CN113462764B - Hairpin-like adaptors, constructs and methods for characterizing double-stranded target polynucleotides - Google Patents

Abstract

The present invention provides hairpin-like adaptors, constructs and methods for characterizing double-stranded target polynucleotides. The hairpin-like adaptor comprises a first single strand and a second single strand for ligation to a template strand and a complementary strand of the double-stranded target polynucleotide, respectively, the first single strand and the second single strand forming at least 1 double-stranded nucleic acid region for bringing the complementary strand into proximity with a transmembrane pore after the template strand has passed through the transmembrane pore. Also provided are constructs comprising the above hairpin-like adaptors, as well as methods, kits, and the like, for characterizing double-stranded target polynucleotides. The characterization methods of the present invention have many advantages, such as balancing translocation speeds of the template strand and the complementary strand of the target polynucleotide, improving sequencing accuracy, and the like.

Description

Hairpin-like adaptors, constructs and methods for characterizing double-stranded target polynucleotides

Technical Field

The present application is in the technical field of bioanalytical detection, and in particular, to hairpin-like adaptors, constructs and methods for characterizing, e.g., sequencing, double-stranded target nucleotides using transmembrane pores.

Background

Currently, nucleic acid sequencing technology has application requirements in a plurality of scenarios. The existing sequencing technology needs to perform complex processing on a sample to be sequenced in the prior period, has long sequencing period, and cannot meet the requirements of rapidness and convenience for the nucleic acid sequencing technology in clinical application scenes and the like.

Transmembrane pores (such as nanopores) have great potential as biosensors to develop new nucleic acid sequencing technologies. When a voltage is applied across the nanopore, a current drop is caused when an analyte (e.g., a nucleotide, a polypeptide) passes through the nanopore, and the degree of current blockage caused by analytes of different structures varies. Nucleotide nanopores can detect a current block of known character and duration caused by the passage of an analyte.

In nanopore sequencing methods, a single polynucleotide is passed through a pore and the nucleotide is directly identified. The strand sequencing method includes a nucleotide binding protein (e.g., helicase, polymerase) as a molecular brake for the passage of the polynucleotide through the nanopore. Since the size limit of nanopores may only allow ssDNA to pass through the pore, dsDNA needs to be melted before passing through the pore to produce ssDNA. Nucleotide binding proteins (e.g., helicases, polymerases) are capable of unwinding dsDNA and controlling the speed of polynucleotide via translocation. However, neither polymerase, helicase, nor exonuclease is used to sequence complementary strands. Thus, only half of the DNA information in dsDNA is sequenced.

Therefore, methods for simultaneously sequencing the template strand and the complementary strand of dsDNA have received much attention. For example, patent CN 103827320 a discloses a method of sequencing the template and complementary strands of dsDNA, which utilizes specially designed hairpin turns of the dsDNA construct. The two strands of dsDNA are connected in a hairpin loop bridge configuration so that the complementary strand can be successively punched after the template strand is punched. However, this method increases the difficulty of sample pre-treatment, increases the operating time, and may result in the loss of precious samples. Even template strands and complementary strands connected by hairpin turns are prone to re-hybridize after the via, which increases translocation speed of the complementary strand through the via, reducing sequencing accuracy.

In addition, patent CN110168104A discloses another method for sequencing the template strand and complementary strand of dsDNA, which is achieved by constructing a nanopore complex. And connecting a nucleotide probe which can be complementarily paired with the terminal part sequence of the complementary strand on the nanopore through chemical modification, wherein when the template strand and the complementary strand are subjected to hole passing after unwinding, the probe can capture the complementary strand to enable the complementary strand to stay near the nanopore, and therefore, the complementary strand can be subjected to hole passing after the template strand is completely subjected to hole passing with a certain probability. However, this method requires chemical modification of the nanopore, increasing the complexity of the preparation process; when the library is constructed, two groups of adapters need to be connected respectively, and purification is performed twice, so that the complexity of library preparation is increased, and the efficiency of library preparation is reduced.

Although methods for simultaneously sequencing the template strand and the complementary strand of dsDNA are disclosed in the prior art, each method has its own drawbacks, and there is still a need to develop a more optimal method for simultaneously and continuously sequencing the template strand and the complementary strand of dsDNA in real time to balance the translocation speed of both strands of a double-stranded target polynucleotide and to improve the sequencing accuracy.

Disclosure of Invention

The present inventors have discovered that sequencing both strands of a double-stranded target polynucleotide by ligating the hairpin-like adaptors has many advantages, such as balancing translocation speeds of the template and complementary strands of the target polynucleotide, increasing sequencing accuracy, and the like.

Accordingly, a first aspect of the invention relates to a hairpin-like adaptor for use in characterising a double-stranded target polynucleotide, which comprises information identifying the double-stranded target polynucleotide, such as base sequencing or sequence modification. The hairpin-like adaptor comprises a first single strand and a second single strand for ligation to a template strand and a complementary strand of the double-stranded target polynucleotide, respectively, the first single strand and the second single strand forming at least 1 double-stranded nucleic acid region for bringing the complementary strand into proximity with a transmembrane pore after the template strand has begun to pass through the transmembrane pore to completely pass through the transmembrane pore.

Preferably, the at least 1 double-stranded nucleic acid region comprises a first double-stranded nucleic acid region distal to the double-stranded target polynucleotide end of the first and second single strands, at least one strand of the first double-stranded nucleic acid region being linked in reverse orientation to the junction of the first or second single strand on which it is located.

Preferably, the second single strand comprises a first segment and a second segment in reverse ligation, the first segment being distal to the complementary strand of the double-stranded target polynucleotide and the second segment being proximal to the complementary strand of the double-stranded target polynucleotide, the first double-stranded nucleic acid region being located at the first segment.

More preferably, the second single strand is a3 '-5' -5 '-3' second single strand or a 5 '-3' -3 '-5' second single strand;

the 3 '-5' -5 '-3' second single strand comprises a3 '-5' first segment and a 5 '-3' second segment, the 5 'end of the 3' -5 'first segment being linked to the 5' end of the 5 '-3' second segment.

The 5 '-3' -3 '-5' second single strand comprises a 5 '-3' first segment and a3 '-5' second segment, the 3 'end of the 5' -3 'first segment being linked to the 3' end of the 3 '-5' second segment.

Preferably, the first and second single strands are both single strands formed from a base group and an abasic group.

Preferably, the double stranded nucleic acid region is a non-covalently linked double stranded region.

Preferably, the first single strand and the second single strand form a second double-stranded nucleic acid region formed at the second segment of the second single strand.

Preferably, the first single strand and the second single strand also form a non-complementary region.

More preferably, the region of the second single strand located in the non-complementary region comprises a binding site for a polynucleotide binding protein, and more preferably the region of the second single strand located in the non-complementary region is located on the first segment distal to the second segment.

Even more preferably, the 2 double-stranded nucleic acid regions formed by the first single strand and the second single strand and the non-complementary region form a hairpin-like structure.

Preferably, the second single strand is formed by reverse ligation of the first segment and the second segment using a click chemistry reaction; alternatively, the second single strand is synthesized directly by using the forward base and the reverse base; preferably, the reagent used in the click chemistry reaction is selected from chemical groups such as DBCO, Azide, Tetrazine or TCO, and the inverted base is selected from inverted thymidine (inverted dTs), inverted adenosine (inverted dAs), inverted guanosine (inverted dGs) or inverted cytidine (inverted dCs).

Preferably, the hairpin-like adaptors comprise polynucleotides, polypeptides or other molecules having intermolecular or intramolecular interactions.

A second aspect of the invention relates to a construct for characterising a double-stranded target polynucleotide, the construct comprising the double-stranded target polynucleotide and a hairpin-like adaptor as described above, the target polynucleotide being linked at or near one end thereof to the hairpin-like adaptor.

Preferably, the construct further comprises at least one polymer attached to the double stranded target polynucleotide at opposite ends of the hairpin-like adaptor.

Preferably, the at least one polymer comprises a leader polymer attached to the template strand of the double-stranded target polynucleotide and a tail polymer attached to the complementary strand of the double-stranded target polynucleotide, and the leader polymer and the tail polymer form part of a complementary double strand; more preferably, the lead polymer comprises binding sites for polynucleotide binding proteins, the lead polymer is a single strand formed by base groups and abasic groups, and the tail polymer is a single strand formed by modified or unmodified base groups.

Preferably, the two strands of the construct are non-covalently linked.

A third aspect of the invention relates to a method of characterising a double-stranded target polynucleotide, the method comprising:

(a) providing a construct comprising a target polynucleotide having a template strand and a complementary strand, wherein the target polynucleotide is ligated at or near one end thereof with a hairpin-like adaptor comprising at least 1 double-stranded nucleic acid region;

(b) contacting the construct with a transmembrane pore and a polynucleotide binding protein;

(c) providing conditions for the template strand and the complementary strand of the same construct to pass sequentially through the transmembrane pore; and

(d) measuring the characteristics of the template strand and the complementary strand of the double-stranded target polynucleotide produced during passage through the transmembrane pore, respectively.

Preferably, the hairpin-like adaptor is as defined above and the construct is as defined above.

Preferably, the method comprises contacting the construct with a first polynucleotide binding protein for separating the two strands of the construct and controlling movement of the template strand through the transmembrane pore and a second polynucleotide binding protein for controlling translocation of the complementary strand through the transmembrane pore such that the entire double stranded target polynucleotide is characterised.

Preferably, the first polynucleotide binding protein binds to a binding site of the polynucleotide binding protein on the leader polymer for sequentially separating the duplex formed by the leader polymer and the tail polymer, the duplex of the target polynucleotide, the duplex nucleic acid region of the hairpin-like adaptor; the second polynucleotide binding protein binds to the polynucleotide binding protein binding site on the second single strand of the hairpin-like adaptor for controlling movement of the complementary strand through the pore.

Preferably, the order in which the first polynucleotide binding protein moves is: the leader polymer, the template strand of the target polynucleotide, and the first single strand; the order in which the second polynucleotide binding protein moves is: a second segment of the second single strand, a complementary strand of the target polynucleotide, and the tail polymer.

Preferably, the method further comprises providing at least one germline strand for bringing the construct into proximity with the transmembrane pore; preferably, each said tether comprises a capture region for capturing said construct and an anchor region for binding to said transmembrane pore or to a membrane anchor in which said transmembrane pore is located.

More preferably, the at least one tether comprises a first tether having a capture region that specifically binds to the hairpin-like adaptor of the construct for bringing the complementary strand into proximity with the transmembrane pore, and more preferably, the specific binding is at a location that comprises at least part of the second segment of the second single-stranded second segment of the hairpin-like adaptor after the hairpin-like adaptor is located in the double-stranded nucleic acid region of the second segment.

More preferably, the at least one tether further comprises a second tether that specifically binds to the tail polymer of the construct for bringing the construct into proximity with the transmembrane pore.

More preferably, the anchoring region is for anchoring binding to a membrane in which the transmembrane pore is located.

Preferably, the template strand moves through the aperture by: one end of the leader polymer enters the pore first, and then the leader polymer, the template strand of the double-stranded target polynucleotide and the first single strand of the hairpin-like adaptor pass through the pore in sequence, with the complementary strand passing through the pore in such a way that: one end of the first segment of the second single-stranded first segment of the hairpin-like adaptor enters the pore before the first segment, the second segment, the complementary strand of the double-stranded target polynucleotide and the tail polymer pass through the pore in sequence.

Preferably, the second tether is attached to the tail polymer, and one end of the leading polymer enters the hole under the force of an electric field and the second tether.

The first tether is coupled to a second segment of the second single strand of the hairpin-like adaptor, one end of the first segment of the second single strand of the hairpin-like adaptor entering the well under the influence of an electric force and the first tether.

Preferably, the capture region of the first tether has 15-30 bases and the anchor region of the first tether has 30-60 iospc 3 or 8 iSp 18.

Preferably, the polynucleotide binding protein is selected from the group consisting of: a polymerase, helicase or exonuclease or derived from said polymerase, said helicase or said exonuclease.

More preferably, the polynucleotide binding protein is a helicase, more preferably, the helicase is selected from a helicase that unwinds in the 5 '-3' direction or a helicase that unwinds in the 3 '-5' direction;

preferably, when the second single strand of the hairpin-like adaptor is a3 '-5' -5 '-3' second single strand, the helicase used is a helicase that unwinds in the 5 '-3' direction; when the second single strand of the hairpin-like adaptor is a 5 '-3' -3 '-5' second single strand, the helicase used is a helicase that unwinds in the 3 '-5' direction.

Preferably, the transmembrane pore is a protein pore or a solid state pore; preferably, the protein pore is derived from Msp, a-hemolysin (a-HL), lysenin, CsgG, ClyA, Sp1 or FraC; and/or, the membrane is an amphiphilic layer or a solid state layer; preferably, the amphiphilic layer is a lipid bilayer.

Preferably, the construct further comprises one or more markers which, when they interact with the pore, generate a characteristic current; preferably, the one or more markers are abasic groups or specific nucleotide sequences.

More preferably, the one or more markers are in or near the hairpin-like adaptor; more preferably, the one or more markers are located at or near the double-stranded nucleic acid region of the hairpin-like adaptor; more preferably, the one or more markers identify the source of the target polynucleotide.

A fourth aspect of the invention relates to a kit for characterising a double-stranded target polynucleotide, the kit comprising:

the first single strand and the second single strand used to prepare the hairpin-like adaptors described above;

preferably, the kit further comprises: at least one polymer as described above;

preferably, the kit further comprises: a polynucleotide binding protein capable of separating and controlling the movement of both strands of the target polynucleotide through a transmembrane pore, respectively, preferably the polynucleotide binding protein is as described above;

optionally, the kit further comprises: at least one tether as described above.

Preferably, the target polynucleotide and the hairpin-like adaptor are ligated by a sticky end or T-A;

and/or, the kit further comprises one or more markers that produce a characteristic current when the marker interacts with the pore; preferably, the one or more markers are abasic groups or specific nucleotide sequences.

The technical scheme of the invention achieves the following technical effects:

the present invention provides for the ligation of two strands of a target polynucleotide by means of a hairpin-like adaptor provided, and the separation of the template and complementary strands of the target polynucleotide by contacting a construct containing the target polynucleotide with a polynucleotide binding protein to effect effective perforation of the antisense single-stranded polynucleotide under the combined action of electrical forces, such that the template and complementary strands of the target polynucleotide can be sequenced by the transmembrane pore, respectively. This approach can double the amount of information obtained from a single double-stranded target polynucleotide construct. Since the sequence in the complementary strand is necessarily orthogonal to the sequence of the template strand, information from the two strands can be efficiently combined, thereby providing an orthogonally corrected read capability with higher reliability.

In addition, the hairpin-like structure of the invention has a first single strand and a second single strand consisting of a first segment and a second segment that are linked in reverse, the region formed by the combination of the first single strand and the second single strand comprising a double-stranded nucleic acid region and a non-complementary region, such that upon sequencing of a construct comprising the hairpin-like adaptor and a double-stranded target polynucleotide, the template strand and the complementary strand of the target polynucleotide separate and the template strand passes through a transmembrane pore under the action of a polynucleotide-binding protein, and the hairpin-like adaptor can then bring the complementary strand into proximity with the transmembrane pore and translocate the complementary strand through the transmembrane pore under the action of the polynucleotide-binding protein. In the process, the template strand and the complementary strand can be physically separated, the probability of re-hybridization on the reverse side of the transmembrane pore can be obviously reduced, an electric signal is easy to detect, and the accuracy of sequencing is improved.

The hairpin-like adaptors of the invention can also be used with a tether, and to some extent and with some probability, can achieve that after the double-stranded nucleic acid region formed by the second segment of the first single strand and the second single strand is unwound, the second single strand located in the double-stranded nucleic acid region will be exposed and captured by the capture region of the tether, thereby allowing the second single strand of the hairpin-like adaptor, to which the complementary strand of the target polynucleotide is attached, to remain in the vicinity of the transmembrane pore for a period of time and increasing the probability that the complementary strand will pass through the transmembrane pore under the influence of an electric field force.

In the invention, the tether used together with the hairpin-like adaptor is directly anchored on the membrane, so that complex modification on the transmembrane pore is not needed, on one hand, the difficulty in synthesizing the tether is low, on the other hand, the influence on the performance of the transmembrane pore protein is small, and the transmembrane pore protein can better play a role.

The hairpin-like adaptor provided by the invention is used for sequencing double-stranded target polynucleotides, and the translocation speed of two strands of the double-stranded target polynucleotides can be balanced, so that the electric signals of complementary strands can be better detected, and the sequencing accuracy is further improved.

Drawings

FIG. 1 shows a schematic diagram of the structure of hairpin-like adaptors according to one embodiment of the invention. Wherein the symbols are represented as follows: (A) a first 5 '-3' single strand having the corresponding sequence of SEQ ID NO 1 containing 4, 6 or 8 iSP 18; (B) a3 '-5' first segment of the second single strand having the corresponding sequence of SEQ ID NO 2 to which 30T or iSPC3 can be ligated at the 3 'end and which can be modified at the 5' end by Azide (N3) or DBCO (DB); (C) a second 5 ' -3 ' segment of the second single strand having the corresponding sequence of SEQ ID NO 3 comprising 4 iSP18 modified at the 5 ' end by DBCO (DB) or Azide (N3) and a polynucleotide binding protein, e.g., helicase, loaded onto segment C; the 5 ' end of B is linked to the 5 ' end of C to form a second 3 ' -5 ' -3 ' single strand.

FIG. 2 shows a gel diagram of hairpin-like adaptors bound to an enzyme according to one embodiment of the invention.

FIG. 3 shows a schematic structural diagram of a construct containing a hairpin-like structure in combination with a second tether, according to one embodiment of the invention. Wherein marker E represents a double stranded target polynucleotide; e, connecting a hairpin-like adaptor at the right end; the left end is connected with a Y adaptor, wherein the corresponding sequence of Y-Top-1 is SEQ ID NO. 7, the sequence can be separated by 4 iSP18, and the 5' end is connected with 30 iSPC 3; the corresponding sequence of Y-Top-2 is SEQ ID NO 8; the corresponding sequence of Y-Bottom is SEQ ID NO 9; FLT _1D corresponds to the sequence SEQ ID NO. 10, the 5' end of the sequence is connected with 20 iSPC3 and is modified by cholesterol, and the sequence is a second chain; the Y-Top-1 of the Y adaptor is loaded with a polynucleotide binding protein, such as helicase.

FIG. 4 shows a schematic of the structure of a construct not containing hairpin-like adaptors of the invention. Wherein both the left and right ends of the double stranded target polynucleotide are ligated to a Y adaptor, the structure of which is defined in FIG. 3.

FIG. 5 shows an electropherogram of a construct containing hairpin-like adaptors of the invention, according to one embodiment of the invention.

FIG. 6 shows a schematic representation of a process for sequencing a construct containing hairpin-like adaptors using a transmembrane pore, according to one embodiment of the invention. The mode of the template chain passing through the transmembrane pore is as follows: the first polynucleotide binding protein loaded on the Y adaptor (Y-Top-1) unwinds the target polynucleotide duplex E first, until it reaches the duplex nucleic acid region formed by A and C; the polynucleotide binding protein then crosses the non-complementary region of a (i.e. 4, 6 or 8 issp 18) under the influence of an electric field; the double-stranded nucleic acid region formed by A and B is then unwound and the polynucleotide binding protein is washed out, and after this step is completed, the template strand and the complementary strand are completely separated and the template strand passes completely through the transmembrane pore. At the instant after the polynucleotide binding protein washes out, the 3' end of B (i.e., 30T or iSPC 3) is very close to the transmembrane pore and can drive the complementary strand through the transmembrane pore under the influence of an electric field. The way the complementary strand passes through the transmembrane pore is: the second polynucleotide binding protein on C will translocate and control the rate on the complementary strand single strand primarily by means of Translocation.

D is a first tether, the corresponding sequence of which is SEQ ID NO. 4, to which 30-60 iSPC3 or 8 iSP18 are linked at the 5 'or 3' end and which is modified with cholesterol. If D is present, the Chol cholesterol tag on D is anchored to the membrane beforehand, the 8 iSP18 or 30-60 iSPC3 regions on D are longer than the conventional tethers and more accessible to the porin coronary open area, making it easier for the leading end of the complementary strand to remain around the porin and to achieve a via under electrical forces. After the first polynucleotide binding protein unwinds into the double-stranded nucleic acid region formed by A and C, the C in the double-stranded nucleic acid region is exposed and has a certain probability of complementary binding with the D sequence. If C forms a region of complementarity with D, then the second polynucleotide binding protein on C will unwind the double strand formed by C and D when it passes through the complementary strand.

FIG. 7 shows a schematic of a process for sequencing the construct of FIG. 4 without hairpin-like adaptors of the invention using a transmembrane pore. Wherein a polynucleotide (e.g., DNA) construct translocates through a nanopore under the control of an enzyme, and a complementary strand cannot pass through the pore after the template strand enters and passes through the transmembrane pore.

FIG. 8 shows a double stranded real time sequencing electrical signal for sequencing a hairpin adaptor-like construct according to one embodiment of the invention, where (a) is a complete signal diagram and (b) is an enlarged view of the signal in box in (a).

Figure 9 shows a schematic of the process of sequencing constructs containing standard hairpin adapters using transmembrane pores. Wherein the template strand and the complementary strand are linked by a standard hairpin adaptor (SEQ ID NO: 5) and the template strand comprises a 5' leader sequence. A polynucleotide (e.g., DNA) construct is translocated through the nanopore under the control of an enzyme, the template strand enters the nanopore and the same enzyme advances around the hairpin to control the motion of the complementary strand behind the template strand, which can reform on the opposite side of the nanopore once the hairpin region is translocated through the nanopore.

Figure 10 shows an accuracy profile of real-time double-stranded sequencing signals for constructs containing standard hairpin adaptors.

FIG. 11 shows an accuracy profile of real-time double-stranded sequencing signals for constructs containing hairpin-like adaptors.

FIG. 12 shows a schematic of the structure of a nanopore sequencer used in the present invention.

Description of sequence listing:

SEQ ID NO:1 shows the 5 '-3' (left to right in FIG. 1) first single strand of the hairpin-like adaptor

5’- P- AAGC GTCAG AGAGG TTCCA AGTCA GAGAG GTTCC-(iSp18)₈ -TACTG ATAGC GTCTG CATCT -3’

SEQ ID NO:2 shows the 3 '-5' (right to left in FIG. 1) first segment of the second single strand of the hairpin-like adaptor

5’- ^N3 AGATG CAGAC GCTAT CAGTA -T₃₀ -3’

SEQ ID NO 3 shows the 5 '-3' (right to left in FIG. 1) second segment of the second single strand of the hairpin-like adaptor

5’- ^DBTTTTT TTTTT - (iSp18)₄ - GGAAC CTCTC TGACT TGGAA CCTCT CTGAC -3’

3 '-5' -5 '-3' (right to left in FIG. 1) second single strand of hairpin-like adaptor

3’- T₃₀- ATGAC TATCG CAGAC GTAGA^N3-5’ - 5’-^DBTTTTT TTTTT - (iSp18)₄ - GGAAC CTCTC TGACT TGGAA CCTCT CTGAC -3’

SEQ ID NO 4 shows the first tether bound to a hairpin-like adaptor

5’- GTCAG AGAGG TTCCA AGTCA GAGAG GTTCC- (iSpC3)₄₀ -Chol -3’

SEQ ID NO 5 shows a standard hairpin adaptor

5’P- AAGC g tagtc gtggt aggca ggttg gtgac -(iSp18)₄-TTTTT TTTTT-(iSp18)₄-gtc ac caacc tgcct accac gacta c -3’

SEQ ID NO 6 shows the template sequence of the plasmid DNA

SEQ ID NO 7 shows Y-Top-1 of the Y adaptor

5’-(iSpC3)₃₀- GCGTG ACTAT CGGAC TCGTG GTC TTTTT TTTTT-(iSp18)₄-GTCAG TTCGC TTCTT ACGCA TCACA CT T-3’

SEQ ID NO 8 shows Y-Top-2 of the Y adaptor

5’- GAC CACGA GTCCG ATAGT CACGC -3’

SEQ ID NO 9 shows Y-Bottom of the Y adaptor

5’- P-AG TGTGA AGTCC AGCAC CGACCTGCGT AAGAA GCGAA CTGAC T-3’

SEQ ID NO 10 shows the second tether FLT _1D of the Y adaptor

5’- Chol-(iSpC3)₂₀- GGTCG GTGCT GGACT -3’

BsaI forward primer is shown in SEQ ID NO. 11

5’-TCGCC ATTCA GGCTG CGC-3’

BsaI reverse primer is shown in SEQ ID NO. 12

5’-GCTTA GAGAC CTGTG CGG-3’

The amino acid sequence of the T4 Dda helicase is shown in SEQ ID NO 13.

Detailed Description

It is understood that different applications of the disclosed products and methods can be tailored to the specific needs in the art. It is also 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.

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

Definition of

In order to more clearly explain the embodiments of the present invention, some scientific terms and terminology are used herein. Unless explicitly defined herein, all such terms and nouns shall be understood to have the meanings that are commonly understood by those skilled in the art. For the sake of clarity, the following definitions are made for certain terms used herein.

Double stranded target polynucleotides

The method of the invention is used for sequencing double-stranded polynucleic acids.

The polynucleotide may be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target polynucleotide may comprise one RNA strand hybridized to one DNA strand. The polynucleotide may be any synthetic nucleic acid known in the art, such as Peptide Nucleic Acid (PNA), Glycerol Nucleic Acid (GNA), Threose Nucleic Acid (TNA), Locked Nucleic Acid (LNA), or other synthetic polymers having nucleotide side chains.

The polynucleotide is preferably DNA, RNA or a hybrid of DNA and RNA, most preferably DNA. The target polynucleotide may be double stranded. The target polynucleotide may comprise single stranded regions and regions having other structures, such as hairpin loops, triplexes, and/or quadruplexes. A DNA/RNA hybrid may comprise DNA and RNA on the same strand. Preferably, the DNA/RNA hybrid comprises one DNA strand hybridized to an RNA strand.

The target polynucleotide can be of any length. For example, a 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 nucleotide pairs in length. The polynucleotide may be 1000 or more nucleotide pairs, 5000 or more nucleotide pairs in length or 100000 or more nucleotide pairs in length.

The target polynucleotide may be present in any suitable sample. The invention is generally performed on samples known to contain, or suspected of containing, the target polynucleotide. Alternatively, the invention may be performed on a sample to determine the identity of one or more target polynucleotides known or expected to be present in the sample.

Hair-like adaptor

The methods of the invention employ hairpin-like adaptors that are capable of ligating two strands of a target polynucleotide. Hairpin-like adaptors of the invention have a loop similar to a conventional hairpin structure, but the loop is not formed by the back-folding of a single-stranded linear molecule upon itself, as in a conventional hairpin structure, but rather by the inversion of the non-complementary regions of two partially complementary single-stranded linear molecules.

In one embodiment of the invention, the hairpin-like adaptor comprises a first single strand and a second single strand for ligation to a template strand and a complementary strand of the double-stranded target polynucleotide, respectively, the first single strand and the second single strand forming at least 1 double-stranded nucleic acid region for bringing the complementary strand into proximity with a transmembrane pore after the template strand has passed through the transmembrane pore. The second single strand comprises a first segment and a second segment that are linked in reverse. The second single strand is formed by connecting the 5 'end of the first segment along the 3' -5 'direction with the 5' end of the second segment along the 5 '-3' direction; or the second single strand is formed by connecting the 3 'end of the first segment along the 5' -3 'direction with the 3' end of the second segment along the 3 '-5' direction. The first single strand and the second single strand are both single-stranded sequences formed by a base group and an abasic group. The first single strand and the second single strand form 2 double-stranded nucleic acid regions, the 2 double-stranded nucleic acid regions are respectively formed in the first segment and the second segment of the second single strand, and the double-stranded nucleic acid regions are non-covalently linked double-stranded regions. The first single strand and the second single strand also form a non-complementary region. The 2 double-stranded nucleic acid regions and the non-complementary region formed by the first single strand and the second single strand form a hairpin-like structure. The region of the second segment of the second single strand located in the non-complementary region comprises a binding site for a polynucleotide binding protein, and the region of the first segment of the second single strand located in the non-complementary region is located on the first segment distal to the second segment. The second single strand is formed by reverse ligation of the first segment and the second segment using a click chemistry reaction; alternatively, the second single strand is synthesized directly by using the forward base and the reverse base; preferably, the reagent used in the click chemistry reaction is selected from chemical groups such as DBCO, Azide, Tetrazine or TCO, and the inverted base is selected from inverted thymidine (inverted dTs), inverted adenosine (inverted dAs), inverted guanosine (inverted dGs) or inverted cytidine (inverted dCs).

The base groups used in the hairpin-like structure are groups containing a nucleobase to form a base pair. It can be called "nucleotide", nucleotide is a kind of compound composed of purine base or pyrimidine base, ribose or deoxyribose and phosphate, also called as nucleotide. Pentose and organic base synthesize nucleoside, nucleoside and phosphoric acid synthesize nucleotide, 4 kinds of nucleotides compose nucleic acid. Examples of the nucleotide include adenine nucleotide (adenosine, AMP), guanine nucleotide (GMP), cytosine nucleotide (cytidine, CMP), uracil nucleotide (uridylic acid, UMP), thymidylic acid (thymidylic acid, TMP), and hypoxanthine nucleotide (inosinic acid, IMP). Nucleotides are mainly involved in the construction of nucleic acids.

The abasic group used in the hairpin-like structure is an iSP18 group or an iSPC3 group or the like which cannot form a base pair. It may be referred to as an "abasic site" or an "abasic nucleotide". An abasic group is a nucleotide or nucleoside that lacks a nucleobase at the 1' position of the sugar moiety. (see, e.g., U.S. Pat. No. 5,998,203).

Preferably, the hairpin-like adaptors of the invention comprise a3 ' -5 ' -3 ' single stranded linear molecule having the structure of a3 ' -5 ' segment-X-5 ' -3 ' segment, wherein the 5 ' -3 ' segment comprises a plurality of nucleotides and a plurality of iSpC3 groups that are incapable of forming a complementary double strand; x is absent or is a linker for click chemistry; the 3 '-5' segment is the reverse region that is opposite to the direction of reading (5 '-3') of the entire single-stranded linear molecule, and comprises a plurality of nucleotides and a plurality of iSPC3 groups that are not capable of forming complementary double strands. These two segments can be formed by various means, such as synthesizing the 5 ' -3 ' segment, and then synthesizing the remaining 3 ' -5 ' segment at the 5 ' -end of the synthesized 5 ' -3 ' segment using the inverted base as a substrate (in the absence of X); or by separately synthesizing the 3 '-5' segment and the 5 '-3' segment and then joining the two segments by using a click chemistry reagent (in the case where X is a linker for click chemistry).

Hairpin-like adaptors of the invention also include a single-stranded linear molecule comprising a plurality of nucleotides and a plurality of iSpC3 or iSP18 groups, which linear molecule is at least partially complementary to the 3 ' -5 ' -3 ' single-stranded linear molecule described above, forming a double-stranded nucleic acid region, which is a region of hybridized double-stranded region formed by base-complementary pairing of two single strands, such as by base-complementary pairing, and a non-complementary region, which is a region where the two single strands are not paired with each other and do not hybridize, but exist as two free single strands. The complementary double-stranded region includes two, divided into a3 ' -5 ' segment and a 5 ' -3 ' segment formed in a3 ' -5 ' -3 ' single-stranded linear molecule. The 2 double-stranded nucleic acid regions and the non-complementary region formed by the first single strand and the second single strand form a hairpin-like structure. The region on the second single strand located at the non-complementary region facilitates binding of the complementary strand of the double-stranded target polynucleotide to a polynucleotide binding protein that facilitates separation of the double-stranded nucleic acid region of the hairpin-like adaptor and/or controls movement of the single-stranded polynucleotide through the pore. The length of each of the 2 double-stranded nucleic acid regions is preferably 20 bp-50 bp, and the second single strand preferably comprises 20-40 iSPC 3.

Hairpin-like adaptors of the invention are preferably conjugated to a tether (tether), which is a single-stranded polynucleotide composed of a plurality of nucleotides, and which also contains groups that are incapable of forming a complementary double strand (such as iSp18 or iSpC3, etc.). During sequencing of the target polynucleotide through the pore under the force of an electric field, the double-stranded nucleic acid region of the hairpin-like adaptor is separated to expose the two strands of the double-stranded nucleic acid region, one end of the tether is complementarily bound to the exposed one strand to connect the complementary strands of the double-stranded target polynucleotide, and the other end of the tether is preferably attached to the membrane in which the transmembrane pore is located, so that the complementary strands surround the pore.

Click chemistry is the first term proposed by Kolb et al in 2001, which describes an expanded set of powerful selective and modular building blocks that can reliably work in small-scale and large-scale applications (Kolb HC, Finn, MG, sharp KB, Click chemistry: differential chemical function from a raw good reactions, angew. chem. int. ed.40(2001) 2004-. They have defined a series of stringent click chemistry criteria: the reaction must be modular, broad in scope, very high in yield, produce only harmless by-products that can be removed by non-chromatographic methods, and stereospecific (but not necessarily enantioselective). Desirable process characteristics 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 benign solvents (such as water) or easy removal, and simple product isolation. If desired, purification by non-chromatographic methods, such as crystallization or distillation, must be carried out, and the product must be stable under physiological conditions ".

Suitable examples of click chemistry include, but are not limited to, the following:

(a) copper-free variants of 1, 3-dipolar cycloaddition reactions, wherein azides are reacted under strain with alkynes, for example in cyclooctane rings;

(b) reaction of an oxygen nucleophilic reagent on one linker with an epoxide or aziridine reactive moiety on the other; and

(c) staudinger ligation, in which the alkyne moiety can be substituted with an aryl phosphine, results in a specific reaction with the azide to give an amide bond.

Preferably, the click chemistry reaction is a cu (i) -catalyzed 1,3 dipolar cycloaddition reaction between an alkyne and an azide. In a preferred embodiment, the linker attached to the 5 'end of the 3' -5 'segment is an azido group and the linker attached to the 5' end of the 5 '-3' segment is an alkynyl group. Nucleobases which incorporate azido and alkynyl groups at preferred positions have been synthesized (e.g., Kocalka P, El-Sagher AH, Brown T, Rapid and functional DNA strand and cross-linking by click chemistry, Chemiochem. 2008.9 (8): 1280-5). Alkynyl groups are commercially available from Berry Associates (Michigan, USA) and azide groups can be synthesized from ATDBio.

The invention provides a construct comprising a double stranded target polynucleotide to be sequenced. The constructs typically allow sequencing of both strands of a target polynucleotide through a transmembrane pore.

The construct comprises a bridging moiety capable of linking two strands of a target polynucleotide. The bridging moiety typically covalently connects the two strands of the target polynucleotide. The bridging moiety can be any substance capable of linking the two strands of the target polynucleotide, provided that the bridging moiety does not interfere with the movement of the single-stranded polynucleotide through the transmembrane pore. Suitable bridging moieties include, but are not limited to, polymeric linkers, chemical linkers, polynucleotides or polypeptides. Preferably, the bridging moiety comprises DNA, RNA, modified DNA (e.g. abasic DNA), RNA, PNA, LNA or PEG. More preferably, the bridging moiety is DNA or RNA.

In the present invention, the bridging moiety is a hairpin-like adaptor of the invention.

The bridging moiety is attached to the target polynucleotide construct by any suitable method known in the art. The bridging moiety can be synthesized separately and then chemically or enzymatically linked to the target polynucleotide. Alternatively, the bridging moiety is generated during processing of the target polynucleotide.

The bridging moiety is attached to the target polynucleotide at or near one end of the target polynucleotide. The bridging moiety is preferably attached to the target polynucleotide within 10 amino acids of the terminus of the target polynucleotide.

The construct comprising the target polynucleotide also preferably comprises at least one polymer at the end of the target polynucleotide opposite the bridging moiety. As discussed in more detail below, such polymers facilitate the sequencing methods of the invention. Suitable polymers include polynucleotides (DNA/RNA), modified polynucleotides such as modified DNA, PNA, LNA, PEG or polypeptides.

The construct preferably comprises a leader polymer. The leader polymer is attached to the target polynucleotide at an end opposite the bridging moiety. The leader polymer facilitates binding of the double-stranded target polynucleotide to the transmembrane pore or to a polynucleotide binding protein which facilitates separation of the two strands and/or controls movement of the single-stranded polynucleotide through the pore. Transmembrane pores and polynucleotide binding proteins are discussed in more detail below.

The lead polymer may be a polynucleotide (e.g., DNA or RNA), a modified polynucleotide (e.g., abasic DNA), PNA, LNA, PEG, or polypeptide. The leader polymer is preferably a polynucleotide, more preferably a single stranded polynucleotide. The lead polymer may be any of the polynucleotides described above. The single-stranded leader polymer is most preferably a single-stranded DNA. The leader polymer may be of any length, but is typically 27 to 150 nucleotides in length, for example 50 to 150 nucleotides in length.

Portions of a single-stranded polynucleotide can be added to a double-stranded target polynucleotide in a variety of ways. Chemical ligation or enzymatic ligation may be performed. Furthermore, Epicentre's Nextera method is suitable.

The construct preferably further comprises a polymeric tail (also attached to the target polynucleotide at the end opposite the bridging moiety). The polymer tail aids the transmembrane pore in sequencing the target construct. In particular, the polymer tail typically ensures that all of the double stranded target polynucleotide (i.e. all of both strands) can be read and sequenced by the transmembrane pore. As described below, the polynucleotide binding protein can control the movement of the single-stranded polynucleotide through the transmembrane pore. The protein typically slows the movement of the polynucleotide through the pore. For example, Phi29 DNA polymerase acts like a brake, slowing the movement of the polynucleotide through the pore following the applied potential across the membrane. Once the polynucleotide is no longer in contact with the binding protein, it will move freely through the pore at a rate at which sequence information is difficult to obtain. Because the distance from the protein to the pore is typically short, typically about 20 nucleotides, some sequence information (approximately equal to this distance) may be missed. The tail polymer "extends" the length of the single-stranded polynucleotide such that when both strands of the target polynucleotide are passed through the pore and sequenced, its movement can be controlled by the nucleic acid binding protein. Such embodiments ensure that sequence information can be obtained from both strands in the target polynucleotide. The tail polymer may also provide a site for primer binding that enables the nucleic acid binding protein to separate the two strands of the target polynucleotide.

The tail polymer may be a polynucleotide (e.g., DNA or RNA), a modified polynucleotide (e.g., abasic DNA), PNA, LNA, PEG, or polypeptide. The tail polymer is preferably a polynucleotide, more preferably a single stranded polynucleotide. The tail polymer may be any of the polynucleotides described above.

The construct preferably further comprises one or more markers which produce a characteristic current (characteristic marker current) when the construct is passed through the transmembrane pore. The marker is typically used to enable the position of the single stranded polynucleotide relative to the pore to be estimated or determined. For example, a signal from a marker located between the two strands of the target polynucleotide indicates that one strand has been sequenced and that the other strand is about to enter the pore. Thus, such markers can be used to distinguish between the template strand and the complementary strand of the target DNA. One or more markers may also be used to identify the source of the target polynucleotide. Suitable markers include, but are not limited to, abasic regions, specific nucleotide sequences, unnatural nucleotides, fluorophores, or cholesterol. The marker is preferably an abasic region or a specific nucleotide sequence.

One or more markers may be located anywhere in the construct. One or more markers may be located in the bridging moiety. One or more markers may also be located adjacent to the bridging moiety. The vicinity of the bridging moiety preferably means within 10 to 100 nucleotides of the bridging moiety.

The marker may also be placed within the lead polymer or the tail polymer.

The construct may be coupled to the membrane using any known method. If the membrane is an amphiphilic layer, such as a lipid bilayer (as described in detail below), the construct is preferably coupled to the membrane via a polypeptide present in the membrane or a hydrophobic anchor present in the membrane. The hydrophobic anchor is preferably a lipid, fatty acid, sterol, carbon nanotube or amino acid.

The construct may be coupled directly to the membrane. The construct is preferably coupled to the membrane via a linker. Preferred linkers include, but are not limited to, polymers such as polynucleotides, polyethylene glycol (PEG), and polypeptides. If a polynucleotide is coupled directly to the membrane, some sequence data is lost because the sequencing run cannot continue to the end of the polynucleotide due to the distance between the membrane and the detector. If a linker is used, the polynucleotide can be processed to completion. If a linker is used, the linker may be attached to the construct at any position. The linker is preferably attached to the polynucleotide at the tail polymer.

Polynucleotide binding proteins

The polynucleotide binding protein may be any protein that is capable of binding to a polynucleotide and controlling its movement through a pore. It is straightforward in the art to determine whether a protein binds to a polynucleotide. Proteins typically interact with polynucleotides and modify at least one property thereof. Proteins can modify a polynucleotide by cleaving the polynucleotide to form individual nucleotides or shorter strands of nucleotides such as dinucleotides or trinucleotides. The moiety may modify the polynucleotide by localizing or moving the polynucleotide to a specific location (i.e., controlling its movement).

In some embodiments, the polynucleotide binding protein is a polynucleotide unzipping enzyme. Polynucleotide unzipping enzymes are enzymes that are capable of unzipping a double-stranded target polynucleotide into single strands. In some embodiments, the polynucleotide unzipping enzyme is capable of unzipping double-stranded DNA into single strands. In some embodiments, the polynucleotide unzipping enzyme is an enzyme with helicase activity. Examples of polynucleotide unzipping enzymes include, for example, helicases described herein. The helicase is selected from helicase helicating in the 5 '-3' direction or helicase helicating in the 3 '-5' direction.

Polynucleotide binding capacity can be measured using any method known in the art. For example, a protein can be contacted with a polynucleotide, and the ability of the protein to bind to and move along the polynucleotide can be measured. The protein may include modifications that facilitate polynucleotide binding and/or facilitate its activity at high salt concentrations and/or at room temperature. Proteins can be modified such that they bind to a polynucleotide (i.e., retain polynucleotide binding capacity) but do not act as a melting enzyme (i.e., when provided with all the necessary components (e.g., ATP and Mg) to facilitate movement²⁺) While not moving along the polynucleotide). Such modifications are known in the art. For example, Mg in helicases²⁺Binding domainsThe modification(s) of (a) usually results in a variant that does not function as a helicase.

The enzyme may be covalently attached to the pore. Any method may be used to covalently attach the enzyme to the pore.

In strand sequencing, polynucleotides are translocated through a pore along or against an applied potential. Exonuclease which acts gradually or stepwise on a double stranded target polynucleotide can be used on the cis side of the well to supply the remaining single strands at an applied potential or on the reverse side to supply the remaining single strands at a reverse potential. Likewise, helicases that melt double-stranded DNA can also be used in a similar manner. Polymerases may also be used. Sequencing applications that require strand translocation against an applied potential are also possible, but the DNA must first be "captured" by the enzyme at the opposite or no potential. As the potential is subsequently switched upon binding, the chain will pass through the pore in a cis-to-trans fashion and remain in an extended configuration by the current. Single-stranded DNA exonuclease or single-stranded DNA-dependent polymerase can act as a molecular motor to pull back recently translocated single strands from trans to cis from the wells in a controlled stepwise manner against an applied potential.

Any helicase may be used in the present invention. Helicases act on the pore in two modes. First, the method is preferably performed using a helicase, such that the helicase moves the polynucleotide through the pore under the action of a field caused by an applied voltage. In this mode, the 5' end of the polynucleotide is first captured in the pore and the helicase moves the polynucleotide into the pore, passing it through the pore under the action of the field until it finally translocates through to the opposite side of the membrane. Alternatively, the method is preferably performed such that the unzipping enzyme moves the polynucleotide through the pore against the field caused by the applied voltage. In this mode, the 3' end of the polynucleotide is first captured in the pore and the unzipping enzyme moves the polynucleotide through the pore such that it is pulled out of the pore against the applied field until it is finally pushed back to the cis side of the membrane.

The method can also be carried out in the opposite direction. The 3' end of the polynucleotide may be captured first in the well and a unzipping enzyme may move the polynucleotide into the well so that it passes through the well under the influence of the field until it eventually translocates through to the opposite side of the membrane.

When the melting enzyme does not have the necessary components to facilitate movement or is modified to prevent or prevent movement, the melting enzyme can bind to the polynucleotide and act as a brake to slow the movement of the polynucleotide as it is pulled into the well by the applied field. In the inactive mode, it is not important whether the 3 'or 5' of the polynucleotide is trapped, and it is the applied field that pulls the polynucleotide into the pore towards the reverse side under the action of the enzyme acting as a brake. When in the inactive mode, control of polynucleotide movement by the unzipping enzyme can be described in a variety of ways, including ratcheting, slipping, and braking. Melting enzyme variants lacking melting enzyme activity may also be used in this manner.

The polynucleotide and polynucleotide binding protein (e.g., polynucleotide unzipping enzyme) and the pore can be contacted in any order. Preferably, when a polynucleotide is contacted with a polynucleotide binding protein (e.g., a polynucleotide unzipping enzyme) and a pore, such as a helicase, the polynucleotide first forms a complex with the polynucleotide binding protein (e.g., a polynucleotide unzipping enzyme). When a voltage is applied across the pore, the polynucleotide/polynucleotide binding protein (e.g., polynucleotide unzipping enzyme) complex forms a complex with the pore and controls the movement of the polynucleotide through the pore.

Any step in a method of using a polynucleotide binding protein (e.g., a polynucleotide unzipping enzyme) is typically performed in the presence of free nucleotides or free nucleotide analogs and enzyme cofactors that facilitate the action of the polynucleotide binding protein (e.g., a polynucleotide unzipping enzyme). The free nucleotides may be one or more of any individual nucleotide. 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 (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), and deoxycytidine triphosphate (dCTP). The free nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotide is preferably Adenosine Triphosphate (ATP). Enzyme cofactors are factors that allow the construct to function. The enzyme cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg²⁺、Mn²⁺、Ca²⁺Or Co²⁺. The enzyme cofactor is most preferably Mg²⁺。

Film

Any membrane may be used according to the present invention. Suitable membranes are well known in the art. The membrane is preferably an amphiphilic layer. The amphiphilic layer is a layer formed of amphiphilic molecules (e.g., phospholipids) having hydrophilicity and lipophilicity. The amphiphilic molecules may be synthetic or naturally occurring. Non-naturally occurring amphiphiles and monolayer-forming amphiphiles are known in the art and include, for example, block copolymers (Gonzalez-Perez et al, Langmuir,2009,25, 10447-10450). Block copolymers are polymeric materials in which two or more monomer subunits are polymerized together to produce a single polymer chain. Block copolymers generally have properties provided by each monomer subunit. However, block copolymers can have unique properties that are not possessed by polymers formed from individual subunits. The block copolymer can be designed such that: one monomeric subunit is hydrophobic (i.e., lipophilic) while the other subunit or subunits are hydrophilic when in an aqueous medium. In this case, the block copolymer may have amphiphilic properties, and may form a structure simulating a biofilm. The block copolymer may be diblock (consisting of two monomer subunits), but may also be composed of more than two monomer subunits to form a more complex arrangement that behaves as an amphiphile. The copolymer may be a triblock, tetrablock or pentablock copolymer.

Archaebacterial bipolar tetraether lipids are naturally occurring lipids that are configured such that the lipids form a monolayer membrane. These lipids are commonly found in extremophiles, thermophiles, halophiles and acidophiles that live in harsh biological environments. Their stability is believed to come from the fusion properties of the final bilayer. Block copolymer materials that mimic these biological entities can be readily constructed by generating hydrophilic-hydrophobic-hydrophilic triblock polymers with the general motif. The material can form a monomeric membrane that behaves like a lipid bilayer and has a range of phase states from vesicle to membrane. Membranes formed from these triblock copolymers have several advantages over biolipid membranes. Because the triblock copolymers are synthetic, the precise construction can be carefully controlled to provide the correct chain length and properties needed to form a membrane and interact with pores and other proteins.

Block copolymers can also be constructed from subunits that are not classified as lipid biomaterials; for example, the hydrophobic polymer may be made from silicone or other non-hydrocarbon compound based monomers. The hydrophilic sub-portion of the block copolymer may also have low protein binding properties, which enables the production of a membrane that is highly resistant when exposed to unprocessed biological samples. The headgroup unit may also be derived from an atypical lipid headgroup.

Triblock copolymer membranes also have enhanced mechanical and environmental stability, such as much higher operating temperatures or pH ranges, compared to biolipidic membranes. The synthetic nature of the block copolymers provides a platform for tailoring polymer-based films for a wide variety of applications.

The amphipathic molecule may be chemically modified or functionalized to facilitate coupling of the analyte.

The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic layer is generally planar. The amphiphilic layer may be non-planar, e.g. curved.

The amphiphilic layer is typically a lipid bilayer. Lipid bilayers are a model of cell membranes and serve as an excellent platform for a series of experimental studies. For example, lipid bilayers can be used for in vitro studies of membrane proteins using single channel recordings. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of substances. The lipid bilayer may be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, planar lipid bilayers, supported bilayers, or liposomes. The lipid bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers are disclosed in international application No. PCT/GB08/000563 (published as WO 2008/102121), international application No. PCT/GB08/004127 (published as WO 2009/077734) and international application No. PCT/GB2006/001057 (published as WO 2006/100484).

Methods for forming lipid bilayers are known in the art. Suitable methods are disclosed in the examples. Lipid bilayers are typically formed by the method of Montal and Mueller (Proc. Natl. Acad. Sc1.USA., 1972;69: 3561-3566), in which a lipid monolayer is carried at the aqueous solution/air interface and through one side of a pore perpendicular to the interface.

The Montal and Mueller process is popular because it is a cost-effective and relatively easy method of forming high quality lipid bilayers suitable for protein pore insertion. Other commonly used methods of bilayer formation include head-dipping (tip-coating), coating bilayers (lipid bilayers), and patch clamping of liposome bilayers.

In a preferred embodiment, the lipid bilayer is formed as described in international application PCT/GB08/004127 (published as WO 2009/077734). Advantageously in this method, the lipid bilayer is formed from dried lipids. In a most preferred embodiment, a lipid bilayer is formed across the opening as described in WO2009/077734 (PCT/GB 08/004127).

In another preferred embodiment, the membrane is a solid layer. The solid layer is not of biological origin. In other words, the solid-state layer is not derived or isolated from a biological environment, such as an organism or cell, or a biologically useful structure in a synthetically manufactured form. The solid-state layer may be formed from organic or inorganic materials, including, but not limited to, microelectronic materials, insulating materials such as Si₃N₄、Al₂O₃And SiO₂Organic and inorganic polymers such as polyamides, plastics such as plastics or elastomers such as two-component addition-type silicone rubber (two-component addition-silicone rubber) and glass. The solid-state layer may be formed from graphene. Suitable graphene layers are disclosed in international application PCT/US2008/010637 (published as WO 2009/035647).

Transmembrane pore

A transmembrane pore is a structure that allows hydrated ions driven by an applied potential to flow from one side of the membrane to the other side of the membrane.

The transmembrane pore is preferably a transmembrane protein pore. A transmembrane protein pore is a polypeptide or collection of polypeptides that allows hydrated ions (e.g., analyte) to flow from one side of the membrane to the other side of the membrane. In the present invention, the transmembrane protein pore is capable of forming a pore that allows hydrated ions driven by an applied potential to flow from one side of the membrane to the other. Transmembrane protein pores preferably allow the flow of analytes (e.g. nucleotides) from one side of the membrane (e.g. lipid bilayer) to the other. A transmembrane protein pore allows a polynucleotide (e.g. DNA or RNA) to move through the pore.

Transmembrane protein pores may be monomeric or oligomeric. The pore is preferably composed of several repeating subunits (e.g., 6, 7 or 8 subunits). More preferably the pores are heptameric or octameric pores.

Transmembrane protein pores typically comprise a barrel or channel through which the ions can flow. The subunits of the pore generally surround the central axis and provide chains for transmembrane β -barrels or channels or transmembrane α -helical bundles or channels.

The barrel or channel of a transmembrane protein pore typically comprises amino acids that facilitate interaction with an analyte (e.g., a nucleotide, polynucleotide, or nucleic acid). These amino acids are preferably located near the constriction of the barrel or channel. Transmembrane protein pores typically comprise one or more positively charged amino acids (e.g. arginine, lysine or histidine) or aromatic amino acids (e.g. tyrosine or tryptophan). These amino acids typically facilitate the interaction between the pore and the nucleotide or polynucleotide or nucleic acid.

Transmembrane protein pores useful in the present invention may be derived from a β -barrel pore or an α -helix bundle pore, the β -barrel pore comprising a barrel or channel formed by a β -strand. Suitable β -barrel pores include, but are not limited to, β -toxins such as α -hemolysin, anthrax toxin, and leukocidin, and bacterial outer membrane proteins/porins (porins) such as Mycobacterium smegmatis (Mycobacterium smegmatis) porin (MspA), outer membrane porin f (ompf), outer membrane porin g (ompg), outer membrane phospholipase a, and Neisseria (Neisseria) autotransporter lipoprotein (NalP). The alpha-helix bundle bore contains a barrel or channel formed by the alpha-helix. Suitable a-helix bundle pores include, but are not limited to, inner membrane proteins and a-outer membrane proteins, such as WZA and ClyA toxins. The transmembrane pore may be derived from Msp or alpha-hemolysin (alpha-HL).

The transmembrane protein pore is preferably derived from Msp, preferably from MspA. Such pores are oligomers and typically comprise 7, 8, 9 or 10 monomers derived from Msp. The pore may be a homo-oligomeric (homo-oligomeric) pore derived from Msp comprising the same monomer. Alternatively, the pore may be a hetero-oligomeric (heter-oligomeric) pore derived from Msp containing at least one monomer different from the other monomer. The pore may further comprise one or more constructs comprising two or more covalently linked monomers derived from Msp.

The transmembrane protein pore is also preferably derived from alpha-hemolysin (alpha-HL). The wild-type α -HL pore is formed from 7 identical monomers or subunits (i.e. it is a heptamer).

In some embodiments, the transmembrane protein pore is chemically modified. The pores may be chemically modified at any site in any manner. The transmembrane protein pore is preferably chemically modified by binding of the molecule to one or more cysteines (cysteine linkage), binding of the molecule to one or more lysines, binding of the molecule to one or more unnatural amino acids, enzymatic modification of an epitope, or modification of the termini. Suitable methods for making such modifications are well known in the art. The transmembrane protein pore may be chemically modified by binding any molecule. For example, the pore may be chemically modified by the incorporation of a dye or fluorophore.

Any number of monomers in the pore may be chemically modified. Preferably, one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10, of said monomers are chemically modified as described above.

Separate by separating

The two strands of the target polynucleotide are separated using a polynucleotide binding protein.

The polynucleotide binding protein is preferably derived from a polynucleotide processing enzyme. However, the enzyme may be used under conditions where the enzyme does not catalyze the reaction. For example, as described in more detail below, proteins from Phi29 DNA polymerase can be used in melting mode.

A polynucleotide processive enzyme is a polypeptide that is capable of interacting with a polynucleotide and modifying at least one property of the polynucleotide. The enzyme may modify the polynucleotide by cleaving the polynucleotide to form a single nucleotide or shorter nucleotide strand (e.g., a dinucleotide or a trinucleotide). The enzyme may modify the polynucleotide by orienting the polynucleotide or moving the polynucleotide to a particular position. The polynucleotide handling enzyme need not exert enzymatic activity as long as it is capable of binding the target polynucleotide and preferably controlling the movement of the target polynucleotide through the pore. For example, the enzyme may be modified to remove its enzymatic activity, or the enzyme may be used under conditions that prevent it from functioning as an enzyme. Such conditions are detailed in more detail below.

The polynucleotide binding proteins are typically derived from the subfamily picoviridae (Picovirinae family). Suitable viruses include, but are not limited to, AHJD-like viruses and Phi 29-like viruses. The polynucleotide binding protein is preferably derived from Phi29 DNA polymerase or helicase.

The method can preferably be carried out in melting mode using a protein derived from Phi29 DNA polymerase. In this embodiment, steps (b), (c) and (d) are carried out in the absence of free nucleotides and in the absence of enzyme cofactors such that the polymerase controls movement of the single-stranded polynucleotide through the pore (as the polynucleotide is untied) along the field generated by the applied voltage. In this embodiment, the polymerase acts as a brake, preventing the single stranded polynucleotide from moving through the pore too quickly under the influence of the applied voltage.

Move

In the methods of the invention, the two single-stranded polynucleotides of the double-stranded target polynucleotide are moved sequentially through the transmembrane pore. Thus, the entire target polynucleotide is moved through the pore and sequenced. Moving a single stranded polynucleotide through the transmembrane pore means moving the polynucleotide from one side of the pore to the other. Movement of the single stranded polynucleotide through the pore may be driven or controlled by an electric potential or enzymatic action or both. The movement may be unidirectional or may allow for both backward and forward movement.

Preferably the single stranded polynucleotide is controlled to move through the pore using a polynucleotide binding protein. The protein is preferably the same protein that separates the two strands of the polynucleotide.

Method for characterizing double-stranded target polynucleotides

The characterization method may comprise measuring one, two, three, four or five or more characteristics of the target polynucleotide. The characteristic is preferably selected from (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide, and (v) whether the polynucleotide is modified.

The method of the invention comprises moving the single-stranded polynucleotide through the transmembrane pore such that a proportion of the nucleotides of the single-stranded polynucleotide interact with the pore.

The method may be performed using any suitable membrane as described above, preferably a lipid bilayer system, wherein pores are inserted into the lipid bilayer. The process is typically carried out using the following membranes: (i) an artificial bilayer comprising pores, (ii) an isolated naturally occurring lipid bilayer comprising pores, or (iii) cells having pores inserted therein. The method is preferably performed using a bilayer of artificial lipids. In addition to the pore, the bilayer may comprise other transmembrane and/or intramembrane proteins and other molecules. Suitable apparatus and conditions are detailed below with reference to the sequencing embodiments of the invention. The methods of the invention are typically performed in vitro.

The present invention provides methods for characterizing double-stranded target polynucleotides.

These methods are possible because transmembrane protein pores can be used to distinguish nucleotides with similar structures based on their differing effects on the current passing through the pore. Individual nucleotides can be identified at the single molecule level based on their current amplitude as they interact with the pore. A nucleotide is present in the pore if a current is passed through the pore in a manner specific to that nucleotide (i.e. if a characteristic current associated with that nucleotide is detected to flow through the pore). Nucleotides in a target polynucleotide are continuously identified, enabling the sequence of the polynucleotide to be estimated or determined.

The method comprises (a) providing a construct comprising the target polynucleotide, wherein the two strands of the target polynucleotide are ligated by the hairpin-like adaptor; (b) separating the two strands of the target polynucleotide by contacting the construct with a nucleic acid binding protein; (c) moving the two single-stranded polynucleotides sequentially through the transmembrane pore; and (d) measuring the current passing through the pore during each interaction, thereby determining or estimating the sequence of the target polynucleotide. Thus, the method involves sensing a portion of the nucleotides in the target polynucleotide across the pore as the nucleotides pass through the barrel or channel one by one in order to sequence the target polynucleotide. As described above, this is strand sequencing.

This method can be used to sequence all or only a portion of the target polynucleotide. The polynucleotide may be of any length. For example, the 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 nucleotide pairs in length. The polynucleotide may be 1000 or more nucleotide pairs, 5000 or more nucleotide pairs or 100000 or more nucleotide pairs in length. The polynucleotide may be naturally occurring or man-made. For example, the method can be used to verify the sequence of manufactured oligonucleotides. The method is typically performed in vitro.

The single stranded polynucleotide may interact with the pore on either side of the membrane. The single stranded polynucleotide may interact with the pore at any site in any manner.

During the interaction of a nucleotide in the single-stranded polynucleotide with the pore, the nucleotide affects the current flowing through the pore in a manner specific to that nucleotide. For example, a particular nucleotide will decrease the current flowing through the pore for a particular average length of time and to a particular extent. In other words, the current flowing through the pore is characteristic for a particular nucleotide. Control experiments can be performed to determine the effect of a particular nucleotide on the current flowing through the pore. The results obtained by performing the methods of the invention on a test sample can then be compared to the results obtained from such control experiments to determine or estimate the sequence of the target polynucleotide.

The sequencing method may be performed using any suitable membrane/pore system in which pores are inserted into the membrane. The methods are typically performed using membranes comprising naturally occurring or synthetic lipids. The membrane is typically formed in vitro. Preferably the method is performed without using isolated naturally occurring pore-containing membranes, or cells expressing pores. The method is preferably performed using an artificial membrane. In addition to the pore, the membrane may comprise other transmembrane and/or intramembrane proteins and other molecules.

Reagent kit

The invention also provides kits for preparing a nucleic acid for characterizing a double-stranded target polynucleotide. The kit comprises (a) a hairpin-like adaptor capable of ligating two strands of the target polynucleotide; and/or (b) at least one polymer; and/or a polynucleotide binding protein; and/or at least one tether.

In a preferred embodiment, the kit further comprises a lead polymer and a tail polymer. The lead polymer and the tail polymer are described in detail above. The kit preferably further comprises one or more markers which produce a characteristic current upon interaction with the transmembrane pore. Such markers are described in detail above. The kit preferably further comprises means (means) for coupling the target polynucleotide to a membrane. Means for coupling the target polynucleotide to a membrane are described above. The means for coupling preferably comprises a reactive group. Suitable groups include, but are not limited to, sulfhydryl, cholesterol, lipid, and biotin groups. The kit may also comprise components of the membrane, such as phospholipids required to form the lipid bilayer.

Any of the embodiments detailed above with respect to the method of the invention are equally applicable to the kit of the invention.

The kits of the invention may additionally comprise one or more other reagents or instruments which enable the practice of any of the embodiments described above. Such reagents or apparatus include one or more of the following: suitable buffers (aqueous solutions), means for sampling from a subject (e.g. a tube or instrument comprising a needle), means for amplifying and/or expressing a polynucleic acid, a membrane or voltage clamp or patch clamp device as defined above. The reagents may be present in the kit in a dry state, such that the reagents may be resuspended with a fluid sample. Optionally, the kit may further comprise instructions for making the kit useful in the methods of the invention, or detailed instructions as to which patients the methods may be used. Optionally, the kit may include nucleotides.

Method for preparing target polynucleotides for characterization

The invention also provides methods of making double-stranded target polynucleotides for characterization. The method generates a construct that allows the target polynucleotide to be characterized. In this method, the two strands of the target polynucleotide are ligated by a hairpin-like adaptor and a polymer is ligated to one strand at the other end of the target polynucleotide. The polymer is preferably a leader polymer, and the method preferably further comprises ligating a tail polymer to the other strand of the target polynucleotide (i.e. at the same end as the leader polymer). The lead polymer and the tail polymer are detailed above.

The method preferably still further comprises means for coupling the construct to the membrane. Such tools are described above.

The hairpin-like adaptor portions can be synthesized separately and then chemically bound or enzymatically ligated to the target polynucleotide. Methods for doing this are known in the art. Alternatively, the hairpin-like adaptors can be generated during processing of the target polynucleotide. Likewise, suitable methods are known in the art.

Examples

The experimental reagents and instruments used in the following examples were all conventional commercially available reagents or instruments, and the sequences used were synthesized by Biotechnology Ltd of Biotechnology and Beijing Ongchoku Biotechnology Ltd.

Example 1: preparation of hairpin-like adapters

(1) Synthesis of the second Single Strand 3 '-5' -5 '-3' (from right to left in FIG. 1)

Click chemistry was carried out on a3 '-5' (right to left in FIG. 1) first segment (SEQ ID NO:2, 5 'modified with Azide (denoted as N3)) and a 5' -3 '(right to left in FIG. 1) second segment (SEQ ID NO:3, 5' modified with DBCO (denoted as DB)) in a ratio of 3:1 in a solution of 10mM Tris, 50mM NaCl, 0.1mM EDTA at a reaction temperature of 30 ℃ for 12 hours, and the obtained product was purified by PAGE to obtain a3 '-5' -5 '-3' second single-stranded chain.

(2) Synthesis of DNA containing hairpin-like Structure

The 5 '-3' first single strand (SEQ ID NO: 1) and the 3 '-5' -5 '-3' second single strand formed in step (1) were annealed to 4 ℃ at 0.1 ℃ every 10 seconds from 95 ℃ to form a hairpin-like structural DNA, as shown in FIG. 1.

(3) Preparation of helicase-DNA Complex

The hairpin-like DNA obtained in step (2) was mixed with T4 Dda helicase (having the sequence shown in SEQ ID NO:13, containing mutation sites M1G/E94C/C109A/C136A/A360C) from Enterobacteriaceae phage T4 (Enterobacteriacea phase T4) at a ratio of 1: 1 (volume/volume) was mixed in 10mM HEPES pH 8.0, 50mM potassium chloride, 1mM EDTA, and the final concentrations of T4 Dda helicase and hairpin-like DNA were 3000nM and 100nM, respectively. T4 Dda helicase was reacted with the hairpin-like DNA for 1 hour at room temperature, followed by purification by magnetic bead purification to give a complex of helicase and DNA.

(4) Preparation of enzyme-loaded hairpin-like adaptor samples

The purified complex was mixed with a buffer (buffer 1: 20mM HEPES pH 8.0, 500mM potassium chloride, 50mM magnesium chloride, 50mM ATP or buffer 2: 20mM HEPES pH 7.0, 500mM potassium chloride, 50mM magnesium chloride, 3mM ATP) at a ratio of 1: 1 (v/v) at 40 ℃ for 1 hour to obtain enzyme-loaded hairpin-like adaptor samples.

(5) Gel assay for hairpin-like adaptors

The reference DNA standards and the samples of the above steps (2), (3) and (4) were loaded on 4-20% TBE gels, respectively, and run at a voltage of 160V for 1 hour, and then the gels were stained with SYBR gold to observe DNA bands, as shown in FIG. 2.

In fig. 2: lane M shows as reference DNA standards (bands corresponding from lowest molecular mass (bottom of gel) to highest molecular mass (top of gel): 25bp (base pair), 50bp, 75bp, 100bp, 150bp, 200bp, 300bp, 400bp and 500 bp);

lane 1 shows the DNA containing the hairpin-like structure prepared in step (2), i.e., the DNA comprising only annealed first single strands (SEQ ID NO: 1) hybridized with second single strands (formed by step (1) of SEQ ID NO:2 and SEQ ID NO: 3);

lane 2 shows the complex of the non-magnetic bead purified T4 Dda helicase (labeled ED1) from step (3) with DNA, i.e., containing pre-ligated hairpin-like adaptors (unpueled (ATP and MgCl)₂) Helicase of (d);

lane 3 shows the complex of the magnetic bead purified T4 Dda helicase of step (3) with DNA;

lane 4 shows hairpin-like adapters in buffer 2 (3 mM ATP) in step (4);

lane 5 shows the hairpin-like adapters in buffer 1 (50 mM ATP) from step (4).

As shown in FIG. 2, lane 1 shows that a hybrid duplex has been formed, but a partially unhybridized naked single strand remains underneath, and the analysis of lanes 2 and 3 in comparison to lane 1 shows that a polynucleotide binding protein has bound to the hybrid duplex, but a different amount of bound polynucleotide binding protein is present, and lanes 4 and 5 show that a hybrid duplex with only one polynucleotide binding protein is formed under the provision of buffer (containing ATP).

Example 2: preparation of constructs

(1) Preparation of Y adaptors

Y-Top-1 (SEQ ID NO: 7), Y-Top-2 (SEQ ID NO: 8) and Y-Bottom (SEQ ID NO: 9) were annealed to 4 ℃ at a temperature reduced by 0.1 ℃ every 10 seconds from 95 ℃ to form Y adaptors.

(2) Preparation of enzyme-loaded Y adaptors

The T4 Dda helicase was loaded onto the Y adaptor using the method of example 1 steps (2) - (3).

(3) Preparation of constructs

A segment of about 2700 base pairs of plasmid DNA (SEQ ID NO: 6) was amplified using PCR primers containing restriction sites as defined below for the preparation of constructs containing double-stranded target polynucleotide sequences.

BsaI forward primer: 5'-TCGCC ATTCA GGCTG CGC-3' (SEQ ID NO: 11)

BsaI reverse primer: 5'-GCTTA GAGAC CTGTG CGG-3' (SEQ ID NO: 12)

The fragment of about 2700bp was digested with the unmodified dA tail (NEB Cat # E7442), BsaI restriction enzyme (NEB Cat # R3733) and ligated to the hairpin-like adaptor prepared in example 1 at one end and the enzyme-carrying Y adaptor prepared in step (2) at the other end to obtain the hairpin-like adaptor-containing construct 1 of the present invention, the structure of which is shown in FIG. 3 (in this case, the construct 1 does not contain FLT _1D located at the left side of FIG. 3).

The adapter is ligated to the target polynucleotide by sticky ends or T-A ligation. The final product was isolated by magnetic bead purification and quantified by fluorescence.

In the same manner, Y adaptors were ligated to both ends of the BsaI restriction enzyme digested fragment to obtain construct 2 without hairpin-like adaptors, the structure of which is shown in FIG. 4.

The results of 1% agarose gel electrophoresis of marker, and BsaI restriction endonuclease digested product and hairpin-like adaptor containing construct 1, respectively, are shown in FIG. 5.

In fig. 5: the M lane shows the bands corresponding from the lowest molecular mass (bottom of the gel) to the highest molecular mass (top of the gel) as reference DNA standards (1 kb, 2kb, 3kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb and 10 kb);

lane 1 shows the product of the 2.7 Kb-containing fragment digested with the unmodified A, BsaI restriction enzyme;

lane 2 shows construct 1 containing the 2.7 Kb fragment, digested sequentially with the unmodified A, BsaI restriction enzyme, and ligated with hairpin-like adaptors containing complementary ends.

Example 3: methods for double-stranded target polynucleotide sequencing of constructs containing hairpin-like adaptors and constructs not containing hairpin-like adaptors, respectively, are described

Materials and methods:

sequencing buffer (20 mM HEPES pH 8.0, 50mM ATP, 50mM MgCl) was thawed at room temperature₂500mM KCl), a first tether (SEQ ID NO:4, located at D in FIG. 6) and a second tether (SEQ ID NO:10, located at FLT-1D on the left side of FIG. 3). To 196ul sequencing buffer 4ul of the above tether (1 uM) was added and mixed well before incubation in a QIOCC gene sequencer QNome-9604 sequencer for 20 minutes. The sequencer QNome-9604 has a structure shown in FIG. 12, and comprises a fluid chip, a signal processing circuit, and sequencing software. Then, a loading library was prepared, and the components are shown in tables 1 and 2.

Table 1: method for preparing construct sequencing library containing hairpin-like adapters

Table 2: method for preparing construct sequencing library without hairpin-like adapter

200ul of the above sample was added to a sequencer and sequenced using the sequencer. As the DNA strand passes through the transmembrane pore, the change in current through the transmembrane pore is measured and collected, and the sequence of the strand is then determined using a base recognition algorithm (e.g., a Recurrent Neural Network (RNN) algorithm). The sequencing results were analyzed and the differences in efficiency of the resulting double-stranded real-time sequencing events are shown in table 3 below. A schematic representation of the process for sequencing a double stranded target polynucleotide for a construct containing a hairpin-like adaptor is shown in FIG. 6, and a schematic representation of the process for sequencing a double stranded target polynucleotide for a construct not containing a hairpin-like adaptor is shown in FIG. 7.

Results and discussion:

table 3: differential efficiency of double-stranded real-time sequencing events achieved by constructs containing hairpin-like adaptors versus constructs not containing hairpin-like adaptors

The tag is "+" if the sequencing signal is a pure template strand; the sequence signal is marked "-" if it is a pure complementary strand; if the sequencing signal is complementary strand following the template strand, it is marked "+ -", and is defined as a double stranded real time sequencing signal, the signal diagram is shown in FIG. 8. The above results indicate that constructs containing hairpin-like adaptors were 18 times more efficient in achieving real-time sequencing of double strands than constructs not containing hairpin-like adaptors (table 3).

Example 4: double-stranded target polynucleotide sequencing of hairpin-like adaptor-containing constructs and standard hairpin adaptor-containing constructs

Materials and methods:

using the method of example 2, a construct containing standard hairpin adaptors was ligated to BsaI restriction endonuclease digested fragments at one end and the other end to standard hairpin adaptors.

The standard hairpin adaptor is a corner hairpin (SEQ ID NO: 5) with a double-stranded complementary region. To hybridize the template strand region and the complementary strand region, the 1uM DNA solution was heated to 95 ℃ on a hot plate, maintained at 95 ℃ for 10min, and then allowed to cool slowly to 12 ℃ over the course of about 1.5 hours. The process yielded a final solution of 1uM of hybridized DNA hairpin.

A schematic of the process of sequencing a double stranded target polynucleotide for a construct containing hairpin-like adaptors is shown in FIG. 6 of example 3. Constructs containing standard hairpin adapters were sequenced in a manner similar to that described in example 3, and a schematic representation of the sequencing process is shown in FIG. 9.

Results and discussion:

the double-stranded real-time sequencing signal of 50 constructs containing standard hairpin adapters and the double-stranded real-time sequencing signal of constructs containing hairpin-like adapters were randomly sampled, respectively, and the ratio of the rate of the complementary strand portion to the rate of the template strand portion of the double-stranded real-time sequencing signal was analyzed and counted, with the results shown in Table 4.

Table 4: ratio of complementary strand portion to template strand portion of double-stranded real-time sequencing signal

The results in Table 4 show that constructs containing standard hairpin adapters achieved double-stranded real-time sequencing signals with the average of the ratio of the rates of the complementary strand portions to the rate of the template strand portions being 1.44, constructs containing hairpin-like adapters achieved double-stranded real-time sequencing signals with the average of the ratio of the rates of the complementary strand portions to the rate of the template strand portions being 1.23, and T-test P values suggest a significant statistical difference between the two. This reflects that the sequencing rate of the complementary strand portion of the double-stranded real-time sequencing signal for the construct containing the hairpin-like adaptor more closely approximates the sequencing rate of the template strand portion.

FIG. 10 shows an accuracy profile of a template strand portion and a complementary strand portion of a signal for real-time sequencing of a double-stranded construct containing standard hairpin adapters, and FIG. 11 shows an accuracy profile of a template strand portion and a complementary strand portion of a signal for real-time sequencing of a double-stranded construct containing hairpin-like adapters. Experimental data indicate that the accuracy of the complementary strand part of the construct double-strand real-time sequencing signal containing the hairpin-like adaptor is better than that of the construct double-strand real-time sequencing signal containing the standard hairpin adaptor on the whole.

Preferred embodiments and specific examples of the present invention are described herein, but these embodiments and examples are provided by way of illustration only, and are not intended to limit the present invention. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Accordingly, the present invention is also intended to cover any such alternatives, modifications, variations or equivalents.

Sequence listing

<110> Beijing Qicarbo science and technology Co., Ltd

<120> hairpin-like adaptors, constructs and methods for characterizing double-stranded target polynucleotides

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (34)..(35)

<223> nucleotides were isolated by 8 × isp18

<400> 1

aagcgtcaga gaggttccaa gtcagagagg ttcctactga tagcgtctgc atct 54

<210> 2

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

agatgcagac gctatcagta tttttttttt tttttttttt tttttttttt 50

<210> 3

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (10)..(11)

<223> nucleotide was isolated by 4 × isp18

<400> 3

tttttttttt ggaacctctc tgacttggaa cctctctgac 40

<210> 4

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (30)..(30)

<223> nucleotide followed by 40 × iSPC3

<400> 4

gtcagagagg ttccaagtca gagaggttcc 30

<210> 5

<211> 66

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (30)..(31)

<223> nucleotide was isolated by 4 × isp18

<220>

<221> misc_feature

<222> (40)..(41)

<223> nucleotide was isolated by 4 × isp18

<400> 5

aagcgtagtc gtggtaggca ggttggtgac tttttttttt gtcaccaacc tgcctaccac 60

gactac 66

<210> 6

<211> 2730

<212> DNA

<213> plasmid pUC57(plasmid pUC57)

<400> 6

atcctgagac cccttttttt tgccttttgt tttagaattt tattcgccat tcaggctgcg 60

caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 120

gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 180

taaaacgacg gccagtgaat tcgagctcgg tacctcgcga atgcatctag atatcggatc 240

ccgggcccgt cgactgcaga ggcctgcatg caagcttggc gtaatcatgg tcatagctgt 300

ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 360

agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 420

tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 480

cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 540

gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 600

ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 660

ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 720

atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 780

aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 840

gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 900

ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 960

ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 1020

acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 1080

gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 1140

ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 1200

ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 1260

gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 1320

ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 1380

agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 1440

ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 1500

gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 1560

catctggccc cagtgctgca atgataccgc gtgaaccacg ctcaccggct ccagatttat 1620

cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 1680

cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 1740

gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 1800

tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 1860

gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 1920

tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 1980

gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 2040

gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 2100

taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 2160

tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 2220

ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 2280

taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 2340

tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 2400

aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta 2460

ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt 2520

tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc 2580

tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt 2640

gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc 2700

ggtgtgaaat accgcacagg tctctaagca 2730

<210> 7

<211> 61

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> nucleotide preceded by 30 xiSPC 3

<220>

<221> misc_feature

<222> (33)..(34)

<223> nucleotide was isolated by 4 × isp18

<400> 7

gcgtgactat cggactcgtg gtcttttttt tttgtcagtt cgcttcttac gcatcacact 60

t 61

<210> 8

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gaccacgagt ccgatagtca cgc 23

<210> 9

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

agtgtgatgc gtaagaagcg aactgacagt ccagcaccga cct 43

<210> 10

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> nucleotide preceded by 20 xiSPC 3

<400> 10

ggtcggtgct ggact 15

<210> 11

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tcgccattca ggctgcgc 18

<210> 12

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gcttagagac ctgtgcgg 18

<210> 13

<211> 439

<212> PRT

<213> Enterobacter phage T4(Enterobacteria phase T4)

<400> 13

Gly Thr Phe Asp Asp Leu Thr Glu Gly Gln Lys Asn Ala Phe Asn Ile

1 5 10 15

Val Met Lys Ala Ile Lys Glu Lys Lys His His Val Thr Ile Asn Gly

20 25 30

Pro Ala Gly Thr Gly Lys Thr Thr Leu Thr Lys Phe Ile Ile Glu Ala

35 40 45

Leu Ile Ser Thr Gly Glu Thr Gly Ile Ile Leu Ala Ala Pro Thr His

50 55 60

Ala Ala Lys Lys Ile Leu Ser Lys Leu Ser Gly Lys Glu Ala Ser Thr

65 70 75 80

Ile His Ser Ile Leu Lys Ile Asn Pro Val Thr Tyr Glu Cys Asn Val

85 90 95

Leu Phe Glu Gln Lys Glu Val Pro Asp Leu Ala Lys Ala Arg Val Leu

100 105 110

Ile Cys Asp Glu Val Ser Met Tyr Asp Arg Lys Leu Phe Lys Ile Leu

115 120 125

Leu Ser Thr Ile Pro Pro Trp Ala Thr Ile Ile Gly Ile Gly Asp Asn

130 135 140

Lys Gln Ile Arg Pro Val Asp Pro Gly Glu Asn Thr Ala Tyr Ile Ser

145 150 155 160

Pro Phe Phe Thr His Lys Asp Phe Tyr Gln Cys Glu Leu Thr Glu Val

165 170 175

Lys Arg Ser Asn Ala Pro Ile Ile Asp Val Ala Thr Asp Val Arg Asn

180 185 190

Gly Lys Trp Ile Tyr Asp Lys Val Val Asp Gly His Gly Val Arg Gly

195 200 205

Phe Thr Gly Asp Thr Ala Leu Arg Asp Phe Met Val Asn Tyr Phe Ser

210 215 220

Ile Val Lys Ser Leu Asp Asp Leu Phe Glu Asn Arg Val Met Ala Phe

225 230 235 240

Thr Asn Lys Ser Val Asp Lys Leu Asn Ser Ile Ile Arg Lys Lys Ile

245 250 255

Phe Glu Thr Asp Lys Asp Phe Ile Val Gly Glu Ile Ile Val Met Gln

260 265 270

Glu Pro Leu Phe Lys Thr Tyr Lys Ile Asp Gly Lys Pro Val Ser Glu

275 280 285

Ile Ile Phe Asn Asn Gly Gln Leu Val Arg Ile Ile Glu Ala Glu Tyr

290 295 300

Thr Ser Thr Phe Val Lys Ala Arg Gly Val Pro Gly Glu Tyr Leu Ile

305 310 315 320

Arg His Trp Asp Leu Thr Val Glu Thr Tyr Gly Asp Asp Glu Tyr Tyr

325 330 335

Arg Glu Lys Ile Lys Ile Ile Ser Ser Asp Glu Glu Leu Tyr Lys Phe

340 345 350

Asn Leu Phe Leu Gly Lys Thr Cys Glu Thr Tyr Lys Asn Trp Asn Lys

355 360 365

Gly Gly Lys Ala Pro Trp Ser Asp Phe Trp Asp Ala Lys Ser Gln Phe

370 375 380

Ser Lys Val Lys Ala Leu Pro Ala Ser Thr Phe His Lys Ala Gln Gly

385 390 395 400

Met Ser Val Asp Arg Ala Phe Ile Tyr Thr Pro Cys Ile His Tyr Ala

405 410 415

Asp Val Glu Leu Ala Gln Gln Leu Leu Tyr Val Gly Val Thr Arg Gly

420 425 430

Arg Tyr Asp Val Phe Tyr Val

435

Claims (18)

1. A hairpin-like adaptor for characterising a double stranded target polynucleotide, the hairpin-like adaptor comprising first and second single strands for ligation with a template strand and a complementary strand of the double stranded target polynucleotide respectively, the second single strand comprising first and second segments in reverse ligation, the first segment being distal to the complementary strand of the double stranded target polynucleotide and the second segment being proximal to the complementary strand of the double stranded target polynucleotide;

the first single strand and the second single strand form a first double-stranded nucleic acid region for bringing the complementary strand into proximity with a transmembrane pore after the template strand has begun to pass through the transmembrane pore to completely pass through the transmembrane pore, the first nucleic acid double-stranded region being located at the first segment of the second single strand;

the first single strand and the second single strand form a second double-stranded nucleic acid region linked to the double-stranded target polynucleotide, the second double-stranded nucleic acid region being formed at the second segment of the second single strand;

the first single strand and the second single strand further form a non-complementary region, the region of the second single strand located in the non-complementary region comprising a binding site for a polynucleotide binding protein, the region of the second single strand located in the non-complementary region located on the first segment distal to the second segment and being for driving the complementary strand proximate to the transmembrane pore through the transmembrane pore;

the length of the first double-stranded nucleic acid region and the length of the second double-stranded nucleic acid region are both 20 bp-50 bp.

2. The hairpin-like adaptor of claim 1 wherein the second single strand is a3 '-5' -5 '-3' second single strand or a 5 '-3' -3 '-5' second single strand; wherein

The 3 '-5' -5 '-3' second single strand comprises a3 '-5' first segment and a 5 '-3' second segment, the 5 'end of the 3' -5 'first segment is linked to the 5' end of the 5 '-3' second segment;

the 5 '-3' -3 '-5' second single strand comprises a 5 '-3' first segment and a3 '-5' second segment, the 3 'end of the 5' -3 'first segment being linked to the 3' end of the 3 '-5' second segment.

3. The hairpin-like adaptor of claim 1 or 2, wherein the second single strand is formed by reverse ligation of the first segment and the second segment using a click chemistry reaction; alternatively, the second single strand is synthesized directly by using the forward base and the reverse base.

4. A construct for characterizing a double-stranded target polynucleotide, the construct comprising the double-stranded target polynucleotide and the hairpin-like adaptor of any one of claims 1 to 3, the double-stranded target polynucleotide being ligated to the hairpin-like adaptor at one end thereof.

5. The construct for characterizing a double-stranded target polynucleotide according to claim 4, further comprising at least one polymer attached to opposite ends of the hairpin-like adaptor on the double-stranded target polynucleotide;

the at least one polymer comprises a lead polymer attached to the template strand of the double-stranded target polynucleotide and a tail polymer attached to the complementary strand of the double-stranded target polynucleotide, and the lead polymer and the tail polymer form a partially complementary double strand; the leading polymer comprises binding sites of the polynucleotide binding protein, the leading polymer is a single chain formed by base groups and non-base groups, and the tail polymer is a single chain formed by modified or unmodified base groups;

the two strands of the construct are non-covalently linked.

6. A method of characterizing a double-stranded target polynucleotide, the method comprising:

(a) providing a construct comprising a double-stranded target polynucleotide having a template strand and a complementary strand, wherein the double-stranded target polynucleotide is ligated at one end thereof to the hairpin-like adaptor of any one of claims 1 to 3;

(b) contacting the construct with a transmembrane pore and a polynucleotide binding protein;

(c) providing conditions for the template strand and the complementary strand of the same construct to pass sequentially through the transmembrane pore; and

(d) measuring the characteristics of the template strand and the complementary strand of the double-stranded target polynucleotide produced during passage through the transmembrane pore, respectively.

7. The method according to claim 6, wherein the construct is as defined in claim 5.

8. A method according to claim 7, comprising contacting the construct with a first polynucleotide binding protein for separating the two strands of the construct and controlling movement of the template strand through the transmembrane pore and a second polynucleotide binding protein for controlling translocation of the complementary strand through the transmembrane pore such that the entire double stranded target polynucleotide is characterised.

9. The method of claim 8, wherein the first polynucleotide binding protein binds to a binding site of the polynucleotide binding protein on the leader polymer for sequentially separating the duplex formed by the leader polymer and the tail polymer, the duplex of the target polynucleotide, the double-stranded nucleic acid region of the hairpin-like adaptor; the second polynucleotide binding protein binds to the polynucleotide binding protein binding site on the second single strand of the hairpin-like adaptor for controlling movement of the complementary strand through the pore.

10. The method of claim 9, wherein the first polynucleotide binding protein is moved in the order of: the leader polymer, the template strand of the target polynucleotide, and the first single strand; the order in which the second polynucleotide binding protein moves is: a second segment of the second single strand, a complementary strand of the target polynucleotide, and the tail polymer.

11. The method of claim 7, further comprising providing at least one tether for bringing the construct into proximity with the transmembrane pore;

the at least one tether comprises a first tether having a capture region that specifically binds to the hairpin-like adaptor of the construct for bringing the complementary strand into proximity with a transmembrane pore; the timing of the specific binding is such that after the hairpin-like adapter is separated from the double-stranded nucleic acid region of the second segment, the location of the specific binding comprises at least a portion of the second single-stranded second segment of the hairpin-like adapter;

the at least one tether further comprises a second tether that specifically binds to the tail polymer of the construct for bringing the construct into proximity with the transmembrane pore;

the first tether and the second tether are associated with a membrane anchor in which the transmembrane pore is located.

12. The method of claim 11, wherein the template strand moves through the hole by: one end of the leader polymer enters the pore first, and then the leader polymer, the template strand of the double-stranded target polynucleotide and the first single strand of the hairpin-like adaptor pass through the pore in sequence, with the complementary strand passing through the pore in such a way that: one end of the first segment of the second single-stranded first segment of the hairpin-like adaptor enters the pore before the first segment, the second segment, the complementary strand of the double-stranded target polynucleotide and the tail polymer pass through the pore in sequence.

13. The method of claim 12, wherein the second tether is attached to the tail polymer, and wherein an end of the lead polymer enters the hole under the force of an electric field and the second tether;

the first tether is coupled to a second segment of the second single strand of the hairpin-like adaptor, one end of the first segment of the second single strand of the hairpin-like adaptor entering the well under the influence of an electric force and the first tether.

14. The method of claim 6 or 7, wherein the polynucleotide binding protein is selected from the group consisting of: polymerase, helicase or exonuclease;

the helicase is selected from helicase helicating in the 5 '-3' direction or helicase helicating in the 3 '-5' direction;

and/or, the transmembrane pore is a protein pore or a solid state pore; and/or, the membrane is an amphiphilic layer or a solid state layer.

15. The method of claim 6 or 7, wherein the construct further comprises one or more markers that produce a characteristic current when they interact with the pore; the one or more markers are abasic groups or specific nucleotide sequences;

the one or more markers are in the hairpin-like adaptor; the one or more markers identify the source of the target polynucleotide.

16. A kit for characterizing a double-stranded target polynucleotide, the kit comprising: the first single strand and the second single strand used to prepare the hairpin-like adaptor as defined in any one of claims 1 to 3.

17. The kit of claim 16, further comprising: at least one polymer comprising a leader polymer that facilitates binding of the double-stranded target polynucleotide to the transmembrane pore or to a polynucleotide binding protein and a tail polymer that facilitates sequencing of the target construct through the transmembrane pore;

a polynucleotide binding protein capable of separating two strands of the target polynucleotide and controlling the movement of the two strands of the target polynucleotide through transmembrane pores, respectively;

at least one tether for bringing the construct into proximity with the transmembrane pore;

and/or, the kit further comprises one or more markers that produce a characteristic current when the marker interacts with the pore; the one or more markers are abasic groups or specific nucleotide sequences.

18. Use of the hairpin-like adaptor of any one of claims 1 to 3, the construct of claim 4 or 5, the method of any one of claims 6 to 15, or the kit of claim 16 or 17 for the preparation of a product for or for characterising a double stranded target polynucleotide.

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