CN114457145B - Linkers, constructs, methods and uses for characterizing target polynucleotide sequencing - Google Patents

Abstract

The present application relates to linkers, constructs, methods and uses for characterizing target polynucleotide sequencing, wherein a polynucleotide binding protein is simultaneously bound and arrested by a blocking band formed by nucleotides whose nucleobases are benzo-structured. The application has a plurality of advantages, such as simplifying the structure composition of the joint, reducing the difficulty and cost of preparing the joint, and the like.

Description

Linkers, constructs, methods and uses for characterizing target polynucleotide sequencing

Technical Field

The present application relates to the field of nanopore sequencing technology, in particular to a linker, construct, method and application for characterizing target polynucleotide sequencing.

Background

Currently, nucleic acid sequencing technology has application requirements in a variety of contexts. The existing sequencing technology requires complex processing of samples in the early stage, has long sequencing period, and cannot meet the requirements of clinical application scenes and the like on the nucleic acid sequencing technology.

The development of new nucleic acid sequencing technologies using transmembrane pores (such as nanopores) as biosensors has great potential. When a voltage is applied across the nanopore, the analyte (e.g., nucleotide, polypeptide, polysaccharide, lipid) causes a decrease in current as it passes through the nanopore, with different levels of current blocking caused by analytes of different structures. The nanopore detects the blocking of the analyte by a resulting current having a known characteristic and duration.

In nanopore sequencing technology, when no potential is applied, the spacer of the polynucleotide is typically capable of arresting the helicase, preventing further movement of the helicase along the target polynucleotide through the spacer. However, when the helicase contacts the transmembrane pore and an electrical potential is applied, one or more of the stalled helicases may be moved through a spacer on the polynucleotide and along the polynucleotide sequence to be sequenced. Thus, existing polynucleotide linkers are both binding regions for enzymes comprising normal polynucleotides and spacer regions of various structures.

The linker design generally comprises the binding region of the enzyme of the normal polynucleotide and the blocking bands of various structures, which increases the difficulty of preparation of the sequencing sample and increases the cost.

Disclosure of Invention

The inventors have found linkers with blocking bands that combine the functions of the binding and spacer regions, enabling similar blocking effects to be achieved compared to the use of a separate spacer region. In addition, the blocking band of the joint is simpler in structural design, and the difficulty and cost of subsequent synthesis and preparation are reduced.

Accordingly, a first aspect of the present invention relates to a linker for characterising a target polynucleotide, the characterisation of a double stranded target polynucleotide comprising recognition of information such as base sequencing or sequence modification of the double stranded target polynucleotide. The linker comprises a blocking band for simultaneously binding and blocking the polynucleotide binding protein.

Preferably, the adaptor further comprises a first section for guiding the adaptor into a transmembrane pore and a second section for ligating a target polynucleotide, the first and second sections having the blocking tape interposed therebetween.

Preferably, the blocking band comprises a single-stranded nucleotide sequence formed by the ligation of a plurality of nucleotides, the nucleobases of which are benzo structures.

Preferably, the blocking band is used to bind and arrest 1 polynucleotide binding protein.

Preferably, the blocking band is a single-stranded nucleotide sequence formed by joining 7-12, more preferably 8-10 nucleotides, the nucleobases of which are benzo structures.

More preferably, the nucleotide is selected from one or more of the following:

wherein n=0 or 1

When n=0, X is N; y is N, C, CCONH or CCONHMe; r is R ₁ Methyl or phenyl; r is R ₂ ，R ₃ And R is ₄ One or two of which are nitro, fluoro or methyl; r is R ₅ Is an arbitrary group, preferably H, methyl, etc.;

when n=1, X and Y are both C; r is R ₃ And R is ₅ One of them isA methyl group; r is R ₆ Is carbonyl; r is R ₁ 、R ₂ And R is ₄ H, methyl, and the like are preferable as the optional group.

Further preferably, the nucleobase of the nucleotide is selected from one or more of the following: 5-nitroindole, 4, 6-nitroindole, 2-methyl-4-nitroindole, 2-phenyl-4-nitroindole, 5-nitroindole-3-carboxamide derivatives, 3-carboxamide indole, 4-methylbenzimidazole, 4-fluoro-6-methyl-benzimidazole, 2, 4-difluorotoluene, 4, 6-difluoro-1H-benzimidazole, 3-methylisoquinolone and 5-methylisoquinolone.

Preferably, the linker does not comprise an additional region for binding the polynucleotide binding protein.

Preferably, the polynucleotide binding protein is derived from a polynucleotide handling enzyme; the polynucleotide handling enzyme is selected from a polymerase, a helicase or an exonuclease.

More preferably, the helicase is selected from He1308 helicase, recD helicase, XPD helicase, dda helicase, or ED1 helicase.

Preferably, the first segment is a partially double-stranded segment formed from a nucleotide and an abasic group, comprising a first double-stranded portion and a first single-stranded portion joined, wherein one strand of the first double-stranded portion is joined to a blocking tape.

Preferably, the second segment is a partially double stranded region formed by nucleotide ligation, comprising a second double stranded portion and a second single stranded portion joined, wherein one strand of the second double stranded portion is joined to a blocking tape.

A second aspect of the invention relates to a construct for characterising a target polynucleotide, wherein the construct comprises a target polynucleotide and a linker as described above, wherein the linker is attached to either or both ends of the target polynucleotide.

A third aspect of the invention relates to a complex for characterising a target polynucleotide, wherein the complex comprises a polynucleotide binding protein, and a linker as described above or a construct as described above; wherein the polynucleotide binding protein binds to and stagnates at the blocking band.

Preferably, the polynucleotide binding protein is derived from a polynucleotide handling enzyme; the polynucleotide handling enzyme is selected from a polymerase, a helicase or an exonuclease.

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

providing a construct having a target polynucleotide linked at one or both ends to a linker, the linker being inserted with a blocking band for simultaneously binding and arresting the polynucleotide binding protein; and contacting the construct with the polynucleotide binding protein such that the polynucleotide binding protein binds to and stagnates at the blocking band; or (b)

Loading a polynucleotide binding protein onto a linker inserted with a blocking band, wherein the polynucleotide binding protein binds to and stagnates at the blocking band; and ligating the linker loaded with target polynucleotide binding protein to the target polynucleotide.

Preferably, the linker is as defined above.

Preferably, the blocking band binds to and arrests 1 of the polynucleotide binding proteins.

Preferably, the polynucleotide binding protein is derived from a polynucleotide handling enzyme; more preferably, the polynucleotide handling enzyme is selected from the group consisting of a polymerase, an exonuclease, a helicase, a topoisomerase.

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 a target polynucleotide through a transmembrane pore, the method comprising:

(a) Implementing the method of controlling the loading of polynucleotide binding proteins on a target polynucleotide as described above;

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

(c) Applying electricity across the transmembrane pore such that the polynucleotide binding protein moves through the blocking band and controls movement of the target polynucleotide through the transmembrane pore.

Preferably, during movement of the target polynucleotide through the transmembrane pore, the first segment of the linker is directed into the transmembrane pore, and then the blocking band, second segment and target polynucleotide attached to the linker pass through the transmembrane pore sequentially.

Preferably, the method comprises providing a tether for bringing the construct close to the transmembrane pore; the tether includes a capture region for capturing a linker of the construct and an anchor region for anchoring association with the transmembrane pore or a membrane in which the transmembrane pore is located.

A sixth aspect of the invention relates to a method of characterizing a target polynucleotide, the method comprising:

(a) Implementing the above method of controlling movement of a target polynucleotide through a transmembrane pore; and

(b) One or more measurements are obtained as the target polynucleotide moves relative to the transmembrane pore, wherein the measurements represent one or more characteristics of the target polynucleotide and thereby characterize the target polynucleotide.

Preferably, the transmembrane pore is a protein pore or a solid state pore; more preferably, the protein pore is derived from Msp, a-hemolysin (a-HL), cytolysin, csgG, clyA, sp, or FraC.

Preferably, the membrane is an amphiphilic layer or a solid state layer; more preferably, the amphiphilic layer is a lipid bilayer.

Preferably, the target polynucleotide is a fully double-stranded polynucleotide, a partially double-stranded polynucleotide, or a single-stranded polynucleotide.

A seventh aspect of the invention relates to a kit for controlling movement of a target polynucleotide, the kit comprising:

(a) the linker, (b) a polynucleotide binding protein, and (c) a transmembrane pore.

Preferably, the polynucleotide binding protein is capable of controlling movement of the target polynucleotide through a transmembrane pore, preferably the polynucleotide binding protein is as described above.

Optionally, the kit further comprises: a tether for coupling to a transmembrane pore or membrane.

Preferably, the target polynucleotide is linked to the linker by a cohesive end or T-A linkage;

and/or the kit further comprises one or more markers that when interacted with the transmembrane pore generate a characteristic electrical current; preferably, the one or more markers are abasic or specific nucleotide sequences.

Preferably, the polynucleotide binding protein is derived from a polynucleotide handling enzyme; more preferably, the polynucleotide handling enzyme is selected from the group consisting of a polymerase, an exonuclease, a helicase, a topoisomerase.

More preferably, the helicase is selected from He1308 helicase, recD helicase, XPD helicase, dda helicase, or ED1 helicase.

An eighth aspect of the invention relates to the use of the above-described linker, the above-described construct, the above-described complex, the above-described method, or the above-described kit for the preparation of a product for or for characterising a target polynucleotide.

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

in order to effectively arrest the polynucleotide binding protein in a single-stranded binding region when no electric field force is applied during sequencing of a target polynucleotide, and simultaneously rapidly pass through the binding region when the electric field force is applied to exert the normal function of the polynucleotide, a blocking band formed by a segment of nucleotide single chains with nucleobases in a benzo structure is screened. The enzyme does not exert its biological activity when bound to the blocking band, stagnates in the region, but can rapidly pass through the region and exert its normal function under the effect of an electric field. The blocking region blocks movement of the polynucleotide binding protein as well as binding to it. The blocking tape with both binding and stagnation polynucleotide binding proteins is inserted into a linker for sequencing, which can simplify the structural composition of the linker and provide a new idea for linker design. Meanwhile, the difficulty and cost for synthesizing and preparing the joint are reduced due to the simplified structure.

Drawings

FIG. 1 illustrates a mass spectrum of the primer described in example 1.

FIG. 2 shows a gel electrophoresis pattern of the primer described in example 1 with an enzyme in the absence of ATP.

FIG. 3 shows a gel electrophoresis of the primer described in example 1 with an enzyme in the presence of ATP.

FIG. 4 shows a gel electrophoresis pattern of the primer described in example 2 with an enzyme in the absence of ATP.

FIG. 5 shows a gel electrophoresis of the primer described in example 2 with an enzyme in the presence of ATP.

FIG. 6 shows gel electrophoresis patterns of structures 10N, 10N/C3, 10T and 10T-4C18 and enzyme in the presence of ATP formed by primer annealing described in example 3, wherein A shows the structure formed by primer annealing and B shows the gel electrophoresis pattern.

FIG. 7 shows gel electrophoresis patterns of structures 10N, 10N/C3, 10T and 10T/C3 and enzyme in the presence of ATP formed by primer annealing described in example 3, wherein A shows the structure formed by primer annealing and B shows the gel electrophoresis pattern.

FIG. 8 shows gel electrophoresis of the structure formed by annealing after the C3 modification in the primer described in example 3 was replaced with other modifications in the presence of ATP and the enzyme.

FIG. 9 shows the results of the ATP substrate experiments of example 3.

Fig. 10 shows gel electrophoresis patterns of other benzo structures and enzymes of example 4, wherein a illustrates electrophoresis patterns of indole analog sequences, B illustrates electrophoresis patterns of benzimidazole structures, and C illustrates electrophoresis patterns of methylisoquinolone structures.

FIG. 11 shows a structure in which a linker comprising a blocking band binds and stagnates an enzyme, wherein the enzyme stagnates on the blocking band (shown in dotted lines) of the linker, the left side of the blocking band being connected to a first section, and the right side of the blocking band being connected to a second section. The first segment encloses a first single-stranded portion (shown as X) and a first double-stranded portion (shown as double black lines), and the second segment includes a second single-stranded portion (shown as single black lines) and a second double-stranded portion (shown as double black lines).

FIG. 12 shows a signal diagram of sequencing of linker libraries.

Detailed Description

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

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

Definition of the definition

In order to more clearly explain the embodiments of the invention, certain scientific terms and terminology are used herein. Unless defined otherwise herein, all such terms and terminology should be interpreted as having the meaning commonly understood by one of ordinary skill in the art. For clarity, the following definitions are made for certain terms used herein.

Joint

The invention provides linkers for characterizing nucleic acids. The linker comprises a blocking band for simultaneously binding and blocking the polynucleotide binding protein, and the linker does not comprise an additional region for binding the polynucleotide binding protein. The linker also includes a first segment for directing the linker into a transmembrane pore and a second segment for ligating a target polynucleotide, the first segment and the second segment having the dead band interposed therebetween.

The term "blocking tape" refers to a region that functions as both a binding region and a spacer region. A "binding region" is a polynucleotide binding protein that binds to a polynucleotide when the polynucleotide is contacted with the polynucleotide binding protein. A "spacer" is a region that normally causes the polynucleotide-binding protein to arrest, i.e., prevents further movement of the polynucleotide-binding protein along the polynucleotide through the spacer. Typically, the polynucleotide binding protein binds to the binding region first and then moves along the polynucleotide until it reaches the spacer region, and stagnates in the spacer region until the polynucleotide to which the polynucleotide binding protein is bound is brought into contact with the transmembrane pore and an electrical potential is applied, and the polynucleotide binding protein does not move further through the spacer region. In the present invention, however, the polynucleotide binding protein binds to the blocking tape and is directly arrested on the blocking tape before contact with the transmembrane pore and before application of the potential, without the need for additional provision of the region to which the polynucleotide binding protein binds, i.e., without the need for the provision of an additional "binding region". The "blocking tape" of the present invention has the function of a region having both a "binding region" and a "spacer region".

The blocking band comprises a single stranded nucleotide sequence formed by the joining of nucleotides, preferably nucleotides whose nucleobases are benzo structures. The blocking band is a single stranded nucleotide sequence formed by a plurality of, preferably 7, 8, 9, 10, 11 or 12 nucleotides linked, preferably can be used to bind and arrest 1 polynucleotide binding protein.

Nucleotides generally contain a nucleobase, a sugar and at least one phosphate group. The nucleobase and sugar form a nucleoside. Nucleobases are typically heterocyclic. Nucleobases include, but are not limited to: purine and pyrimidine, more specifically adenine (a), guanine (G), thymine (T), uracil (U) and cytosine (C). Depending on the base, the nucleotides include adenine (adenylate, AMP), guanine (guanylate, GMP), cytosine (cytidylate, CMP), uracil (uridylate, UMP), thymine (thymidylate, TMP), inosine (inosinic acid, IMP) and the like. Nucleotides are mainly involved in constituting nucleic acids.

The nucleotides forming the blocking band used in the present invention are preferably nucleotides whose nucleobases are of benzo structure. More preferably, the nucleotides forming the blocking band are selected from one or more of the following formulas:

Wherein n=0 or 1

When n=0, X is N; y is N, C, CCONH or CCONHMe; r is R ₁ Methyl or phenyl; r is R ₂ ，R ₃ And R is ₄ One or two of which are nitro, fluoro or methyl; r is R ₅ Is an arbitrary group, preferably H or methyl;

when n=1, X and Y are both C; r is R ₃ And R is ₅ One of them is methyl; r is R ₆ Is carbonyl; r is R ₁ 、R ₂ And R is ₄ Is an arbitrary group, preferably H or methyl.

In a preferred embodiment, the nucleobases of the nucleotides forming the blocking band are selected from one or more of the following: nitroindole, 4, 6-nitroindole, 2-methyl-4-nitroindole, 2-phenyl-4-nitroindole, 5-nitroindole-3-carboxamide derivatives, 3-carboxamide indole, nitropyrrole, pyrrole-3-carboxamide, imidazole-4-carboxamide, 1,2, 4-triazole-3-carboxamide, 4-methylbenzimidazole, 4-fluoro-6-methyl-benzimidazole, 2, 4-difluorotoluene, 4, 6-difluoro-1H-benzimidazole, 3-methylisoquinolone and 5-methylisoquinolone. These structures can function to bind and arrest the polynucleotide binding protein simultaneously, probably because the benzo structure is similar to the base structure of a normal nucleotide, and can interact with related residues in the enzyme binding pocket to function to bind the polynucleotide binding protein; meanwhile, the benzo structure is not well combined with residues responsible for displacement on the enzyme, and the function of retarding the movement of the polynucleotide binding protein can be exerted.

The two ends of the blocking belt are respectively connected with one end of the first section and one end of the second section. The other end of the first section enters the transmembrane pore and guides the whole linker to enter the transmembrane pore during sequencing, and the other end of the second section is connected with the target polynucleotide, so that the target polynucleotide is sequenced.

The first segment is a partially double-stranded segment formed from a nucleotide and an abasic group, comprising a first double-stranded portion and a first single-stranded portion, wherein the first single-stranded portion is first transmembrane pore when sequenced, and one strand of the first double-stranded portion is attached to a blocking band. Preferably, both ends of the upper strand of the first double-stranded portion are connected to the first single-stranded portion and the blocking tape, respectively.

The second segment is a partially double-stranded region formed by nucleotide ligation, comprising a second double-stranded portion and a second single-stranded portion, wherein one strand of the second double-stranded portion is ligated to the blocking tape and the second single-stranded portion, where the other strand is not ligated to the blocking tape and is proximal to the blocking tape, is a free end for binding to a tether for bringing the linker proximal to the transmembrane pore during sequencing. Preferably, the upper strand of the second double stranded portion is attached to the blocking tape and the lower strand of the second double stranded portion is attached to the second single stranded portion at an end proximal to the blocking tape. Optionally, the second segment further comprises a third single stranded portion located on the upper strand attached to the blocking tape and distal from the blocking tape, the third single stranded portion not complementarily paired with the lower strand.

In some embodiments, the linker is a Y linker.

The linker of the invention is preferably bound to a tether (tether) which is a single stranded polynucleotide consisting of a plurality of nucleotides, and which further comprises abasic groups which are not capable of forming a complementary double strand. One end of the tether is complementarily conjugated to the linker and the other end is anchored to the membrane or the pore, thereby surrounding the pore around the target polynucleotide to which the linker is attached.

The adaptors of the present invention may be used alone or in combination with other adaptors (such as hairpin adaptors, hairpin-like adaptors, or conventional Y adaptors, etc.) to form constructs for sequencing. In some embodiments, the adaptors of the present invention ligate both ends of the target polynucleotide to form a construct for characterizing the target polynucleotide. In some embodiments, the linker of the invention is attached to the 5 'end of the target polynucleotide, and the 3' end of the target polynucleotide is attached to a conventional Y linker other than the linker of the invention, forming a construct. In some embodiments, if it is desired to characterize both strands of a double-stranded target polynucleotide, the adaptor of the invention can be ligated to the 5 'end of the double-stranded target polynucleotide, and the 3' end of the double-stranded target polynucleotide ligated to a hairpin adaptor or hairpin-like adaptor to form a construct. Hairpin-like linkers are loops that have a similarity to conventional hairpin structures, but are not formed by a single-stranded linear molecule folded back on itself as in conventional hairpin structures, and are capable of linking the two strands of the target polynucleotide. See CN113462764a for a specific example of hairpin-like linkers.

The non-base group used in the linker or tether is an iss 18 group or an issc 3 group or the like that cannot form a base pair. It may also be referred to as an "abasic site" or "abasic nucleotide". An abasic group is a nucleotide or nucleoside lacking a nucleobase at the 1' position of the sugar moiety. Preferably, 1 blocking band binds to and arrests 1 polynucleotide binding protein.

Target polynucleotide

The method of the invention is used for sequencing single-stranded, partially double-stranded and double-stranded polynucleic acids.

The polynucleotide may be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target polynucleotide may comprise an RNA strand hybridized to a DNA strand. The polynucleotide may be any synthetic nucleic acid known in the art, such as Peptide Nucleic Acid (PNA), glycerol Nucleic Acid (GNA), threose Nucleic Acid (TNA), locked Nucleic Acid (LNA), or other synthetic polymer having a nucleotide side chain.

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

The target polynucleotide may be of any length. For example, a polynucleotide may be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotide pairs in length. The polynucleotide may be 1000 or more nucleotide pairs, 5000 or more nucleotide pairs in length or 100000 or more nucleotide pairs in length.

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

Polynucleotide binding proteins

The polynucleotide binding protein may be any protein capable of binding to a polynucleotide and controlling its movement through a pore. It is straightforward in the art to determine whether a protein binds to a polynucleotide. Proteins typically interact with polynucleotides and modify at least one property thereof. The protein may modify the polynucleotide by cleaving the polynucleotide to form individual nucleotides or shorter nucleotide chains such as di-or tri-nucleotides. The moiety may modify the polynucleotide by positioning or moving the polynucleotide to a specific position (i.e., controlling its movement).

In some embodiments, the polynucleotide binding protein is a polynucleotide melting enzyme, such as an ED1 enzyme. Polynucleotide melting enzymes are enzymes capable of melting a double-stranded target polynucleotide into single strands. In some embodiments, the polynucleotide melting enzyme is capable of melting double-stranded DNA into single strands. In some embodiments, the polynucleotide melt enzyme is an enzyme having helicase activity. Examples of polynucleotide melting enzymes include, for example, the helicases described herein.

The polynucleotide binding capacity may be measured using any method known in the art. For example, a protein may be contacted with a polynucleotide, and the ability of the protein to bind to and move along the polynucleotide may be measured. Proteins may include modifications that facilitate polynucleotide binding and/or facilitate their activity at high salt concentrations and/or room temperature. The protein may be modified so that it binds to the polynucleotide (i.e., retains the ability of the polynucleotide to bind) but does not act as a melting enzyme (i.e., when provided with all essential components that facilitate movement (e.g., ATP and Mg ²⁺ ) Not along the polynucleotide). Such modifications are known in the art. For example, mg in helicase ²⁺ Modification of the binding domain typically results in variants that do not function as helicases.

The enzyme may be covalently attached to the well. The enzyme may be covalently attached to the well using any method.

In strand sequencing, polynucleotides translocate through a pore, either along or against an applied potential. Exonucleases acting gradually or stepwise on double-stranded target polynucleotides may be used on the forward side of the pore to supply the remaining single strands at an applied potential, or on the reverse side to supply the remaining single strands at a reverse potential. Likewise, helicases that melt double stranded DNA may also be used in a similar manner. Polymerase may also be used. Sequencing applications that require strand translocation against an externally applied potential are also possible, but the DNA must first be "captured" by the enzyme at the opposite or no potential. As the potential subsequently switches after binding, the chain will pass through the pore in cis to trans fashion and remain in an extended configuration by the current. Single-stranded DNA exonucleases or single-stranded DNA-dependent polymerases can act as molecular motors to pull recently translocated single strands back from the pore in a controlled, stepwise manner against an applied potential from trans to cis.

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

The method may also be performed in the opposite direction. The 3' end of the polynucleotide may be captured first in the pore and the melting enzyme may move the polynucleotide into the pore so that it passes through the pore under the influence of the field until it finally translocates through to the opposite side of the membrane.

When the melting enzyme is not provided with the necessary components to facilitate movement or is modified to prevent or inhibit movement thereof, the melting enzyme may bind to the polynucleotide and act as a brake to slow movement of the polynucleotide as it is pulled into the well by an applied field. In the inactive mode, it is not important whether the 3 'or 5' of the polynucleotide is captured, and it is the applied field that pulls the polynucleotide into the well towards the opposite side under the action of the enzyme acting as a brake. When in inactive mode, control of movement of the polynucleotide by the melting enzyme can be described in a variety of ways, including ratcheting, sliding, and braking. In this way, variants of the melting enzyme lacking melting enzyme activity can also be used.

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

Any step in a method of using a polynucleotide binding protein (e.g., a polynucleotide melt enzyme) is typically performed in the presence of free nucleotides or free nucleotide analogs and an enzyme cofactor that facilitates the action of the polynucleotide binding protein (e.g., a polynucleotide melt enzyme). The free nucleotides may be one or more of any individual nucleotides. Free nucleotides include, but are not limited to: adenosine Monophosphate (AMP), adenosine Diphosphate (ADP), adenosine Triphosphate (ATP), guanosine Monophosphate (GMP), guanosine Diphosphate (GDP), guanosine Triphosphate (GTP), thymidine Monophosphate (TMP), thymidine Diphosphate (TDP), thymidine Triphosphate (TTP), uridine Monophosphate (UMP), uridine Diphosphate (UDP), uridine Triphosphate (UTP), cytidine Monophosphate (CMP), cytidine Diphosphate (CDP), cytidine Triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dabp), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), triphosphateDeoxyguanosine (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (ddudp), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP). The free nucleotide is preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotide is preferably Adenosine Triphosphate (ATP). Enzyme cofactors are factors that allow the construct to function. The enzyme cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg ²⁺ 、Mn ²⁺ 、Ca ²⁺ Or Co ²⁺ . Most preferably the enzyme cofactor is Mg ²⁺ 。

Film and method for producing the same

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

Archaebacteria bipolar tetraether lipids are naturally occurring lipids that are configured such that the lipids form a monolayer film. These lipids are typically found in the most polar organisms, thermophiles, halophiles and acidophiles that survive in harsh biological environments. Their stability is believed to result from the fusion properties of the final bilayer. Block copolymer materials that mimic these biological entities are readily constructed by creating a triblock polymer with the general motif hydrophilic-hydrophobic-hydrophilic. The material may form a monomeric membrane that behaves like a lipid bilayer and has a range of phase states from vesicles to the membrane. Membranes formed from these triblock copolymers have several advantages over biolipid membranes. Because the triblock copolymers are synthetic, the precise construction can be carefully controlled to provide the correct chain length and properties required to form films and interact with pores and other proteins.

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

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

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

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

The amphiphilic layer is typically a lipid bilayer. Lipid bilayers are a model of cell membranes and serve as an excellent platform for a series of experimental studies. For example, lipid bilayers can be used for in vitro studies of membrane proteins using single channel recordings. Alternatively, the lipid bilayer may be used as a biosensor to detect the presence of a range of substances. The lipid bilayer may be any lipid bilayer. Suitable lipid bilayers include, but are not limited to planar lipid bilayers, supported bilayers, or liposomes. The lipid bilayer is preferably a planar lipid bilayer.

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

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

In another preferred embodiment, the film is a solid layer. The solid layer is not of biological origin. In other words, the solid state layer is not derived or isolated from a biological environment, such as an organism or cell, or a biologically useful structure in a synthetically manufactured form. The solid layer may be formed of an organic or inorganic material 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 type silicone rubber (two-component addition-cure silicone rubber) and glass. The solid layer may be formed of graphene.

Transmembrane pore

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

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

The transmembrane protein pore may be monomeric or oligomeric. The pore is preferably composed of several repeating subunits (e.g., 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 generally surround the central axis and provide a chain for the transmembrane β -barrel or channel or transmembrane α -helical bundle or channel.

The barrels or channels of transmembrane protein pores typically contain amino acids that facilitate interactions with analytes (e.g., nucleotides, polynucleotides, or nucleic acids). 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 (e.g., arginine, lysine, or histidine) or aromatic amino acids (e.g., tyrosine or tryptophan). These amino acids generally promote interactions between the pore and a nucleotide or polynucleotide or nucleic acid.

Transmembrane protein pores useful in the present invention may be derived from β -barrel pores comprising barrels or channels formed from β -strands or α -helical bundle pores. Suitable beta-barrel wells include, but are not limited to, beta-toxins, such as alpha-hemolysin, anthrax toxin, and leukocidal proteins, and bacterial outer membrane proteins/porins (porins), such as mycobacterium smegmatis (Mycobacterium smegmatis) porins (Msp) (e.g., mspA), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase a, and Neisseria (neissenia) self-transport lipoproteins (NalP). The alpha-helical bundle holes comprise barrels or channels formed by alpha-helices. Suitable alpha-helical bundle pores include, but are not limited to, inner membrane proteins and alpha-outer membrane proteins, such as WZA and ClyA toxins. The transmembrane pore may be derived from Msp or α -hemolysin (α -HL).

The transmembrane protein pore is preferably derived from Msp, preferably from MspA. Such pores are oligomers and typically comprise 7, 8, 9 or 10 monomers derived from Msp. The pore may be a homo-oligomer (homo-oligo) pore derived from Msp comprising the same monomer. Alternatively, the pore may be a hetero-oligomeric (hetero) pore derived from Msp containing at least one monomer different from 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 (alpha-HL). The wild-type a-HL pore is formed from 7 identical monomers or subunits (i.e., it is a heptamer).

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

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

Movement of

In the methods of the invention, the target polynucleotide is moved through the transmembrane pore and sequenced. Moving a target polynucleotide through the transmembrane pore refers to moving the polynucleotide from one side of the pore to the other. The movement of the target polynucleotide through the pore may be driven or controlled by an electric potential or an enzymatic action or both. The movement may be unidirectional or may allow for backward and forward movement.

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

Polynucleotide characterization

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

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

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

The present invention provides a method of characterizing a linker, the method comprising:

(a) Providing a linker inserted with a blocking band for simultaneously binding and arresting the polynucleotide binding protein;

(b) Contacting a linker with a polynucleotide binding protein such that the polynucleotide binding protein binds to the blocking band and arrests the polynucleotide binding protein at the blocking band.

(c) Contacting the adaptor and the arrested polynucleotide binding protein with the pore; and

(d) Applying an electrical application across the pore such that the polynucleotide binding protein moves through the blocking band and controls movement of the entire linker through the pore;

(e) One or more measurements are taken as the linker moves relative to the transmembrane pore.

The invention provides methods of characterizing a target polynucleotide.

These methods are possible because transmembrane protein pores can be used to distinguish between nucleotides having similar structures, based on their different effects on the current through the pore. Individual nucleotides can be identified at the single molecule level based on their current amplitudes as they interact with the pore. If a current flows through the pore in a manner specific for a nucleotide (i.e., if a characteristic current associated with the nucleotide is detected flowing through the pore), then the nucleotide is present in the pore. The nucleotides in the target polynucleotide are continuously identified so that the sequence of the polynucleotide can be estimated or determined.

The method comprises the following steps: (a) Providing a construct having a target polynucleotide linked at one or both ends to a linker, the linker being inserted with a blocking band for simultaneously binding and arresting the polynucleotide binding protein;

(b) Contacting the construct with the polynucleotide binding protein such that the polynucleotide binding protein binds to the blocking band and arrests the polynucleotide binding protein at the blocking band;

(c) Contacting the construct and the arrested polynucleotide binding protein with the pore; and

(d) Applying an electrical application across the pore such that the polynucleotide binding protein moves through the blocking band and controls movement of the target polynucleotide through the pore;

(e) One or more measurements are obtained as the target polynucleotide moves relative to the transmembrane pore, wherein the measurements represent one or more characteristics of the target polynucleotide and thereby characterize the target polynucleotide.

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

In some embodiments, the method comprises providing a tether for bringing the construct in proximity to the transmembrane pore; the tether includes a capture region for capturing a linker of the construct and an anchor region for anchoring association with the transmembrane pore or a membrane in which the transmembrane pore is located.

All or only a portion of the target polynucleotide may be sequenced using this method. The polynucleotide may be of any length. For example, the polynucleotide may be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotide pairs in length. The polynucleotide may be 1000 or more nucleotide pairs, 5000 or more nucleotide pairs, or 100000 or more nucleotide pairs in length. The polynucleotide may be naturally occurring or man-made. For example, the method can be used to verify the sequence of the oligonucleotide produced. The method is typically performed in vitro.

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

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

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

Kit for detecting a substance in a sample

The invention also provides kits for preparing a kit for characterizing a target polynucleotide. The kit comprises (a) a linker of the invention, (b) a polynucleotide binding protein, (c) a transmembrane pore, and optionally a tether.

The kit preferably further comprises one or more markers that when interacted with the transmembrane pore generate a characteristic current. Such markers are described in detail above. The kit preferably further comprises means (means) for coupling the target polynucleotide to a membrane. Means for coupling the target polynucleotide to a membrane are described above. The means of coupling preferably comprises 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 phospholipids required to form lipid bilayers.

Any of the embodiments detailed above in relation to the methods of the invention are equally applicable to the kits of the invention.

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

Method for preparing target polynucleotides for characterization

The invention also provides methods of preparing target polynucleotides for characterization. The method generates a construct that allows the target polynucleotide to be characterized. In this method, one end of the target polynucleotide is linked to the linker of the invention and the other end is linked to any linker.

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

The linker moiety of the invention may be synthesized separately and then chemically bound or enzymatically linked to the target polynucleotide. Methods for doing this are known in the art. Alternatively, the linker of the invention may be generated during processing of the target polynucleotide. Also, suitable methods are known in the art.

Examples

Details of experimental procedures not specifically noted in the examples below can be found in the references cited herein, using conventional commercially available reagents or apparatus, and using nucleotide sequences synthesized by the biological engineering Co., ltd and Beijing engine biosciences Co., ltd. Nitro in the nucleotide sequence used in the examples represents a nucleotide whose nucleobase is 5-nitroindole.

Example 1: the modified single-stranded nucleotide sequence can effectively prevent enzyme movement under the action of no electric field force

1. The primer sequences were designed as follows:

(1) BS 7-2Nitro (shown as SEQ ID NO. 1)

5 ^， -30/iSpC3/-7T-2Nitro-CCGTTCTCATTGGTGC- 3 ^，

(2) BS 7-4Nitro (shown as SEQ ID NO. 2)

5 ^， -30/iSpC3/-7T-4Nitro-CCGTTCTCATTGGTGC- 3 ^，

(3) BS 7-5Nitro (shown as SEQ ID NO. 3)

5 ^， -30/iSpC3/-7T-5Nitro-CCGTTCTCATTGGTGC- 3 ^，

(4) BS 7-8Nitro (as shown in SEQ ID NO. 4)

5 ^， -30/iSpC3/-7T-8Nitro-CCGTTCTCATTGGTGC- 3 ^，

(5) BD 7-4Nitro-4C18 (shown in SEQ ID NO. 5)

5 ^， -30/iSpC3/-7T-4Nitro-7T-4/iSpC18/-CCGTTCTCATTGGTGC- 3 ^，

(6) Anneal C (shown as SEQ ID NO. 6)

5 ^， -FAM-GCACCAATGAGAACGG-3 ^，

After purifying the sequences (1) to (5) by PAGE, mass spectrometry detection is carried out, and the mass spectrometry detection results of the primers (1) to (4) are shown in FIG. 1. The results showed that only the sequence (5. BD.times.7-4 Nitro-4C18 primer failed to synthesize.

2. Primer binding experiments

The primers (1) to (4) were annealed to the primer (6) in a ratio of 1:0.8. 60nM annealing primer (6), ED1 enzyme (30X, 50X, 70X, 90X, 100X), 2X incubation buffer (50 mM KCl, 10mM HEPES pH 7.0, 5mM Mg) were added to 20. Mu.l of the incubation systems of primers (1) - (4) ²⁺ ) 1000 XTMAD was added after incubation at 30℃for 1 h. The incubated products were subjected to gel electrophoresis, and the results are shown in FIG. 2. The results showed that each primer was found to be 1: 30-1: 100 Under the condition of ED1 enzyme and TMAD, 1 ED1 enzyme can be combined.

3. ATP unwinding experiment

Each of the primers (1) to (4) and the primer (6) was annealed at a ratio of 1:0.8. Mu.l of the incubation system of primers (1) - (4) was added with 60nM of annealed primer (6), ED1 enzyme (30X), 2X incubation buffer (50 mM KCl, 10mM HEPES pH 7.0, 5mM Mg) ²⁺ ) After incubation at 30℃for 1h, 1000 XTMAD was added, incubation was continued for 30min, 3mM ATP was added to the incubation system, and the incubation was performed at 30℃for 10min. The treated product was subjected to gel electrophoresis, and the results are shown in FIG. 3.

From the above results, it can be seen that nitroindole can simply act as a spacer to block the enzyme movement under conditions of only electric field, but the binding band disappears when ATP is added, but there is no obvious single strand, which may be a single strand after the primer is unwound and annealed back, or a simple enzyme is released. In summary, in the presence of ATP, none of the 2, 4, 5 or 8 nitroindoles act as spacers to block the enzymatic movement.

Example 2: single strands containing nitroindole nucleobases bind and block enzyme movement

1. The new primer sequences were designed as follows:

(1) Seq-4-10N (as shown in SEQ ID NO. 7):

5’-Nitro- Nitro- Nitro- Nitro- Nitro- Nitro- Nitro- Nitro- Nitro- Nitro- TGGACTTCGCGTAGGTCAGTTCAGG-3’

(2) abasic Cy5 (shown as SEQ ID NO. 8):

5’-Cy5 CCTGAACTGACCTACGCGAAGTCCA-3’

(3) 5-3L1-7 (as shown in SEQ ID NO. 9):

5’-TTTTTTT TCGCTGCTCCACAGGTCTCAGCTTGAGCAGCGA-3’

(4) 5-3S1-FAM (as shown in SEQ ID NO. 10):

5’-FAM-TCGCTGCTCAAGCTGAGACCTGTGGAGCAGCGA-3’

2. primer binding experiments

Primer (1) and primer (2) were annealed in a ratio of 1:0.8. Into 20. Mu.l of the incubation system of primer (1), 60nM of annealed primer (2), ED1 enzyme (10X, 20X, 30X, 40X, 50X, 60X, 70X), 2X incubation buffer (50 mM KCl, 10mM HEPES Ph 7.0, 5mM Mg) ²⁺ ) After incubation at 30℃for 1h 1000 XTMAD was added and incubation continued for 30min. The result of gel electrophoresis of the incubated product, and the result of electrophoresis after annealing of primer (1) and primer (2) is shown in FIG. 4.

The results show that when 10 nitroindoles are used as single-chain binding domains for helicases, 1 ED1 enzyme can be bound, and we have experiments that show that 10 nitroindoles have no significant difference in their ability to bind to helicases from 10T.

3. ATP unwinding experiment

ATP experiments were performed using primers (3)5-3L 1-7) as controls. 3mM ATP was added to the incubation system and treated at 30℃for 10min. The treated product was subjected to gel electrophoresis, and the results are shown in FIG. 5. The results show that 10 nitroindoles act as spacers to block the enzyme's movement. The reason for this is that the helicase depends on the presence of single stranded DNA and the hydrolysis is induced by magnesium ions and ATP to generate energy to unwind the double stranded DNA, so that if the presence of nitroindole does not block the movement of the helicase, no band of helicase binding to single stranded DNA will occur at the time of electrophoretic detection.

Example 3: optimizing the blocking enzyme effect of single strands comprising nitroindole nucleobases

1. The new primer sequences were designed as follows:

(1) Seq-4-10N (as shown in SEQ ID NO. 7):

5’-Nitro- Nitro- Nitro- Nitro- Nitro- Nitro- Nitro- Nitro- Nitro- Nitro- TGGACTTCGCGTAGGTCAGTTCAGG-3’

(2) SEQ4-FAM (as shown in SEQ ID NO. 11):

5’- FAM CCTGAACTGACCTACGCGAAGTCCA-3’

(3) SEQ4-FAM-2 (as shown in SEQ ID NO. 12):

5’- FAM CCTGAACTGACCTACGCGAAGTCCA-4/iSpC3/-3’

(4) Seq-10T (as shown in SEQ ID NO. 13):

5’-10T - TGGACTTCGCGTAGGTCAGTTCAGG-3’

(5) Seq-10T-4C18 (as shown in SEQ ID NO. 14):

5’-10T –4/iSpC18/TGGACTTCGCGTAGGTCAGTTCAGG-3’

2. ATP unwinding experiment

The primers were annealed in a 1:0.8 ratio. 100nM annealing primer, 30 XED 1 enzyme and 2 Xincubation buffer (50mM KCl,20mM HEPES,pH 7.0,1mM EDTA) are added into 10 μl incubation system, 1000 XTMAD is added after incubation for 1h at 30 ℃, incubation is continued for 30min at 30 ℃, and finally 10mM ATP is added for 20min, and the products after ATP treatment are subjected to gel electrophoresis. The double-stranded structure formed by primer annealing is shown in FIG. 6A, and the electrophoresis result is shown in FIG. 6B. Wherein 10N represents the structure formed by annealing of the primers (1) and (2), 10N/C3 (i.e., 10N-FAM2 in FIG. 6B) represents the structure formed by annealing of the primers (1) and (3), 10T represents the structure formed by annealing of the primers (4) and (2), and 10T-4C18 represents the structure formed by annealing of the primers (5) and (2).

The results show that the band of the helicase binding single-stranded DNA of the incubated sample is still clear under the ATP treatment, so that the 10N/C3 primer structure is more advantageous in blocking effect.

3. It was verified whether the blocking of nitroindole is associated with C3 on the complementary strand.

Verification was performed under the same experimental conditions, the double-stranded structure formed by primer annealing is shown in FIG. 7A, and the electrophoresis result is shown in FIG. 7B. Wherein 10N represents a structure formed by annealing the primers (1) and (2), 10N/C3 represents a structure formed by annealing the primers (1) and (3), 10T represents a structure formed by annealing the primers (4) and (2), and 10T/C3 represents a structure formed by annealing the primers (4) and (3).

The results showed that when the main chain contained no modified nucleotide sequence and only the complementary strand contained modified nucleotides, the binding band completely disappeared after ATP addition, indicating that the C3 modification on the complementary strand did not have a blocking effect. However, when the backbone contains a modified nucleotide sequence, the binding band does not disappear upon addition of ATP. This may illustrate the role of blocking enzyme movement when the backbone contains nitroindole modifications.

Under the same experimental conditions, the complementary strand C3 modification was replaced with another modification, and verification was performed. The result of electrophoresis is shown in FIG. 8. The results show that there is no significant difference in blocking effect when the C3 modification of the complementary strand is replaced with another modification, such as C18, dspacer.

This result is similar to that of the ATP substrate assay. ATP substrate experiments were performed by the following manner: to 100nM dsDNA was added 4. Mu.M ED1 enzyme in binding buffer (10mM HEPES,50mM KCL,5mM MgCl) ₂ ) After 60min of reaction at 30 ℃, an unbinding buffer (10mM HEPES,600mM KCL,5mM MgCl2) containing Picogreen Reagent was added, after 10min of reaction, 480/520nm readings were performed, and after addition of ATP, readings were performed every 30 s. The experimental results are shown in FIG. 9.

In the ATP substrate experiments, the blocking effect of the complementary strand with or without C3 on ED1 enzyme is observed to be not great, and the efficiency is slightly lower than that of 10T-4C 18.

Example 4: blocking effects of other analog sequences

In this example, the sequences of some other similar structures (i.e., other benzo structures) were also tested for blocking effect.

(1) Seq-10N-N (as shown in SEQ ID NO. 15):

5’-XXXXXXXXXX TGGACTTCGCGTAGGTCAGTTCAGG-3’

n=1: x is 4, 6-nitroindole;

n=2: x is 2-methyl-4-nitroindole;

n=3: x is 5-nitroindole-3-carboxamide;

n=4: x is 3-carboxamide indole;

n=5: x is 4-methylbenzimidazole;

n=6: x is methyl isoquinolone;

(2) seq4 (as shown in SEQ ID NO. 16):

5’- CCTGAACTGACCTACGCGAAGTCCA-3’

primers of each analogue were annealed (sequenceAnd (2)), 100nM annealing primer, 25 XED 1 enzyme and 2 Xincubation buffer (50mM KCl,20mM HEPES,pH 7.0,1mM EDTA) are added into 10 μl incubation system, 1000 XTMAD is added after incubation for 1h at 30 ℃, incubation is continued for 30min at 30 ℃, and finally 10mM ATP is added for 20min, and the ATP treated product is subjected to gel electrophoresis. The result of electrophoresis is shown in FIG. 10. After enzyme binding to the corresponding structurally modified ds DNA, the binding band did not show significant change in brightness after treatment with 10mM ATP, indicating that the structure blocked the movement of the enzyme on the polynucleotide strand. FIG. 10A is an experimental result of indole analog sequences, showing that all four indole analogs have binding/blocking enzyme effects in the presence of ATP. FIGS. 10B and 10C are, respectively, benzimidazole structures and methylisoquinolones The sequence of the ketone structure also shows a binding/blocking enzyme effect in the presence of ATP.

Example 5: design nitroindole joint, build up library test and combine/block effect

1. The sequence of the linker structure is as follows:

(1) Y1-Nitro (as shown in SEQ ID NO. 17): 5 ^, -XXXXX XXXXX XXXXX XXXXX (iSp18)4 ATCCT TTTTA GAATT TT AGAGATTC AGAGATTC AGAGATTC AGAGATTC Nitro/ Nitro/ Nitro/ Nitro/ Nitro/ Nitro/ Nitro/ Nitro/ Nitro/ Nitro/AGAGATTC AGAGATTC AGAGATTC AGAGATTC TGACA TGCA GTTA GCGA GACT CTTG AGCA T -3’

(2) Y2-Nitro (as shown in SEQ ID NO. 18): 5 ^, -GAATCTCT GAATCTCT GAATCTCT GAATCTCT AA AATTC TAAAA AGGAT -3

(3) Y-Bottom-Nitro (shown in SEQ ID NO. 19): 5 ^, P-TGCT CAAG AGTC TCGC TAAC TGCA TGTCA GAATCTCT GAATCTCT GAATCTCT GAATCTCT AGTCC AGCAC CGACC -3

(4) The other-Nitro (shown as SEQ ID NO. 20): 5 ^, -Chol-(iSpC3)20-GGTCG GTGCT GGACT-3’

Using the above sequences, a linker as shown in fig. 11 was synthesized. The synthesis method is as follows: annealing the primers (1), (2) and (3) according to the proportion of 1:2.5:2.5, wherein the final concentration of primer annealing is 4 mu M, and the annealing procedure is carried out at 98 ℃ for 10min;6s/-0.1 ℃,300x Cys;65 ℃ for 5min;6s/-0.1 ℃,400x Cys;12 ℃, hold. The annealed primers were incubated according to the system in table 1 below:

table 1: incubation system for annealed primers

dsDNA（4μM）	12.5μl
		ED1-212(28.5μM)	26.3μl
NaAC(3M pH7.0)	5.83μl
		TMAD(1M)	1.5μl
NF-water	53.87μl
		Total	100μl

The sample was added to a 1.5ml low adsorption centrifuge tube (tinfoil wrapped in dark), gently mixed (vortex shaker not available), and placed in a 30 ℃ metal bath for 30min. Finally, the incubated product is subjected to magnetic bead purification to obtain a compound combined with helicase.

2. Sequencing Using synthetic adaptors

Using a QNome9604 sequencing platform, 4. Mu.L of 1. Mu.M primer 4 was added to 200. Mu.L sequencing buffer (600 mM KCl, 10mM HEPES pH8.0, 3mM Mg) ²⁺ 3mM ATP), mixing by vortex, instantaneous centrifuging, adding the mixed solution into a sequencing chip, and standing for 15min; then 40fmol of the prepared joint is added into 200 mu L of sequencing buffer, the mixture is gently mixed upside down, the mixture is subjected to instantaneous centrifugation, and the mixture is added into a sequencing chip for standing for 15min, and then sequencing is started. At 600mM KCl, 10mM HEPES pH8.0, 3mM Mg ²⁺ The signal captured during the signal acquisition is shown in FIG. 12, which is a test performed at 3mM ATP and 35 ℃. From this figure, the step signal of each part of the nitroindole linker primer can be seen. The spacing between the nanopore and helicase is 20nt, and the length of the leading chain is more than or equal to 20nt. The (iSp 18) 4 step captured by the signal capture marks that the linker primer enters the nanopore to start sequencing under the guidance of the leader because there is no helicase at the leaderMost of the signals obtained from the test start from nitroindole where the helicase binds and blocks, the clear signals may be obtained because the interaction of the helicase with the nanopore allows the adaptor primer to be slowed down as it passes through the nanopore, thus allowing the front signal to be acquired. And when the rest of the joint primer passes through the nanopore under the control of helicase, the second half of the signal step is obtained.

As described above, the linker with the inserted blocking band can be sequenced in the nanopore under the control of the helicase, while the double stranded portion of the second segment of the linker is a conventional nucleotide sequence (i.e., a nucleotide sequence consisting of A, G, T, C), which can be sequenced. Thus, when a target polynucleotide to be detected (e.g., a double-stranded target polynucleotide of about 200 bp), also comprised of A, G, T, C, is ligated to the right of the second segment of the adaptor (i.e., to the right of the complex shown in FIG. 11), the helicase will continue to move and control the sequencing of the target polynucleotide.

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

Sequence listing

<110> Chengdu carbon technology Co., ltd

<120> linkers, constructs, methods and uses for characterizing target polynucleotide sequencing

<130> 21NI1030

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> nucleotide was preceded by 30×iSpC3

<220>

<221> misc_feature

<222> (7)..(8)

<223> ligation between nucleotides 2 XNitro

<400> 1

tttttttccg ttctcattgg tgc 23

<210> 2

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)

<223> nucleotide was preceded by 30×iSpC3

<220>

<221> misc_feature

<222> (7)..(8)

<223> ligation between nucleotides 4 XNitro

<400> 2

tttttttccg ttctcattgg tgc 23

<210> 3

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> nucleotide was preceded by 30×iSpC3

<220>

<221> misc_feature

<222> (7)..(8)

<223> ligation between nucleotides 5 XNitro

<400> 3

tttttttccg ttctcattgg tgc 23

<210> 4

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> nucleotide was preceded by 30×iSpC3

<220>

<221> misc_feature

<222> (7)..(8)

<223> ligation between nucleotides 8 XNitro

<400> 4

tttttttccg ttctcattgg tgc 23

<210> 5

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> nucleotide was preceded by 30×iSpC3

<220>

<221> misc_feature

<222> (7)..(8)

<223> ligation between nucleotides 4 XNitro

<220>

<221> misc_feature

<222> (14)..(15)

<223> ligation between nucleotides 4×iSpC18

<400> 5

tttttttttt ttttccgttc tcattggtgc 30

<210> 6

<211> 16

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

gcaccaatga gaacgg 16

<210> 7

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> nucleotide was preceded by 10 XNitro

<400> 7

tggacttcgc gtaggtcagt tcagg 25

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

cctgaactga cctacgcgaa gtcca 25

<210> 9

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

ttttttttcg ctgctccaca ggtctcagct tgagcagcga 40

<210> 10

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

tcgctgctca agctgagacc tgtggagcag cga 33

<210> 11

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

cctgaactga cctacgcgaa gtcca 25

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (25)..(25)

<223> nucleotide followed by 4×iSpC3

<400> 12

cctgaactga cctacgcgaa gtcca 25

<210> 13

<211> 35

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

tttttttttt tggacttcgc gtaggtcagt tcagg 35

<210> 14

<211> 35

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (10)..(11)

<223> ligation between nucleotides 4×iSpC18

<400> 14

tttttttttt tggacttcgc gtaggtcagt tcagg 35

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

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<400> 15

tggacttcgc gtaggtcagt tcagg 25

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

cctgaactga cctacgcgaa gtcca 25

<210> 17

<211> 111

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

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<220>

<221> misc_feature

<222> (49)..(50)

<223> ligation between nucleotides 10 XNitro

<400> 17

atccttttta gaattttaga gattcagaga ttcagagatt cagagattca gagattcaga 60

gattcagaga ttcagagatt ctgacatgca gttagcgaga ctcttgagca t 111

<210> 18

<211> 49

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

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<210> 19

<211> 76

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

tgctcaagag tctcgctaac tgcatgtcag aatctctgaa tctctgaatc tctgaatctc 60

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Claims (34)

1. A linker for characterizing target polynucleotide sequencing, the linker comprising a blocking band, a first segment and a second segment, the blocking band interposed between the first segment and the second segment, the blocking band for simultaneously binding and arresting a helicase;

wherein the blocking band comprises a single stranded nucleotide sequence formed by the ligation of a plurality of nucleotides, the nucleobases of which are selected from one or more of the following: 5-nitroindole, 4, 6-nitroindole, 2-methyl-4-nitroindole, 5-nitroindole-3-carboxamide, 3-carboxamide indole, 4-methylbenzimidazole and methylisoquinolone.

2. The linker of claim 1 wherein said first segment is for guiding said linker into a transmembrane pore and said second segment is for ligating a target polynucleotide.

3. A linker according to claim 1 or 2 wherein the blocking band is used to bind and arrest 1 of the helicases.

4. The linker of claim 1 or 2, wherein the blocking band is a single stranded nucleotide sequence formed by 7-12 nucleotide ligation.

5. The linker of claim 4 wherein the blocking band is a single stranded nucleotide sequence formed by 8-10 nucleotide ligation.

6. A linker according to claim 1 or 2, wherein the linker does not comprise an additional region for binding the helicase.

7. A construct for characterizing target polynucleotide sequencing, wherein the construct comprises a target polynucleotide and the linker of any one of claims 1-6, wherein the linker is attached to either or both ends of the target polynucleotide.

8. A complex for characterising sequencing of a target polynucleotide, wherein the complex comprises a helicase and a linker according to any one of claims 1 to 6 or a construct according to claim 7;

Wherein the helicase binds to and stagnates at the blocking band.

9. A kit for controlling movement of a target polynucleotide, the kit comprising:

(a) the linker of any one of claims 1 to 6, (b) a helicase, and/or (c) a transmembrane pore.

10. Use of a linker according to any one of claims 1 to 6, a construct according to claim 7, a complex according to claim 8, or a kit according to claim 9 in the preparation of a product for or in characterization of target polynucleotide sequencing.

11. A method of controlling the loading of a target polynucleotide with a helicase, the method comprising:

providing a construct having a target polynucleotide, wherein the target polynucleotide is linked at one or both ends thereof to a linker, the linker having a blocking tape inserted therein; and contacting the construct with the helicase such that the helicase binds to and stagnates at the blocking band; or (b)

Loading a helicase onto a linker inserted with a blocking band, wherein the helicase binds to and stagnates at the blocking band; and ligating the helicase loaded adaptor to the target polynucleotide;

Wherein the blocking band comprises a single stranded nucleotide sequence formed by the ligation of a plurality of nucleotides, the nucleobases of which are selected from one or more of the following: 5-nitroindole, 4, 6-nitroindole, 2-methyl-4-nitroindole, 5-nitroindole-3-carboxamide, 3-carboxamide indole, 4-methylbenzimidazole and methylisoquinolone.

12. The method of claim 11, wherein the linker further comprises a first segment for guiding the linker into a transmembrane pore and a second segment for ligating a target polynucleotide, the first segment and the second segment having the blocking band interposed therebetween.

13. A method according to claim 11 or 12, wherein the blocking band binds to and arrests 1 of the helicases.

14. The method of claim 11 or 12, wherein the blocking band is a single stranded nucleotide sequence formed by 7-12 nucleotide ligation.

15. The method of claim 14, wherein the blocking band is a single stranded nucleotide sequence formed by 8-10 nucleotide ligation.

16. A method according to claim 11 or 12, wherein the linker does not comprise an additional region for binding the helicase.

17. A method of controlling movement of a target polynucleotide through a transmembrane pore, the method comprising:

(a) Implementing the method of any one of claims 11 to 16;

(b) Contacting the helicase-loaded target polynucleotide provided in step (a) with the transmembrane pore; and

(c) An electrical application is applied across the transmembrane pore such that the helicase moves through the blocking band and controls movement of the target polynucleotide through the transmembrane pore.

18. The method of claim 17, wherein the method comprises providing a tether for bringing the construct close to the transmembrane pore; the tether includes a capture region for capturing a linker of the construct and an anchor region for anchoring association with the transmembrane pore or a membrane in which the transmembrane pore is located.

19. A method of characterizing target polynucleotide sequencing, the method comprising:

(a) Implementing the method of claim 17 or 18; and

(b) One or more measurements are obtained as the target polynucleotide moves relative to the transmembrane pore, wherein the measurements represent one or more characteristics of the target polynucleotide and thereby characterize the target polynucleotide.

20. The method of claim 19, 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.

21. The method of claim 20, wherein the target polynucleotide is a fully double-stranded polynucleotide, a partially double-stranded polynucleotide, or a single-stranded polynucleotide.

22. A complex for characterizing sequencing of a target polynucleotide, the complex comprising a helicase, and a linker or construct;

wherein the linker comprises a blocking band for simultaneously binding and retaining the helicase; the blocking band comprises a single-stranded nucleotide sequence formed by the ligation of a plurality of nucleotides, the nucleobases of which are selected from one or more of the following: 5-nitroindole, 4, 6-nitroindole, 2-methyl-4-nitroindole, 5-nitroindole-3-carboxamide, 3-carboxamide indole, 4-methylbenzimidazole and methylisoquinolone;

the construct comprises a target polynucleotide and the linker attached to either or both ends of the target polynucleotide;

the helicase binds to and stagnates at the blocking band.

23. The complex of claim 22, wherein the linker further comprises a first segment for guiding the linker into a transmembrane pore and a second segment for ligating a target polynucleotide, the first segment and the second segment having the blocking band interposed therebetween.

24. A complex according to claim 22 or 23, wherein the blocking band is used to bind to and arrest 1 of the helicases.

25. The complex of claim 22 or 23, wherein the blocking band is a single stranded nucleotide sequence formed by 7-12 nucleotide ligation.

26. The complex of claim 25, wherein the blocking band is a single stranded nucleotide sequence formed by 8-10 nucleotide ligation.

27. A complex according to claim 22 or 23 wherein the linker does not comprise an additional region for binding the helicase.

28. Use of the method of any one of claims 11-21 or the complex of claims 22-27 in the preparation of a product for or in characterization of target polynucleotide sequencing.

29. Use of a linker, construct or kit for the preparation of a product for or for characterising target polynucleotide sequencing, said linker comprising a blocking band for simultaneous binding and arrest of helicase;

wherein the blocking band comprises a single stranded nucleotide sequence formed by the ligation of a plurality of nucleotides, the nucleobases of which are selected from one or more of the following: 5-nitroindole, 4, 6-nitroindole, 2-methyl-4-nitroindole, 5-nitroindole-3-carboxamide, 3-carboxamide indole, 4-methylbenzimidazole and methylisoquinolone;

The construct comprises a target polynucleotide and the linker attached to either or both ends of the target polynucleotide;

the kit comprises: (a) the linker, (b) a helicase, and/or (c) a transmembrane pore.

30. The use of claim 29, wherein the linker further comprises a first segment for guiding the linker into a transmembrane pore and a second segment for ligating a target polynucleotide, the first segment and the second segment having the blocking band interposed therebetween.

31. The use according to claim 29 or 30, wherein the blocking band is used to bind and arrest 1 of the helicases.

32. The use of claim 29 or 30, wherein the blocking band is a single stranded nucleotide sequence formed by 7-12 nucleotide ligation.

33. The use of claim 32, wherein the blocking band is a single stranded nucleotide sequence formed by 8-10 nucleotide ligation.

34. The use according to claim 29 or 30, wherein the linker does not comprise an additional region for binding the helicase.