到-5V，例如从+4V到-4V，+3V到-3V或+2V到-2V。通常使用的电压通常为-600mV到+600mV，或-400mV到+400mV。使用的电压可选地在具有以下下限和上限的范围内，所述下限选自在-400mV，-300mV，-200mV，-150mV，-100mV，-50mV，-20mV和0mV，所述上限独立地选自+10mV，+20mV，+50mV，+100mV，+150mV，+200mV，+300mV和+400mV。所用的电压更可选在100mV到240mV的范围内并最可选在120mV到220mV的范围内。可通过对孔施加提高的电势来提高对不同核糖核苷酸的分辨力。The methods of the present application may comprise measuring the current flowing through the pore as the RNA polynucleotide moves relative to the pore. The current flowing through the pore as the polynucleotide moves relative to the pore is used to determine the sequence of the target RNA polynucleotide. This is strand sequencing. Suitable conditions for measuring ionic currents through transmembrane protein pores are known in the art and disclosed in the Examples. The method is carried out by applying a voltage across the membrane and pore. The voltage used is usually +5V to -5V, for example from +4V to -4V, +3V to -3V or +2V to -2V. Commonly used voltages are typically -600mV to +600mV, or -400mV to +400mV. The voltage used is optionally within a range having a lower limit selected from -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV and 0mV, and an upper limit independently Select from +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300mV and +400mV. The voltage used is more preferably in the range of 100 mV to 240 mV and most preferably in the range of 120 mV to 220 mV. The resolution of different ribonucleotides can be increased by applying an increased potential to the well.
该方法通常在任何载荷子，如金属盐，例如碱金属盐，卤盐，例如氯盐，如碱金属氯盐的存在下实施。载荷子可包括离子型液体或有机盐，例如四甲基氯化铵，三甲苯基氯化铵，苯基三甲基氯化苯，或1-乙基-3-甲基咪唑鎓氯化物。在上面讨论的示例性设备中，所述盐存在于所述室中的水 溶液中。通常使用氯化钾(KCl)，氯化钠(NaCl)或氯化铯(CsCl)或亚铁***和铁***的混合物。可选氯化钾，氯化钠和亚铁***和铁***的混合物。所述载荷子可以非对称地穿过所述膜。例如，在所述膜的各侧上载荷子的类型和/或浓度可能不同。The method is generally carried out in the presence of any charge carrier, such as a metal salt, eg an alkali metal salt, a halide salt, eg a chloride salt, eg an alkali metal chloride salt. Chargers may include ionic liquids or organic salts such as tetramethylammonium chloride, tricresyl ammonium chloride, phenyltrimethylbenzene chloride, or 1-ethyl-3-methylimidazolium chloride. In the exemplary apparatus discussed above, the salt is present in an aqueous solution in the chamber. Usually potassium chloride (KCl), sodium chloride (NaCl) or cesium chloride (CsCl) or a mixture of potassium ferrocyanide and potassium ferricyanide is used. Potassium chloride, sodium chloride and mixtures of potassium ferrocyanide and potassium ferricyanide are optional. The charge carriers may pass through the membrane asymmetrically. For example, the type and/or concentration of charge carriers may be different on each side of the membrane.
盐浓度可为饱和的。盐浓度可以是3M或更低，通常为0.1M至2.5M，0.3M至1.9M，0.5M至1.8M，0.7M至1.7M，0.9M至1.6M或1M至1.4M。可选盐浓度为150mM到1M。所述方法可选使用至少为0.3M，例如至少为0.4M，至少为0.5M，至少为0.6M，至少为0.8M，至少为1.0M，至少为1.5M，至少为2.0M，至少为2.5M，或者至少为3.0M的盐浓度进行实施。高盐浓度提供了高信噪比，并使得在正常电流波动的背景下，代表核糖核苷酸存在的电流能被识别。The salt concentration can be saturated. The salt concentration may be 3M or less, typically 0.1M to 2.5M, 0.3M to 1.9M, 0.5M to 1.8M, 0.7M to 1.7M, 0.9M to 1.6M or 1M to 1.4M. The optional salt concentration is 150mM to 1M. The method optionally uses at least 0.3M, such as at least 0.4M, at least 0.5M, at least 0.6M, at least 0.8M, at least 1.0M, at least 1.5M, at least 2.0M, at least 2.5 M, or at least a salt concentration of 3.0M for implementation. The high salt concentration provides a high signal-to-noise ratio and allows currents representing the presence of ribonucleotides to be identified against the background of normal current fluctuations.
所述方法通常在缓冲剂存在下实施。在上面讨论的示例性设备中，缓冲剂在所述室中的水溶液中存在。本申请的方法可使用任何缓冲剂。通常地，缓冲剂是磷酸盐缓冲液。其他合适的缓冲剂为HEPES和Tris-HCl缓冲剂。该方法通常在pH值为4.0至12.0，4.5至10.0，5.0至9.0，5.5至8.8，6.0至8.7，7.0至8.8，或7.5至8.5下实施。所使用的pH可选约为7.5。The methods are generally performed in the presence of a buffer. In the exemplary apparatus discussed above, the buffer is present in the aqueous solution in the chamber. Any buffer may be used in the methods of the present application. Typically, the buffer is phosphate buffered saline. Other suitable buffers are HEPES and Tris-HCl buffers. The method is typically carried out at a pH of 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7, 7.0 to 8.8, or 7.5 to 8.5. The pH used is optionally about 7.5.
所述方法可在0℃至100℃，15℃至95℃，16℃至90℃，17℃至85℃，18℃至80℃，19℃至70℃，或20℃至60℃下实施。所述方法通常在室温下进行。该方法可选地在支持酶功能的温度下，例如约37℃实施。The method can be performed at 0°C to 100°C, 15°C to 95°C, 16°C to 90°C, 17°C to 85°C, 18°C to 80°C, 19°C to 70°C, or 20°C to 60°C. The method is generally carried out at room temperature. The method is optionally performed at a temperature that supports enzyme function, such as about 37°C.
所述方法可在游离核苷酸或游离核苷酸类似物和/或有利于发挥解旋酶或构建体的功能的酶辅因子的存在下实施。所述方法也可在游离核苷酸或游离核苷酸类似物不存在和酶辅因子不存在下实施。所述游离核苷酸可以是任何单个核苷酸的一种或多种。所述游离核苷酸包括，但不限于，单磷酸腺苷(AMP)，二磷酸腺苷(ADP)，三磷酸腺苷(ATP)，单磷酸鸟苷(GMP)，二磷酸鸟苷(GDP)，三磷酸鸟苷(GTP)，单磷酸胸苷(TMP)，二磷酸胸苷(TDP)，三磷酸胸苷(TTP)，单磷酸尿苷(UMP)，二磷酸尿苷(UDP)，三磷酸尿苷(UTP)，单磷酸胞苷(CMP)，二磷酸胞苷(CDP)，三磷酸胞苷(CTP)，单磷酸环腺苷(cAMP)，单磷酸环鸟苷(cGMP)，单磷酸脱氧腺苷(dAMP)，二磷酸脱氧腺苷(dADP)，三磷酸脱氧腺苷(dATP)，单磷酸脱氧鸟苷 (dGMP)，二磷酸脱氧鸟苷(dGDP)，三磷酸脱氧鸟苷(dGTP)，单磷酸脱氧胸苷(dTMP)，二磷酸脱氧胸苷(dTDP)，三磷酸脱氧胸苷(dTTP)，单磷酸脱氧尿苷(dUMP)，二磷酸脱氧尿苷(dUDP)，三磷酸脱氧尿苷(dUTP)，单磷酸脱氧胞苷(dCMP)，二磷酸脱氧胞苷(dCDP)和三磷酸脱氧胞苷(dCTP)。所述游离核苷酸可选选自AMP，TMP，GMP，CMP，UMP，dAMP，dTMP，dGMP或dCMP。所述游离核苷酸可选腺苷三磷酸(ATP)。所述酶辅因子是使解旋酶或构建体发挥功能的因子。所述酶辅因子可选是二价金属阳离子。所述二价金属阳离子可选为Mg 2+，Mn 2+，Ca 2+或Co 2+。所述酶辅因子最可选为Mg 2+。 The method may be performed in the presence of free nucleotides or free nucleotide analogs and/or enzyme cofactors that facilitate the function of the helicase or construct. The method can also be performed in the absence of free nucleotides or free nucleotide analogs and in the absence of enzyme cofactors. The free nucleotides may be one or more of any single nucleotide. The free nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), triphosphate Guanosine phosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate Cytidine monophosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxygenate monophosphate Adenosine (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP) , deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate Cytidine monophosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP). The free nucleotides may be selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotide may be adenosine triphosphate (ATP). The enzyme cofactor is the factor that enables the function of the helicase or construct. The enzyme cofactor is optionally a divalent metal cation. The divalent metal cation may be Mg 2+ , Mn 2+ , Ca 2+ or Co 2+ . The enzyme cofactor is most preferably Mg 2+ .
目标RNA多核苷酸target RNA polynucleotide
RNA是包含两个或多个核糖核苷酸的大分子。所述目标RNA多核苷酸可以是真核或原核RNA。所述目标RNA多核苷酸可以包含任何核糖核苷酸的任何组合。所述核糖核苷酸可以是天然存在的或人造的。所述目标RNA多核苷酸中的一个或多个核糖核苷酸可被氧化或甲基化。所述目标RNA中的一个或多个核糖核苷酸可被损伤。例如，所述目标RNA可以包含嘧啶二聚体，例如尿嘧啶二聚体。此类二聚体通常与紫外线导致的损伤相关，并是皮肤黑色素瘤的首要原因。所述目标RNA多核苷酸中的一个或多个核糖核苷酸可被修饰，例如用标记物或标签修饰。合适的标记物如下所述。所述目标RNA可包含一个或多个阻断链。RNA is a macromolecule comprising two or more ribonucleotides. The target RNA polynucleotide can be eukaryotic or prokaryotic RNA. The target RNA polynucleotide may comprise any combination of ribonucleotides. The ribonucleotides may be naturally occurring or man-made. One or more ribonucleotides in the target RNA polynucleotide may be oxidized or methylated. One or more ribonucleotides in the target RNA may be damaged. For example, the target RNA may comprise a pyrimidine dimer, such as a uracil dimer. Such dimers are often associated with UV-induced damage and are the leading cause of cutaneous melanoma. One or more ribonucleotides in the target RNA polynucleotide may be modified, eg, with a label or tag. Suitable markers are described below. The target RNA may comprise one or more blocker strands.
核糖核苷酸含有碱基、核糖和至少一个磷酸基团。所述碱基通常为杂环的。碱基包括但不限于：嘌呤和嘧啶，更具体地，腺嘌呤、鸟嘌呤、胸腺嘧啶、尿嘧啶和胞嘧啶。所述核苷酸通常含有单磷酸，二磷酸或三磷酸。磷酸可被连接在核苷酸的5’或3’侧。Ribonucleotides contain a base, ribose sugar and at least one phosphate group. The bases are typically heterocyclic. Bases include, but are not limited to, purines and pyrimidines, more specifically, adenine, guanine, thymine, uracil, and cytosine. The nucleotides generally contain monophosphates, diphosphates or triphosphates. Phosphate can be attached to the 5' or 3' side of the nucleotide.
核糖核苷酸包括但不限于，单磷酸腺苷(AMP)，单磷酸鸟苷(GMP)，单磷酸胸苷(TMP)，单磷酸尿苷(UMP)，单磷酸胞苷(CMP)，单磷酸5-甲基胞苷，二磷酸5-甲基胞苷，三磷酸5-甲基胞苷，单磷酸5-羟甲基胞苷，二磷酸5-羟甲基胞苷和三磷酸5-羟甲基胞苷。所述核苷酸可选选自AMP，TMP，GMP，CMP和UMP。Ribonucleotides include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), 5-Methylcytidine Phosphate, 5-Methylcytidine Diphosphate, 5-Methylcytidine Triphosphate, 5-Hydroxymethylcytidine Monophosphate, 5-Hydroxymethylcytidine Diphosphate, and 5-Hydroxymethylcytidine Triphosphate Hydroxymethylcytidine. The nucleotides may optionally be selected from AMP, TMP, GMP, CMP and UMP.
核糖核苷酸可以是脱碱基的(即缺少碱基)。核糖核苷酸也可缺少碱基 和糖(即C3阻断链)。Ribonucleotides may be abasic (ie, lack a base). Ribonucleotides can also lack bases and sugars (i.e. C3 blocks the chain).
所述目标RNA多核苷酸的核糖核苷酸可以任何方式彼此连接。如在核酸中一样，所述核糖核苷酸通常通过它们的糖和磷酸基团连接。如嘧啶二聚体中一样，所述核糖核苷酸可通过它们的碱基连接。The ribonucleotides of the target RNA polynucleotide may be linked to each other in any manner. As in nucleic acids, the ribonucleotides are usually linked through their sugar and phosphate groups. As in pyrimidine dimers, the ribonucleotides may be linked via their bases.
RNA是非常多样的分子。所述目标RNA多核苷酸可以是任何天然产生的或合成的核糖核苷酸分子，例如，RNA，信使RNA(mRNA)，核糖体RNA(rRNA)，核不均一RNA(hnRNA)，转移RNA(tRNA)，转移信使RNA(tmRNA)，微小RNA(miRNA)，小核RNA(snRNA)，小核仁RNA(snoRNA)，信号识别颗粒(SRP RNA)，SmY RNA，小卡侯氏体(Small Cajal body-speicifc)RNA(scaRNA)，向导RNA(gRNA)，剪接前导RNA(SL RNA)，反义RNA(asRNA)，长非编码RNA(lncRNA)，Piwi-相互作用(Piwi-interacting)RNA(piRNA)，小分子干扰RNA(siRNA)，反式作用siRNA(tasiRNA)，重复联系siRNA(rasiRNA)，Y RNA，病毒性RNA或染色体的RNA，在适当情况下所有的RNA可以是单链的，双链的或三链的。RNA is a very diverse molecule. The target RNA polynucleotide can be any naturally occurring or synthetic ribonucleotide molecule, for example, RNA, messenger RNA (mRNA), ribosomal RNA (rRNA), nuclear heterogeneous RNA (hnRNA), transfer RNA ( tRNA), transfer messenger RNA (tmRNA), microRNA (miRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), signal recognition particle (SRP RNA), SmY RNA, small Cajal body-speicifc) RNA (scaRNA), guide RNA (gRNA), splicing leader RNA (SL RNA), antisense RNA (asRNA), long non-coding RNA (lncRNA), Piwi-interacting (Piwi-interacting) RNA (piRNA ), small interfering RNA (siRNA), trans-acting siRNA (tasiRNA), repeat linkage siRNA (rasiRNA), Y RNA, viral RNA or chromosomal RNA, where appropriate all RNAs can be single-stranded, double-stranded chained or triple chained.
所述目标RNA多核苷酸可选是信使RNA(mRNA)。所述目标mRNA可以是交替剪接变体(alternate splice variant)。mRNA和/或交替mRNA剪接变体的变异量(或等级)可能与疾病或健康状况有关。The target RNA polynucleotide is optionally messenger RNA (mRNA). The target mRNA may be an alternate splice variant. The amount (or grade) of variation in mRNA and/or alternate mRNA splice variants may be associated with a disease or health condition.
或者所述目标RNA多核苷酸是微小RNA(或miRNA)。很难在低浓度下检测到的一组RNAs是微小核糖核酸(micro-RNA或miRNAs)。miRNAs是高度稳定的RNA寡聚体，其能转录后调节蛋白质产物。它们通过两个机理中的一个起作用。在植物中，miRNAs已被证明主要通过引导信使RNA的***来起作用，然而在动物中，通过miRNAs的基因调控通常包括miRNAs到信使RNAs的3’UTRs的杂交，这阻碍了翻译(Lee et al.,Cell75,843-54(1993)；Wightman等，Cell 75,855-62(1993)；和Esquela-Kerscher等，Cancer 6,259-69(2006))。miRNAs经常以有缺陷的互补与它们的目标结合。它们已被预测能与多达200个或更多的基因目标分别结合，并调节全人类超过三分之一的基因(Lewis等，Cell 120,15-20(2005))。Alternatively, the target RNA polynucleotide is a microRNA (or miRNA). A group of RNAs that are difficult to detect at low concentrations are microRNAs (micro-RNAs or miRNAs). miRNAs are highly stable RNA oligomers that post-transcriptionally regulate protein production. They work by one of two mechanisms. In plants, miRNAs have been shown to function primarily by directing the cleavage of messenger RNAs, whereas in animals, gene regulation by miRNAs often involves hybridization of miRNAs to the 3'UTRs of messenger RNAs, which hinders translation (Lee et al ., Cell 75, 843-54 (1993); Wightman et al., Cell 75, 855-62 (1993); and Esquela-Kerscher et al., Cancer 6, 259-69 (2006)). miRNAs often bind their targets with defective complementarity. They have been predicted to individually bind as many as 200 or more gene targets and regulate more than one-third of all human genes (Lewis et al., Cell 120, 15-20 (2005)).
用于本申请的合适的miRNAs是本领域已知的。例如，在公众可获得的数据库上存储的合适的miRNAs(Jiang Q.,Wang Y.,Hao Y.,Juan L.,Teng  M.,Zhang X.,Li M.,Wang G.,Liu Y.,(2009)miR2Disease:a manually curated database for microRNA deregulation in human disease.Nucleics Acides Res.)。已知在肿瘤中某些microRNAs的表达水平会改变，产生了不同肿瘤类型特征模式的microRNA表达(Rosenfeld,N.等，Nature Biotechnology 26,462-9(2008))。另外，已知miRNA表达谱能够比信使RNA表达谱更精确地揭示肿瘤发展的阶段(Lu等，Nature 435,834-8(2005)和Barshack等，The International Journal of Biochemistry&Cell Biology 42,1355-62(2010))。这些发现，结合miRNAs的高稳定性，和在血清和血浆中检测循环的miRNAs的能力(Wang等，Bio化学and Biophysical Research Communications394,184-8(2010)；Gilad等，PloS One 3,e3148(2008)；和Keller等，Nature Methods 8,841-3(2011))，引起了将microRNAs作为癌症生物标记物的潜在应用的大量兴趣。为了有效地治疗，癌症需要被精确地分类并不同地治疗，但是由于许多不同类型的癌症共享形态学特征这一事实，作为分类的方法的肿瘤形态评估的功效被削弱了。miRNAs提供了潜在地更可靠并低创伤性的解决方案。Suitable miRNAs for use in this application are known in the art. For example, suitable miRNAs deposited on publicly available databases (Jiang Q., Wang Y., Hao Y., Juan L., Teng M., Zhang X., Li M., Wang G., Liu Y. , (2009) miR2Disease: a manually curated database for microRNA deregulation in human disease. Nucleics Acides Res.). The expression levels of certain microRNAs are known to be altered in tumors, resulting in patterns of microRNA expression characteristic of different tumor types (Rosenfeld, N. et al., Nature Biotechnology 26, 462-9 (2008)). In addition, miRNA expression profiling is known to reveal the stage of tumor development more precisely than messenger RNA expression profiling (Lu et al., Nature 435, 834-8 (2005) and Barshack et al., The International Journal of Biochemistry & Cell Biology 42, 1355-62 (2010) ). These findings, combined with the high stability of miRNAs, and the ability to detect circulating miRNAs in serum and plasma (Wang et al., Biochemistry and Biophysical Research Communications 394, 184-8 (2010); Gilad et al., PloS One 3, e3148 (2008 ); and Keller et al., Nature Methods 8,841-3 (2011)), have aroused a great deal of interest in the potential application of microRNAs as cancer biomarkers. To be effectively treated, cancers need to be accurately classified and treated differently, but the power of tumor morphology assessment as a method of classification is diminished by the fact that many different types of cancer share morphological features. miRNAs offer a potentially more reliable and less invasive solution.
mRNAs和miRNAs对诊断或预测疾病或身体状况的用途更详细地讨论如下。The use of mRNAs and miRNAs to diagnose or predict a disease or condition is discussed in more detail below.
可研究任何数量的RNA。例如，本申请的方法可关注于确定3，4，5，6，7，8，9，10，20，30，50，100或更多个RNA分子的存在，缺失或一个或多个特征。Any amount of RNA can be studied. For example, the methods of the present application may focus on determining the presence, absence or one or more characteristics of 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more RNA molecules.
所述多核苷酸可以是天然存在的或人工合成的。例如，所述方法可用于核实两个或多个人工制造的寡核苷酸的序列。所述方法通常在体外实施。The polynucleotides may be naturally occurring or synthetic. For example, the method can be used to verify the sequence of two or more artificially produced oligonucleotides. The methods are typically performed in vitro.
所述目标RNA多核苷酸可以是任意长度。例如，所述RNA多核苷酸可以是至少10、至少50、至少100、至少150、至少200、至少250、至少300、至少400或至少500个核糖核苷酸长度。所述目标RNA可以是1000或更多个核糖核苷酸，5000或更多个核苷酸或在100000或更多核糖核苷酸长度。全部或只有部分的所述目标RNA可以使用这种方法表征。要被测序的RNA部分可选包含全部目标分子，但可以例如比全部分子少，例如，4个碱基到1kb，例如，4到100个碱基。The target RNA polynucleotide can be of any length. For example, the RNA polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 ribonucleotides in length. The target RNA can be 1000 or more ribonucleotides, 5000 or more nucleotides or at 100000 or more ribonucleotides in length. All or only a portion of the target RNA can be characterized using this method. The portion of RNA to be sequenced optionally comprises the entire target molecule, but can eg be less than the entire molecule, eg 4 bases to 1 kb, eg 4 to 100 bases.
所述目标RNA多核苷酸通常存在于或来源于任何合适样本中。本申请通常在已知含有或怀疑含有所述目标RNA多核苷酸的样品中实施。替代地，可对样品实施本申请，以确认在样品中的存在是已知的或期望的一个或多个目标RNAs身份。The target RNA polynucleotide is typically present in or derived from any suitable sample. The application is generally practiced on samples known to contain or suspected to contain the RNA polynucleotide of interest. Alternatively, the present application can be performed on a sample to confirm the presence in the sample of one or more target RNAs whose identity is known or expected.
所述样品可以是生物样品。本申请可以针对从任何生物体或微生物中获得或提取的样品在体外实施。所述生物体或微生物通常是古细菌的(archaeal)，原核的或真核的，并且通常属于以下五界中的一个：植物界，动物界，真菌，原核生物和原生生物。所述目标RNA多核苷酸可来源于真核细胞或可来源于使用真核细胞的转录机制的病毒。本申请可以针对从任何病毒中获得或提取的样品在体外实施。The sample can be a biological sample. The present application may be practiced in vitro on samples obtained or extracted from any organism or microorganism. The organisms or microorganisms are generally archaeal, prokaryotic or eukaryotic, and generally belong to one of the following five kingdoms: Plantae, Animalia, Fungi, Prokaryotes and Protista. The RNA polynucleotide of interest may be derived from a eukaryotic cell or may be derived from a virus that uses the transcriptional machinery of a eukaryotic cell. The present application can be practiced in vitro on samples obtained or extracted from any virus.
所述样品可选是液体样品。样品通常包括病人的体液。所述样品可以是尿液，淋巴液，唾液，粘液或羊水，但可选血液，血浆或血清。通常，所述样品来源于人，但替代地可以是来自其他哺乳动物，如自商业上养殖的动物如马，牛，羊或猪或替代地可以是宠物如猫或狗。或者，来源于植物的样品通常从商业作物，如谷类，豆类，水果或蔬菜，例如小麦，大麦，燕麦，菜籽油(canola)，玉米，大豆，水稻，香蕉，苹果，番茄，土豆，葡萄，烟草，菜豆(beans)，小扁豆，甘蔗，可可或棉花。The sample is optionally a liquid sample. Samples typically include bodily fluids of patients. The sample may be urine, lymph, saliva, mucus or amniotic fluid, but may alternatively be blood, plasma or serum. Typically, the sample will be of human origin, but may alternatively be from other mammals, such as from a commercially bred animal such as a horse, cow, sheep or pig or alternatively may be a pet such as a cat or dog. Alternatively, samples of plant origin are usually obtained from commercial crops such as cereals, legumes, fruits or vegetables such as wheat, barley, oats, rapeseed oil (canola), corn, soybeans, rice, bananas, apples, tomatoes, potatoes, Grapes, tobacco, beans, lentils, sugar cane, cocoa or cotton.
所述样品可以是非生物样品。所述非生物样品可选为流体样品。非生物样品的实例包括手术液，水如饮用水、海水或河水，以及用于实验室试验的试剂。The sample can be a non-biological sample. The non-biological sample can optionally be a fluid sample. Examples of non-biological samples include surgical fluids, water such as drinking water, sea water or river water, and reagents used in laboratory tests.
所述样品通常是在被分析前处理，例如通过离心，或通过膜过滤掉不需要的分子或细胞，例如红细胞。所述样品可在采集后立即测量。样品也可通常在分析前被存储，可选低于-70℃存储。所述目标RNA多核苷酸在用于本申请的方法之前通常从所述样品中提取。RNA提取试剂盒是可从例如，New England和商业获得的。The sample is typically processed before being analyzed, for example by centrifugation, or by membrane filtration to remove unwanted molecules or cells, such as red blood cells. The samples can be measured immediately after collection. Samples can also usually be stored prior to analysis, optionally below -70°C. The target RNA polynucleotides are typically extracted from the sample prior to use in the methods of the present application. RNA extraction kits are available, eg, from New England and commercially.
连接connect
所述衔接体连接到所述目标RNA多核苷酸，以形成修饰的RNA多核苷酸。The adapter is ligated to the target RNA polynucleotide to form a modified RNA polynucleotide.
在具体的实施方案中，通过共价键将所述衔接体连接到所述目标RNA 多核苷酸，所述共价键形成在所述RNA多核苷酸和所述衔接体的各自至少一个反应基团之间；和/或In specific embodiments, the adapter is linked to the target RNA polynucleotide by a covalent bond formed at least one reactive group on each of the RNA polynucleotide and the adapter. between groups; and/or
通过化学或酶促连接将所述衔接体连接到所述RNA多核苷酸。The adapter is ligated to the RNA polynucleotide by chemical or enzymatic ligation.
所述目标RNA多核苷酸可化学地连接到所述衔接体，例如通过共价键。所述目标RNA多核苷酸可通过化学或酶结合连接到所述衔接体。所述目标RNA多核苷酸可通过杂交和/或合成方法连接到所述衔接体。可使用拓扑异构酶将所述RNA多核苷酸连接到所述衔接体。所述RNA多核苷酸可在不止一个，例如两个或三个位点连接到所述衔接体。连接方法可包括一个，两个，三个，四个，五个或更多不同的连接方法。根据本申请可使用任何以下描述的连接方法的组合。The target RNA polynucleotide can be chemically linked to the adapter, for example by a covalent bond. The target RNA polynucleotide can be ligated to the adapter by chemical or enzymatic conjugation. The target RNA polynucleotide can be ligated to the adapter by hybridization and/or synthetic methods. The RNA polynucleotide can be ligated to the adapter using a topoisomerase. The RNA polynucleotide may be linked to the adapter at more than one, eg two or three positions. Connection methods may include one, two, three, four, five or more different connection methods. Combinations of any of the attachment methods described below may be used in accordance with the present application.
所述RNA多核苷酸和所述衔接体可分别制备然后再连接到一起。这两个组分可以以任何构造连接。例如，它们可以通过它们的末端(即5'或3')连接。合适的构造包括，但不限于，RNA多核苷酸的5'末端连接到衔接体的3'末端和反之亦然。或者，这两组分可通过它们序列内部的核苷酸连接。The RNA polynucleotide and the adapter can be prepared separately and then ligated together. These two components can be linked in any configuration. For example, they can be linked via their ends (ie 5' or 3'). Suitable configurations include, but are not limited to, ligation of the 5' end of the RNA polynucleotide to the 3' end of the adapter and vice versa. Alternatively, the two components can be linked via nucleotides within their sequences.
所述RNA多核苷酸可使用一个或多个化学交联剂或一个或多个肽连接体连接到所述衔接体。合适的化学交联剂是本领域众所周知的。合适的化学交联剂包括但不限于，包括以下功能性基团的化学交联剂：马来酰亚胺，活性酯，琥珀酰亚胺，叠氮化物，炔烃(例如二苯并环辛醇(DIBO或DBCO)，二氟脂环烃和线性炔烃)，磷化氢(例如用在无痕和非无痕施陶丁格连接中的磷化氢)，卤代乙酰(例如碘乙酰胺)，光气型试剂，磺酰氯试剂，异硫氰酸酯，酰基卤类，肼，二硫化物，乙烯砜类，氮杂环丙烷和光敏试剂(例如芳香叠氮化物，双氮杂环丙烷)。The RNA polynucleotide can be linked to the adapter using one or more chemical cross-linkers or one or more peptide linkers. Suitable chemical crosslinkers are well known in the art. Suitable chemical cross-linking agents include, but are not limited to, chemical cross-linking agents comprising the following functional groups: maleimides, active esters, succinimides, azides, alkynes (e.g. dibenzocyclooctyl alcohols (DIBO or DBCO), difluoroalicyclic hydrocarbons and linear alkynes), phosphines (such as those used in traceless and non-traceless Staudinger linkages), haloacetyls (such as iodoethyl Amides), phosgene reagents, sulfonyl chloride reagents, isothiocyanates, acid halides, hydrazines, disulfides, vinyl sulfones, aziridines and photosensitive reagents (such as aromatic azides, diazacyclones propane).
所述RNA多核苷酸和所述衔接体之间的反应可为自发的，例如半胱氨酸/马来酰亚胺，或可能需要外部试剂，例如用于连接叠氮化物和线性炔烃的Cu(I)。The reaction between the RNA polynucleotide and the adapter may be spontaneous, such as cysteine/maleimide, or may require external reagents, such as for ligation of azide and linear alkyne Cu(I).
可选的交联剂包括2,5-二氧代吡咯烷-1-基3-(吡啶-2-基二硫烷基)丙酸酯、2,5-二氧代吡咯烷-1-基4-(吡啶-2-基二硫烷基)丁酸酯和2,5-二氧代吡咯烷-1-基8-(吡啶-2-基二硫烷基)辛酸酯、二马来酰亚胺PEG 1k、二马来酰亚胺PEG 3.4k、二马来酰亚胺PEG 5k、二马来酰亚胺PEG 10k、双(马来酰 亚胺基)乙烷(BMOE)、双马来酰亚胺己烷(BMH)、1,4-双马来酰亚胺丁烷(BMB)、1,4-双马来酰亚胺基-2,3-二羟基丁烷(BMDB)、BM[PEO]2(1,8-双马来酰亚胺二乙二醇)、BM[PEO]3(1,11-双马来酰亚胺三乙二醇)、三[2-马来酰亚氨基乙基]胺(TMEA)、DTME二硫代二马来酰亚胺乙烷、双马来酰亚胺PEG3、双马来酰亚胺PEG11、DBCO-马来酰亚胺、DBCO-PEG4-马来酰亚胺、DBCO-PEG4-NH2、DBCO-PEG4-NHS、DBCO-NHS、DBCO-PEG-DBCO 2.8kDa、DBCO-PEG-DBCO 4.0kDa、DBCO-15原子-DBCO、DBCO-26原子-DBCO、DBCO-35原子-DBCO、DBCO-PEG4-S-S-PEG3-生物素、DBCO-S-S-PEG3-生物素和DBCO-S-S-PEG11-生物素。最可选的交联剂是3-(2-吡啶基二硫代)丙酸琥珀酰亚胺酯(SPDP)和马来酰亚胺-PEG(2kDa)-马来酰亚胺(α,ω-双马来酰亚胺基聚乙二醇)。Optional crosslinkers include 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanyl)propionate, 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butyrate and 2,5-dioxopyrrolidin-1-yl 8-(pyridin-2-yldisulfanyl)octanoate, dimale Imide PEG 1k, bismaleimide PEG 3.4k, bismaleimide PEG 5k, bismaleimide PEG 10k, bis(maleimide) ethane (BMOE), bis Maleimide hexane (BMH), 1,4-bismaleimide butane (BMB), 1,4-bismaleimide-2,3-dihydroxybutane (BMDB) , BM[PEO]2 (1,8-bismaleimide diethylene glycol), BM[PEO]3 (1,11-bismaleimide triethylene glycol), three [2-horse Leimidoethyl]amine (TMEA), DTME dithiobismaleimide ethane, bismaleimide PEG3, bismaleimide PEG11, DBCO-maleimide, DBCO -PEG4-maleimide, DBCO-PEG4-NH2, DBCO-PEG4-NHS, DBCO-NHS, DBCO-PEG-DBCO 2.8kDa, DBCO-PEG-DBCO 4.0kDa, DBCO-15atom-DBCO, DBCO- 26 atoms-DBCO, DBCO-35 atoms-DBCO, DBCO-PEG4-S-S-PEG3-biotin, DBCO-S-S-PEG3-biotin and DBCO-S-S-PEG11-biotin. The most preferred crosslinkers are succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and maleimide-PEG (2kDa)-maleimide (α,ω - bismaleimide polyethylene glycol).
所述连接体可被标记。合适的标签包括但不限于，荧光分子(例如Cy3或555)，放射性同位素，如125I，35S，酶，抗体，抗原，多核苷酸和配体例如生物素。这种标签使得可确定连接体的量。所述标签也可是能断裂的纯化标签，例如生物素，或鉴定方法中出现的具体序列。The linker can be labeled. Suitable labels include, but are not limited to, fluorescent molecules (eg Cy3 or 555), radioisotopes such as125I,35S, enzymes, antibodies, antigens, polynucleotides and ligands such as biotin. This tag allows the amount of the linker to be determined. The tag may also be a cleavable purification tag, such as biotin, or a specific sequence present in the identification method.
通过保持连接体的溶度大量过剩于RNA多核苷酸和/或所述衔接体可防止RNA多核苷酸或所述衔接体自身的交联。或者，在其中使用两个连接体的情况下，可使用“锁和钥匙”的设置。各连接体仅一个末端可一起反应以形成更长的连接体，所述各连接体的另一端与构建体(即RNA多核苷酸或所述衔接体)的不同部分反应。Cross-linking of the RNA polynucleotide or the adapter itself can be prevented by maintaining the linker in a large excess in solubility over the RNA polynucleotide and/or the adapter. Alternatively, where two connectors are used, a "lock and key" arrangement may be used. Only one end of each linker can be reacted together to form a longer linker, the other end of each linker reacting with a different part of the construct (ie, the RNA polynucleotide or the adapter).
点击化学click chemistry
所述目标RNA多核苷酸可共价地连接到所述衔接体。所述衔接体可能包含或可能不包含预先结合的DNA解旋酶。在可选实施例中，可使用游离铜点击化学或铜催化的点击化学来制得所述RNA多核苷酸和所述衔接体间的共价键。由于点击化学令人满意的性质和其对于在多种构建块(building blocks)之间生成共价连接的范围，使得在这些应用中使用点击化学。例如，它是快速的，清洁的并且无毒的，只产生无害的副产物。点击化学是由Kolb等在2001首次介绍的术语，为描述更广泛的一系列强大，有选择性的和模块化的构建块，所述构建块可靠地用于小规模和大规模应 用(Kolb HC,Finn,MG,Sharpless KB，点击chemistry:diverse chemical function from a few good reactions,Angew.Chem.Int.Ed.40(2001)2004–2021)。他们定义了如下一系列严格标准用于点击化学：“反应必须是模块化的，宽的范围，给出非常高的产量，只产生无害的可通过非色谱法去除的副产物，并且是立体定向的(但不必然是对映选择性)。所要求的方法特征包括简单的反应条件(理想地所述方法应对氧气和水不敏感)，容易获得的起始物质和试剂，无溶剂或溶剂的使用，所述溶剂是温和的(例如水)或容易去除的，和简单的产物分离。纯化如果需要必须是通过非色谱法，例如结晶或蒸馏，并且所述产物在生理状态下必须是稳定的”。The target RNA polynucleotide can be covalently linked to the adapter. The adapter may or may not contain a pre-bound DNA helicase. In alternative embodiments, free copper click chemistry or copper catalyzed click chemistry can be used to make a covalent bond between the RNA polynucleotide and the adapter. Click chemistry is used in these applications because of its desirable properties and its scope for generating covalent linkages between a variety of building blocks. For example, it is fast, clean and non-toxic, producing only harmless by-products. Click chemistry is a term first introduced by Kolb et al. in 2001 to describe a broader set of robust, selective and modular building blocks that are reliably usable for both small-scale and large-scale applications (Kolb HC , Finn, MG, Sharpless KB, click chemistry:diverse chemical function from a few good reactions, Angew.Chem.Int.Ed.40(2001)2004–2021). They defined the following set of stringent criteria for click chemistry: "The reaction must be modular, broad in scope, give very high yields, produce only harmless by-products that can be removed by non-chromatographic methods, and be stereogenic. Directional (but not necessarily enantioselective). Required process features include simple reaction conditions (ideally the process should be insensitive to oxygen and water), readily available starting materials and reagents, solvent-free or use, the solvent is mild (e.g. water) or easily removed, and simple product isolation. Purification must be by non-chromatographic methods if necessary, such as crystallization or distillation, and the product must be stable under physiological conditions of".
下列实施例说明本申请。The following examples illustrate the application.
实施例1：2’-F-RNA能与DNA解旋酶结合Example 1: 2'-F-RNA can bind to DNA helicase
将带有Cy3标记的2’-F取代修饰的RNA(具体RNA序列为：5’-Substitute the modified RNA with Cy3-labeled 2'-F (the specific RNA sequence is: 5'-
GCCAGAAACG-3’，序列长度：大于6nt即可，序列没有偏好性)以及相同长度和碱基序列的带Cy3标记的DNA(100nM)与20或30倍物质的量的DNA解旋酶。GCCAGAAACG-3', sequence length: greater than 6nt, no sequence preference) and Cy3-labeled DNA (100nM) of the same length and base sequence with 20 or 30 times the amount of DNA helicase.
T4 Dda-M1G/E94C/C109A/C136A/A360C(3μM)和DNA解旋酶Hel308在缓冲液(20mM HEPES(pH 7.0)；50mM NaCl；0.5mM TMAD)中混合并室温孵育60分钟。然后用TBE(天然的)PAGE凝胶分析其结合效率，TBE(天然的)PAGE为4-20％凝胶，160V下运行40分钟，然后用SYBR金染料对核酸进行染色。T4 Dda-M1G/E94C/C109A/C136A/A360C (3 μM) and DNA helicase Hel308 were mixed in buffer (20 mM HEPES (pH 7.0); 50 mM NaCl; 0.5 mM TMAD) and incubated at room temperature for 60 min. Then use TBE (native) PAGE gel to analyze its binding efficiency, TBE (native) PAGE is 4-20% gel, run at 160V for 40 minutes, and then use SYBR gold dye to stain the nucleic acid.
结果分别如图1和图2所示，从图中可以看出，DNA解旋酶T4 Dda-M1G/E94C/C109A/C136A/A360C和DNA解旋酶Hel308均能与2’-F-RNA良好的结合，并且结合效果不逊于该酶与DNA的结合。因此，2’-F-RNA序列可以用于纳米孔RNA测序的衔接体制备。The results are shown in Figure 1 and Figure 2, respectively, as can be seen from the figure, DNA helicase T4 Dda-M1G/E94C/C109A/C136A/A360C and DNA helicase Hel308 can be well with 2'-F-RNA , and the binding effect is not inferior to the binding of the enzyme to DNA. Therefore, 2'-F-RNA sequences can be used for adapter preparation for nanopore RNA sequencing.
DNA解旋酶T4 Dda-M1G/E94C/C109A/C136A/A360C的氨基酸序列如下SEQ ID NO.1所示：The amino acid sequence of DNA helicase T4 Dda-M1G/E94C/C109A/C136A/A360C is shown in SEQ ID NO.1 below:
Figure PCTCN2022139679-appb-000001
Figure PCTCN2022139679-appb-000001
Figure PCTCN2022139679-appb-000002
Figure PCTCN2022139679-appb-000002
DNA解旋酶Hel308的氨基酸序列如SEQ ID NO.2所示：The amino acid sequence of DNA helicase Hel308 is shown in SEQ ID NO.2:
Figure PCTCN2022139679-appb-000003
Figure PCTCN2022139679-appb-000003
实施例2：含2’-F-RNA前导链的测序接头复合物的孵育和制备Example 2: Incubation and preparation of sequencing adapter complexes containing 2'-F-RNA leading strand
合成如下序列：Synthesize the following sequence:
RNA-Y1:RNA-Y1:
Figure PCTCN2022139679-appb-000004
Figure PCTCN2022139679-appb-000004
Figure PCTCN2022139679-appb-000005
Figure PCTCN2022139679-appb-000005
RNA-YB:RNA-YB:
Figure PCTCN2022139679-appb-000006
Figure PCTCN2022139679-appb-000006
RNA-Y2:5'- CGCCTCAGTTTGCCATCTTCAGC-3' RNA-Y2:5'- CGCCTCAGTTTGCCATCTTCAGC -3'
分别合成RNA-Y1；RNA-YB以及RNA-Y2链将这三条链分别以1:1.1:1.1在退火缓冲液中进行退火处理形成Y型接头，退火处理具体为从95℃缓慢降温到25℃，降温幅度不超过0.1℃/s。退火缓冲液包括160mM HEPES 7.0，200mM NaCl。Synthesize RNA-Y1; RNA-YB and RNA-Y2 strands respectively. Anneal the three strands in the annealing buffer at a ratio of 1:1.1:1.1 to form a Y-shaped joint. The annealing process is to slowly cool down from 95°C to 25°C , the cooling range does not exceed 0.1°C/s. Annealing buffer includes 160mM HEPES 7.0, 200mM NaCl.
需要注意的是：糖环2’-F修饰，是一种较为常见的技术，在本申请实施例中2’-F修饰的碱基都是U，不需要过多考虑形成二级结构的问题，15个U的长度根据具体酶所占空间的大小确定，经验证，可以至少结合1个酶，且不是2个酶。It should be noted that the 2'-F modification of the sugar ring is a relatively common technique. In the examples of this application, the bases of the 2'-F modification are all U, and there is no need to consider the formation of secondary structures too much. , the length of 15 U is determined according to the size of the space occupied by the specific enzyme. It has been verified that at least one enzyme can be combined, not two enzymes.
在本申请实施例中，i2OMe代表的是糖环修饰的一种，即2’-甲氧基修饰。In the examples of this application, i2OMe represents a kind of sugar ring modification, that is, 2'-methoxy modification.
在本申请实施例中，iXNA，是一种LNA，与Y1中的iSp18一起用于联合阻滞酶。In the embodiment of the present application, iXNA, which is a kind of LNA, is used together with iSp18 in Y1 to block enzymes.
取500nM Y型接头、15倍物质的量的DNA解旋酶Hel308(其移动方向是3’到5’端)混合并室温孵育30分钟，然后加入1500倍物质的量的M-P-M并且室温孵育1h；制备得到测序接头复合物，用TBE PAGE凝胶160V下运行40分钟进行分析，并且用SYBR金染料进行染色，其孵育结合效果如图3所示。图3结果显示，形成了测序接头复合物。Take 500nM Y-type linker, DNA helicase Hel308 (the moving direction is 3' to 5' end) with 15 times the amount of substance, mix and incubate at room temperature for 30 minutes, then add M-P-M with 1500 times the amount of substance and incubate at room temperature for 1h; The sequencing adapter complex was prepared, analyzed by running TBE PAGE gel at 160V for 40 minutes, and stained with SYBR gold dye. The incubation binding effect is shown in Figure 3. The results in Figure 3 show that a sequencing adapter complex was formed.
之后将测序接头复合物加入DNAPac PA200柱，用洗脱缓冲液进行纯化，以将没有结合到测序接头复合物上的酶从柱子上洗脱掉。然后用10倍柱体积的缓冲液A和缓冲液B的混合物对测序接头复合物进行洗脱。然 后汇集主洗脱峰，测量其浓度，获得RNA测序接头并用TBE PAGE凝胶160V下运行40分钟。其中，缓冲液A：20mMNa-CHES，250mM NaCl，4％(W/V)甘油，pH 8.6；缓冲液B：20mM Na-CHES，1MNaCl，4％(W/V)甘油，pH 8.6，最终结果如图4所示。The sequencing adapter complex was then added to a DNAPac PA200 column and purified with elution buffer to elute the enzymes not bound to the sequencing adapter complex from the column. The sequencing adapter complex was then eluted with 10 column volumes of a mixture of buffer A and buffer B. Then the main elution peaks were pooled, their concentrations were measured, and the RNA sequencing adapters were obtained and run for 40 minutes with a TBE PAGE gel at 160V. Among them, buffer A: 20mMNa-CHES, 250mM NaCl, 4% (W/V) glycerol, pH 8.6; buffer B: 20mM Na-CHES, 1MNaCl, 4% (W/V) glycerol, pH 8.6, the final result As shown in Figure 4.
实施例3：2’-F-RNA前导链的测序接头复合物的上机测试Example 3: On-machine testing of the sequencing adapter complex of the 2'-F-RNA leading strand
用牛津纳米孔科技公司的RNA直接建库试剂盒SQK-RNA002，其中将RMX组分替换成实施例2中所制备的纯化后的含有2’-F-RNA前导链的测序接头复合物，最终在ONT的MinION平台上进行测试收集信号如图5所示。结果表明，实施例2的接头或接头复合物可以用于纳米孔测序。Using the RNA direct library construction kit SQK-RNA002 of Oxford Nanopore Technology Company, wherein the RMX component was replaced with the purified sequencing adapter complex containing the 2'-F-RNA leading strand prepared in Example 2, and finally The signals collected during the test on the ONT's MinION platform are shown in Figure 5. The results show that the linker or linker complex of Example 2 can be used for nanopore sequencing.
另外，本文中术语“和/或”，仅仅是一种描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示：单独存在A，同时存在A和B，单独存在B这三种情况。另外，本文中字符“/”，一般表示前后关联对象是一种“或”的关系。In addition, the term "and/or" in this article is only an association relationship describing associated objects, which means that there may be three relationships, for example, A and/or B may mean: A exists alone, A and B exist at the same time, There are three cases of B alone. In addition, the character "/" in this article generally indicates that the contextual objects are an "or" relationship.
应理解，在本申请实施例中，“与A相应的B”表示B与A相关联，根据A可以确定B。但还应理解，根据A确定B并不意味着仅仅根据A确定B，还可以根据A和/或其它信息确定B。It should be understood that in this embodiment of the present application, "B corresponding to A" means that B is associated with A, and B can be determined according to A. However, it should also be understood that determining B according to A does not mean determining B only according to A, and B may also be determined according to A and/or other information.
以上所述，仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到各种等效的修改或替换，这些修改或替换都应涵盖在本申请的保护范围之内。因此，本申请的保护范围应以权利要求的保护范围为The above is only a specific embodiment of the application, but the scope of protection of the application is not limited thereto. Any person familiar with the technical field can easily think of various equivalents within the scope of the technology disclosed in the application. Modifications or replacements, these modifications or replacements shall be covered within the scope of protection of this application. Therefore, the protection scope of the present application should be based on the protection scope of the claims

Claims (19)

  1. 一种用于表征目标多核苷酸的衔接体，包含解旋酶的结合区域，所述结合区域包含修饰的RNA多核苷酸，用于结合或装载所述解旋酶。An adapter for characterizing a polynucleotide of interest comprising a helicase binding region comprising a modified RNA polynucleotide for binding or loading the helicase.
  2. 根据权利要求1所述的衔接体，其中，The adapter according to claim 1, wherein,
    所述解旋酶包括DNA解旋酶；和/或The helicase comprises a DNA helicase; and/or
    所述修饰的RNA多核苷酸选自糖环2’-F修饰的RNA；和/或The modified RNA polynucleotide is selected from sugar ring 2'-F modified RNA; and/or
    所述解旋酶的结合区域不包含DNA。The binding region of the helicase does not contain DNA.
  3. 根据权利要求1或2所述的衔接体，其中，所述衔接体包含优先地穿入纳米孔的前导序列；The adapter according to claim 1 or 2, wherein said adapter comprises a leader sequence that preferentially penetrates into a nanopore;
    可选地，所述解旋酶的结合区域位于所述前导序列。Optionally, the binding region of the helicase is located in the leader sequence.
  4. 根据权利要求1或2所述的衔接体，其中，所述目标多核苷酸为目标RNA多核苷酸和/或目标DNA多核苷酸；The adapter according to claim 1 or 2, wherein the target polynucleotide is a target RNA polynucleotide and/or a target DNA polynucleotide;
    所述目标多核苷酸为单链或双链；The target polynucleotide is single-stranded or double-stranded;
    可选地，通过共价键将所述衔接体连接到所述目标多核苷酸，所述共价键形成在所述RNA多核苷酸和所述非核苷酸的各自至少一个反应基团之间；和/或Optionally, the adapter is linked to the target polynucleotide by a covalent bond formed between the RNA polynucleotide and at least one reactive group each of the non-nucleotides ;and / or
    通过化学或酶促连接将所述衔接体连接到所述目标多核苷酸。The adapter is ligated to the polynucleotide of interest by chemical or enzymatic ligation.
  5. 一种表征目标多核苷酸的方法，所述方法使用如权利要求1至4中任一项所述的衔接体。A method of characterizing a polynucleotide of interest using the adapter of any one of claims 1-4.
  6. 根据权利要求5所述的方法，其中，所述方法包括：The method according to claim 5, wherein said method comprises:
    a)提供(i)多核苷酸构建体和(ii)解旋酶，所述多核苷酸构建体包含所述目标多核苷酸和如权利要求1至4中任一项所述的衔接体；所述解旋酶包括DNA解旋酶；A) providing (i) a polynucleotide construct and (ii) a helicase, the polynucleotide construct comprising the target polynucleotide and the adapter according to any one of claims 1 to 4; The helicase comprises a DNA helicase;
    b)将a)中提供的所述多核苷酸构建体和所述解旋酶与跨膜孔接触，使得所述解旋酶控制所述目标多核苷酸相对于所述跨膜孔的移动；b) contacting the polynucleotide construct provided in a) and the helicase with a transmembrane pore such that the helicase controls movement of the polynucleotide of interest relative to the transmembrane pore;
    c)随着所述目标多核苷酸相对于所述跨膜孔移动，获取一个或多个测量值，其中所述测量值代表所述目标多核苷酸的一个或多个特征，并由此表征所述目标多核苷酸。c) taking one or more measurements as the target polynucleotide moves relative to the transmembrane pore, wherein the measurements represent one or more characteristics of the target polynucleotide and thereby characterize The target polynucleotide.
  7. 根据权利要求6所述的方法，其特征在于，所述一个或多个特征选自(i)所述目标多核苷酸的长度，(ii)所述目标多核苷酸的同一性，(iii)所述目标多核苷酸的序列，(iv)所述目标多核苷酸的二级结构和(v)所述目标多核苷酸是否是修饰的。The method of claim 6, wherein the one or more characteristics are selected from (i) the length of the target polynucleotide, (ii) the identity of the target polynucleotide, (iii) The sequence of the target polynucleotide, (iv) the secondary structure of the target polynucleotide and (v) whether the target polynucleotide is modified.
  8. 根据权利要求6或7所述的方法，其中，所述目标多核苷酸的一个或多个特征可以通过电测量和/或光测量来测量。The method of claim 6 or 7, wherein one or more characteristics of the target polynucleotide are measurable by electrical and/or optical measurements.
  9. 根据权利要求6或7所述的方法，其中，步骤c)包括随着所述目标多核苷酸相对于所述跨膜孔移动，测量流过所述跨膜孔的电流，其中所述电流代表所述目标多核苷酸的一个或多个特征，并由此表征所述目标多核苷酸。The method according to claim 6 or 7, wherein step c) comprises measuring the current flowing through the transmembrane pore as the target polynucleotide moves relative to the transmembrane pore, wherein the current represents One or more characteristics of the target polynucleotide, thereby characterizing the target polynucleotide.
  10. 根据权利要求6或7所述的方法，其中，所述目标多核苷酸额外地或进一步通过甲基化、氧化、损伤、用一个或多个蛋白，或用一个或多个标记物、标签或阻断链进行修饰。The method according to claim 6 or 7, wherein the target polynucleotide is additionally or further detected through methylation, oxidation, damage, with one or more proteins, or with one or more markers, tags or Block chains for modification.
  11. 根据权利要求6或7所述的方法，其中，所述目标多核苷酸可以使用一个或多个锚耦合到所述膜。The method of claim 6 or 7, wherein the target polynucleotide is coupled to the membrane using one or more anchors.
  12. 根据权利要求6或7所述的方法，其中，所述解旋酶包含修饰，以减小多核苷酸结合域中开口的大小，所述目标多核苷酸可以在至少一个构象状态下穿过所述开口从所述解旋酶上解绑。The method of claim 6 or 7, wherein the helicase comprises a modification to reduce the size of the opening in the polynucleotide binding domain through which the target polynucleotide can pass in at least one conformational state. The opening is unbound from the helicase.
  13. 根据权利要求6或7所述的方法，其中，所述解旋酶为：The method according to claim 6 or 7, wherein the helicase is:
    a)Hel308解旋酶、RecD解旋酶、XPD解旋酶、Dda解旋酶、Tral解旋酶、TrwC解旋酶；a) Hel308 helicase, RecD helicase, XPD helicase, Dda helicase, Tral helicase, TrwC helicase;
    b)衍生自a)中所述任何解旋酶的解旋酶；或b) a helicase derived from any of the helicases described in a); or
    c)a)和/或b)中所述解旋酶的任意组合。c) Any combination of the helicases described in a) and/or b).
  14. 根据权利要求6或7所述的方法，其中，所述跨膜孔是蛋白孔或固态孔。The method according to claim 6 or 7, wherein the transmembrane pore is a protein pore or a solid pore.
  15. 根据权利要求8至14中任一项所述的方法，其中，所述跨膜蛋白质孔是蛋白孔，并衍生自如下任一种或多种：溶血素、杀白细胞素，耻垢分枝杆菌(Mycobacterium smegmatis)孔蛋白A(MspA)、MspB、MspC、MspD、胞溶素(lysenin)、CsgG、外膜孔蛋白F(OmpF)、外膜孔蛋白G(OmpG)，外膜磷脂酶A、奈瑟球菌属(Neisseria)自转运脂蛋白(NalP)和WZA。The method according to any one of claims 8 to 14, wherein the transmembrane protein pore is a protein pore derived from any one or more of: hemolysin, leukocidin, Mycobacterium smegmatis (Mycobacterium smegmatis) porin A (MspA), MspB, MspC, MspD, lysenin, CsgG, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP) and WZA.
  16. 一种目标多核苷酸相对于跨膜孔移动的方法，所述移动被解旋酶控制，所述方法包括：A method for the movement of a target polynucleotide relative to a transmembrane pore, the movement being controlled by a helicase, the method comprising:
    a)提供(i)目标RNA多核苷酸或目标DNA多核苷酸，和(ii)解旋酶，所述目标RNA多核苷酸或目标DNA多核苷酸被修饰以包含用于结合或装载所述解旋酶的修饰的RNA多核苷酸区域充当DNA解旋酶的结合区域；a) providing (i) a target RNA polynucleotide or a target DNA polynucleotide, and (ii) a helicase, the target RNA polynucleotide or target DNA polynucleotide being modified to comprise The modified RNA polynucleotide region of the helicase acts as a binding region for the DNA helicase;
    其中，所述修饰的RNA多核苷酸包含2’-F修饰的RNA；Wherein, the modified RNA polynucleotide comprises 2'-F modified RNA;
    b)将a)中提供的所述目标RNA多核苷酸或目标DNA多核苷酸、和所述解旋酶与跨膜孔接触，使得所述解旋酶控制所述RNA多核苷酸相对于所述跨膜孔的移动。b) contacting the target RNA polynucleotide or the target DNA polynucleotide provided in a), and the helicase with a transmembrane pore such that the helicase controls the relative relation of the RNA polynucleotide to the Movement of the transmembrane pore.
  17. 一种复合物，所述复合物包含权利要求1至4中任一项所述的衔接体和解旋酶；A complex comprising the adapter and helicase of any one of claims 1 to 4;
    所述解旋酶包括所述的DNA解旋酶；The helicase includes the DNA helicase;
    可选地，所述DNA解旋酶选自：Optionally, the DNA helicase is selected from:
    a)Hel308解旋酶、RecD解旋酶、XPD解旋酶、Dda解旋酶、Tral解旋酶、或TrwC解旋酶；a) Hel308 helicase, RecD helicase, XPD helicase, Dda helicase, Tral helicase, or TrwC helicase;
    b)衍生自a)中所述任何解旋酶的解旋酶；或b) a helicase derived from any of the helicases described in a); or
    c)a)和/或b)中所述解旋酶的任意组合。c) Any combination of the helicases described in a) and/or b).
  18. 一种用于表征目标多核苷酸的试剂盒，所述试剂盒包含权利要求1至4中任一项所述的衔接体和所述解旋酶或权利要求18所述的复合物；A kit for characterizing a target polynucleotide, the kit comprising the adapter according to any one of claims 1 to 4 and the helicase or the complex according to claim 18;
    所述目标多核苷酸为目标RNA多核苷酸或目标DNA多核苷酸。The target polynucleotide is a target RNA polynucleotide or a target DNA polynucleotide.
  19. 一种分离的多核苷酸，所述多核苷酸包含RNA多核苷酸或DNA多核苷酸，和修饰的RNA多核苷酸区域，所述修饰的RNA多核苷酸区域用于结合解旋酶；an isolated polynucleotide comprising an RNA polynucleotide or a DNA polynucleotide, and a modified RNA polynucleotide region for binding a helicase;
    其中，所述修饰的RNA多核苷酸包含2’-F修饰的RNA；Wherein, the modified RNA polynucleotide comprises 2'-F modified RNA;
    所述解旋酶包括DNA解旋酶。The helicases include DNA helicases.
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