CN116497102A - Adaptors, methods for characterizing polynucleotides of interest and uses thereof - Google Patents

Abstract

The invention provides an adapter, a method and application for representing target polynucleotide. The invention provides an adapter for characterizing a target polynucleotide, the adapter comprising a binding region for a DNA helicase, the binding region comprising a modified RNA polynucleotide for binding to the DNA helicase. The invention also provides methods of characterizing polynucleotides of interest using the adaptors. The invention provides modified RNA capable of combining with DNA, which can be used for preparing an adapter for sequencing nano-pore RNA, and the adapter can greatly enrich the diversity of RNA sequencing and provide a good foundation for the further development of nano-pore RNA sequencing.

Description

Adaptors, methods for characterizing polynucleotides of interest and uses thereof

Cross Reference to Related Applications

The present application claims priority from chinese patent application 202111573545.8 entitled "adaptors, methods for characterising polynucleotides of interest, and uses thereof," filed on 12/21 of 2021, the entire contents of which are incorporated herein by reference.

Technical Field

The invention belongs to the field of gene sequencing, relates to an adapter used in characterization of polynucleotides, and also relates to a method for characterizing polynucleotides by using the adapter.

Background

The nanopore sequencing technology has the characteristics of long reading length, direct reading of modification information and parallel analysis of real-time data production, and has obvious advantages in detection of long fragment nucleic acid detection variation (including but not limited to point mutation, indels, inversion translocation, gene fusion, RNA abnormal shearing, RNA editing and other nucleic acid related variation) and modification information (including but not limited to methylation, acetylation and the like) compared with a second generation sequencing platform or other sequencing platforms. The platform supports the parallel characteristics of data production and analysis, realizes real-time variation/modification detection and diagnosis, and has wide application prospect due to the portable design.

When a voltage is applied across the nanopore, the current drops as the analyte (e.g., polynucleotide, polypeptide) passes through the nanopore, and the degree of current interruption by analytes of different structures varies. The current will change when the analyte stays in the nanopore barrel (barrel) for a period of time. Nanopore detection nucleotides give a current change of known characteristics and duration.

There is a need for a rapid and inexpensive polynucleotide (e.g., DNA or RNA) sequencing and identification technique that has a wide range of applications. The prior art is slow and expensive, mainly because they rely on amplification techniques to produce large amounts of polynucleotides, and require large amounts of specific fluorescent chemicals for signal detection.

Messenger RNA provides an observation of organism dynamics and the benefits and applications of direct RNA sequencing are enormous, including for health screening; such as metastatic processes and heart disease for certain cancers. Direct RNA sequencing can be used in investigating disease resistance of crops, determining stress factors such as drought, ultraviolet light and salinity responses of crops, and in cell differentiation and determination of embryo development processes.

The problem in the direct sequencing of RNA, particularly 500 or more nucleotides, is to find a suitable molecular motor capable of controlling the translocation of RNA through a transmembrane pore. To date, molecular motors for RNA and providing continuous movement have not been presented. For characterization or sequencing of polynucleotides, continuous movement of the RNA polymer and the ability to read long fragment polymers are required.

International patent application No. pct/GB2014/053121 (WO 2015/056028) discloses a method of characterizing ribonucleic acid (RNA) of interest comprising forming complementary polynucleotides, and then characterizing the complementary polynucleotides using a transmembrane pore. Such indirect RNA characterization is prone to error and may lead to loss of important information of the methylation status of the RNA. Other important modifications in the conversion of RNA to cDNA may also be hidden.

International patent application WO2016059436A1 discloses a method for the characterization of nanopore RNA. It characterizes RNA using a DNA helicase that reads the target RNA sequence "tricked" in nature by the presence of a non-RNA leader sequence. Once the movement of the DNA helicase is initiated by the non-RNA polynucleotide (which may comprise DNA or DNA analogs), it may continue to move along the RNA.

Clearly, both of these approaches define that current nanopore RNA sequencing must provide RNA polynucleotides comprising DNA modified leader sequences, which greatly limits the sequence diversity of existing RNA sequencing adaptors.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a novel adapter, and also provides a preparation method of the adapter and application of the adapter in nanopore sequencing. The adapter of the invention directly uses modified RNA to combine with helicase, greatly enriches the diversity of RNA sequencing, and provides a good foundation for the further development of nanopore RNA sequencing.

The invention aims at realizing the following technical scheme:

in a first aspect the invention provides an adaptor for characterising a target polynucleotide, the adaptor comprising a binding region for a helicase, the binding region for a helicase comprising a modified RNA polynucleotide for binding or loading the helicase.

The adaptor according to the present invention, wherein,

the helicase comprises a DNA helicase; and/or

The modified RNA polynucleotide is selected from sugar ring 2' -F modified RNA; and/or

The binding region of the helicase does not comprise DNA.

The adaptor according to the invention, wherein the adaptor comprises a leader sequence that preferentially penetrates into the nanopore;

preferably, the binding region of the helicase is located in the leader sequence.

The adaptor according to the present invention, wherein the target polynucleotide is a target RNA polynucleotide and/or a target DNA polynucleotide, preferably a target RNA polynucleotide;

the target polynucleotide is single-stranded or double-stranded;

preferably, the adaptor is attached to the target polynucleotide by a covalent bond formed between the RNA polynucleotide and the respective at least one reactive group of the non-nucleotide; and/or

Ligating the adaptor to the RNA polynucleotide by chemical or enzymatic ligation. The adaptor according to the present invention, wherein the DNA helicase is:

a) Hel308 helicase, recD helicase, XPD helicase, dda helicase, tral helicase, or TrwC helicase;

b) A helicase derived from any of the helicases described in a); or (b)

c) any combination of the helicases described in a) and/or b).

In a second aspect the invention provides a method of characterising a target polynucleotide, the method using the adaptor.

The method according to the invention, wherein the target polynucleotide is a target RNA polynucleotide and/or a target DNA polynucleotide, preferably a target RNA polynucleotide;

the target polynucleotide is single-stranded or double-stranded;

preferably, the method comprises:

a) Providing (i) a polynucleotide construct comprising the polynucleotide of interest and the adaptor, and (ii) a helicase; the helicase comprises the DNA helicase;

b) Contacting the polynucleotide construct provided in a) and the helicase with a transmembrane pore such that the helicase controls movement of the target polynucleotide relative to the transmembrane pore;

c) One or more measurements are obtained 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.

The method of the invention, 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.

The method according to the invention, wherein one or more characteristics of the target polynucleotide may be measured by electrical and/or optical measurements.

The method according to the invention, 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 is representative of one or more characteristics of the target polynucleotide and thereby characterising the target polynucleotide.

The method according to the invention, wherein the target RNA polynucleotide is additionally or further modified by methylation, oxidation, damage, with one or more proteins, or with one or more markers, tags or blocking chains.

The method of the invention, wherein the target polynucleotide may be coupled to the membrane using one or more anchors.

The method of the invention, wherein the helicase comprises a modification to reduce the size of an opening in a polynucleotide binding domain through which the target polynucleotide can be unbound from the helicase in at least one conformational state.

The method according to the invention, wherein the one or more helicases are as described previously.

The method according to the invention, wherein the method further comprises the use of one or more molecular brakes derived from the helicase, which molecular brakes are modified such that they bind to the polynucleotide but do not function as helicase.

The method according to the invention, wherein the transmembrane pore may be a protein pore or a solid state pore.

The method of the invention, wherein the transmembrane protein pore is a protein pore and is derived from any one or more of the following: haemolysin, leukocidal, mycobacterium smegmatis (Mycobacterium smegmatis) porin a (MspA), mspB, mspC, mspD, lysin (lyserin), csgG, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase a, neisseria (Neisseria) self-transporter lipoproteins (NalP) and WZA.

The third aspect of the invention also provides a method of moving a target polynucleotide relative to a transmembrane pore, the movement being controlled by a helicase, the method comprising:

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 a modified RNA polynucleotide region for binding or loading the helicase acting as a binding region for the DNA helicase;

Wherein the modified RNA polynucleotide comprises a 2' -F modified RNA;

the helicase comprises the DNA helicase;

b) Contacting the target RNA polynucleotide or target DNA polynucleotide provided in a), and the helicase with a transmembrane pore such that the helicase controls movement of the RNA polynucleotide relative to the transmembrane pore.

In a fourth aspect the invention provides a complex comprising said adaptor and helicase;

the helicase comprises the DNA helicase;

preferably, the DNA helicase is selected from:

a) Hel308 helicase, recD helicase, XPD helicase, dda helicase, tral helicase, or TrwC helicase;

b) A helicase derived from any of the helicases described in a); or (b)

c) any combination of the helicases described in a) and/or b).

In a fifth aspect the invention provides a kit for characterising a polynucleotide of interest, said kit comprising said adaptor and said helicase or said complex;

the target polynucleotide is target RNA polynucleotide or target DNA polynucleotide.

In a sixth aspect the invention provides an isolated polynucleotide comprising an RNA polynucleotide or a DNA polynucleotide, and a modified RNA polynucleotide region, the modified RNA polynucleotide and/or non-nucleotide region being for binding a helicase;

Wherein the modified RNA polynucleotide comprises a 2' -F modified RNA;

the helicase comprises the DNA helicase.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the modified RNA which can be combined with DNA helicase is less prone to degradation compared with RNA in the related technology, can be used for preparing adapters for sequencing nanopore polynucleotides including RNA and DNA, greatly enriches the diversity of RNA sequencing by using the adapters, and provides a good basis for further development of the sequencing of the nanopore RNA.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort to a person of ordinary skill in the art.

FIG. 1 shows the binding of DNA helicase T4 Dda to ssDNA of the same length and 2' -F-RNA;

FIG. 2 shows the binding of the DNA helicase Hel308 to ssDNA of the same length and 2' -F-RNA;

FIG. 3 shows an electrophoretically detected pattern of the complex formed by the DNA helicase Hel308 binding to the Y-type adaptor;

FIG. 4 shows an electrophoretogram of the DNA helicase Hel308 after purification to form a complex after binding to the Y-adapter;

FIG. 5 shows a signal diagram that is a complex that can be used for nanopore sequencing.

Detailed Description

It will be appreciated that the different applications of the disclosed products and methods may be adapted to the specific needs of the art. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

In addition, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes two or more such proteins, reference to "a polynucleotide binding protein includes two or more such proteins, reference to" a helicase "includes two or more helicases, reference to" a monomer "refers to two or more monomers, reference to" a pore "includes two or more pores, and the like.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Adapter body

The invention first provides an adapter for characterizing a target polynucleotide, the adapter comprising a binding region for a DNA helicase, the binding region comprising a modified RNA polynucleotide for binding to the DNA helicase.

In one embodiment of the invention, the helicase comprises a DNA helicase. The helicase may be a multimeric or oligomeric helicase. It will be appreciated that the helicase may need to form a multimer or oligomer such as a dimer to function. In such embodiments, two or more portions cannot be on different monomers. The helicase is preferably monomeric. It will be appreciated that the helicase preferably does not need to form a multimer or oligomer such as a dimer to function. For example, hel308, recD, traI and XPD helicases are all monomeric helicases.

The monomeric helicase may comprise several domains attached together. For example, the TraI helicase and TraI subgroup helicase can contain two RecD helicase domains, a release enzyme domain, and a C-terminal domain. These domains typically form a monomeric helicase that is capable of functioning without forming oligomers.

In one embodiment of the invention, the modified RNA polynucleotide is selected from sugar ring 2' -F modified RNA.

In one embodiment of the invention, the binding region of the gyrase comprises a non-deoxyribonucleic acid.

In one embodiment of the invention, the adapter comprises a leader sequence that preferentially penetrates into the nanopore;

in a specific embodiment, the binding region of the DNA helicase is located in the leader sequence.

The adaptors of the present invention are more suitable for the characterization of target RNA polynucleotides. In particular embodiments, the adaptor can be attached to the target RNA polynucleotide by a covalent bond formed between the RNA polynucleotide and at least one respective reactive group of the adaptor; and/or ligating the adaptor to the RNA polynucleotide by chemical or enzymatic ligation.

The target RNA polynucleotide is preferably modified by ligating an adapter of the invention to the RNA. The adaptors of the present invention facilitate the characterization method of the present invention. The adaptors of the present invention are designed to preferentially penetrate the pores and thus facilitate movement of the polynucleotide through the pores. The adaptors of the present invention may also be used to ligate the target RNA polynucleotide to one or more anchors as described below. The adaptors of the present invention may be ligated to the target RNA polynucleotides.

The adaptors of the present invention generally comprise a polymeric region. The polymer region is preferably negatively charged. The polymer is preferably a polynucleotide, such as DNA, modified polynucleotide (e.g., abasic DNA), PNA, LNA, polyethylene glycol (PEG), or polypeptide.

The adaptors of the present invention preferably comprise one or more blocking strands.

Blocking chain

One or more blocking strands are included in the target polynucleotide. One or more blocking strands are included in the target RNA polynucleotide and/or the target DNA polynucleotide. The blocking strand or strands are preferably part of the polynucleotide of interest, for example it/they interrupt the polynucleotide sequence. The one or more blocking strands are preferably not part of one or more block molecules, such as a deceleration strip, that hybridizes to the polynucleotide of interest.

There are any number of blocking strands in the target polynucleotide, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more blocking strands. Preferably there are 2, 4 or 6 blocking strands in the target polynucleotide. The target polynucleotide may have a blocking strand in a different region, such as a blocking strand in a leader sequence and a blocking strand in a 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. The one or more blocking strands may arrest the one or more helicases by reducing their drag (e.g., by removing bases from nucleotides in the target polynucleotide) or physically blocking their movement (e.g., with bulky chemical groups).

The one or more blocking strands may include any molecule or combination of any molecules that arrest one or more helicases. The one or more blocking strands may include any molecule or combination of any molecules that prevent the one or more helicases from moving along the target polynucleotide. It directly determines whether one or more helicases remain at one or more blocking strands in the absence of a transmembrane pore and an applied potential. For example, the tests shown in the examples, such as the ability of helicase to pass through a blocked strand and displace the complementary strand of DNA, can be measured by PAGE.

The one or more blocking chains typically comprise a linear molecule such as a polymer. The one or more blocking strands generally have a different structure than the target polynucleotide. For example, if the target polynucleotide is DNA, one or more of the blocked strands is typically not deoxyribonucleic acid. In particular, if the target polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), the one or more blocking strands preferably include Peptide Nucleic Acid (PNA), glycerol Nucleic Acid (GNA), threose Nucleic Acid (TNA), locked Nucleic Acid (LNA) or synthetic polymers having nucleotide side chains.

The one or more blocking chains preferably comprise one or more nitroindoles, e.g. one or more 5-nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuracils, one or more inverted thymidines (inverted dTs), one or more inverted deoxythymidines (ddTs), one or more dideoxycytidines (ddCs), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2 'alkoxy-modified ribonucleotides (preferably 2' methoxy-modified ribonucleotides), one or more isodeoxycytidines (iso-dCs), one or more isodeoxyguandines (iso dGs), one or more irpc 3 groups (i.e. nucleotides lacking sugar and base groups), one or more Photocleavage (PC) groups, one or more hexanediol groups, one or more blocking chains (i.e.9, or more thiol groups, one or more blocking chains (i.18). The one or more blocking chains may include any combination of these groups. Many of these groups are available from (Integrated DNA).

The one or more blocking chains may comprise any number of these groups. For example, for 2-aminopurine, 2-6-diaminopurine, 5-bromodeoxyuridine, inverse dTs, ddTs, ddCs, 5-methylcytidine, 5-hydroxymethylcytidine, 2 'alkoxy-modified ribonucleotides (preferably 2' methoxy-modified ribonucleotides), iso dCs, iso dGs, iSpC3 groups, PC groups, hexanediol groups and thiol linkages, one or more of the blocked chains preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. The one or more blocking chains preferably comprise 2, 3, 4, 5, 6, 7, 8 or more iss 9 groups. The one or more blocking chains preferably comprise 2, 3, 4, 5 or 6 or more iss 18 groups. The most preferred blocking groups are 4 iss 18 groups.

The polymer is preferably a polypeptide or polyethylene glycol (PEG). The polypeptide preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acids. The PEG preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more monomer units.

The one or more blocked strands preferably include one or more abasic nucleotides (i.e., nucleotides lacking nucleobases), such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more abasic nucleotides. Nucleobases can be replaced by-H (idSp) or-OH in abasic nucleotides. An abasic blocking strand may be inserted into a target polynucleotide by removing nucleobases from one or more adjacent nucleotides.

The one or more blocking strands preferably comprise one or more chemical groups that physically lead to the arrest of the one or more helicases. The one or more chemical groups are preferably one or more pendant chemical groups. The one or more chemical groups may be attached to one or more nucleobases in the target polynucleotide. The one or more chemical groups may be attached to the backbone of the target polynucleotide. Any number, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more of these chemical groups may be present. Suitable groups include, but are not limited to, fluorophores, streptavidin and/or biotin, cholesterol, methylene blue, dinitrophenol (DNPs), digitoxin and/or anti-digitoxin and diphenylcyclooctyne groups.

Different blocking strands in a target polynucleotide may comprise different stuttering molecules. For example, one blocking strand may comprise one linear molecule as discussed above, and another blocking strand may comprise one or more chemical groups that physically result in the arrest of one or more helicases. The blocking strand may include any of the linear molecules discussed above and one or more chemical groups that physically result in the arrest of one or more helicases, such as one or more abasic and fluorophores.

Method for characterizing a target polynucleotide

The problem addressed by the methods of the present invention is how to characterize a target polynucleotide.

A method of characterizing a polynucleotide of interest, comprising:

a) Providing (i) a polynucleotide construct comprising the polynucleotide of interest and an adaptor according to any one of claims 1 to 4, and (ii) a helicase; the helicase comprises a DNA helicase;

b) Contacting the polynucleotide construct provided in a) and the helicase with a transmembrane pore such that the helicase controls movement of the target polynucleotide relative to the transmembrane pore;

c) One or more measurements are obtained 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.

The adaptors of the present invention are used in the methods described above, bind to the DNA helicase and ligate to the target polynucleotide such that the target polynucleotide is transported to a transmembrane pore and the pore is used to characterize the target polynucleotide. The present invention provides methods for characterizing a ribonucleic acid (RNA and/or DNA) polynucleotide of interest by obtaining one or more measurements as the polynucleotide of interest moves relative to the transmembrane pore under the control of a DNA helicase.

Illustratively, the target polynucleotide includes a target RNA polynucleotide and a target DNA polynucleotide.

Because 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 generally do not include Polymerase Chain Reaction (PCR) or reverse transcription PCR (RT-PCR). This greatly reduces the amount of effort required to characterize the target polynucleotide. This also avoids any bias and artefacts caused by PCR.

The methods of the invention 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.

Illustratively, the one or more characteristics are preferably selected from (i) the length of the RNA polynucleotide, (ii) the identity of the RNA polynucleotide, (iii) the sequence of the RNA polynucleotide, (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 the invention, for example { i }, { ii }, { iii }, { iv }, { v }, { i, ii }, { i, iii }, { i, iv }, { i, v }, { ii, iii }, { ii, v }, { iii, iv }, { iii, 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) may be measured, including any of the combinations listed above. The method of the invention preferably comprises estimating the sequence of the RNA polynucleotide or sequencing the RNA polynucleotide.

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 well, or the duration of interactions between the target RNA polynucleotide and the well.

For (ii), identity of the target RNA polynucleotide can be determined in a variety of ways. The identity of the target RNA polynucleotide may be determined in conjunction with or without sequence determination of the target RNA polynucleotide. The former is direct; the polynucleotides are sequenced and thereby identify the identity of the target RNA polynucleotide. 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 specific sources.

For (iii), the sequence of the target RNA polynucleotide may be determined as described above. Suitable sequencing methods, particularly 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 International application WO 2000/28312.

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 a change in residence time through the pores or a change in current. This allows regions of single-stranded and double-stranded RNA polynucleotides to be distinguished.

For (v), the presence or absence of any modification can be determined. The method preferably comprises determining whether the polynucleotide has been modified by methylation, oxidation, damage, with one or more proteins, or with one or more markers, tags or blocking strands. Specific modifications will result in specific interactions with the wells, which can be determined using the methods described below. For example, cytosine can be distinguished from methylcytosine based on the current through the pore during its interaction with each nucleotide. The method of the invention can be used to distinguish between RNA and DNA, even in a single sample: RNA and DNA can be distinguished from each other as a function of average amplitude and range even when the RNA and DNA sequences are identical.

The method may be implemented using any apparatus suitable for studying a membrane/pore system in which pores are present in a membrane. The method may be implemented using any device suitable for transmembrane pore sensing. For example, the apparatus comprises a chamber comprising an aqueous solution and a barrier (barrier) dividing the chamber into two parts. The barrier typically has a slit, wherein a membrane comprising pores is formed in the slit. Or the barrier forms a membrane in which pores are present.

The process may be carried out using the apparatus described in International application No. PCT/GB08/000562 (WO 2008/102120).

The method may comprise measuring the current through the pore as the RNA polynucleotide moves relative to the pore. The device may thus also include circuitry capable of applying an electrical potential across the membrane and the well and measuring a current signal. The method may be performed using patch clamp or voltage clamp. The method preferably comprises the use of a voltage clamp.

The methods of the invention 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 current through a transmembrane protein pore are known in the art and are disclosed in the examples. The method is performed by applying a voltage across the membrane and the pores. The voltages used are generally +5V to-5V, for example from +4V to-4V, +3V to-3V or +2V to-2V. The voltages typically used are typically-600 mV to +600mV, or-400 mV to +400mV. The voltage used is preferably within a range having a lower limit selected from the group consisting of-400 mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV and 0mV and an upper limit independently selected from the group consisting of +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300mV and +400mV. The voltage used is more preferably in the range of 100mV to 240mV and most preferably in the range of 120mV to 220 mV. The resolution of different ribonucleotides can be improved by applying an increased potential to the well.

The process is generally carried out in the presence of any charge carrier, such as a metal salt, e.g. an alkali metal salt, a halogen salt, e.g. a chloride salt, e.g. an alkali metal chloride salt. The charge carriers may include ionic liquids or organic salts, such as tetramethyl ammonium chloride, trimethyl ammonium chloride, phenyl trimethyl benzene 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. Preferably potassium chloride, sodium chloride and mixtures of potassium ferrocyanide and potassium ferricyanide. The charge carriers may asymmetrically pass through the membrane. For example, the type and/or concentration of charge carriers may be different on each side of the membrane.

The salt concentration may 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. Preferably the salt concentration is 150mM to 1M. The process is preferably carried out using a salt concentration of at least 0.3M, for example 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 2.5M, or at least 3.0M. The high salt concentration provides a high signal to noise ratio and allows the current representing the presence of ribonucleotides to be identified in the context of normal current fluctuations.

The process is typically carried out 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 invention. Typically, the buffer is a phosphate buffer. Other suitable buffers are HEPES and Tris-HCl buffers. The process is generally carried out at a pH of from 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 from 7.5 to 8.5. The pH used is preferably about 7.5.

The process may be carried out at 0 ℃ to 100 ℃,15 ℃ to 95 ℃,16 ℃ to 90 ℃,17 ℃ to 85 ℃,18 ℃ to 80 ℃,19 ℃ to 70 ℃, or 20 ℃ to 60 ℃. The process is usually carried out at room temperature. The method is optionally carried out at a temperature that supports the function of the enzyme, for example at about 37 ℃.

The method may be carried out in the presence of free nucleotides or free nucleotide analogs and/or enzyme cofactors which facilitate the function of the helicase or construct. The method may 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), guanosine Triphosphate (GTP), thymidine Monophosphate (TMP), thymidine Diphosphate (TDP), thymidine Triphosphate (TTP), uridine Monophosphate (UMP), uridine Diphosphate (UDP), uridine Triphosphate (UTP), cytidine Monophosphate (CMP), cytidine Diphosphate (CDP), cytidine Triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dabp), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dGTP), deoxythymidine monophosphate (dGDP), deoxythymidine diphosphate (dTTP), deoxyuridine diphosphate (dGTP), deoxycytidine diphosphate (dGTP), and deoxycytidine triphosphate (dGTP). The free nucleotide is preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotide is preferably Adenosine Triphosphate (ATP). The enzyme cofactor is a factor that functions the helicase or construct. The enzyme is assisted The factor is preferably a divalent metal cation. The divalent metal cation is preferably Mg ²⁺ ，Mn ²⁺ ，Ca ²⁺ Or Co ²⁺ . Most preferably the enzyme cofactor is Mg ²⁺ 。

Target RNA polynucleotides

RNA is a macromolecule comprising two or more ribonucleotides. The target RNA polynucleotide may be eukaryotic or prokaryotic RNA. The target RNA polynucleotide may comprise any combination of any 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 can be damaged. For example, the target RNA may comprise a pyrimidine dimer, such as 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, e.g. with a label or tag. Suitable labels are described below. The target RNA may comprise one or more blocking strands.

Ribonucleotides contain a base, ribose and at least one phosphate group. The bases are typically heterocyclic. Bases include, but are not limited to: purine and pyrimidine, more specifically adenine, guanine, thymine, uracil and cytosine. The nucleotides typically contain a monophosphate, a diphosphate or a triphosphate. The phosphate may 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 monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate and 5-hydroxymethylcytidine triphosphate. The nucleotide is preferably selected from AMP, TMP, GMP, CMP and UMP.

Ribonucleotides can be abasic (i.e., lack bases). Ribonucleotides may also lack bases and sugars (i.e., C3 blocking chains).

The ribonucleotides of the target RNA polynucleotide may be linked to each other in any manner. As in nucleic acids, the ribonucleotides are typically linked by their sugar and phosphate groups. As in pyrimidine dimers, the ribonucleotides may be linked by their bases.

RNA is a very diverse molecule. The target RNA polynucleotide may 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), microRNA (snRNA), micronucleolar RNA (snorRNA), signal recognition particle (SRP RNA), smY RNA, small Cajan body-specfc RNA (scaRNA), guide RNA (gRNA), splicing leader RNA (SL RNA), antisense RNA (asRNA), long non-coding RNA (lncRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), trans-acting siRNA (tasiRNA), repeated association siRNA (rasiRNA), Y RNA, viral RNA or chromosomal RNA, where appropriate all of which may be single-stranded, double-stranded or triple-stranded.

The target RNA polynucleotide is preferably messenger RNA (mRNA). The target mRNA may be an alternate splice variant (alternate splice variant). The amount (or grade) of variation in mRNA and/or alternative mRNA splice variants may be related to disease or health.

Or the target RNA polynucleotide is a microrna (or miRNA). One group of RNAs that are difficult to detect at low concentrations are micro ribonucleic acids (micro-RNAs or miRNAs). miRNAs are highly stable RNA oligomers that are capable of post-transcriptional regulation of protein products. They act through one of two mechanisms. In plants, miRNAs have been shown to function primarily by directing the division of messenger RNAs, whereas in animals gene regulation by miRNAs generally involves hybridization of miRNAs to the 3' utrs of messenger RNAs, which prevents translation (Lee et al, cell75,843-54 (1993); wightman et al, cell75,855-62 (1993); and esquesa-Kerscher et al, cancer 6,259-69 (2006)). miRNAs often bind their targets with defective complementarity. They have been predicted to bind to up to 200 or more gene targets, respectively, and regulate more than one third of the genes in all humans (Lewis et al, cell 120,15-20 (2005)).

Suitable miRNAs for use in the present invention are known in the art. For example, suitable 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. It is known that the expression levels of certain microRNAs in tumors will change, resulting in microRNA expression in patterns characteristic of different tumor types (Rosenfeld, N.et al, nature Biotechnology 26,462-9 (2008)). In addition, miRNA expression profiles are known to reveal the stage of tumor progression more precisely than messenger RNA expression profiles (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, the high stability of binding to miRNAs, and the ability to detect circulating miRNAs in serum and plasma (Wang et al, bio chemistry and Biophysical Research communications394,184-8 (2010); gilad et al, ploS One 3, e3148 (2008); and Keller et al, nature Methods 8,841-3 (2011)), have led to a great deal of interest in the potential use of microRNAs as cancer biomarkers. For effective treatment, cancers need to be precisely classified and treated differently, but the efficacy of tumor morphology assessment as a method of classification is impaired due to the fact that many different types of cancers share morphological features. miRNAs provide a potentially more reliable and less invasive solution.

The use of mRNAs and miRNAs for diagnosing or prognosticating diseases or conditions is discussed in more detail below.

Any number of RNAs can be studied. For example, the methods of the invention may be directed to 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 polynucleotide may be naturally occurring or synthetic. For example, the method can be used to verify the sequence of two or more artificially created oligonucleotides. The method is typically performed in vitro.

The target RNA polynucleotide may 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 may be 1000 or more ribonucleotides, 5000 or more nucleotides or at 100000 or more ribonucleotide length. All or only a portion of the target RNA can be characterized using this method. The RNA portion to be sequenced preferably comprises all target molecules but may for example be less than all molecules, for example 4 bases to 1kb, for example 4 to 100 bases.

The target RNA polynucleotide is typically present in or derived from any suitable sample. The invention is generally practiced in samples known to contain or suspected of containing the target RNA polynucleotide. Alternatively, the invention may be practiced on a sample to confirm that the presence in the sample is known or desired of one or more target RNAs identity.

The sample may be a biological sample. The invention may be practiced in vitro on samples obtained or extracted from any organism or microorganism. The organism or microorganism is typically archaeal (prokaryotic), prokaryotic or eukaryotic, and typically belongs to one of the five kingdoms: the kingdom phytoales, zookingdom, fungi, prokaryotes and protozoa. The target RNA polynucleotide may be derived from eukaryotic cells or may be derived from viruses that use the transcriptional machinery of eukaryotic cells. The invention may be practiced in vitro on samples obtained or extracted from any virus.

The sample is preferably a liquid sample. The sample typically comprises a body fluid of the patient. The sample may be urine, lymph, saliva, mucus or amniotic fluid, but is preferably blood, plasma or serum. Typically, the sample is of human origin, but alternatively may be from other mammals, such as from commercially farmed animals such as horses, cattle, sheep or pigs or alternatively may be pets such as cats or dogs. Alternatively, samples derived from plants are typically derived from commercial crops such as cereals, legumes, fruits or vegetables, for example wheat, barley, oats, canola (canola), corn, soybean, rice, banana, apple, tomato, potato, grape, tobacco, beans (beans), lentils, sugarcane, cocoa or cotton.

The sample may be a non-biological sample. The non-biological sample is preferably a fluid sample. Examples of non-biological samples include surgical fluids, water such as drinking water, seawater or river water, and reagents for laboratory tests.

The sample is typically treated prior to being analyzed, such as by centrifugation, or by filtration through a membrane to filter out unwanted molecules or cells, such as erythrocytes. The sample may be measured immediately after collection. The sample may also be stored generally prior to analysis, preferably below-70 ℃. The target RNA polynucleotide is typically extracted from the sample prior to use in the methods of the invention. RNA extraction kits are commercially available from, for example, new England and.

Connection

The adaptors are ligated to the target RNA polynucleotides to form modified RNA polynucleotides.

In particular embodiments, the adaptor is attached to the target RNA polynucleotide by a covalent bond formed between the RNA polynucleotide and at least one respective reactive group of the adaptor; and/or

Ligating the adaptor to the RNA polynucleotide by chemical or enzymatic ligation.

The target RNA polynucleotide may be chemically linked to the adapter, for example by covalent bonds. The target RNA polynucleotide may be linked to the adapter by chemical or enzymatic binding. The target RNA polynucleotide may be linked 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 ligated to the adapter at more than one, e.g., two or three, positions. The connection methods may include one, two, three, four, five or more different connection methods. Any combination of the connection methods described below may be used in accordance with the present invention.

The RNA polynucleotide and the adaptor may be prepared separately and then ligated together. The two components may be connected in any configuration. For example, they may be linked by their ends (i.e., 5 'or 3'). Suitable constructs include, but are not limited to, ligating the 5 'end of the RNA polynucleotide to the 3' end of the adapter and vice versa. Alternatively, the two components may be linked by nucleotides within their sequences.

The RNA polynucleotide may be linked to the adapter using one or more chemical cross-linkers or one or more peptide linkers. Suitable chemical cross-linking agents are well known in the art. Suitable chemical crosslinkers include, but are not limited to, chemical crosslinkers that include the following functional groups: maleimide, active esters, succinimides, azides, alkynes (e.g., dibenzocyclooctanol (DIBO or DBCO), difluoroalicyclic hydrocarbons and linear alkynes), phosphides (e.g., phosphides used in traceless and non traceless staudinger ligation), haloacetyl (e.g., iodoacetamide), phosgene reagents, sulfonyl chloride reagents, isothiocyanates, acid halides, hydrazine, disulfides, vinyl sulfones, aziridines and photoactive reagents (e.g., aromatic azides, diazacyclopropanes).

The reaction between the RNA polynucleotide and the adaptor may be spontaneous, such as cysteine/maleimide, or may require external reagents, such as Cu (I) for ligating azide and linear alkyne.

Preferred cross-linking agents include 2, 5-dioxopyrrolidin-1-yl 3- (pyridin-2-yl-dithioyl) propionate, 2, 5-dioxopyrrolidin-1-yl 4- (pyridin-2-yl-dithioyl) butyrate and 2, 5-dioxopyrrolidin-1-yl 8- (pyridin-2-yl-dithioyl) octanoate, bismaleimide PEG 1k, bismaleimide PEG 3.4k, bismaleimide PEG 5k, bismaleimide PEG 10k, bis (maleimido) ethane (BMOE), bismaleimide 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), tris [ 2-maleimidoethyl ] amine (TMEA), DTME dithiobismaleimide ethane, bismaleimide PEG3, bismaleimide PEG11, DBCO-maleimide, DBCO-PEG4-NH2, DBCO-PEG4-NHS, DBCO-PEG-DBCO 2.8kDa, DBCO-PEG-DBCO 4.0kDa, DBCO-15-atom-DBCO, DBCO-26-atom-DBCO, DBCO-35-atom-DBCO, DBCO-PEG4-S-S-PEG 3-biotin, DBCO-S-S-PEG 3-biotin and DBCO-S-S-PEG 11-biotin. Most preferred cross-linking agents are succinimidyl 3- (2-pyridyldithio) propionate (SPDP) and maleimide-PEG (2 kDa) -maleimide (alpha, omega-bismaleimidolyethylene glycol).

The linker may be labeled. Suitable labels include, but are not limited to, fluorescent molecules (e.g., cy3 or 555), radioisotopes such as 125i,35s, enzymes, antibodies, antigens, polynucleotides, and ligands such as biotin. Such a tag allows the amount of linker to be determined. The tag may also be a cleavable purification tag, such as biotin, or a specific sequence that occurs in the identification method.

Crosslinking of the RNA polynucleotide or the adaptor itself can be prevented by maintaining a substantial excess of the solubility of the adaptor over the RNA polynucleotide and/or the adaptor. Alternatively, in the case 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 being reacted with a different portion of the construct (i.e., the RNA polynucleotide or the adaptor).

Click chemistry

The target RNA polynucleotide may be covalently linked to the adapter. The adaptors may or may not comprise a pre-bound DNA helicase. In preferred embodiments, free copper click chemistry or copper catalyzed click chemistry can be used to make covalent bonds between the RNA polynucleotide and the adaptor. Click chemistry is used in these applications due to its desirable nature and its scope for creating covalent linkages between the various building blocks. For example, it is fast, clean and non-toxic, producing only harmless by-products. Click chemistry is the term first introduced by Kolb et al at 2001 to describe a broader array of powerful, selective and modular building blocks that are reliably used for small 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-range, give very high yields, produce only harmless by-products which can be removed by non-chromatography, and be stereospecific (but not necessarily enantioselective). The claimed process features include simple reaction conditions (ideally the process should be insensitive to oxygen and water), readily available starting materials and reagents, no use of solvents or solvents that are mild (e.g., water) or easy to remove, and simple product isolation. Purification must be by non-chromatography, e.g. crystallization or distillation, if desired, and the product must be stable in physiological conditions.

The following examples illustrate the invention.

Example 1:2' -F-RNA binds to DNA helicase

RNA with Cy3 marked 2' -F substitution modification (specific RNA sequence is 5'-GCCAGAAACG-3', the sequence length is more than 6nt, the sequence has no preference) and DNA helicase with Cy3 marked DNA (100 nM) and 20 or 30 times of the mass of the same length and base sequence.

T4 Dda-M1G/E94C/C109A/C136A/A360C (3. Mu.M) and DNA helicase Hel308 were mixed in buffer (20 mM HEPES (pH 7.0); 50mM NaCl;0.5mM TMAD) and incubated at room temperature for 60 minutes. The binding efficiency was then analyzed by TBE (native) PAGE gels, which were 4-20% gels, run at 160V for 40 min, and then stained with SYBR Jin Ranliao.

As a result, as shown in FIGS. 1 and 2, respectively, it was revealed that both the DNA helicase T4 Dda-M1G/E94C/C109A/C136A/A360C and the DNA helicase Hel308 were able to bind well to 2' -F-RNA, and the binding effect was no worse than that of the enzyme to DNA. Thus, the 2' -F-RNA sequence can be used for adaptor preparation for nanopore RNA sequencing.

The amino acid sequence of the DNA helicase T4 Dda-M1G/E94C/C109A/C136A/A360C is shown in SEQ ID NO. 1:

GTFDDLTEGQKNAFNIVMKAIKEKKHHVTINGPAGTGKTTLTKFIIEALISTGETGIILAAPTHAAKKILSKLSGKEASTIHSILKINPVTYECNVLFEQKEVPDLAKARVLICDEVSMYDRKLFKILLSTIPPWATIIGIGDNKQIRPVDPGENTAYISPFFTHKDFYQCELTEVKRSNAPIIDVATDVRNGKWIYDKVVDGHGVRGFTGDTALRDFMVNYFSIVKSLDDLFENRVMAFTNKSVDKLNSIIRKKIFETDKDFIVGEIIVMQEPLFKTYKIDGKPVSEIIFNNGQLVRIIEAEYTSTFVKARGVPGEYLIRHWDLTVETYGDDEYYREKIKIISSDEELYKFNLFLGKTCETYKNWNKGGKAPWSDFWDAKSQFSKVKALPASTFHKAQGMSVDRAFIYTPCIHYADVELAQQLLYVGVTRGRYDVFYV*

The amino acid sequence of the DNA helicase Hel308 is shown in SEQ ID NO. 2:

MKIESLDLPDEVKQFYLDSGILELYPPQAEAVEKGLLEGRNLLAAIPTASGKTLLAELAMLKSILNGGKALYIVPLRALASEKFKRFREFSKLGIRVGISTGDYDLRDEGLGVNDIIVATSEKTDSLLRNETVWMQEISVVVADEVHLIDSPDRGPTLEITLAKLRKMNPSCQILALSATIGNADELAAWLEAGLVLSEWRPTELREGVFFNGTFYCKDREKSIEQSTKDEAVNLVLDTLREDGQCLVFENSRKNCMAFAKKASSAVKKILSAEDKEALAEIADEVLENSETDTSAALAACIRSGTAFHHAGLTTPLRELVEDGFRAGKIKLISSTPTLAAGLNLPARRVVIRSYRRYSSEDGMQPIPVIEYKQMAGRAGRPRLDPYGEAVLVAKSYEEFVFLFRNYIEADAEDIWSKLGTENALRTHVLSTISNGFARTKEELMEFLEATFFAFQYSNFGLSTVVDECLNFLRQEEMLEKTDTLISTSFGKLVSKLYIDPLSAARIVKGLKEAKILTELTLLHLVCSTPDMRLLYMRNQDYQDINDYVIAHADEFVRVPSPFNYTEYEWFLGEVKTSLLLVDWIHEKSENEICLKFGIGEGDIHAIADIAEWLMHVTAQLARLLELKGAKEAAELEKRIHYGASPELMDLLDIRGIGRMRARKLYESGFRSSAELAGADPVKVAALLGPKIADRIFKQIGRREVLPEIAEPTLPEKSPSSGQKTINDY*

example 2: incubation and preparation of sequencing linker complexes containing 2' -F-RNA leaderThe following sequences were synthesized:

RNA-Y1:

5'-P-CAGTCGTCCTGGCTTACTCGTCA/iSp18//i2FU//i2FU//i2FU//i2FU//i2FU//i2FU//i2FU//i2FU//i2FU//i2FU//i2FU//i2FU//i2FU//i2FU//i2FU/GCTGAAGATG GCAAACTGAGGCG/iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSp C3//iSpC3//iSpC3/-3'

RNA-YB:

5'-

/i2OMeC//i2OMeC//i2OMeA//i2OMeG//i2OMeC//i2OMeC//i2OMeA//i2OMeC//i2OMeG//i2OMeA//i2OMeC//i2OMeC//i2OMeU//i2OMeG//i2OMeA//iXNA_T//iXNA_G//iXNA_A//iXNA_C//iXNA_G/AGTAAGCCAGGACGACTGGC-3'

RNA-Y2:5'-CGCCTCAGTTTGCCATCTTCAGC-3'

respectively synthesizing RNA-Y1; the three chains are annealed in an annealing buffer solution according to a ratio of 1:1.1:1.1 respectively by the RNA-YB and the RNA-Y2 chains to form a Y-shaped joint, and the annealing treatment is specifically to slowly cool the temperature from 95 ℃ to 25 ℃ with the cooling amplitude not exceeding 0.1 ℃/s. The annealing buffer included 160mM HEPES 7.0, 200mM NaCl.

It should be noted that: sugar ring 2'-F modification is a common technology, in the embodiment of the application, bases modified by 2' -F are all U, the problem of secondary structure formation is not needed to be considered excessively, the length of 15U is determined according to the space occupied by specific enzyme, and at least 1 enzyme can be combined instead of 2 enzymes through verification.

In the examples herein, i2OMe represents one of the sugar ring modifications, i.e. the 2' -methoxy modification.

In the examples herein, iXNA is an LNA that is used in combination with the iss 18 in Y1 to block enzymes.

Mixing and incubating at room temperature for 30 minutes with 500nM type Y linker, 15-fold amount of DNA helicase Hel308 (the direction of movement is 3 'to 5') and then adding 1500-fold amount of M-P-M and incubating at room temperature for 1h; sequencing adapter complexes were prepared and analyzed by running a TBE PAGE gel at 160V for 40 minutes and stained with SYBR gold dye, the incubation binding effect of which is shown in FIG. 3. The results in FIG. 3 show that a sequencing adapter complex is formed.

The sequencing adapter complex was then applied to a dnappa PA200 column and purified with elution buffer to elute the enzyme from the column that was not bound to the sequencing adapter complex. The sequencing adapter complex was then eluted with 10 column volumes of a mixture of buffer a and buffer B. The main elution peaks were then pooled and their concentrations were measured to obtain RNA sequencing adaptors and run for 40 minutes with TBE PAGE gels at 160V. Wherein, buffer a:20mM Na-CHES,250mM NaCl,4% (W/V) glycerol, pH 8.6; buffer B:20mM Na-CHES,1M NaCl,4% (W/V) glycerol, pH 8.6, and the final results are shown in FIG. 4.

Example 3: on-machine testing of sequencing adapter complexes of 2' -F-RNA leader

The RMX component was replaced with the purified sequencing linker complex containing the 2' -F-RNA leader prepared in example 2 using the oxford nanopore technology RNA direct library kit SQK-RNA002, and finally the signal was collected by testing on the protein platform of ONT as shown in fig. 5. The results indicate that the linker or linker complex of example 2 can be used for nanopore sequencing.

In addition, the term "and/or" herein is merely an association relationship describing an association object, and means that three relationships may exist, for example, a and/or B may mean: a exists alone, A and B exist together, and B exists alone. In addition, the character "/" herein generally indicates that the front and rear associated objects are an "or" relationship.

It should be understood that in embodiments of the present invention, "B corresponding to a" means that B is associated with a, from which B may be determined. It should also be understood that determining B from a does not mean determining B from a alone, but may also determine B from a and/or other information.

While the invention has been described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes and substitutions of equivalents may be made and equivalents will be apparent to those skilled in the art without departing from the scope of the invention. Therefore, the protection scope of the invention is subject to the protection scope of the claims.

Claims (19)

1. An adaptor for characterizing a polynucleotide of interest, comprising a binding region of a helicase, the binding region comprising a modified RNA polynucleotide for binding or loading the helicase.

2. The adapter according to claim 1, wherein,

the helicase comprises a DNA helicase; and/or

The modified RNA polynucleotide is selected from sugar ring 2' -F modified RNA; and/or

The binding region of the helicase does not comprise DNA.

3. The adaptor according to claim 1 or 2, wherein the adaptor comprises a leader sequence that preferentially penetrates into a nanopore;

Preferably, the binding region of the helicase is located in the leader sequence.

4. The adaptor according to claim 1 or 2, wherein the target polynucleotide is a target RNA polynucleotide and/or a target DNA polynucleotide;

the target polynucleotide is single-stranded or double-stranded;

preferably, the adaptor is attached to the target polynucleotide by a covalent bond formed between the RNA polynucleotide and the respective at least one reactive group of the non-nucleotide; and/or

Ligating the adaptor to the target polynucleotide by chemical or enzymatic ligation.

5. A method of characterizing a polynucleotide of interest using the adaptor of any one of claims 1 to 4.

6. The method according to claim 5, characterized in that the method comprises:

a) Providing (i) a polynucleotide construct comprising the polynucleotide of interest and an adaptor according to any one of claims 1 to 4, and (ii) a helicase; the helicase comprises a DNA helicase;

b) Contacting the polynucleotide construct provided in a) and the helicase with a transmembrane pore such that the helicase controls movement of the target polynucleotide relative to the transmembrane pore;

c) One or more measurements are obtained 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. The method of claim 6, wherein the one or more characteristics are selected from the group consisting of (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. The method of claim 6 or 7, wherein one or more characteristics of the target polynucleotide can be measured by electrical and/or optical measurement.

9. The method of 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 is representative of one or more characteristics of the target polynucleotide and thereby characterizes the target polynucleotide.

10. The method of claim 6 or 7, wherein the target polynucleotide is additionally or further modified by methylation, oxidation, damage, with one or more proteins, or with one or more markers, tags or blocking chains.

11. The method of claim 6 or 7, wherein the target polynucleotide can be coupled to the membrane using one or more anchors.

12. A method according to claim 6 or claim 7, wherein the helicase comprises a modification to reduce the size of an opening in the binding domain of the polynucleotide through which the target polynucleotide can be unbound from the helicase in at least one conformational state.

13. The method according to claim 6 or 7, wherein the helicase is:

a) Hel308 helicase, recD helicase, XPD helicase, dda helicase, tral helicase, trwC helicase;

b) A helicase derived from any of the helicases described in a); or (b)

c) any combination of the helicases described in a) and/or b).

14. The method of claim 6 or 7, wherein the transmembrane pore is a protein pore or a solid state pore.

15. The method of any one of claims 8 to 14, wherein the transmembrane protein pore is a protein pore and is derived from any one or more of: haemolysin, leukocidal, mycobacterium smegmatis (Mycobacterium smegmatis) porin a (MspA), mspB, mspC, mspD, lysin (lyserin), csgG, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase a, neisseria (Neisseria) self-transporter lipoproteins (NalP) and WZA.

16. A method of moving a target polynucleotide relative to a transmembrane pore, the movement being controlled by a helicase, the method comprising:

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 a modified RNA polynucleotide region for binding or loading the helicase acting as a binding region for the DNA helicase;

wherein the modified RNA polynucleotide comprises a 2' -F modified RNA;

b) Contacting the target RNA polynucleotide or target DNA polynucleotide provided in a), and the helicase with a transmembrane pore such that the helicase controls movement of the RNA polynucleotide relative to the transmembrane pore.

17. A complex comprising the adaptor of any one of claims 1 to 4 and a helicase;

the helicase comprises the DNA helicase;

preferably, the DNA helicase is selected from:

a) Hel308 helicase, recD helicase, XPD helicase, dda helicase, tral helicase, or TrwC helicase;

b) A helicase derived from any of the helicases described in a); or (b)

c) any combination of the helicases described in a) and/or b).

18. A kit for characterizing a polynucleotide of interest, the kit comprising the adaptor of any one of claims 1 to 4 and the helicase or the complex of claim 18;

the target polynucleotide is target RNA polynucleotide or target DNA polynucleotide.

19. An isolated polynucleotide comprising an RNA polynucleotide or a DNA polynucleotide, and a modified RNA polynucleotide region for binding a helicase;

wherein the modified RNA polynucleotide comprises a 2' -F modified RNA;

the helicase comprises a DNA helicase.