CN116200477A - Adaptors, constructs, methods and uses comprising a combination blocker - Google Patents

Abstract

The present invention relates to adaptors, constructs, methods and uses for characterizing analyte sequencing, wherein arrest of polynucleotide binding proteins is achieved by a combined blocking element. The present invention has many advantages such as the ability to obtain good binding proteins for stagnant polynucleotides, and the widening of the range of applications for spacers.

Description

Adapters, constructs, methods and uses comprising associated arresting elements

Technology area

The present application relates to the technical field of nanopore sequencing, in particular to an adapter, a construct, a method and an application for characterizing an analyte comprising a combined blocking element.

Background technique

Sequencing technologies for analytes such as polynucleotides (eg, RNA or DNA) are widely used. Sequencing technologies for polynucleotides, especially DNA, have also been updated.

DNA sequencing technology has developed to the fourth generation sequencing technology since 1977. In addition to the first generation sequencing technology, the second generation, third generation, 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 Sanger sequencing method to the second-generation sequencing-by-synthesis, the third-generation single-molecule sequencing, and the fourth-generation nanopore sequencing. Compared with the first-generation sequencing technology, NGS technology is more widely used due to the advantages of increased throughput, lower cost and shorter sequencing cycle.

The fourth generation of nanopore sequencing refers to that when DNA molecules pass through nanopores driven by electrophoresis, characteristic current changes will be caused due to the different size and shape of each base of DNA molecules, so that DNA can be determined by electron conduction detection. The base type and arrangement sequence of molecules realize single-molecule sequencing.

In nanopore sequencing techniques, it is often necessary to immobilize a polynucleotide-binding protein on a polynucleotide when no potential is applied, preventing further movement of the polynucleotide-binding protein along the polynucleotide. However, when the polynucleotide is in contact with the polynucleotide binding protein and the transmembrane pore and an electric potential is applied, the stagnant polynucleotide binding protein can move along the polynucleotide sequence to be tested, thereby achieving the purpose of sequencing.

In the prior art, single-stranded nucleic acid formed by abasic groups such as iSp18 or iSpC9 is usually used as a spacer to inhibit the translocation function of polynucleotide-binding proteins so as to achieve the effect of stagnation of polynucleotide-binding proteins, and then pass the electric field Force the polynucleotide-binding protein to cross the spacer to begin normal sequencing. However, in the prior art, there are very limited spacers that can effectively arrest polynucleotide-binding proteins, and some groups as spacers will cause high shedding rates or low data throughput of polynucleotide-binding proteins.

Contents of the invention

The present inventors have surprisingly found that some single-stranded nucleic acid modifications can not play a good blocking effect as a spacer, but if they are combined with another to improve double-strand stability (such as increasing the difficulty of melting the double-strand or increasing the double-strand Tm value) ) blocking elements are used in combination, a good blocking effect can be achieved, even equal or better than the traditional spacer in the prior art, and can be used in nanopore sequencing.

Accordingly, a first aspect of the present invention relates to an adapter for characterizing an analyte, said adapter comprising one or more first blocking elements and one or more second blocking elements different from said first blocking elements. two arresting elements, and after the adapter is contacted with a polynucleotide binding protein, the one or more first arresting elements and the one or more second arresting elements are capable of acting in combination to arrest the polynucleotide binding protein.

Preferably, said adapter comprises a complementary host strand and a barrier strand, said one or more first blocker elements being covalently linked to said host strand; said one or more second blocker elements Covalently modified to or non-covalently interacting with the host chain or the barrier chain.

Preferably, said one or more second arresting elements are complementary to said barrier strand or said host strand, wherein when said one or more second arresting elements are covalently modified to said host strand When the one or more second blocking elements are complementary to the blocking chain; when the one or more second blocking elements are covalently modified to the blocking chain, the one or more a plurality of second barrier elements complementary to the host strand; when one or more second barrier elements non-covalently interact with the host strand and/or the barrier strand, the second barrier An element may be complementary to the barrier strand or the host strand.

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

Preferably, said first arresting element comprises one or more elements selected from the group consisting of organic oligocations, iSpC3, iSp18, iSp9, nitroindole, inosine, acridine, 2-aminopurine, 2-6-diaminopurine , 5-bromo-deoxyuridine, reverse thymidine (reverse dT), reverse dideoxythymidine (ddT), dideoxycytidine (ddC), 5-methylcytidylic acid, 5-hydroxymethyl Group consisting of cytidine, 2'-O-methyl RNA base, isodeoxycytidine (iso-dC), isodeoxyguanosine (iso-dG), photocleavable (PC) group, or hexanediol The second block element comprises one or more selected from the group consisting of locked nucleic acid (LNA), peptide nucleic acid (PNA), methoxy (OMe), bicyclic nucleoside (BNA), glycerol nucleic acid (GNA), threose Nucleic acid (TNA), cyclic pyrrolidazole polyamide (cPIP) or double-strand binding protein modified nucleotide group.

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

-HPO ₃ -R ¹ -(XR ² _n ) _n1 -XR ³ -O-, wherein R ¹ , R ² _n and R ³ are the same or different C1-C5 lower alkylene, 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 C1-C5 lower alkylene, R ⁵ and R ⁶ are the same or different C1-C5 lower alkylene , X ¹ is putrescine, spermidine or spermine residues,

-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 natural amino acids with cationic side chains, n2=2-20;

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

Preferably, said first retardation element is not complementary to said second retardation element.

Preferably, the adapter further comprises a third strand that is partially complementary to the main body strand; more preferably, the main body strand is complementary to part of the barrier strand and the main body strand is partially complementary to the main body strand The complementary end of the barrier strand is used for direct or indirect attachment to the analyte.

Preferably, the analyte is selected from polynucleotides, polypeptides, lipids or polysaccharides, preferably polynucleotides, which are fully double-stranded polynucleotides, partially double-stranded polynucleotides or single-stranded polynucleotides .

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

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

A third aspect of the present invention relates to a complex for characterizing an analyte, said complex comprising a polynucleotide binding protein and an adapter of the first aspect of the present invention or a construct of the second aspect of the present invention; wherein said The polynucleotide binding protein is arrested on the adapter by the combined action of the first arresting element and the second arresting element.

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

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

A construct is provided having an analyte, wherein the analyte is directly or indirectly linked at one or both ends to an adapter comprising one or more first arresting elements and one or more a second arresting element different from the arresting element; and contacting the construct with the polynucleotide binding protein such that the polynucleotide binding protein is between the one or more first arresting elements and the arresting on said adapter in combination with said one or more second arresting elements; or

loading the polynucleotide binding protein onto an adapter comprising one or more first arrest elements and one or more second arrest elements different from the first arrest elements, the multinuclear a nucleotide binding protein stalls on the adapter under the combined action of the one or more first arresting elements and the one or more second arresting elements; and causing the loading polynucleotide to bind Protein adapters are attached to the analyte.

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

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

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

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

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

(c) applying a potential across said transmembrane pore such that said polynucleotide binding protein moves through said one or more first blocking elements, optionally (i.e., through or not through) said one or a partial region of the plurality of second blocking elements, and controls the movement of the analyte through the transmembrane pore.

Preferably, said second blocking element comprises covalently modified nucleotides or non-covalently modified nucleotides; when said polynucleotide binding protein moves through said one or more second blocking elements When the arresting element is selected, the polynucleotide binding protein moves through the nucleotides of the one or more second arresting elements without passing through the covalent or non-covalent nucleotides of the one or more second arresting elements. Covalent modifiers.

Preferably, 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 It is used to capture the adapter, and the anchor region is used to anchor the transmembrane pore or the membrane where the transmembrane pore is located.

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

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

(b) taking 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 assay thing.

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

Preferably, the analyte is selected from polynucleotides, polypeptides, lipids or polysaccharides, preferably polynucleotides, which are fully double-stranded polynucleotides, partially double-stranded polynucleotides or single-stranded polynucleotides .

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

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

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

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

An eighth aspect of the present invention relates to the use of an organic oligocation having the general formula Bj, Bj being the organic oligocation moiety of a j-mer, j = 1-50, wherein B is selected from the group comprising:

-HPO ₃ -R ¹ -(XR ² _n ) _n1 -XR ³ -O-, wherein R ¹ , R ² _n and R ³ are the same or different C1-C5 lower alkylene, 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 C1-C5 lower alkylene, R ⁵ and R ⁶ are the same or different C1-C5 lower alkylene , X ¹ is putrescine, spermidine or spermine residues,

-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 natural amino acids with cationic side chains, n2=2-20;

Preferably, the organic oligocation is selected from spermine.

The ninth aspect of the present invention relates to the adapter 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 seventh aspect of the present invention The kit of the aspect is used in the preparation of a product for characterizing an analyte or in characterizing an analyte.

Technical scheme of the present invention has obtained following technical effect:

In the present invention, the enzyme is stagnated by using more than one blocking element in combination, which further expands the application range of the spacer, so that when some blocking elements are used alone, they cannot achieve a good blocking effect or even have no blocking effect. However, when combined with another retarding element, a good retarding effect can be achieved.

In addition, various spacers used in the prior art, such as single-stranded nucleic acid formed by abasic groups such as iSp18 or iSpC9, are used as spacers. Although the action of an electric field force will cause the enzyme to cross the spacer, the blocking element will It always exists and will not disappear, so this spacer is only suitable for entry sequencing, not for outry sequencing. However, in the present invention, the combined blocking structure formed by the first blocking element and the second blocking element will be destroyed or eliminated due to the sequence passing through the nanopore, so its application can be further expanded, so that it can not only be used for internal sequencing It can also be used for pull-out sequencing. "Introduction sequencing" refers to the method in which the direction of the electric field force is the same as that of the enzyme movement so that the enzyme passes through and then the analyte is sequenced. "External pull sequencing" refers to the direction of the electric field force is opposite to the movement direction of the enzyme so that the enzyme A method by which the analyte is then sequenced.

Description of drawings

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

Figure 2 shows schematic diagrams of exemplary structures of various Y adapters containing two arresting elements. The combination relationship of 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 the 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 TCATTCGGTC CTGCT GACT-3'

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

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

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

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

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

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

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

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

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

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

SEQ ID NO: 7 shows the main strand Y1-4C18 comprising 4C18 of the Y linker:

5'-(iSpC3)30-GCGGAGTCAAACGGT AGAAG TCG TTTTT TTTTT-(iSpC18)4-ACTGCTCATT CGGTC CTGCT GACT-3'

SEQ ID NO:8 shows the fluorescent chain Y2 of the Y linker, the 3' end of the sequence is connected with 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 OMe-modified to prevent it 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 CAGGACCGAA TGA(GCAGT) _LNA (AGTCC AGCAC CGACC) _OMe -3'

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

5'-(GTCAG CAGGACCGAA 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 the helicase El:

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

Detailed ways

It will be appreciated that various applications of the disclosed products and methods may be tailored to specific needs within 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 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 proper nouns are used herein. Unless explicitly defined herein, all these terms and nouns should be understood to have the meanings commonly understood by those skilled in the art. For greater clarity, certain terms used herein are defined below.

Adapter

The present invention provides adapters for characterizing nucleic acids that are capable of linking an analyte. Adapters are also referred to as adapters, adapters, and the like. The adapters of the present invention comprise one or more first arresting elements and one or more second arresting elements, the first and second arresting elements are completely different in structure and composition. The adapter comprises a complementary, preferably partially complementary, main strand and a blocking strand, both of which are single-stranded structures or partial nucleotides formed by linking nucleotides, nucleotide analogs or modified nucleotides Modified single-stranded structure. Any number of first retardation elements, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more first retardation elements for a total of Valence bonds are connected to the main chain. Any number of second retardation elements, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more second retardation elements for a total of The valence bond is modified to the main chain or the blocking chain (such as LNA, PNA, etc.) or interacts with the main chain, the blocking chain or the double chain formed by the main chain and the blocking chain through a non-covalent bond (such as cPIP, etc.).

When one or more second blocker elements are covalently bonded to the host strand, the one or more second blocker elements are complementary to the blocker strand; when one or more second blocker elements are covalently bonded When on the blocking chain, one or more second blocking elements are complementary to the main body chain; when one or more second blocking elements interact with the main body chain and/or blocking chain with non-covalent bonds, the second blocking Elements can be complementary to barrier strands or host strands.

The second arresting element comprises nucleotides modified by modifiers, when one or more nucleotide moieties of the second arresting element are located on the main strand and the modifier moiety is non-covalently bonded to the main strand and/or to the barrier When the strands interact, the second blocker element is complementary to the barrier strand; when the nucleotide moiety of one or more second blocker elements is located on the blocker strand and the modifier portion is non-covalently bonded to the main strand and/or the blocker strand When the chains interact, the second arresting element is complementary to the main chain.

The second block element comprises nucleotides modified by modifiers, and "the second block element is located on the main strand or the barrier strand" as described herein means that the nucleotides contained in the second block element are connected to the main strand or On the blocking chain, not a restriction on the position of the modifier.

In some embodiments, the one or more first blocker elements may not associate with the one or more second blocker elements in a complementary base pairing manner. In some embodiments, the one or more second blocker elements associate with the barrier strand or the host strand in complementary base pairing.

The one or more first blocking elements and the one or more second blocking elements may be located adjacently or spaced apart.

In some embodiments, one or more first retardation elements are located in close proximity to one or more second retardation elements. At least one first arresting element is disposed immediately before or after the at least one second arresting element or before or after a segment complementary to the at least one second arresting element in the direction of travel of the polynucleotide binding protein in the subject strand , there is no group between them.

In some embodiments, one or more second retarding elements are positioned adjacently together. The one or more first retarding elements may be positioned immediately adjacent each other, or one or more second retarding elements, or a section of the main body strand that is complementary to the second retarding elements.

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

In some embodiments, one or more first retardation elements are spaced apart from one or more second retardation elements. There are one or more bases between the at least one first arresting element and the at least one second arresting element or a segment complementary to the at least one second arresting element in the direction of movement of the main strand of the polynucleotide binding protein. group.

In some embodiments, one or more second retardation elements are spaced apart. The one or more first retardation elements may be spaced apart from, or spaced from, the one or more second retardation elements, or spaced from a segment on the main body strand that is complementary to the second retardation element.

The meaning of "the first blocker elements are arranged at intervals" herein means that there are one or more groups between one or more first blocker elements on the main body chain, such as 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10 or more groups. The meaning of "the first blocking element and the second blocking element are spaced apart" in this article refers to the first blocking element on the main body chain and the second blocking element on the main body chain, or the first blocking element on the main body chain. One or more groups, such as 1, 2, 3, 4, 5, 6, 7, 8, are present between a blocker element and a segment on the main body strand that is complementary to a second blocker element located on the barrier strand , 9, 10 or more groups. The meaning of "the second retardation element spaced apart" as used herein means that there are one or more groups between one or more second retardation elements on the host chain or barrier chain, such as 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10 or more groups.

As shown in FIG. 2, one or more first retardation elements and one or more second retardation elements may be disposed on the adapter body in any combination or position. In some embodiments, the adapter includes a first arresting element and a second arresting element disposed adjacently or spaced apart in the direction of movement of the polynucleotide binding protein. In some embodiments, the adapter includes a first arresting element, a second arresting element, and a first arresting element disposed adjacently or spaced apart in the direction of movement of the polynucleotide binding protein. In some embodiments, the adapter comprises a first arresting element, a second arresting element, a first arresting element and 1 second blocking element. In some embodiments, the adapter includes a second arresting element and a first arresting element disposed adjacently or spaced apart in the direction of movement of the polynucleotide binding protein. The direction of movement of polynucleotide binding proteins is usually 5'-3', and some polynucleotide binding proteins move in the direction of 3'-5'.

After the adapter is contacted with the polynucleotide binding protein, the one or more first arresting elements and the one or more second arresting elements can act in conjunction to arrest the polynucleotide binding protein. The polynucleotide binding protein may be arrested before the first arresting element or the second arresting element, preferably before the first first arresting element or the first second arresting element.

The first arresting element may be of any molecule or combination of molecules that arrests one or more polynucleotide binding proteins, preferably having or consisting of a structure distinct from nucleotides. More preferably, the first arresting 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 muscle Glycoside, one or more acridine, one or more 2-aminopurine, one or more 2-6-diaminopurine, one or more 5-bromo-deoxyuridine, one or more reverse thymidine (reverse 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 multiple photocleavable (PC) groups or one or more hexanediol linkages.

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

-HPO ₃ -R ¹ -(XR ² _n ) _n1 -XR ³ -O-, wherein R ¹ , R ² _n and R ³ are the same or different C1-C5 lower alkylene, 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 C1-C5 lower alkylene, R ⁵ and R ⁶ are the same or different C1-C5 lower alkylene , X ¹ is putrescine, spermidine or spermine residues,

-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 natural amino acids with cationic side chains, n2=2-20.

More preferably, the organic oligocation is selected from spermine.

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

The second retardation element has one or more groups that increase the stability of the double strand, such as any group or combination of arbitrary groups that increase the difficulty of unzipping the double strand or increase the Tm value of the double strand, preferably with a modified Nucleotides or consist of modified nucleotides. Preferably, the second block element comprises one or more locked nucleic acid (LNA) modified nucleotides, one or more peptide nucleic acid (PNA) modified nucleotides, one or more methoxy ( OMe) modified nucleotides, one or more bicyclic nucleoside (BNA) modified nucleotides, one or more glycerol nucleic acid (GNA) modified nucleotides, one or more threose nucleic acid ( TNA) modified nucleotides, one or more cyclic pyrrolimidazole polyamide (cPIP) modified nucleotides or one or more double stranded binding protein modified nucleotides linked.

More preferably, the second retardation 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 block 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 body chain, the 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 at intervals are respectively Connected to one end of the first section and one end of the second section. The other end of the first segment first enters the transmembrane pore during sequencing 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, and then to the analyte. Analytes were characterized. 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 linking nucleotides.

In some embodiments, on the blocking chain, two ends of one or more second blocking elements arranged adjacently or at intervals are respectively connected to one end of the third section and one end of the fourth section. The other end of the third segment is a free end, which is used for combining with a tether during sequencing and for bringing 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, not connected to any group. The third segment is a single-stranded segment formed by linking nucleotides and/or modified nucleotides, and the fourth segment is a single-stranded segment formed by nucleotides. The second segment of the subject strand can combine with the fourth strand of the barrier strand in a complementary base pairing manner to form a double strand or a partially double strand. Partially double-stranded refers to a structure containing both double-stranded and single-stranded.

The adapter also includes a third strand, which is a single strand formed of nucleotides. The third strand is also called the fluorescent strand. The third strand can combine with the first segment of the main strand in complementary base pairing to form a double strand or a partially 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 that a handle connects two arms, in which the second section of the main body chain is combined with the fourth section of the blocking chain to form the handle of the Y-connector. One end of the handle connects the two arms, and the other end is connected to the analysis connection. One arm is formed by the free end of the barrier chain and the other arm is formed by combining the first section of the main body chain with the third chain.

The adapters of the present invention are preferably combined with a tether, which is a single-stranded polynucleotide composed of multiple nucleotides, and also contains an abasic 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 pore, thereby surrounding the analyte to which the adapter is attached around the pore.

The adapter of the present invention can be used independently, and can also be used in combination with other adapters (such as hairpin adapters, hairpin-like adapters or conventional Y adapters, etc.) to form constructs for sequencing. In some embodiments, an adapter of the invention joins the two ends of an 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 strands of the 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 the A clip adapter or a hairpin-like adapter to form a construct. The hairpin-like adapter is a loop similar to the conventional hairpin structure, but the loop is not the same as the conventional hairpin structure, but is formed by a single-stranded linear molecule folded back on itself, which can connect the two strands of the polynucleotide. For specific examples of hairpin-like adapters, see CN113462764A.

The abasic group used in the adapter or tether is a group that cannot form a base pair such as iSp18, iSpC3 or iSp9, which can also be called "abasic site" or "abasic core". Glycolic acid". An abasic group is a nucleotide or nucleoside that lacks a nucleobase at the 1' position of the sugar moiety.

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). A polynucleotide can be single-stranded or double-stranded. A polynucleotide can be circular. The polynucleotide can be an aptamer, a probe that hybridizes to the microRNA or the microRNA itself. A 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. A polynucleotide can be 1000 or more nucleotide pairs, 5000 or more nucleotide pairs in length, or 100000 or more nucleotide pairs in length.

Analytes can be present in any suitable sample. The invention is generally practiced on samples known to contain or suspected to contain an analyte. The invention can be practiced on samples containing one or more analytes of unknown species. Alternatively, the invention may be practiced on a sample to identify the species of one or more analytes known or expected to be present in said sample. Those skilled in the art can expect that "providing the analyte" in the present invention refers to providing a sample containing the analyte, and "connecting the sequencing adapter to the analyte" in the present invention refers to connecting the sequencing adapter to the analyte present in the sample .

polynucleotide

A polynucleotide can be any polynucleotide. Polynucleotides such as nucleic acids are macromolecules containing two or more nucleotides. The polynucleotide or nucleic acid may comprise any combination of nucleotides. Nucleotides can be naturally occurring or synthetic.

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

Nucleobases 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).

Sugars are typically pentoses. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar is preferably deoxyribose.

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

The nucleotides typically contain monophosphates, diphosphates or triphosphates. Phosphatases can be attached to the 5' or 3' side of the nucleotide.

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

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

A polynucleotide may 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. A polynucleotide may comprise single-stranded regions and regions with other structures, such as hairpin loops, triplexes and/or quadruplexes. A DNA/RNA hybrid can contain DNA and RNA on the same strand. Preferably, a DNA/RNA hybrid comprises a DNA strand hybridized to an RNA strand.

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

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

polynucleotide binding protein

A polynucleotide binding protein can be any protein capable of binding a polynucleotide and controlling its movement through a pore. It is straightforward in the art to determine whether a protein is bound to a polynucleotide. Proteins typically interact with and modify at least one property of a polynucleotide. Proteins can modify polynucleotides by cleaving them to form individual nucleotides or shorter chains of nucleotides such as dinucleotides or trinucleotides. The moieties can modify a polynucleotide by positioning or moving the polynucleotide to a specific location (ie, controlling its movement).

In some embodiments, 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 He1308 helicase, RecD helicase, XPD helicase, Dda helicase or ED1 helicase.

In some embodiments, the polynucleotide binding protein is a polynucleotide helicase. Polynucleotide helicases are enzymes capable of melting double-stranded polynucleotides into single strands. In some embodiments, the polynucleotide helicase is capable of melting double-stranded DNA into single strands. In some embodiments, the polynucleotide helicase is an enzyme having helicase activity. Examples of polynucleotide helicases include, eg, the helicases described herein.

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. A protein may include modifications that facilitate polynucleotide binding and/or facilitate its activity at high salt concentrations and/or at room temperature. A protein can be modified such that it binds polynucleotides (i.e. retains polynucleotide binding ability) but does not act as a helicase (i.e. does not move along polynucleotides when all the necessary components to facilitate movement such as ATP and Mg ²⁺ are present. acid movement). Such modifications are known in the art. For example, modification of the Mg2 ⁺ binding domain in a helicase often results in a variant that does not function as a helicase.

Enzymes can be covalently attached to the pores. Enzymes can be covalently attached to the pores using any method.

In strand sequencing, polynucleotides translocate through the pore either along or against an applied potential. Exonucleases that act gradually or stepwise on double-stranded polynucleotides can be used on the cis side of the pore to supply the remaining single strands at an applied potential, or on the trans side to supply the remaining single strands at a reverse potential . Likewise, helicases that unwind double-stranded DNA can also be used in a similar manner. Polymerases can 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 then switched after binding, the chain will pass through the pore in a cis-to-trans fashion and remain in the elongated configuration by the current. ssDNA exonucleases or ssDNA-dependent polymerases act as molecular motors to pull recently translocated ssDNA back out of the pore in a controlled stepwise fashion from trans to cis against an applied potential.

Any helicase can be used in the present invention. Helicases can act on the pore in two modes. First, the method is preferably carried out 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, allowing it to pass through the pore under the force of a field until it finally translocates through, reaching to the opposite side of the membrane. Alternatively, the method is preferably carried out such that the helicase 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 helicase moves the polynucleotide through the pore such that it is pulled out of the pore against an applied field until it is finally pushed back up to the cis side of the membrane.

It is also possible to carry out the method in the opposite direction. The 3' end of the polynucleotide can be captured in the pore first, and the helicase can move the polynucleotide into the pore so that it passes through the pore under the action of a field until it finally translocates through, reaching the trans side of the membrane .

When the helicase does not have the necessary components to facilitate movement or is modified to impede or prevent its movement, the helicase can bind to the polynucleotide and act as a brake when the polynucleotide is pulled into the pore by an applied field Slow down the movement of polynucleotides. In the inactive mode, it does not matter whether the 3' or 5' of the polynucleotide is trapped, it is the applied field that pulls the polynucleotide into the pore towards the trans side under the action of the enzyme acting as a brake. When in the inactive mode, the helicase's control of the movement of the polynucleotide can be described in a variety of ways, including ratcheting, sliding, and braking. Helicase variants lacking helicase activity can also be used in this way.

The polynucleotide can be contacted with the polynucleotide binding protein (eg, polynucleotide helicase) and the pore in any order. Preferably, when contacting a polynucleotide with a polynucleotide binding protein such as a helicase (e.g., polynucleotide helicase) and the pore, the polynucleotide is first contacted with the polynucleotide binding protein (e.g., polynucleotide Acid 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., polynucleotide unwinding enzyme) is typically between free nucleotides or free nucleotide analogs and a polynucleotide binding protein (e.g., polynucleotide unwinding enzyme). Enzyme) acts in the presence of an enzyme cofactor. A free nucleotide 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), monophosphate Deoxythymidine (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). 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. Enzyme cofactors are preferably divalent metal cations. The divalent metal cation is preferably Mg ²⁺ , Mn ²⁺ , Ca ²⁺ or Co ²⁺ . The enzyme cofactor is most preferably Mg ²⁺ .

stagnation

A polynucleotide binding protein is stalled if it has stopped moving along the analyte, eg, polynucleotide. Associated arresting elements are used to arrest polynucleotide binding proteins. The polynucleotide binding protein can be arrested prior to the arresting element.

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

This allows the position of the polynucleotide binding protein on the polynucleotide to be controlled. Until the stalled polynucleotide binding protein and polynucleotide are brought into contact with the transmembrane pore and an electric potential is applied, the polynucleotide binding protein stays where they are stalled. Even in the presence of the necessary components (such as ATP and Mg2+) to facilitate the movement of the polynucleotide binding protein, the polynucleotide binding protein will not move across the associated arresting element on the polynucleotide until there is a transmembrane The presence of pores and the applied potential.

membrane

Any membrane can be used in accordance with 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 (such as phospholipids) having hydrophilicity and lipophilicity. Amphiphiles can be synthetic or naturally occurring. Non-naturally occurring amphiphiles and monolayer-forming amphiphiles are known in the art and include, for example, block copolymers. Block copolymers are polymeric materials in which two or more monomeric subunits are aggregated together to create a single polymer chain. Block copolymers generally have properties provided by each monomer subunit. However, block copolymers can have unique properties not found in polymers formed from individual subunits. Block copolymers can be designed such that one monomeric subunit is hydrophobic (ie, lipophilic), while the other subunit or subunits are hydrophilic in aqueous media. In this case, the block copolymers can have amphiphilic properties and can form structures that mimic biological membranes. Block copolymers can be diblocks (composed of two monomeric subunits), but can also be composed of more than two monomeric subunits to form more complex arrangements that behave as amphiphiles. The copolymers may be triblock, tetrablock or pentablock copolymers.

Archaeal bipolar tetraether lipids are naturally occurring lipids that are structured such that the lipids form monolayer membranes. These lipids are commonly found in extremophiles, thermophiles, halophiles and acidophiles that live in harsh biological environments. Their stability is thought to arise from the fused nature of the final bilayer. Block copolymer materials that mimic these biological entities are readily constructed by generating triblock polymers with a universal motif hydrophilic-hydrophobic-hydrophilic. The material forms monomeric membranes that behave similarly to lipid bilayers and have a range of phase states from vesicles to lamellar membranes. Membranes formed from these triblock copolymers have several advantages over biological lipid membranes. Because the triblock copolymers are synthetic, the precise architecture can be carefully controlled to provide the correct chain lengths and properties needed to form membranes and interact with pores and other proteins.

Block copolymers can also be constructed from subunits not classified as lipid submaterials; for example hydrophobic polymers can be made from siloxane or other non-hydrocarbon compound based monomers. The hydrophilic subportions of the block copolymers can also have low protein binding properties, which enables the creation of membranes that are highly resistant when exposed to raw biological samples. The headgroup unit can also be derived from atypical lipid headgroups.

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

The amphiphilic molecules can be chemically modified or functionalized to facilitate conjugation of analytes.

The amphiphilic layer can be a monolayer or a bilayer. The amphiphilic layer is usually planar. The amphiphilic layer may be non-planar, eg curved.

The amphiphilic layer is usually a lipid bilayer. Lipid bilayers are models of cell membranes and serve as excellent platforms for a range 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 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 state 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 usable structure in a synthetically manufactured form. Solid layers can be formed from 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 the flow of hydrated ions driven by an applied potential from one side of the membrane to the other.

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

Transmembrane protein pores can be monomeric or oligomeric. The pore is preferably composed of several repeating subunits (eg 6, 7 or 8 subunits). The pores are more preferably heptameric or octameric pores.

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

The barrel or channel of a transmembrane protein pore typically contains amino acids that facilitate interaction with an analyte (eg, nucleotide, polynucleotide, or nucleic acid). These amino acids are preferably located near the constriction of the barrel or channel. Transmembrane protein pores typically contain one or more positively charged amino acids (such as arginine, lysine, or histidine) or aromatic amino acids (such as tyrosine or tryptophan). These amino acids generally facilitate the interaction between the pore and nucleotides or polynucleotides or nucleic acids.

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

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

The transmembrane protein pore is also preferably derived from alpha-hemolysin (α-HL). The wild-type α-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 pores can be chemically modified at any point in any manner. Preferably via 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, epitope Enzyme modification or terminal modification chemically modifies the transmembrane protein pore. Suitable methods for making such modifications are well known in the art. The transmembrane protein pore can be chemically modified by binding any molecule. For example, the pores can be chemically modified by the incorporation of dyes or fluorophores.

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

move

In the methods of the invention, an analyte, such as a polynucleotide, is moved through a transmembrane pore and sequenced. Moving a polynucleotide through a transmembrane pore refers to moving a polynucleotide from one side of the pore to the other. Movement of polynucleotides through the pore can be driven or controlled by an electrical potential or an enzymatic action or both. Movement can be unidirectional, or backward and forward movement can be allowed.

Polynucleotide binding proteins are preferably used to control the movement of polynucleotides through the pore.

Analyte Characterization

The characterization method may comprise measuring one, two, three, four or five or more characteristics of an analyte, such as a polynucleotide. The method includes controlling the movement of the analyte through the transmembrane pore, and taking 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) length of the polynucleotide, (ii) identity of the polynucleotide, (iii) sequence of the polynucleotide, (iv) secondary structure of the polynucleotide, and (v) polynucleotide Whether the nucleotide is modified.

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

The methods of the invention comprise moving the single-stranded polynucleotide through the transmembrane pore such that a portion of the nucleotides of the single-stranded polynucleotide interacts with the pore.

The method may be performed using any suitable membrane as described above, preferably a lipid bilayer system in which pores are inserted into the lipid bilayer. The methods are typically performed using membranes (i) artificial bilayers comprising pores, (ii) isolated naturally occurring pore-containing lipid bilayers, or (iii) cells with pores inserted therein. The method is preferably performed using an artificial lipid bilayer. In addition to the pores, the bilayer may comprise other transmembrane and/or intramembrane proteins as well as 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 invention provides methods of characterizing polynucleotides comprising:

(a1) providing a construct having a polynucleotide linked at one or both ends to an adapter of the invention comprising a co-arrest element; and contacting the construct with a polynucleotide binding protein such that the polynucleotide the nucleotide-binding protein is arrested on the adapter in conjunction with arresting elements on the adapter; or

(a2) loading the polynucleotide binding protein onto the adapter comprising an associated arresting element of the present invention, the polynucleotide binding protein is arrested on the adapter under the combined action of the arresting elements on the adapter; and loading an adapter for the polynucleotide binding protein is attached 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 electrical potential across the transmembrane pore such that the polynucleotide binding protein moves through the first arresting element, a portion of the second arresting element, and controls movement of the polynucleotide through the transmembrane pore;

(d) taking one or more measurements representing one or more characteristics of the polynucleotide as the polynucleotide moves relative to the transmembrane pore, and thereby characterizing the polynucleotide.

In some embodiments, when the adapter only comprises a first blocking element and a second blocking element arranged in close proximity, the polynucleotide binding protein stops at the first blocking element of the adapter under the joint action of the blocking elements. Before the hysteresis element, it then moves through the host strand of the adapter under the action of an electric potential. The sequence of movement on the main strand is: the first arresting element, the segment complementary to the second arresting element, the second segment, and then passing through the polynucleotide chain connected with the main strand. In some embodiments, the polynucleotide binding protein does not cross the barrier strand of the adapter. In some embodiments, the polynucleotide binding protein also passes through the blocking strand of the adapter, and when the second blocking element is located on the blocking strand, the order of movement on the blocking strand is as follows: the fourth segment, the second blocking element, and the second blocking element. Nucleotides for hysteresis elements. The polynucleotide binding protein does not pass through the modification of the second arresting element.

These approaches are possible because transmembrane protein pores can be used to distinguish between nucleotides of similar structure 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 as they interact with the pore. A nucleotide is present in the pore if the current flows through the pore in a manner specific for that nucleotide (i.e. if a characteristic current flow through the pore associated with that nucleotide is detected). exists in. The serial identification of nucleotides in a polynucleotide allows the sequence of the polynucleotide to be estimated or determined.

Thus, the method involves passing a portion of the nucleotides in the polynucleotide across the membrane pore as they pass individually through the barrel or channel in order to sequence the polynucleotide. feel. As mentioned above, this is strand sequencing.

In some embodiments, the method includes providing a tether for bringing the construct close to the transmembrane pore; the tether includes a capture region and an anchor region, the capture region for capturing the construct The adapter, the anchoring region is used to anchor and combine with the transmembrane pore or the membrane where the transmembrane pore is located.

All or only a portion of the polynucleotide can be sequenced using this method. 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 may be 1000 or more nucleotide pairs, 5000 or more nucleotide pairs, or 100000 or more nucleotide pairs in length. The polynucleotides may be naturally occurring or man-made. For example, the methods can be used to verify the sequence of manufactured oligonucleotides. The methods are typically performed in vitro.

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

During the interaction of the nucleotides in the single stranded polynucleotide with the pore, the nucleotides affect the current flow through the pore in a manner specific for that nucleotide. For example, a particular nucleotide will reduce the current flowing through the pore for a particular average time and to a particular degree. 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 particular nucleotides on the current flowing through the pore. The results obtained from performing the methods of the invention on the test samples can then be compared with 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 pores are inserted into the membrane. The methods are typically performed using membranes comprising naturally occurring or synthetic lipids. The membrane is usually formed in vitro. The method is preferably performed without the use of isolated naturally occurring pore-containing membranes, or cells expressing pores. The method is preferably carried out using artificial membranes. In addition to the pores, the membrane may comprise other transmembrane and/or intramembrane proteins as well as 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 linked to a nucleic acid to obtain a nucleic acid-polypeptide linker, and then the adapter of the present invention is combined with the nucleic acid-polypeptide linker linked to characterize the peptide. The other steps of the method of characterizing a polypeptide are similar to the steps of characterizing a polynucleotide. Reagent test kit

The invention also provides kits for the preparation of kits for the characterization of analytes, such as polynucleotides. The kit comprises (a) an adapter of the invention, and (b) a polynucleotide binding protein, and/or (c) a transmembrane pore, and optionally a tether.

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

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

The kits of the invention may additionally comprise one or more other reagents or instruments that enable any of the above-described embodiments to be practiced. Such reagents or instruments include one or more of the following: a suitable buffer (aqueous solution), means for sampling from a subject (eg, a tube or device containing a needle), for amplification and/or Means for expressing polynucleic acids, such as membranes or voltage-clamp or patch-clamp devices as defined above. Reagents may be present in the kit in a dry state such that the reagents can be resuspended with a fluid sample. Optionally, the kit may also include instructions enabling the kit to be used in the methods of the invention, or details as to which patients the method may be used in. Optionally, the kit may include nucleotides.

Example

The details of the experimental operation not specifically indicated in the following examples can be referred to herein as the cited references, the experimental reagents and equipment used are conventional commercially available reagents or instruments, and the sequences used are synthesized by biological companies .

Example 1: Preparation and quality inspection of E1 joints containing retardation elements

The main chain, fluorescent chain and blocking chain were synthesized respectively, and the main chain, fluorescent chain and blocking chain were annealed at a ratio of 1:1.1:1.1 to form a Y-shaped joint as shown in Figure 1. Specifically, the annealing treatment is to slowly lower the temperature from 95°C to 25°C, and the temperature drop rate does not exceed 0.1°C/s. The annealing treatment system includes 160mM HEPES 7.0, 200mM NaCl, and the concentration of the main chain in the annealing system is 4-8uM.

Get 500nM Y-type linker, helicase E1 (SEQ ID NO.:14 with M1G/E94C/C109A/C136A/A360C mutation) and 1.5mM TMAD (azodicarbonamide) in an amount of 6 times the amount of the substance, mix and cool at room temperature Incubate for 30 minutes to prepare the sequencing adapter complex QMX1-QMX10. The sequencing adapter complex was added to a DNAPac PA200 column and purified with an elution buffer to elute enzymes not bound to the sequencing adapter complex from the column. The sequencing adapter complex was then eluted with 10 column volumes of a mixture of buffer A and buffer B. The main eluting peaks were then pooled, their concentrations measured, and run with a TBEPAGE 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 strand, fluorescent strand and blocking strand used in the linker of this embodiment are as follows:

Subject 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: Take 0.2pmol of the sequencing adapter complex and add it to the sequencing buffer system (containing 10mM HEPES7.0, 500mM KCl, 100mM MgCL2, 50mM ATP), in which cPIP group is added 6 times the amount of cPIP, and placed at 40°C , incubate at room temperature for 30 minutes, and run TBE PAGE gel at 160V for 40 minutes, use Cy5 mode to sweep the gel, and then perform gray scale calculation: the ratio of unbound enzyme bands/(bound enzyme bands + unbound enzyme bands) is The enzyme shedding rate thereof, the results are shown in Table 1 below.

Table 1: Enzyme shedding rate results of QMX1-QMX10

serial number Connector type (Y1/Y2/B/E1) Quality inspection drop rate 1 QMX-1(Y1/Y2/B-PNA/E1) 100% 2 QMX-2(Y1/Y2/B-LNA/E1) 100% 3 QMX-3(Y1-3C3/Y2/B/E1) 70.9% 4 QMX-4(Y1-5C3/Y2/B/E1) 60.9% 5 QMX-5(Y1-3C3/Y2/B-PNA/E1) 36.1% 6 QMX-6(Y1-5C3/Y2/B-PNA/E1) 28.7% 7 QMX-7(Y1-3C3/Y2/B-LNA/E1) 51.8% 8 QMX-8(Y1-5C3/Y2/B-LNA/E1) 50.1% 9 QMX-9(Y1-5C3-cPIP/B-cPIP/Y2/E1) 60.5% 10 QMX-10(Y1-5C3-cPIP/B-cPIP/Y2/E1/cPIP) 51.8%

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

Example 2: Preparation and quality inspection of E2 joints containing retardation elements

The main chain, fluorescent chain and blocking chain were synthesized respectively, and the main chain, fluorescent chain and blocking chain were annealed at a ratio of 1:1.1:1.1 to form a Y-shaped joint. Specifically, the annealing treatment is to slowly lower the temperature from 95°C to 25°C, and the temperature drop rate does not exceed 0.1°C/s. The annealing treatment system includes 160mM HEPES 7.0, 200mM NaCl, and the concentration of the main chain in the annealing system is 4-8uM.

Get 500nM Y-type linker, helicase E2 (with D99C/A366C/C308T/C419D/E286K/F246Y/S293N/V422H mutation SEQ ID NO.: 15) and 1.5mM TMAD (azobis formamide) and incubated at room temperature for 30 minutes to prepare the sequencing adapter complex QMX11-QMX14. The sequencing adapter complex was added to a DNAPac PA200 column, and purified with an elution buffer to elute enzymes not bound to the sequencing adapter complex from the column. The sequencing adapter complex was then eluted with 10 column volumes of a mixture of buffer A and buffer B. The main eluting peaks were then pooled, their concentrations were 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.

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

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: Add 0.2pmol of the sequencing adapter complex to the sequencing buffer system (containing 10mM HEPES7.0, 500mM KCl, 100mM MgCL2, 50mM ATP), place it at 34°C, incubate at room temperature for 30min, and run it with TBEPAGE gel at 160V for 40 Minutes, scan the gel with Cy5 mode, and then perform grayscale calculation: the ratio of unbound enzyme bands/(bound enzyme bands+unbound enzyme bands) is the enzyme shedding rate, and the results are shown in Table 2 below.

Table 2: Enzyme shedding rate results of QMX11-QMX14

serial number Connector type (Y1/Y2/B/E2) Quality inspection drop rate 1 QMX-11(Y1-Sp/Y2/B/E2) 35.2% 2 QMX-12(Y1-2Sp/Y2/B/E2) 18.1%% 3 QMX-13(Y1-Sp/Y2/B-LNA/E2) 13.5% 4 QMX-14(Y1-2Sp/Y2/B-LNA/E2) 8.6%

Interpretation of the results: Samples 11 and 12 used 1 Sp and 2 Sp as the blocking element on the main chain, and when there was no blocking element on the blocking chain, the drop-off rates of the adapter quality inspection enzymes were 35.2% and 18.1%, respectively. ; Sample 13 and sample 14, when there are 1 Sp and 2 Sp on the main chain as the blocking element on the main chain, and there is LNA modification on the blocking chain, the quality inspection enzyme shedding rate drops to 13.5% and 8.6 respectively %.

Example 3: Chip-mixed library testing of joints containing a single blocking element and joints containing joint blocking

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) prepared in Example 2 were used ), QMX-13(Y1-Sp/Y2/B-LNA/E2), QMX-14(Y1-2Sp/Y2/B-LNA/E2) were connected with the library to build the library. The QMX-11, QMX-12, QMX-13, and QMX-14 libraries with equal amounts of substances were evenly loaded onto three different Qitan Technology Co., Ltd. QNome-9604 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 the results: the better the blocking effect, the more data will be generated in the mixed test, that is, the more the number of sequencing signals. It can be seen from the table that when there is only Sp or 2Sp 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, adapters containing two combined blocking elements were significantly more effective in blocking during sequencing than adapters containing only a single blocking element.

Example 4: Chip Mixed Library Test of Linkers Containing Joint Blocking Elements and Commercial Linkers

The method of Example 1 was used to prepare the commercialized linker complex QMX-15 (Y1-4C18/Y2/B/E2) containing 4C18. The linker in the linker complex was composed of the main chain Y1-4C18 (such as SEQ ID NO.: 7), a fluorescent chain (shown in SEQ ID NO.:8) and a blocking chain (shown in SEQ ID NO.:9) are formed.

A 10kb 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 used to connect to the library Build a library. The QMX-13 and QMX-15 libraries with equal amounts of substances were evenly loaded onto three different Qitan Technology Co., Ltd. QNome-9604 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

Number of sequencing signals Number of sequencing signals Number of sequencing signals QMX-15 9008 14484 12858 QMX-13 12045 18461 16690

Interpretation of the results: The mixed test data of 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 in the sequencing process was not inferior to that of commercial products Linkers containing 4C18 showed even better blocking effects than commercial linkers.

Claims (22)

1. 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 the one or more first blocking elements and the one or more second blocking elements being capable of acting in combination to arrest a polynucleotide binding protein after the adaptor is contacted with the polynucleotide binding protein.

2. The adaptor of claim 1, wherein the adaptor comprises complementary host and blocking strands, the one or more first blocking elements being covalently linked to the host strand; the one or more second blocking elements are covalently modified to or non-covalently interacted with the subject chain or the blocking chain;

preferably, the one or more second blocking elements are complementary to the blocking strand or the main body strand.

3. The adaptor of claim 1 or 2, wherein the one or more first blocking elements have a structure different from a nucleotide; the one or more second blocking elements have a structure for improving double-strand stability.

4. The adaptor of any one of claims 1 to 3, wherein the first blocking element comprises one or more groups selected from the group consisting of organic oligocations, iSpC3, iss 18, iss 9, nitroindole, inosine, acridine, 2-aminopurine, 2-6-diaminopurine, 5-bromo-deoxyuracil, reverse thymidine (reverse dT), reverse dideoxythymidine (ddT), dideoxycytidine (ddC), 5-methylcytidine, 5-hydroxymethylcytidine, 2' -O-methyl RNA bases, isodeoxycytidine (iso-dC), isodeoxyguanosine (iso-dG), photocleavage (PC), or hexanediol; the second blocking element comprises one or more nucleotides selected from the group consisting of 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 modified nucleotides.

5. The adaptor of claim 4, wherein the organic oligocation has the general formula Bj, bj being an organic oligocation moiety of j-mer, j = 1-50, wherein B is selected from the group comprising:

-HPO ₃ -R ¹ -(X-R ² _n ) _n1 -X-R ³ -O-, wherein R ¹ 、R ² _n And R is ³ Is the same or different C1-C5 lower alkylene, 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 C1-C5 lower alkylene, R ⁵ And R is ⁶ C1-C5 lower alkylene, X, which are identical or different ¹ Is 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 a C1-C5 lower alkylene, serine, natural amino alcohol, (aa) _n2 Is a peptide containing a natural amino acid having a cationic side chain, n2=2-20;

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

6. The adapter of any one of claims 1-5, wherein the first blocking element is non-complementary to the second blocking element.

7. The adapter of any one of claims 1-6, wherein the adapter further comprises a third strand that is partially complementary to the subject strand; preferably, the host strand is complementary to a portion of the blocking strand and the complementary ends of the host strand and the blocking strand are for direct or indirect attachment to the analyte.

8. The adaptor 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, which is a fully double stranded polynucleotide, a partially double stranded polynucleotide or a single stranded polynucleotide.

9. A construct for characterizing an analyte, the construct comprising the analyte and the adapter of any one of claims 1-8, wherein the adapter is directly or indirectly attached to either or both ends of the analyte.

10. A complex for characterizing an analyte, the complex comprising a polynucleotide binding protein, and the adapter of any one of claims 1-8 or the construct of claim 9;

wherein the polynucleotide binding protein is arrested on the adapter by the combined action of the first blocker and the second blocker;

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.

11. A method of 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 linked at one or both ends to an adapter comprising one or more first blocking elements and one or more second blocking elements different from the first blocking elements; and contacting the construct with the polynucleotide binding protein such that the polynucleotide binding protein is arrested on the adapter by the combined action of the one or more first blocking elements and the one or more second blocking elements; or (b)

Loading a polynucleotide binding protein onto an adaptor, the adaptor comprising one or more first blocking elements and one or more second blocking elements different from the first blocking elements, the polynucleotide binding protein being arrested on the adaptor by the combination of the one or more first blocking elements and the one or more second blocking elements; and ligating the adaptor loaded with the polynucleotide binding protein to the analyte.

12. The method of claim 11, wherein the adaptor is as defined in any one of claims 1 to 8.

13. The method of claim 11 or 12, wherein 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.

14. A method of controlling movement of an analyte through a transmembrane pore, the method comprising:

(a) Implementing the method of 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) An electrical potential is applied across the transmembrane pore such that the polynucleotide binding protein moves through a partial region of the one or more first blocking elements, optionally the one or more second blocking elements, and controls movement of the analyte through the transmembrane pore.

15. The method of claim 14, wherein the second blocker 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, the polynucleotide binding protein moves through the nucleotides of the one or more second blocking elements, but not through the covalent or non-covalent modifications of the one or more second blocking elements.

16. The method of claim 14 or 15, wherein the method comprises providing a tether for bringing the analyte loaded with the polynucleotide binding protein into proximity with the transmembrane pore; the tether includes a capture region for capturing the adaptor and an anchor region for anchoring association with the transmembrane pore or a membrane in which the transmembrane pore is located.

17. A method of characterizing an analyte, the method comprising:

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

(b) One or more measurements are obtained 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.

18. The method of 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.

19. The method of claim 17 or 18, wherein 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.

20. A kit for characterizing an analyte, the kit comprising:

(a) the adapter of any one of claims 1 to 8, and (b) a polynucleotide binding protein, and/or (c) a transmembrane pore.

21. Use of an organic oligocation as a blocking element for a arrested polynucleotide binding protein, the organic oligocation having the general formula Bj, bj being an organic oligocation moiety of a j-mer, j = 1-50, wherein B is selected from the group comprising:

-HPO ₃ -R ¹ -(X-R ² _n ) _n1 -X-R ³ -O-, wherein R ¹ 、R ² _n And R is ³ Is the same or different C1-C5 lower alkylene, 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 C1-C5 lower alkylene, R ⁵ And R is ⁶ C1-C5 lower alkylene, X, which are identical or different ¹ Is 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 a C1-C5 lower alkylene, serine, natural amino alcohol, (aa) _n2 Is a peptide containing a natural amino acid having a cationic side chain, n2=2-20;

preferably, the organic oligocation is selected from spermine.

22. Use of an adapter according to any one of claims 1 to 8, a construct according to claim 9, a complex according to claim 10, a method according to any one of claims 11 to 19, or a kit according to claim 20 for the preparation of a product for or for the characterization of an analyte.

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