WO2023116575A1 - Adaptateur pour la caractérisation d'un polynucléotide cible, procédé et utilisation de celui-ci - Google Patents

Adaptateur pour la caractérisation d'un polynucléotide cible, procédé et utilisation de celui-ci 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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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

L'invention concerne un adaptateur pour caractériser un polynucléotide cible, un procédé et une utilisation de celui-ci. L'invention concerne un adaptateur pour caractériser un polynucléotide cible. L'adaptateur comprend une région de liaison d'une ADN hélicase. La région de liaison comprend un polynucléotide d'ARN modifié pour lier l'ADN hélicase. L'invention concerne en outre un procédé de caractérisation du polynucléotide cible. Le procédé utilise l'adaptateur.
PCT/CN2022/139679 2021-12-21 2022-12-16 Adaptateur pour la caractérisation d'un polynucléotide cible, procédé et utilisation de celui-ci WO2023116575A1 (fr)

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Citations (7)

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WO2006074334A2 (fr) * 2005-01-05 2006-07-13 Biohelix Corporation Identification de cibles d'arn au moyen d'helicases
US20120195936A1 (en) * 2009-07-31 2012-08-02 Ethris Gmbh Rna with a combination of unmodified and modified nucleotides for protein expression
US20170073743A1 (en) * 2014-10-14 2017-03-16 MS+hu 2 +l ARRAY LLC Fluorous oligonucleotide microarray
US20170253923A1 (en) * 2014-10-17 2017-09-07 Oxford Nanopore Technologies Ltd. Method for nanopore rna characterisation
US20180030525A1 (en) * 2014-09-29 2018-02-01 The Regents Of The University Of California Nanopore sequencing of polynucleotides with multiple passes
WO2021036995A1 (fr) * 2019-08-23 2021-03-04 Nanjing University Séquençage direct de micro-arn à l'aide d'un séquençage assisté par enzyme
US20210348224A1 (en) * 2018-05-23 2021-11-11 The Regents Of The University Of California Methods of Analyzing Capped Ribonucleic Acids

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WO2006074334A2 (fr) * 2005-01-05 2006-07-13 Biohelix Corporation Identification de cibles d'arn au moyen d'helicases
US20120195936A1 (en) * 2009-07-31 2012-08-02 Ethris Gmbh Rna with a combination of unmodified and modified nucleotides for protein expression
US20180030525A1 (en) * 2014-09-29 2018-02-01 The Regents Of The University Of California Nanopore sequencing of polynucleotides with multiple passes
US20170073743A1 (en) * 2014-10-14 2017-03-16 MS+hu 2 +l ARRAY LLC Fluorous oligonucleotide microarray
US20170253923A1 (en) * 2014-10-17 2017-09-07 Oxford Nanopore Technologies Ltd. Method for nanopore rna characterisation
US20210348224A1 (en) * 2018-05-23 2021-11-11 The Regents Of The University Of California Methods of Analyzing Capped Ribonucleic Acids
WO2021036995A1 (fr) * 2019-08-23 2021-03-04 Nanjing University Séquençage direct de micro-arn à l'aide d'un séquençage assisté par enzyme

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