WO2024125342A1

WO2024125342A1 - Adapter containing blocking elements used in combination, construct, method and use

Info

Publication number: WO2024125342A1
Application number: PCT/CN2023/136344
Authority: WO
Inventors: 刘先宇; 刘铮; 刘少伟; 王玉博; 罗江燕
Original assignee: 成都齐碳科技有限公司
Priority date: 2022-12-13
Filing date: 2023-12-05
Publication date: 2024-06-20
Also published as: CN116200477A

Abstract

Provided in the present invention are an adapter for characterizing an analyte, a construct comprising the analyte and the adapter, and a method for characterizing the analyte using same. Further provided is the use of an organic oligocation such as spermine as a blocking element for blocking the binding of polynucleotide to protein. Specifically, the adapter contains two different blocking elements. By the combined use of the two blocking elements, blocking the binding of polynucleotide to protein can be achieved and the application range of a spacer region is broadened.

Description

Adaptors, constructs, methods and uses comprising combined blocking elements

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application No. 202211595915.2, filed on December 13, 2022, entitled “Adapters, constructs, methods and applications comprising combined blocking elements,” the entire contents of which are incorporated herein by reference.

Technology

The present application relates to the technical field of nanopore sequencing, and in particular to adaptors, constructs, methods and applications comprising a combined blocking element for characterizing analytes.

Background technique

Analytes such as polynucleotides (such as RNA or DNA) sequencing technologies are widely used. The sequencing technologies for polynucleotides, especially DNA, are also constantly being updated.

DNA sequencing technology has developed to the fourth generation of sequencing technology since 1977. In addition to the first generation of sequencing technology, the second, third, and fourth generation sequencing technologies are collectively referred to as next generation sequencing (NGS) technology. Gene sequencing has mainly experienced the development process from the first generation of Sanger sequencing, to the second generation of sequencing by synthesis, the third generation of single molecule sequencing, and the fourth generation of nanopore sequencing. Compared with the first generation of sequencing technology, NGS technology has been more widely used due to its advantages of increased throughput, reduced costs, and shortened sequencing cycles.

The fourth-generation nanopore sequencing refers to the situation where when a DNA molecule passes through a nanopore driven by electrophoresis, the different sizes and shapes of each base in the DNA molecule will cause characteristic current changes. Therefore, the base type and arrangement order of the DNA molecule can be determined by electron conduction detection, thus achieving single-molecule sequencing.

In nanopore sequencing technology, when no potential is applied, the polynucleotide binding protein is usually required to be stagnant on the polynucleotide to prevent the polynucleotide binding protein from moving further along the polynucleotide. However, when the polynucleotide contacts the polynucleotide binding protein and the transmembrane pore and a potential is applied, the stagnant polynucleotide binding protein can be moved along the polynucleotide sequence to be tested, thereby achieving the purpose of sequencing.

In the prior art, single-stranded nucleic acids formed by abasic groups such as iSp18 or iSpC9 are usually used as spacers to inhibit the translocation function of polynucleotide binding proteins, thereby achieving the effect of stagnating polynucleotide binding proteins, and then the polynucleotide binding proteins are allowed to cross the spacers through the electric field force to start normal sequencing. However, the spacers that can effectively stagnate polynucleotide binding proteins in the prior art are very limited, and some groups as spacers will cause the polynucleotide binding proteins to have a high shedding rate or low data throughput.

Summary of the invention

The inventors surprisingly found that when certain single-stranded nucleic acid modifications are used as spacers, they cannot achieve a good blocking effect. However, if they are used in combination with another blocking element that improves the stability of the double strand (such as increasing the difficulty of double-stranded unzipping or increasing the double-stranded Tm value), then a good blocking effect can be achieved, even the same or better blocking effect than the traditional spacers in the prior art, and can be used in nanopore sequencing.

Therefore, a first aspect of the present invention relates to an adaptor for characterizing an analyte, the adaptor comprising one or more first blocking elements and one or more second blocking elements different from the first blocking elements, and after the adaptor is contacted with a polynucleotide binding protein, the one or more first blocking elements and the one or more second blocking elements can act in combination to stop The polynucleotide binding protein is retained.

Preferably, the linker comprises a complementary main chain and a blocking chain, and the one or more first blocking elements are covalently connected to the main chain; the one or more second blocking elements are covalently modified to the main chain or the blocking chain or interact with the main chain and/or the blocking chain by non-covalent bonds.

Preferably, the one or more second blocking elements are complementary to the blocking chain or the main chain, wherein, when the one or more second blocking elements are modified to the main chain by covalent bonds, the one or more second blocking elements are complementary to the blocking chain; when the one or more second blocking elements are modified to the blocking chain by covalent bonds, the one or more second blocking elements are complementary to the main chain; when the one or more second blocking elements interact with the main chain and/or the blocking chain by non-covalent bonds, the second blocking elements may be complementary to the blocking chain or the main chain.

Preferably, the one or more first retarding elements have a structure different from that of nucleotides; and the one or more second retarding elements have a structure for improving double-strand stability.

Preferably, the first blocking element comprises one or more selected from the group consisting of organic oligocations, iSpC3, iSp18, iSp9, nitroindole, inosine, acridine, 2-aminopurine, 2-6-diaminopurine, 5-bromo-deoxyuracil, inverted thymidine (inverted dT), inverted dideoxythymidine (ddT), dideoxycytidine (ddC), 5-methylcytidylic acid, 5-hydroxymethylcytidine, 2'-O-methyl RNA base, isodeoxycytidine (iso-dC), isodeoxyguanosine (iso-dG), a photocleavable (PC) group or hexanediol; the second blocking element comprises one or more selected from the group consisting of nucleotides modified with locked nucleic acid (LNA), peptide nucleic acid (PNA), methoxy (OMe), bicyclic nucleoside (BNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), cyclopyrrolimidazole polyamide (cPIP) or double-stranded binding protein.

Preferably, the organic oligocation has the general formula Bj, Bj is the organic oligocation part of a j-mer, j=1-50, wherein B is selected from the group comprising the following groups:

-HPO ₃ -R ¹ -(X-R ² _n ) _n1 -X-R ³ -O-, wherein R ¹ , R ² _n and R ³ are the same or different C1-C5 lower alkylene groups, X is NH or NC(NH ₂ ) ₂ , n1＝2-20,

-HPO ₃ -R ⁴ -CH(R ⁵ X ¹ )-R ⁶ -O-, wherein R ⁴ is a C1-C5 lower alkylene group, R ⁵ and R ⁶ are the same or different C1-C5 lower alkylene groups, and X ¹ is a putrescine, spermidine or spermine residue,

‐HPO ₃ ‐R ⁷ ‐(aa) _n2 ‐R ⁸ ‐O‐, wherein R ⁷ is C1‐C5 lower alkylene, R ⁸ is C1‐C5 lower alkylene, serine, natural amino alcohol, (aa) _n2 is a peptide containing a natural amino acid having a cationic side chain, and n2=2‐20;

More preferably, the organic oligocation is selected from spermine (Sp).

Preferably, the first blocking element is not complementary to the second blocking element.

Preferably, the adapter further comprises a third strand, which is partially complementary to the main strand; more preferably, the main strand is partially complementary to the blocking strand and the complementary ends of the main strand and the blocking strand are used to directly or indirectly connect to the analyte.

Preferably, the analyte is selected from a polynucleotide, a polypeptide, a lipid or a polysaccharide, preferably a polynucleotide, which is a fully double-stranded polynucleotide, a partially double-stranded polynucleotide or a single-stranded polynucleotide.

Preferably, the polynucleotide binding protein is derived from a polynucleotide processing enzyme; the polynucleotide processing enzyme is selected from a polymerase, a helicase or a nuclease exonuclease. More preferably, the helicase is selected from He1308 helicase, RecD helicase, XPD helicase, Dda Helicase or ED1 helicase.

The second aspect of the present invention relates to a construct for characterizing an analyte, the construct comprising the analyte and the adaptor of the first aspect of the present invention, wherein the adaptor is directly or indirectly linked to either or both ends of the analyte.

The third aspect of the present invention relates to a complex for characterizing an analyte, which comprises a polynucleotide binding protein and a linker of the first aspect of the present invention or a construct of the second aspect of the present invention; wherein the polynucleotide binding protein is arrested on the linker under the combined action of the first blocking element and the second blocking element.

Preferably, the polynucleotide binding protein is derived from a polynucleotide handling enzyme; the polynucleotide handling enzyme is selected from a polymerase, a helicase or an exonuclease. More preferably, the helicase is selected from a He1308 helicase, a RecD helicase, a XPD helicase, a Dda helicase or an ED1 helicase.

A fourth aspect of the present invention relates to a method for controlling the loading of a polynucleotide binding protein on an analyte, the method comprising:

Providing a construct having an analyte, wherein the analyte is directly or indirectly connected to an adaptor at one or both ends thereof, the adaptor comprising one or more first retarding elements and one or more second retarding elements different from the first retarding elements; and contacting the construct with the polynucleotide binding protein, such that the polynucleotide binding protein is arrested on the adaptor under the combined action of the one or more first retarding elements and the one or more second retarding elements; or

The polynucleotide binding protein is loaded onto a linker, wherein the linker comprises one or more first blocking elements and one or more second blocking elements different from the first blocking elements, and the polynucleotide binding protein is arrested on the linker under the combined action of the one or more first blocking elements and the one or more second blocking elements; and the linker loaded with the polynucleotide binding protein is connected to the analyte.

Preferably, the adapter is as defined in the first aspect of the present invention.

A fifth aspect of the invention relates to a method of controlling the movement of an analyte through a transmembrane pore, the method comprising:

(a) implementing the method for controlling the loading of a polynucleotide binding protein on an analyte according to the fourth aspect of the present invention;

(b) contacting the analyte loaded with the polynucleotide binding protein provided in step (a) with the transmembrane pore; and

(c) applying an electric potential across the transmembrane pore so that the polynucleotide binding protein moves through the one or more first retardation elements, optionally (i.e., through or not through) a portion of the one or more second retardation elements, and controls the movement of the analyte through the transmembrane pore.

Preferably, the second blocking element comprises covalently modified nucleotides or non-covalently modified nucleotides; when the polynucleotide binding protein moves through the one or more second blocking elements, the polynucleotide binding protein moves through the nucleotides of the one or more second blocking elements without passing through the covalent or non-covalent modifications of the one or more second blocking elements.

Preferably, the method includes providing a tether for bringing the analyte loaded with the polynucleotide binding protein close to the transmembrane pore; the tether includes a capture region and an anchor region, the capture region is used to capture the adapter, and the anchor region is used to anchor and bind to the transmembrane pore or the membrane where the transmembrane pore is located.

A sixth aspect of the present invention relates to a method for characterizing an analyte, the method comprising:

(a) implementing the method of controlling the movement of an analyte through a transmembrane pore according to the fifth aspect of the present invention; and

(b) obtaining one or more measurements as the analyte moves relative to the transmembrane pore, wherein the measurements represent one or more characteristics of the analyte and thereby characterize the analyte.

Preferably, 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.

A seventh aspect of the present invention relates to a kit for characterizing an analyte, the kit comprising:

(a) the adaptor according to the first aspect of the invention, and (b) a polynucleotide binding protein, and/or (c) a transmembrane pore.

Preferably, the transmembrane pore is a protein pore or a solid-state pore.

The eighth aspect of the present invention relates to the use of an organic oligocation as a blocking element for arresting a polynucleotide binding protein, wherein the organic oligocation has the general formula Bj, Bj is the organic oligocation part of a j-mer, j=1-50, wherein B is selected from the group comprising the following groups:

Preferably, the organic oligocation is selected from spermine.

The ninth aspect of the present invention relates to the use of the linker of the first aspect of the present invention, the construct of the second aspect of the present invention, the complex of the third aspect of the present invention, the methods of the third to sixth aspects of the present invention, or the kit of the seventh aspect of the present invention in the preparation of a product for characterizing an analyte or in characterizing an analyte.

The technical solution of the present invention achieves the following technical effects:

The present invention uses more than one blocking element to jointly act to arrest the enzyme, further expanding the application range of the spacer region, so that some blocking elements cannot play a good blocking effect or even have no blocking effect when used alone, but can achieve a good blocking effect when used in combination with another blocking element.

In addition, the prior art uses a variety of spacers, such as single-stranded nucleic acids formed by a base-free group such as iSp18 or iSpC9 as spacers. Although the electric field force causes the enzyme to cross the spacer, the blocking element will always exist and will not disappear. Therefore, this spacer is only suitable for entry sequencing and cannot be used for outry sequencing. In the present invention, the combined blocking structure formed by the first blocking element and the second blocking element will be destroyed or eliminated as the sequence passes through the nanopore, so it can be Further expand its application, so that it can be used not only for internal push sequencing, but also for external pull sequencing. "Internal push sequencing" refers to a method in which the direction of the electric field force is the same as the direction of the enzyme movement, so that the enzyme passes through and then the analyte is sequenced, and "external pull sequencing" refers to a method in which the direction of the electric field force is opposite to the direction of the enzyme movement, so that the enzyme passes through and then the analyte is sequenced.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a schematic diagram of a Y adapter containing two blocking elements and an enzyme passing through the blocking element 1 according to an embodiment. The labels are as follows: (Y1) the main strand containing the blocking element 1; (Y2) the third strand, also known as the fluorescent strand; (B) the blocking strand containing the blocking element 2. Part of the sequence Y1 forms a complementary double-stranded region with Y2, part of the sequence Y1 is complementary to the B part, and the nucleotides of the blocking element 2 on B are complementary to Y1.

Fig. 2 shows a schematic diagram of the exemplary structures of various Y adapters containing two blocking elements. The combination relationship of the two blocking elements is blocking element 1 + blocking element 2, blocking element 1 + blocking element 2 + blocking element 1, blocking element 1 + blocking element 2 + blocking element 1 + blocking element 2, and blocking element 2 + blocking element 1.

Description of sequence listing:

SEQ ID NO: 1 shows the main chain Y1 of the Y linker:

5'-(iSpC3)30-GCGGA GTCAA ACGGT AGAAG TCG TTTTT TTTTT ACTGC TCATT CGGTC CTGCT GACT-3'

SEQ ID NO: 2 shows the main chain Y1-3C3 containing 3C3 of the Y linker:

5'-(iSpC3)30-GCGGA GTCAA ACGGT AGAAG TCG TTTTT TTTTT-(iSpC3)3-ACTGC TCATT CGGTC CTGCT GACT-3'

SEQ ID NO: 3 shows the main chain Y1-5C3 containing 5C3 of the Y linker:

5'-(iSpC3)30-GCGGA GTCAA ACGGT AGAAG TCG TTTTT TTTTT-(iSpC3)5-ACTGC TCATT CGGTC CTGCT GACT-3

SEQ ID NO: 4 shows the main chain Y1-5C3-cPIP containing 5C3 of the Y linker:

5'-GCGGA GTCAA ACGGT AGAAG TCG TTTTT TTTTT-(iSpC3)5-CGAGC AGCAC GCGAG CAGCA CG GACT-3'

SEQ ID NO:5 shows the main chain Y1-Sp containing Sp of the Y linker:

5'-(iSpC3)30-GCGGA GTCAA ACGGT AGAAG TCG TTTTT TTTTT-Sp-ACTGC TCATT CGGTC CTGCT GACT-3'

SEQ ID NO:6 shows the main chain Y1-2Sp containing 2Sp of the Y linker:

5'-(iSpC3)30-GCGGA GTCAA ACGGT AGAAG TCG TTTTT TTTTT-(Sp)2-ACTGC TCATT CGGTC CTGCT GACT-3'

SEQ ID NO:7 shows the main chain Y1-4C18 containing 4C18 of the Y linker:

5'-(iSpC3)30-GCGGA GTCAA ACGGT AGAAG TCG TTTTT TTTTT-(iSpC18)4-ACTGC TCATT CGGTC CTGCT GACT-3'

SEQ ID NO: 8 shows the fluorescent chain Y2 of the Y linker, and the 3' end of the sequence is connected to the fluorescent group CY5:

5'-CGACT TCTAC CGTTT GACTC CGC-CY5-3'

SEQ ID NO:9 shows the blocking strand B of the Y linker, in which some nucleotides are modified with OMe to prevent them from binding to the helicase:

5'-P-GTCAG CAGGA CCGAA TGA(GC AGTAG TCCAG CACCG ACC) _OMe -3'

SEQ ID NO: 10 shows the blocking strand B-LNA of the Y linker comprising LNA-modified nucleotides:

5'-P-GTCAG CAGGA CCGAA TGA(GCAGT) _LNA (AGTCC AGCAC CGACC) _OMe -3'

SEQ ID NO: 11 shows the blocking strand B-PNA of the Y linker comprising a PNA-modified nucleotide:

5'-(GTCAG CAGGA CCGAA TGAGC AGT) _PNA- 3'

SEQ ID NO: 12 shows the blocking strand sequence B-cPIP of the Y linker:

5'-GTCCG TGCTG CTCGC GTGCT GCTCG-3'

SEQ ID NO: 13 shows a tether with cholesterol attached to the 5' end of the chain:

5’-Chol-(iSpC3)20-TTGGTGGGTGGGTGGG-3’

SEQ ID NO: 14 shows the wild-type sequence of helicase E1:

SEQ ID NO: 15 shows the wild-type sequence of helicase E2:

Detailed ways

It should be understood that different applications of the disclosed products and methods can be adjusted according to specific needs in the art. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments of the present invention and is not intended to be limiting.

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

definition

In order to explain the embodiments of the present invention more clearly, some scientific terms and special terms are used herein. Unless clearly defined herein, all these terms and special terms should be understood to have the meanings commonly understood by those skilled in the art. For the sake of clarity, some terms used in this article are defined below.

Connector

The present invention provides an adapter for characterizing nucleic acids, wherein the adapter is capable of connecting an analyte. An adapter is also referred to as a joint, an adapter, etc. The adapter of the present invention comprises one or more first retarding elements and one or more second retarding elements, and the first retarding element and the second retarding element are completely different in structure and composition. The adapter comprises a complementary, preferably partially complementary main chain and a blocking chain, and both the main chain and the blocking chain are single-stranded structures formed by connecting nucleotides, nucleotide analogs or modified nucleotides, or single-stranded structures in which some nucleotides are modified. Any number of first retarding elements, such as 1, 2, 3, 4, 5, 6, 7, 8 and, 9, 10 or more first retarding elements are covalently connected to the main chain. Any number of second blocking elements, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more second blocking elements are modified to the main chain or the blocking chain with a covalent bond (such as LNA, PNA, etc.) or interact with the main chain, the blocking chain or the double chain formed by the main chain and the blocking chain with a non-covalent bond (such as cPIP, etc.).

When one or more second retarding elements are modified to the main chain by covalent bonds, the one or more second retarding elements are complementary to the blocking chain; when one or more second retarding elements are modified to the blocking chain by covalent bonds, the one or more second retarding elements are complementary to the main chain; when one or more second retarding elements interact with the main chain and/or the blocking chain by non-covalent bonds, the second retarding elements may be complementary to the blocking chain or the main chain.

The second blocking element comprises a nucleotide modified by a modifier, and when the nucleotide portion of one or more second blocking elements is located on the main chain and the modifier portion interacts with the main chain and/or the blocking chain by a non-covalent bond, the second blocking element is complementary to the blocking chain; when the nucleotide portion of one or more second blocking elements is located on the blocking chain and the modifier portion interacts with the main chain and/or the blocking chain by a non-covalent bond, the second blocking element is complementary to the main chain.

The second blocking element comprises nucleotides modified with a modifier. The phrase "the second blocking element is located on the main strand or the blocking strand" herein means that the nucleotides comprised by the second blocking element are connected to the main strand or the blocking strand, rather than limiting the position of the modifier.

In some embodiments, the one or more first retarding elements may not bind to the one or more second retarding elements in a complementary base pairing manner. In some embodiments, the one or more second retarding elements may bind to the barrier strand or the main strand in a complementary base pairing manner.

The one or more first blocking elements and the one or more second blocking elements may be disposed adjacent to each other or spaced apart from each other.

In some embodiments, one or more first retarding elements are disposed adjacent to one or more second retarding elements. In the direction in which the polynucleotide binding protein moves in the main chain, at least one first retarding element is disposed adjacent to at least one second retarding element or to a segment complementary to at least one second retarding element, and no group exists between the two.

In some embodiments, one or more second blocking elements are disposed adjacent to each other. One or more first blocking elements may be disposed adjacent to each other, or adjacent to one or more second blocking elements, or adjacent to a segment on the main chain that is complementary to a second blocking element.

The meaning of "the first blocking element is arranged in close proximity" in this article is that there is no The meaning of "the first blocking element and the second blocking element are arranged in close proximity" as described herein refers to that there is no gap formed by any group between the first blocking element on the main chain and the second blocking element located on the main chain, or between the first blocking element on the main chain and the segment on the main chain that is complementary to the second blocking element located on the blocking chain, but they are directly connected together. The meaning of "the second blocking element is arranged in close proximity" as described herein refers to that there is no gap formed by any group between one or more second blocking elements located on the main chain or the blocking chain, but they are directly connected together.

In some embodiments, one or more first retarding elements are spaced apart from one or more second retarding elements. In the direction in which the polynucleotide binding protein moves along the main strand, one or more groups exist between at least one first retarding element and at least one second retarding element or a segment complementary to at least one second retarding element.

In some embodiments, one or more second blocking elements are spaced apart. One or more first blocking elements may be spaced apart, or spaced apart from one or more second blocking elements, or spaced apart from a segment on the main chain that is complementary to the second blocking element.

The meaning of "the first retarding element is arranged at intervals" as described herein refers to the presence of one or more groups, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more groups, between one or more first retarding elements on the main chain. The meaning of "the first retarding element and the second retarding element are arranged at intervals" as described herein refers to the presence of one or more groups, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more groups, between the first retarding element on the main chain and the second retarding element located on the main chain, or between the first retarding element on the main chain and the segment on the main chain that is complementary to the second retarding element located on the blocking chain. The meaning of "the second retarding element is arranged at intervals" as described herein refers to the presence of one or more groups, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more groups, between one or more second retarding elements located on the main chain or the blocking chain.

As shown in Figure 2, one or more first retarding elements and one or more second retarding elements can be arranged on the adapter in any combination or position. In some embodiments, the adapter includes 1 first retarding element and 1 second retarding element that are arranged adjacent to or spaced in the direction of movement of the polynucleotide binding protein. In some embodiments, the adapter includes 1 first retarding element, 1 second retarding element and 1 first retarding element that are arranged adjacent to or spaced in the direction of movement of the polynucleotide binding protein. In some embodiments, the adapter includes 1 first retarding element, 1 second retarding element, 1 first retarding element and 1 second retarding element that are arranged adjacent to or spaced in the direction of movement of the polynucleotide binding protein. In some embodiments, the adapter includes 1 second retarding element and 1 first retarding element that are arranged adjacent to or spaced in the direction of movement of the polynucleotide binding protein. The direction of movement of the polynucleotide binding protein is usually 5'-3', and some polynucleotide binding proteins move in the 3'-5' direction.

After the adaptor contacts the polynucleotide binding protein, one or more first blocking elements and one or more second blocking elements can work together to arrest the polynucleotide binding protein. The polynucleotide binding protein can be arrested before the first blocking element or the second blocking element, preferably before the first first blocking element or the first second blocking element.

The first blocking element may have any molecule or any combination of molecules that arrest one or more polynucleotide binding proteins, preferably having a structure different from nucleotides or consisting of a structure different from nucleotides. More preferably, the first blocking element has one or more organic oligocations, one or more iSpC3, one or more iSp18, one or more iSp9, one or more nitroindoles, one or more inosine, one or more acridine, one or more 2-aminopurine, one or more 2-6-diaminopurine, one or more 5-bromo-deoxyuracil, one or more inverted thymidine (inverted dT), one or more inverted dideoxythymidine (ddT), one or more dideoxycytidine (ddC), one or more 5-methylcytidylic acid, one or more 5-hydroxymethylcytidine, one or more 2'-O-methyl RNA bases, one or more isodeoxycytidine (iso-dC), one or more isodeoxyguanosine (iso-dG), one or more photocleavable (PC) groups or one or more hexanediol linkages.

‐HPO ₃ ‐R ⁷ ‐(aa) _n2 ‐R ⁸ ‐O‐, wherein R ⁷ is C1‐C5 lower alkylene, R ⁸ is C1‐C5 lower alkylene, serine, natural amino alcohol, (aa) _n2 is a peptide containing a natural amino acid having a cationic side chain, and n2=2‐20.

More preferably, the organic oligocation is selected from spermine.

More preferably, the first retarding element comprises 1, 2, 3, 4, 5, 6, 7, 8 or more spermine, iSpC3, iSp18 or iSp9. Most preferably, the first retarding element is 3, 4 or 5 iSpC3, or 1, 2, 3, 4 or 5 spermine.

The second blocking element has one or more groups that improve the stability of the double-stranded chain, such as any group or combination of any groups that increases the difficulty of unwinding the double-stranded chain or increases the Tm value of the double-stranded chain, preferably has a modified nucleotide or is composed of a modified nucleotide. Preferably, the second blocking element comprises one or more nucleotides modified by locked nucleic acid (LNA), one or more nucleotides modified by peptide nucleic acid (PNA), one or more nucleotides modified by methoxy (OMe), one or more nucleotides modified by bicyclic nucleoside (BNA), one or more nucleotides modified by glycerol nucleic acid (GNA), one or more nucleotides modified by threose nucleic acid (TNA), one or more nucleotides modified by cyclic pyrrolimidazole polyamide (cPIP) or one or more nucleotides modified by double-stranded binding protein.

More preferably, the second blocking element comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 15, 20, 23, 25 or more LNA-modified nucleotides, PNA-modified nucleotides, OMe-modified nucleotides. Most preferably, the second blocking element is 3, 4, 5 or 6 LNA-modified nucleotides, or 15, 20, 23 or 25 PNA-modified nucleotides, 5, 10, 15 or 20 OMe-modified nucleotides.

In some embodiments, on the main chain, two ends of one or more first blocking elements, second blocking elements, or a combination of the first blocking element and the second blocking element that are arranged adjacently or spaced apart are connected to one end of the first segment and one end of the second segment, respectively. During sequencing, the other end of the first segment first enters the transmembrane pore and guides the entire adapter into the transmembrane pore, and the other end of the second segment is directly connected to the analyte or indirectly connected to the analyte through nucleotides, thereby characterizing the analyte. The first segment is a single-stranded segment formed by nucleotides and abasic groups, and the second segment is a single-stranded segment formed by nucleotides.

In some embodiments, on the blocking strand, two ends of one or more second blocking elements disposed adjacently or spaced apart are connected to one end of the third segment and one end of the fourth segment, respectively. The other end of the third segment is a free end, which is used to bind to the tether during sequencing to bring the adapter close to the transmembrane pore. The other end of the fourth segment is directly connected to the analyte or indirectly connected to the analyte through nucleotides, thereby characterizing the analyte. In some embodiments, the other end of the fourth segment is a free end and is not connected to any group. The third segment is a single-stranded segment formed by connecting nucleotides and/or modified nucleotides, and the fourth segment is a single-stranded segment formed by nucleotides. The second segment of the main strand can be combined with the fourth strand of the blocking strand in a base complementary pairing manner to form a double strand or a partial double strand. A partial double strand refers to a structure that contains both a double strand and a single strand.

The adapter also includes a third strand, which is a single strand formed by nucleotides. The third strand is also called a fluorescent strand. The third strand can be combined with the first segment of the main strand in a base complementary pairing manner to form a double strand or a partial double strand.

In some embodiments, the main strand, the blocking strand and the third strand form a Y-shaped adapter. The structure of the Y-shaped adapter is a handle connecting two arms, wherein the second segment of the main strand is combined with the fourth segment of the blocking strand to form the handle of the Y-shaped adapter, one end of the handle is connected to the two arms, and the other end is connected to the analyte. One arm is composed of the free end of the blocking strand, and the other arm is formed by the first segment of the main strand combined with the third strand.

The adapter of the present invention is preferably combined with a tether, which is a single-stranded polynucleotide composed of multiple nucleotides and also contains a baseless group that cannot form a complementary double strand. One end of the tether is complementary to the adapter and the other end is anchored to the membrane or the hole, thereby surrounding the analyte connected to the adapter around the hole.

The adapter of the present invention can be used independently, or it can be used in combination with other adapters (such as hairpin adapters, hairpin-like adapters or conventional Y adapters, etc.) to form a construct for sequencing. In some embodiments, the adapter of the present invention connects the two ends of the analyte, such as a polynucleotide, to form a construct for characterizing the polynucleotide. In some embodiments, the adapter of the present invention is connected to the 5' end of the polynucleotide, and the 3' end of the polynucleotide is connected to a conventional Y adapter other than the adapter of the present invention to form a construct. In some embodiments, if it is necessary to characterize the two chains of a double-stranded polynucleotide, the adapter of the present invention can be connected to the 5' end of the double-stranded polynucleotide, and the 3' end of the double-stranded polynucleotide can be connected to a hairpin adapter or a hairpin-like adapter to form a construct. A hairpin-like adapter is a loop having a similar structure to a conventional hairpin, but the loop is not the same as a conventional hairpin structure but is formed by a single-stranded linear molecule folding back on itself, and can connect the two chains of a polynucleotide. For specific examples of hairpin-like adapters, see CN113462764A.

The abasic groups used in the adapter or tether are groups such as iSp18, iSpC3 or iSp9 that cannot form base pairs, which may also be referred to as "abasic sites" or "abasic nucleotides." Abasic groups are nucleotides or nucleosides that lack a nucleobase at the 1' position of the sugar portion.

Analyte

The analyte is selected from one or more of polynucleotides, polypeptides, polysaccharides and lipids. The analyte is preferably a polynucleotide such as a nucleic acid, including deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA). The polynucleotide can be single-stranded or double-stranded. The polynucleotide can be circular. The polynucleotide can be an aptamer, a probe hybridized with a microRNA, or the microRNA itself. The polynucleotide can 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 can be 1000 or more nucleotide pairs, 5000 or more nucleotide pairs in length or 100000 or more nucleotide pairs in length.

The analyte may be present in any suitable sample. The present invention is generally implemented on a sample known to contain or suspected of containing the analyte. The present invention may be implemented on a sample containing one or more analytes of unknown species. Alternatively, the present invention may be implemented on a sample to confirm the species of one or more analytes known or expected to be present in the sample. It is contemplated by those skilled in the art that "providing an analyte" of the present invention refers to providing a sample containing the analyte, and "connecting a sequencing adapter to an analyte" of the present invention refers to connecting a sequencing adapter to a sequencing adapter. Binds to the analyte present in the sample.

Polynucleotide

A polynucleotide can be any polynucleotide. A polynucleotide such as a nucleic acid is a macromolecule containing two or more nucleotides. The polynucleotide or nucleic acid can include any combination of nucleotides. Nucleotides can be naturally occurring or artificially synthesized.

Nucleotides generally contain a nucleobase, a sugar and at least one phosphate group. The nucleobase and sugar form a nucleoside. Nucleotides can be natural nucleotides or non-natural nucleotides.

Nucleoside bases are typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, more specifically, adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C).

The sugar is usually a pentose. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar is preferably deoxyribose.

The nucleotides in the polynucleotide are usually ribonucleotides or deoxyribonucleotides. The polynucleotide may contain the following nucleosides: adenosine, uridine, guanosine and cytidine. The nucleotides are preferably deoxyribonucleotides. The polynucleotide preferably contains the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and/or thymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC).

The nucleotides typically contain monophosphate, diphosphate or triphosphate. The phosphatase can be attached to the 5' or 3' side of the nucleotide.

The nucleotides in a polynucleotide can be linked to each other in any manner. As in nucleic acids, the nucleotides are usually linked by their sugar and phosphate groups. As in pyrimidine dimers, the nucleotides can be linked by their nucleobases.

The polynucleotide may be single-stranded or double-stranded. At least a portion of the polynucleotide is preferably double-stranded.

A polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

The polynucleotide is preferably DNA, RNA or a DNA or RNA hybrid. The polynucleotide may comprise a single-stranded region and a region with other structures, such as a hairpin loop, a triplex and/or a quadruplex. A DNA/RNA hybrid may comprise DNA and RNA on the same strand. Preferably, the DNA/RNA hybrid comprises a DNA strand hybridized to an RNA strand.

The polynucleotide can 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 can be 1000 or more nucleotide pairs in length, 5000 or more nucleotide pairs in length or 100000 or more nucleotide pairs in length.

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

Polynucleotide binding protein

The polynucleotide binding protein can be any protein that can bind to the polynucleotide and control its movement through the hole. It is very simple to determine whether a protein binds to a polynucleotide in the art. Proteins usually interact with polynucleotides and modify at least one of their properties. Proteins can modify polynucleotides by cleaving polynucleotides to form a single nucleotide or a shorter nucleotide chain such as a dinucleotide or trinucleotide. The part can modify the polynucleotide by positioning or moving the polynucleotide to a specific position (i.e., controlling its movement).

In some embodiments, the polynucleotide binding protein is derived from a polynucleotide processing enzyme; the polynucleotide processing enzyme is selected from a polymerase, a helicase or a nuclease exonuclease. More preferably, the helicase is selected from a He1308 helicase, a RecD helicase, a XPD helicase, a Dda helicase or an ED1 helicase.

In some embodiments, the polynucleotide binding protein is a polynucleotide helicase. A polynucleotide helicase is an enzyme that can melt a double-stranded polynucleotide into a single strand. In some embodiments, a polynucleotide helicase can melt a double-stranded DNA into a single strand. In some embodiments, a polynucleotide helicase is an enzyme with helicase activity. Examples of polynucleotide helicases include, for example, helicases as described herein.

The polynucleotide binding ability 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 the polynucleotide and move along the polynucleotide can be measured. The protein can include modifications that contribute to polynucleotide binding and/or contribute to its activity at high salt concentrations and/or room temperature. The protein can be modified so that it binds to the polynucleotide (i.e., retains the polynucleotide binding ability) but does not act as a helicase (i.e., does not move along the polynucleotide when all the necessary components (e.g., ATP and Mg ²⁺ ) are available for movement). Such modifications are known in the art. For example, modifications of the Mg ²⁺ binding domain in a helicase typically produce variants that do 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 chain sequencing, polynucleotides are translocated through the hole along or against the applied potential. Exonucleases that gradually or stepwise work on double-stranded polynucleotides can be used on the cis side of the hole to supply the remaining single strand under the applied potential, or on the trans side to supply the remaining single strand under the reverse potential. Similarly, helicases that unwind the double-stranded DNA can also be used in a similar manner. Polymerases can also be used. Sequencing applications that require chain translocation against the applied potential are also possible, but DNA must first be "captured" by the enzyme at the opposite potential or without potential. As the potential is subsequently converted after binding, the chain will pass through the hole in a cis-to-trans manner and be in an extended configuration by current retention. Single-stranded DNA exonucleases or single-stranded DNA-dependent polymerases can serve as molecular motors to pull the recently translocated single strand back from the hole in a controlled, stepwise manner against the applied potential from trans to cis.

Any helicase can be used in the present invention. The helicase can act on the hole in two modes. First, the method is preferably carried out using a helicase so that the helicase moves the polynucleotide through the hole under the action of the field caused by the applied voltage. In this mode, the 5' end of the polynucleotide is first captured in the hole, and the helicase moves the polynucleotide into the hole so that it passes through the hole under the action of the field until it finally translocates through and reaches the reverse side of the membrane. Alternatively, the method is preferably carried out in this way, and the helicase moves the polynucleotide through the hole against the field caused by the applied voltage. In this mode, the 3' end of the polynucleotide is first captured in the hole, and the helicase moves the polynucleotide through the hole so that it is pulled out of the hole against the applied field until it is finally pushed back to the cis side of the membrane.

The method can also be performed in the reverse direction. The 3' end of the polynucleotide can first be captured in the pore, and the helicase can move the polynucleotide into the pore, allowing it to pass through the pore under the influence of the field until it finally translocates through and reaches the opposite side of the membrane.

When the helicase does not possess the essential component that is convenient to move or is modified to stop or prevent it from moving, the helicase can be combined with the polynucleotide and serve as a brake to slow down the movement of the polynucleotide when the polynucleotide is pulled into the hole by the external field. In the inactive mode, it is not important whether the 3 ' or 5 ' of the polynucleotide is captured, and it is the external field that pulls the polynucleotide into the hole towards the opposite side under the effect of the enzyme that serves as the brake. When in the inactive mode, the control of the movement of the helicase to the polynucleotide can be described in a variety of ways, including toothing, sliding and braking. The helicase variant lacking helicase activity can also be used in this way.

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

Any step in the method using a polynucleotide binding protein (e.g., a polynucleotide helicase) is typically performed in the presence of free nucleotides or free nucleotide analogs and an enzyme cofactor that promotes the action of the polynucleotide binding protein (e.g., a polynucleotide helicase). The free nucleotides can be one or more of any individual nucleotides. 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), deoxyguanosine monophosphate (DAMP), deoxyguanosine monophosphate (DGP), deoxyguanosine monophosphate (DTP), deoxyguanosine monophosphate (DMP), deoxyguanosine diphosphate (DTP ... The free nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotides are preferably adenosine triphosphate (ATP). The enzyme cofactor is a factor that allows the construct to work. The enzyme cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg ²⁺ , Mn ²⁺ , Ca ²⁺ or Co ²⁺ . The enzyme cofactor is most preferably Mg ²⁺ .

Stagnation

If the polynucleotide binding protein has stopped moving along the analyte, such as a polynucleotide, it is arrested. The combination of an arresting element is used to arrest the polynucleotide binding protein. The polynucleotide binding protein can be arrested before the arresting element.

The stalled helicase and polynucleotide are contacted with a transmembrane pore and an electric potential is applied. Under the action of the field generated by the applied electric potential, the polynucleotide moves through the pore. The polynucleotide-binding protein is usually too large to move through the pore. When a portion of the polynucleotide enters the pore and moves along the field generated by the applied electric potential, the polynucleotide-binding protein moves through the pore through the associated blocking element as the polynucleotide moves through the pore.

This allows the position of the polynucleotide binding protein on the polynucleotide to be controlled. Before the stalled polynucleotide binding protein and polynucleotide are contacted with the transmembrane pore and an electric potential is applied, the polynucleotide binding protein stays at the position where they are stalled. Even in the presence of necessary components (e.g., ATP and Mg2+) to promote the movement of the polynucleotide binding protein, the polynucleotide binding protein will not move through the combined blocking element on the polynucleotide until there is a transmembrane pore and an applied electric potential.

membrane

Any membrane may be used in accordance with the present invention. Suitable membranes are well known in the art. The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed by amphiphilic molecules (e.g., phospholipids) having both hydrophilic and lipophilic properties. The amphiphilic molecules may be synthetic or naturally occurring. Non-naturally occurring amphiphiles and amphiphiles that form monolayers are known in the art and include, for example, block copolymers. Block copolymers are polymeric materials in which two or more monomer subunits are polymerized together to produce a single polymer chain. Block copolymers typically have properties provided by each monomer subunit. However, block copolymers may have unique properties that polymers formed by individual subunits do not have. Block copolymers may be designed such that one monomer 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 membrane. The structure of the mimicking biofilm. The block copolymers can be diblock (composed of two monomer subunits), but can also be composed of more than two monomer subunits to form more complex arrangements that behave as amphiphiles. The copolymers can be triblock, tetrablock or pentablock copolymers.

Archaeal bipolar tetraether lipids are naturally occurring lipids that are constructed so that the lipids form monolayer membranes. These lipids are commonly found in extremophiles, thermophiles, halophiles, and acidophiles that survive in harsh biological environments. It is believed that their stability comes from the fusogenic nature of the final bilayer. It is easy to construct block copolymer materials that mimic these biological entities by creating triblock polymers with the general motif hydrophilic-hydrophobic-hydrophilic. The material can form a monomeric membrane that behaves similarly to a lipid bilayer and has a range of phase states from vesicles to laminae. The membranes formed by these triblock copolymers have some advantages over biological lipid membranes. Because the triblock copolymers are synthetic, the precise construction can be carefully controlled to provide the correct chain length and properties required 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 submaterials; for example, hydrophobic polymers can be made from monomers based on siloxane or other non-hydrocarbon compounds. The hydrophilic subportion of the block copolymer can also have low protein binding properties, which enables the production of membranes that are highly resistant when exposed to raw biological samples. The head group unit can also be derived from atypical lipid head groups.

Compared to biological lipid membranes, triblock copolymer membranes also have enhanced mechanical and environmental stability, such as much higher operating temperature or pH range.The synthetic nature of the block copolymers provides a platform to tailor polymer-based membranes for a wide range of applications.

The amphiphilic molecules may be chemically modified or functionalized to facilitate coupling of the analyte.

The amphiphilic layer can be a single layer or a double layer. The amphiphilic layer is usually planar. The amphiphilic layer can be non-planar, for example curved.

The amphiphilic layer is typically a lipid bilayer. The lipid bilayer is a model for a cell membrane and serves as an excellent platform for a range of experimental studies. For example, lipid bilayers can be used for in vitro studies of membrane proteins using single channel recording. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of substances. The lipid bilayer can 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.

In another preferred embodiment, the film is a solid layer. The solid layer is not of biological origin. In other words, the solid layer is not derived from or separated from a biological environment, such as an organism or a cell, or a biologically usable structure in a synthetically manufactured form. The solid layer can be formed with organic or inorganic materials, including but not limited to microelectronic materials, insulating materials such as Si ₃ N ₄ , Al ₂ O ₃ , and SiO ₂ , organic and inorganic polymers such as polyamides, plastics such as or elastomers such as two-component addition-cure silicone rubber and glass. The solid layer can be formed with graphene.

Transmembrane pore

Transmembrane pores are structures that allow hydrated ions to flow from one side of the membrane to the other side driven by an applied electrical potential.

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

The transmembrane protein pore may be a monomer or an oligomer. The pore is preferably composed of several repeating subunits (e.g. 6, 7 or 8 subunits). The pore is more preferably a heptamer or octamer pore.

Transmembrane protein pores typically comprise a barrel or channel through which the ions can flow. The subunits of the pore typically surround a central axis and provide strands for a transmembrane beta barrel or channel or a transmembrane alpha-helical bundle or channel.

The barrel or channel of a transmembrane protein pore typically comprises amino acids that promote interaction with an analyte (e.g., a nucleotide, a polynucleotide, or a 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 promote interaction between the pore and a nucleotide, polynucleotide, or nucleic acid.

Transmembrane protein pores useful in the present invention may be derived from β-barrel pores or α-helical bundle pores, β-barrel pores comprising a barrel or channel formed by β-strands. 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, such as Mycobacterium smegmatis porins (Msp) (e.g., MspA), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A and Neisseria autotransporter lipoprotein (NalP). α-helical bundle pores comprise a barrel or channel formed by α-helices. Suitable α-helical bundle pores include, but are not limited to inner membrane proteins and α-outer membrane proteins, such as WZA and ClyA toxins. Transmembrane pores may be derived from Msp or α-hemolysin (α-HL).

The transmembrane protein pore is preferably derived from Msp, preferably derived 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 pore derived from Msp comprising the same monomers. Alternatively, the pore may be a hetero-oligomeric pore derived from Msp containing at least one monomer different from the other monomers. The pore may also 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 alpha-HL pore is formed by 7 identical monomers or subunits (ie it is a heptamer).

In some embodiments, the transmembrane protein pore is chemically modified. The pore can be chemically modified at any site in any manner. Preferably, the transmembrane protein pore is chemically modified by binding of a molecule to one or more cysteines (cysteine ligation), binding of a molecule to one or more lysines, binding of a molecule to one or more non-natural amino acids, enzymatic modification of an epitope, or modification of a terminal end. Suitable methods for performing such modifications are well known in the art. The transmembrane protein pore can be chemically modified by binding any molecule. For example, the pore can be chemically modified by binding a dye or a fluorophore.

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

move

In the method of the present invention, the analyte, such as the polynucleotide, is moved through the transmembrane pore and sequenced. Moving the polynucleotide through the transmembrane pore refers to moving the polynucleotide from one side of the pore to the other side. The movement of the polynucleotide through the pore may be driven or controlled by an electric potential or an enzymatic action or both an electric potential and an enzymatic action. The movement may be unidirectional, or backward and forward movement may be allowed.

Preferably a polynucleotide binding protein is used to control the movement of the polynucleotide through the pore.

Analyte characterization

The characterization method may include measuring one, two, three, four or five or more characteristics of an analyte, such as a polynucleotide. The method includes controlling movement of an analyte through a transmembrane pore and obtaining one or more measurements as the analyte moves relative to the pore, wherein the measurements represent one or more characteristics of the analyte.

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.

In some embodiments, the analyte is a polynucleotide. Any number of polynucleotides can be characterized. For example, the method of the present invention can involve characterizing 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more polynucleotides. The polynucleotide can be naturally occurring or artificial. For example, the method can be used to test the sequence of the manufactured oligonucleotide. The method is generally performed in vitro.

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

The method can be performed using any suitable membrane as described above, preferably a lipid bilayer system, wherein the pore is inserted into the lipid bilayer. The method is typically performed using a membrane that includes (i) an artificial bilayer containing a pore, (ii) an isolated naturally occurring lipid bilayer containing a pore, or (iii) a cell having a pore inserted therein. The method is preferably performed using an artificial lipid bilayer. In addition to the pore, the bilayer may include other transmembrane proteins and/or intramembrane proteins and other molecules. Suitable apparatus and conditions are described in detail below with reference to the sequencing embodiment of the present invention. The method of the present invention is typically performed in vitro.

The present invention provides a method for characterizing a polynucleotide, the method comprising:

(a1) providing a construct having a polynucleotide, wherein the polynucleotide is connected to an adaptor comprising a combined blocking element of the present invention at one or both ends thereof; and contacting the construct with a polynucleotide binding protein, so that the polynucleotide binding protein is arrested on the adaptor under the combined action of the blocking element on the adaptor; or

(a2) loading the polynucleotide binding protein onto the adapter comprising the combined blocking element of the present invention, the polynucleotide binding protein being arrested on the adapter under the combined action of the blocking element on the adapter; and connecting the adapter loaded with the polynucleotide binding protein to the polynucleotide;

(b) contacting the polynucleotide loaded with the polynucleotide binding protein provided in step (a) with the transmembrane pore; and

(c) applying an electric potential across the transmembrane pore to allow the polynucleotide binding protein to move through the first blocking element and a partial region of the second blocking element, and controlling the movement of the polynucleotide through the transmembrane pore;

(d) obtaining one or more measurements as the polynucleotide moves relative to the transmembrane pore, wherein the measurements represent one or more characteristics of the polynucleotide, and thereby characterize the polynucleotide.

In some embodiments, when the adapter only comprises a first retarding element and a second retarding element that are arranged adjacent to each other, the polynucleotide binding protein stops in front of the first retarding element of the adapter under the combined action of the retarding elements, and then moves through the main chain of the adapter under the action of the electric potential. The order of movement on the main chain is: the first retarding element, the segment complementary to the second retarding element, the second segment, and then through the polynucleotide chain connected to the main chain. In some embodiments, the polynucleotide binding protein does not pass through the blocking chain of the adapter. In some embodiments, the polynucleotide binding protein also passes through the blocking chain of the adapter, and when the second retarding element is located on the blocking chain, the order of movement on the blocking chain is: the fourth segment, the nucleotide of the second retarding element. The polynucleotide binding protein does not pass through the modifier of the second retarding element.

These methods are possible because transmembrane protein pores can be used to distinguish nucleotides with similar structures based on their different effects on the current passing through the pore. Individual nucleotides can be identified at the single molecule level based on their current amplitude when they interact with the pore. If the current flows through the pore in a manner specific for a certain nucleotide (i.e., if a characteristic current associated with the nucleotide is detected flowing through the pore), then the nucleotide is present in the pore. Continuous identification of nucleotides in a polynucleotide enables the sequence of the polynucleotide to be estimated or determined.

Thus, the method involves sensing a portion of the nucleotides in the polynucleotide across a membrane pore as they pass through the barrel or channel one by one in order to sequence the polynucleotide. As described above, this is strand sequencing.

In some embodiments, the method includes providing a tether for bringing the construct into proximity with the transmembrane pore; the tether includes a capture region and an anchor region, wherein the capture region is used to capture the adaptor of the construct, and the anchor region is used to anchor and bind to the transmembrane pore or the membrane in which the transmembrane pore is located.

The method can be used to sequence all or only a portion of the polynucleotide. The polynucleotide can 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 can be 1000 or more nucleotide pairs, 5000 or more nucleotide pairs or 100000 or more nucleotide pairs in length. The polynucleotide can be naturally occurring or artificial. For example, the method can be used to verify the sequence of the oligonucleotide manufactured. The method is usually 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.

In the process of the nucleotide in the single-stranded polynucleotide interacting with the hole, the nucleotide affects the current flowing through the hole in a manner specific to the nucleotide. For example, a specific nucleotide will reduce the current flowing through the hole, and this reduction lasts for a specific average duration and reaches a specific degree. In other words, the current flowing through the hole is characteristic for a specific nucleotide. Control experiments can be performed to determine the influence of a specific nucleotide on the current flowing through the hole. Then, the results obtained by performing the method of the present invention on the test sample can be compared with the results obtained from such control experiments to determine or estimate the sequence of the polynucleotide.

The sequencing method can be performed using any suitable membrane/pore system in which the pore is inserted into the membrane. The method is typically performed using a membrane comprising naturally occurring or synthetic lipids. The membrane is typically formed in vitro. The method is preferably not performed using an isolated naturally occurring pore-containing membrane, or a cell expressing a pore. The method is preferably performed using an artificial membrane. In addition to the pore, the membrane may contain other transmembrane proteins and/or intramembrane proteins and other molecules.

When the analyte is an analyte other than a polynucleotide, such as a polypeptide, in the method for characterizing the polypeptide, the polypeptide is first connected to a nucleic acid to obtain a nucleic acid-polypeptide conjugate, and then the adaptor of the present invention is connected to the nucleic acid-polypeptide conjugate, thereby characterizing the polypeptide. The other steps of the method for characterizing the polypeptide are similar to the steps for characterizing the polynucleotide. Kit

The present invention also provides a kit for preparing a method for characterizing an analyte such as a polynucleotide, wherein the kit comprises (a) an adaptor of the present invention, and (b) a polynucleotide binding protein, and/or (c) a transmembrane pore, and optionally a tether.

The kit preferably also comprises one or more markers that produce a characteristic current when interacting with the transmembrane pore. Such markers are described in detail above. The kit preferably also comprises a means for coupling the polynucleotide to the membrane. The means for coupling the polynucleotide to the membrane are described above. The means for coupling preferably comprises a reactive group. Suitable groups include but are not limited to Limited to sulfhydryl, cholesterol, lipid and biotin groups. The kit may also contain components of membranes, such as phospholipids required for lipid bilayer formation.

Any of the embodiments detailed above with respect to the methods of the invention are equally applicable to the kits of the invention.

The kit of the present invention may additionally comprise one or more other reagents or instruments that enable any of the above embodiments to be implemented. Such reagents or instruments include one or more of the following: a suitable buffer (aqueous solution), a tool for sampling from a subject (e.g., a tube or apparatus comprising a needle), a tool for amplifying and/or expressing polynucleic acids, such as a membrane or voltage clamp or patch clamp device as defined above. The reagents may be present in the kit in a dry state so that the reagents can be resuspended with a fluid sample. Optionally, the kit may also include instructions for making the kit useful in the method of the present invention, or detailed instructions on which patients the method may be used for. Optionally, the kit may include nucleotides.

Example

For experimental operation details not specifically noted in the following examples, references may be made to the references cited herein. The experimental reagents and instruments used are all conventional commercially available reagents or instruments, and the sequences used are synthesized by a biological company.

Example 1: Preparation and quality inspection of E1 linker containing blocking element

The main chain, fluorescent chain and blocking chain were synthesized separately, and the main chain, fluorescent chain and blocking chain were annealed at a ratio of 1:1.1:1.1 to form a Y-type connector as shown in Figure 1. The annealing treatment was specifically to slowly cool from 95°C to 25°C, with the cooling amplitude not exceeding 0.1°C/s. The annealing treatment system included 160mM HEPES 7.0, 200mM NaCl, and the concentration of the main chain in the annealing system was 4-8uM.

500nM Y-type adapter, 6 times the amount of substance of helicase E1 (SEQ ID NO.: 14 with M1G/E94C/C109A/C136A/A360C mutations) and 1.5mM TMAD (azodicarbonamide) were mixed and incubated at room temperature for 30 minutes to prepare sequencing adapter complexes QMX1-QMX10. The sequencing adapter complex was added to a DNAPac PA200 column and purified with elution buffer to elute the enzyme that was not bound to the sequencing adapter complex from the column. The sequencing adapter complex was then eluted with a mixture of buffer A and buffer B with 10 times the column volume. The main elution peaks were then pooled, their concentrations measured, and run on a TBE PAGE gel at 160V for 40 minutes. Among them, buffer A: 20mM Na-CHES, 250mM NaCl, 4% (W/V) glycerol, pH 8.6; buffer B: 20mM Na-CHES, 1M NaCl, 4% (W/V) glycerol, pH 8.6.

The sequences of the main chain, fluorescent chain and blocking chain used in the linker of this example are as follows:

Main chain sequence: Y1 (as shown in SEQ ID NO.: 1), Y1-3C3 (as shown in SEQ ID NO.: 2), Y1-5C3 (as shown in SEQ ID NO.: 3), Y1-5C3-cPIP (as shown in SEQ ID NO.: 4)

Fluorescent chain sequence: Y2 (as shown in SEQ ID NO.: 8)

Blocking chain sequence: B (as shown in SEQ ID NO.: 9), B-LNA (as shown in SEQ ID NO.: 10), B-PNA (as shown in SEQ ID NO.: 11), B-cPIP (as shown in SEQ ID NO.: 12)

Quality inspection: 0.2 pmol of sequencing adapter complex was added to the sequencing buffer system (containing 10 mM HEPES 7.0, 500 mM KCl, 100 mM MgCL2, 50 mM ATP), where 6 times the amount of cPIP was added to the cPIP group, placed at 40°C, incubated at room temperature for 30 min, and run TBE PAGE gel at 160 V for 40 min, scanned with Cy5 mode, and then calculated the grayscale: the ratio of unbound enzyme band/(bound enzyme band + unbound enzyme band) is the enzyme detachment rate. The results are shown in Table 1 below.

Table 1: Enzyme shedding rate results of QMX1-QMX10

Interpretation of results: For samples 1 and 2, when only PNA and LNA modified nucleotides were used as retarding elements on the blocking strand, and there was no retarding element on the main strand, the quality inspection enzyme shedding rates were 100% and 100%, respectively. For samples 3 and 4, when only 3C3 and 5C3 were used as retarding elements on the main strand, and there was no retarding element on the blocking strand, the quality inspection enzyme shedding rates were 70.9% and 60.9%, respectively. When 3C3 and 5C3 were used as retarding elements on the main strand, and the blocking strand was modified with LNA to enhance the double-stranded Tm value, the quality inspection enzyme shedding rates dropped to 51.8% and 50.1, respectively (samples 7 and 8). When 3C3 and 5C3 were used as retarding elements on the main strand, and there was PNA modification on the blocking strand, the quality inspection enzyme shedding rates dropped to 36.1% and 28.7%, respectively (samples 5 and 6). Sample No. 9 is a sample with 5C3 on the main chain but no cPIP; Sample No. 10 is a sample with 5C3 on the main chain as a blocking element and cPIP interacting with the double chain as another blocking element. It can be seen from samples No. 9 and No. 10 that in the presence of cPIP, the enzyme quality inspection shedding rate dropped from 60.5% to 51.8%.

Example 2: Preparation and quality inspection of E2 linker containing blocking element

The main chain, fluorescent chain and blocking chain were synthesized separately, and the main chain, fluorescent chain and blocking chain were annealed at a ratio of 1:1.1:1.1 to form a Y-type connector. The annealing treatment was specifically to slowly cool from 95°C to 25°C, with the cooling amplitude not exceeding 0.1°C/s. The annealing treatment system included 160mM HEPES 7.0, 200mM NaCl, and the concentration of the main chain in the annealing system was 4-8uM.

500 nM Y-type adapter, 5 times the amount of substance of helicase E2 (SEQ ID NO.: 15 with D99C/A366C/C308T/C419D/E286K/F246Y/S293N/V422H mutations) and 1.5 mM TMAD (azodicarbonamide) were mixed and incubated at room temperature for 30 minutes to prepare sequencing adapter complexes QMX11-QMX14. The sequencing adapter complex complex was added to a DNAPac PA200 column and purified with elution buffer to elute the enzyme that was not bound to the sequencing adapter complex from the column. The sequencing adapter complex was then eluted with a mixture of buffer A and buffer B with 10 times the column volume. The main elution peak was then pooled, its concentration was measured, and a TBE PAGE gel was run at 160V for 40 minutes. Among them, buffer A: 20mM Na-CHES, 250mM NaCl, 4% (W/V) glycerol, pH 8.6; buffer B: 20mM Na-CHES, 1M NaCl, 4% (W/V) glycerol, pH 8.6.

Main chain sequence: Y1-Sp (as shown in SEQ ID NO.: 5), Y1-2Sp (as shown in SEQ ID NO.: 6)

Fluorescent chain sequence: Y2 (as shown in SEQ ID NO.: 8)

Blocking chain sequence: B (as shown in SEQ ID NO.: 9), B-LNA (as shown in SEQ ID NO.: 10)

Quality inspection: Take 0.2 pmol of sequencing adapter complex and add it to the sequencing buffer system (containing 10mM HEPES 7.0, 500mM KCl, 100mM MgCL2, 50mM ATP), place it at 34℃, incubate it at room temperature for 30min, run TBE PAGE gel at 160V for 40min, scan the gel in Cy5 mode, and then calculate the grayscale: the ratio of unbound enzyme band/(bound enzyme band + unbound enzyme band) is the enzyme detachment rate. The results are shown in Table 2 below.

Table 2: Enzyme shedding rate results of QMX11-QMX14

Interpretation of results: When samples 11 and 12 used 1 Sp and 2 Sp as blocking elements on the main chain, respectively, and there was no blocking element on the blocking chain, their linker quality inspection enzyme shedding rates were 35.2% and 18.1%, respectively; for samples 13 and 14, when there were 1 Sp and 2 Sp as blocking elements on the main chain and LNA modification on the blocking chain, their quality inspection enzyme shedding rates dropped to 13.5% and 8.6%, respectively.

Example 3: Chip mixed library test of linkers containing single blocking elements and linkers containing combined blocking elements

Four types of libraries with a length of 10 kb were prepared by end repair, and QMX-11 (Y1-Sp/Y2/B/E2), QMX-12 (Y1-2Sp/Y2/B/E2), QMX-13 (Y1-Sp/Y2/B-LNA/E2), and QMX-14 (Y1-2Sp/Y2/B-LNA/E2) prepared in Example 2 were connected to the library for construction. The same amount of QMX-11, QMX-12, QMX-13, and QMX-14 libraries were evenly loaded onto three different QNome-9604s of Qi Carbon Technology Co., Ltd. for sequencing. The sequence of the tether used in the sequencing process is shown in SEQ ID NO: 13. The comparison of the mixed test data throughput is shown in Table 3 below.

Table 3: Mixed test data throughput of three chips

Interpretation of results: The better the blocking effect, the more data will be obtained during the mixed test, that is, the more sequencing signals will be obtained. From the table, it can be seen that when only Sp or 2Sp is used as a single blocking element, the amount of library data measured in the mixed test chip is significantly lower than that of the library with combined blocking elements such as 1Sp/LNA or 2Sp/LNA. Therefore, the blocking effect of the connector containing two combined blocking elements during sequencing is significantly better than that of the connector containing only a single blocking element.

Example 4: Chip mixed library test of adapters containing joint blocking elements and commercial adapters

The commercial 4C18-containing linker complex QMX-15 (Y1-4C18/Y2/B/E2) was prepared using the method of Example 1. The linker in the linker complex was formed by a main chain Y1-4C18 (as shown in SEQ ID NO.: 7), a fluorescent chain (as shown in SEQ ID NO.: 8) and a blocking chain (as shown in SEQ ID NO.: 9).

A 10 kb library was prepared by end repair, and the QMX-13 prepared in Example 2 and the commercial linker QMX-15 (Y1-4C18/Y2/B/E2) containing the 4C18 blocking element were connected to the library. Equal amounts of QMX-13 and QMX-15 libraries were evenly loaded onto three different QNome-9604s of Qi Carbon Technology Co., Ltd. for sequencing. The sequence of the tether used in the sequencing process is shown in SEQ ID NO: 13. The comparison of the mixed test data throughput is shown in Table 4 below.

Table 4: Mixed test data throughput of three chips

Interpretation of results: The data of mixed tests using adapters containing combined blocking elements and commercial adapters containing 4C18 blocking elements showed that the blocking effect of the adapters containing combined blocking elements of the present invention during sequencing is not inferior to that of commercial adapters containing 4C18, and is even better than that of commercial adapters.

Claims

A linker for characterizing an analyte, the linker comprising one or more first blocking elements and one or more second blocking elements different from the first blocking elements, and after the linker is contacted with a polynucleotide binding protein, the one or more first blocking elements and the one or more second blocking elements can work together to arrest the polynucleotide binding protein.
The adapter according to claim 1, wherein the adapter comprises a complementary main chain and a blocking chain, the one or more first blocking elements are covalently linked to the main chain; the one or more second blocking elements are covalently modified to the main chain or the blocking chain or interact with the main chain and/or the blocking chain by non-covalent bonds;

Preferably, the one or more second blocking elements are complementary to the barrier chain or the main chain.
The adapter according to claim 1 or 2, wherein the one or more first blocking elements have a structure different from that of nucleotides; and the one or more second blocking elements have a structure for improving double-stranded stability.
The adapter according to any one of claims 1 to 3, wherein the first blocking element comprises one or more selected from the group consisting of organic oligocations, iSpC3, iSp18, iSp9, nitroindole, inosine, acridine, 2-aminopurine, 2-6-diaminopurine, 5-bromo-deoxyuracil, inverted thymidine (inverted dT), inverted dideoxythymidine (ddT), dideoxycytidine (ddC), 5-methylcytidylic acid, 5-hydroxymethylcytidine, 2'-O-methyl RNA base, isodeoxycytidine (iso-dC), isodeoxyguanosine (iso-dG), a photocleavable (PC) group or hexanediol; the second blocking element comprises one or more selected from the group consisting of nucleotides modified with locked nucleic acid (LNA), peptide nucleic acid (PNA), methoxy (OMe), bicyclic nucleoside (BNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), cyclopyrrolimidazole polyamide (cPIP) or double-stranded binding protein.
The adapter according to claim 4, wherein the organic oligocation has the general formula Bj, Bj is the organic oligocation part of the j-mer, j=1-50, wherein B is selected from the group including the following groups:

-HPO 3 -R 1 -(X-R 2 n ) n1 -X-R 3 -O-, wherein R 1 , R 2 n and R 3 are the same or different C1-C5 lower alkylene groups, X is NH or NC(NH 2 ) 2 , n1＝2-20,

-HPO 3 -R 4 -CH(R 5 X 1 )-R 6 -O-, wherein R 4 is a C1-C5 lower alkylene group, R 5 and R 6 are the same or different C1-C5 lower alkylene groups, and X 1 is a putrescine, spermidine or spermine residue,

‐HPO 3 ‐R 7 ‐(aa) n2 ‐R 8 ‐O‐, wherein R 7 is C1‐C5 lower alkylene, R 8 is C1‐C5 lower alkylene, serine, natural amino alcohol, (aa) n2 is a peptide containing a natural amino acid having a cationic side chain, and n2=2‐20;

Preferably, the organic oligocation is selected from spermine (Sp).
The adapter according to any one of claims 1 to 5, wherein the first blocking element is not complementary to the second blocking element.
The adapter according to any one of claims 1 to 6, wherein the adapter further comprises a third chain, which is partially complementary to the main chain; preferably, the main chain is partially complementary to the blocking chain and the complementary ends of the main chain and the blocking chain are used to directly or indirectly connect to the analyte.
The adapter according to any one of claims 1 to 7, wherein the analyte is selected from a polynucleotide, a polypeptide, a lipid or a polysaccharide, preferably a polynucleotide, and the polynucleotide is a fully double-stranded polynucleotide, a partially double-stranded polynucleotide or a single-stranded polynucleotide.
A construct for characterizing an analyte, the construct comprising the analyte and the adapter according to any one of claims 1 to 8, wherein the adapter is directly or indirectly linked to either or both ends of the analyte.
A complex for characterizing an analyte, the complex comprising a polynucleotide binding protein, and the adaptor according to any one of claims 1 to 8 or the construct according to claim 9;

wherein the polynucleotide binding protein is arrested on the adaptor under the combined action of the first blocking element and the second blocking element;

Preferably, the polynucleotide binding protein is derived from a polynucleotide handling enzyme; the polynucleotide handling enzyme is selected from a polymerase, a helicase or an exonuclease.
A method for controlling the loading of a polynucleotide binding protein onto an analyte, the method comprising:

Providing a construct having an analyte, wherein the analyte is directly or indirectly connected to an adaptor at one or both ends thereof, the adaptor comprising one or more first retarding elements and one or more second retarding elements different from the first retarding elements; and contacting the construct with the polynucleotide binding protein, such that the polynucleotide binding protein is arrested on the adaptor under the combined action of the one or more first retarding elements and the one or more second retarding elements; or

The polynucleotide binding protein is loaded onto a linker, wherein the linker comprises one or more first blocking elements and one or more second blocking elements different from the first blocking elements, and the polynucleotide binding protein is arrested on the linker under the combined action of the one or more first blocking elements and the one or more second blocking elements; and the linker loaded with the polynucleotide binding protein is connected to the analyte.
The method according to claim 11, wherein the adapter is as defined in any one of claims 1 to 8.
The method according to claim 11 or 12, wherein the polynucleotide binding protein is derived from a polynucleotide processing enzyme; the polynucleotide processing enzyme is selected from a polymerase, a helicase or a nuclease.
A method of controlling movement of an analyte through a transmembrane pore, the method comprising:

(a) carrying out the method according to any one of claims 11 to 13;

(b) contacting the analyte loaded with the polynucleotide binding protein provided in step (a) with the transmembrane pore; and

(c) applying an electric potential across the transmembrane pore to allow the polynucleotide binding protein to move through the one or more first retardation elements, optionally a portion of the one or more second retardation elements, and control the movement of the analyte through the transmembrane pore.
The method according to claim 14, wherein the second blocking element comprises a covalently modified nucleotide or a non-covalently modified nucleotide; when the polynucleotide binding protein moves through the one or more second blocking elements When the polynucleotide binding protein is bound to one or more second retardation elements, the polynucleotide binding protein moves through the nucleotides of the one or more second retardation elements without passing through the covalent or non-covalent modifications of the one or more second retardation elements.
A method according to claim 14 or 15, wherein the method comprises providing a tether for bringing the analyte loaded with the polynucleotide binding protein close to the transmembrane pore; the tether comprises a capture region and an anchor region, the capture region being used to capture the adapter, and the anchor region being used to anchor and bind to the transmembrane pore or the membrane where the transmembrane pore is located.
A method of characterizing an analyte, the method comprising:

(a) carrying out the method of any one of claims 14 to 16; and

(b) obtaining one or more measurements as the analyte moves relative to the transmembrane pore, wherein the measurements represent one or more characteristics of the analyte and thereby characterize the analyte.
The method according to claim 17, wherein 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.
The method according to claim 17 or 18, wherein the analyte is selected from polynucleotides, polypeptides, lipids or polysaccharides, preferably polynucleotides, and the polynucleotides are fully double-stranded polynucleotides, partially double-stranded polynucleotides or single-stranded polynucleotides.
A kit for characterizing an analyte, the kit comprising:

(a) the adaptor of any one of claims 1 to 8, and (b) a polynucleotide binding protein, and/or (c) a transmembrane pore.
Use of an organic oligocation as a blocking element for arresting a polynucleotide binding protein, the organic oligocation having the general formula Bj, Bj is the organic oligocation part of a j-mer, j=1-50, wherein B is selected from the group comprising the following groups:

-HPO 3 -R 1 -(X-R 2 n ) n1 -X-R 3 -O-, wherein R 1 , R 2 n and R 3 are the same or different C1-C5 lower alkylene groups, X is NH or NC(NH 2 ) 2 , n1＝2-20,

-HPO 3 -R 4 -CH(R 5 X 1 )-R 6 -O-, wherein R 4 is a C1-C5 lower alkylene group, R 5 and R 6 are the same or different C1-C5 lower alkylene groups, and X 1 is a putrescine, spermidine or spermine residue,

‐HPO 3 ‐R 7 ‐(aa) n2 ‐R 8 ‐O‐, wherein R 7 is C1‐C5 lower alkylene, R 8 is C1‐C5 lower alkylene, serine, natural amino alcohol, (aa) n2 is a peptide containing a natural amino acid having a cationic side chain, and n2=2‐20;

Preferably, the organic oligocation is selected from spermine.
Use of the adapter of any one of claims 1 to 8, the construct of claim 9, the complex of claim 10, the method of any one of claims 11-19, or the kit of claim 20 in the preparation of a product for characterizing an analyte or in characterizing an analyte.