Adapters for characterizing polynucleotides and uses thereof

Abstract

The present invention provides an adapter for characterizing a polynucleotide and its use. The adapter comprises {L-S} n or {S‑L} n ; Moreover, the L double strand comprises a polynucleotide chain L' connected to S and a complementary chain L' of the L', wherein the L' comprises a first segment away from the blocking chain S, and a second segment close to the blocking chain S, the first segment comprises a modified portion, and the second segment comprises a motor protein binding active region, which is blocked by the complementary chain L'; the complementary chain L' comprises a polymer that can competitively bind to the L' chain with the motor protein. The present invention provides a novel Y-shaped adapter, the use of which can greatly avoid ATP consumption in nanopore sequencing and significantly improve sequencing efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of gene sequencing, and relates to an adapter used in characterizing a polynucleotide. The present invention also relates to a method for characterizing a polynucleotide using the adapter. Background Art

[0002] Nanopore sequencing technology offers long read lengths, direct access to modification information, and real-time data production and parallel analysis. It offers significant advantages over next-generation sequencing (NGS) or other sequencing platforms for detecting long-range nucleic acid variants (including but not limited to point mutations, indels, inversions, translocations, gene fusions, aberrant RNA splicing, RNA editing, and other nucleic acid-related variants) and modification information (including but not limited to methylation and acetylation). This platform's parallel data production and analysis capabilities enable real-time variant / modification detection and diagnosis, and its portable design lends itself to broad application prospects.

[0003] When a voltage is applied across the nanopore, the passage of an analyte (e.g., a polynucleotide or peptide) through the nanopore causes a decrease in current. Different analytes cause varying degrees of current blockage. When the analyte remains in the nanopore barrel for a period of time, the current changes. Detection of nucleotides by the nanopore results in a current change of known characteristics and duration.

[0004] In nanopore sequencing, when no electric potential is applied, the blocking strands of a polynucleotide typically stall the helicase, preventing it from moving through the blocking strands and further along the target polynucleotide. However, when the helicase-polynucleotide complex contacts the transmembrane pore and an electric potential is applied, one or more stalled helicases can be mobilized to move through the blocking strands on the polynucleotide and along the sequence of the target polynucleotide, thereby achieving sequencing. Therefore, nanopore sequencing requires the use of an adapter that contains both the enzyme binding region and the blocking strands.

[0005] In nanopore sequencing, nucleic acid adapters such as Y-shaped adapters or hairpin-like adapters are generally used (patent CN202111018113.0), in which motor proteins such as helicases bind to the binding region of the adapter. In actual applications, existing adapters suffer from ATP consumption, that is, adapters that have not undergone through-hole sequencing will also consume a large amount of ATP. Due to the reduction in ATP concentration in the sequencing environment, the sequencing speed will be reduced and the sequencing time will be too short, which will affect the output of sequencing data. Therefore, there is currently a demand for sequencing methods that can reduce ATP consumption. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a new adapter, a method for preparing the adapter, and its use in nanopore sequencing. The adapter of the present invention greatly reduces the ATP consumption in nanopore sequencing.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] In one aspect, the present invention provides an adapter for characterizing a target polynucleotide, wherein the adapter comprises {LS} n or {SL} n ,

[0009] Wherein, L is a modified double-stranded polynucleotide, S is a blocking strand, and n is a positive integer;

[0010] Furthermore, the L double strand comprises a polynucleotide chain L' linked to S and a complementary chain L' of the L'.

[0011] The L' comprises a first segment away from the blocking chain S and a second segment close to the blocking chain S, wherein the first segment comprises a modified portion, and the second segment comprises a motor protein binding active region, which is blocked by the complementary chain L".

[0012] The complementary strand L" comprises a polymer that is capable of competing with the motor protein for binding to the L' strand.

[0013] According to the adapter of the present invention, the adapter comprises {D1-LS} n or {SL-D1} n , D1 is the first double-stranded polynucleotide,

[0014] and / or, the adaptor comprises {LS-D2} in the 5' to 3' direction n or {D2-SL} n , D2 is a second double-stranded polynucleotide;

[0015] Preferably, n is an integer of 1-20, for example, n can be 1, 2, 3, 4, 5, 6, 7, 8 or more.

[0016] According to the adapter of the present invention, the modified portion of the chain L' causes the first segment to have weaker binding ability to the motor protein than the second segment, or causes the first segment to not bind to the motor protein;

[0017] Preferably, the modified portion in the chain L' is a ribonucleotide (RNA) and / or a nucleic acid analog;

[0018] The ribonucleotides include 2′-modified ribonucleotides, preferably 2′-alkoxy-modified ribonucleotides, more preferably 2′-methoxy-modified ribonucleotides;

[0019] The nucleic acid analogue includes any one or any combination of two or more of peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), and bridge nucleic acid (BNA), more preferably, the unmodified portion of the chain L' is a deoxyribonucleotide;

[0020] Preferably, the number of the modified polynucleotides is 1-20, more preferably 1-15, 1-10 or 2-6, further preferably 3, 4 or 5.

[0021] Among them, modification is provided at one end (first segment) away from the blocking chain S in the polynucleotide chain L', and the binding ability of the modified end (first segment) to the motor protein is weaker, so that during the preparation of the adapter, the motor protein is more easily bound to the end (second segment) of the polynucleotide chain L' close to the blocking chain S, thereby being more conducive to the subsequent expulsion of the motor protein. It will be understood by those skilled in the art that the modified portion can be an optional modification, as long as the modification can weaken the binding of the modified portion to the motor protein. The length of the modified portion can be determined according to the length of the polynucleotide chain L'. Without being limited to any particular theory, after ensuring that the end (second segment) of the polynucleotide chain L' close to the blocking chain S has a sufficient number of polynucleotides binding to the motor protein, the remaining parts can be selectively modified without affecting the technical effects of the present application.

[0022] According to the adapter of the present invention, the end of the complementary chain L" close to the blocking chain S comprises a portion with stronger binding force to the polynucleotide chain L' or the second segment; preferably, the portion comprises PNA or LNA.

[0023] The main function of the complementary chain L" is to expel the motor protein by complementing the polynucleotide chain L'. Those skilled in the art will understand that, without being limited to any particular theory, as long as the complementary chain L" has a stronger binding force. In the preparation of the adapter, the motor protein can be expelled to the blocking chain by complementing the polynucleotide chain L'.

[0024] According to the adapter of the present invention, the end of the complementary chain L″ away from the blocking chain S comprises a first part of click chemistry, preferably a click reaction group;

[0025] And / or, the D1 double strand comprises a polynucleotide chain D1' connected to L' and its complementary chain D1", wherein one end of the complementary chain D1" close to the blocking chain S comprises a second part of click chemistry, preferably a click reaction group;

[0026] And / or, the D2 double strand comprises a polynucleotide chain D2' connected to the blocking chain S and its complementary chain D2", wherein the complementary chain D2" comprises a portion that does not hybridize with the adapter; preferably, the portion that does not hybridize with the adapter is located at one end of the complementary chain D2" adjacent to the blocking chain S.

[0027] By introducing click chemistry groups, the binding of this part can be made stronger.

[0028] According to the adapter of the present invention, the blocking strand has a structure different from that of the polynucleotide and is used to block the motor protein;

[0029] Preferably, the blocking strand comprises one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuracils, one or more inverted thymidines (inverted dTs), one or more inverted dideoxythymidines (ddTs), one or more dideoxycytidines (ddCs), one or more 5-methylcytidylic acid, one or more 5-hydroxymethylcytidines, one or more 2'alkoxy modified ribonucleotides (preferably 2'methoxy modified ribonucleotides), one or more isodeoxycytidines (iso-dCs), one or more isodeoxyguanosines (iso-dGs), one or more C3 groups, one or more photocleavable (PC) groups, one or more hexanediols, one or more iSp9 groups, one or more iSp18 groups, a polymer or one or more thiol linkages.

[0030] In a preferred embodiment, the number of 2'alkoxy-modified ribonucleotides is 1-10, more preferably 2-6; more preferably,

[0031] The 2' alkoxy modified ribonucleotides are evenly distributed on the blocking strand.

[0032] According to the adapter of the present invention, the end of L' away from S comprises a leading strand sequence;

[0033] The end of S away from L' is used to connect to the target polynucleotide;

[0034] The polynucleotide chain L' connected to S comprises a motor protein binding active region at one end close to the blocking chain S, and the active region is blocked by the complementary chain L".

[0035] The motor protein is a protein that can bind to a polynucleotide and control its movement through the pore; preferably, the motor protein is selected from one or more of a polymerase, an exonuclease, a helicase and a topoisomerase, more preferably, the helicase is selected from one or more of a Hel308 helicase, a RecD helicase, a Tral helicase, a TrwC helicase, an XPD helicase and a DDA helicase.

[0036] In another aspect, the present invention provides a complex comprising the adaptor of the present invention, and the motor protein and / or the target polynucleotide, wherein the motor protein is located on the blocking strand.

[0037] In another aspect, the present invention provides a method for preparing the composite, comprising:

[0038] S1: The Y1 chain containing L'-S binds to the motor protein, and the binding region is located in the L' chain;

[0039] S2: adding a PNA-R chain containing a complementary chain L″ to obtain the complex, wherein the motor protein is driven to the blocking chain by the PNA-R chain;

[0040] Preferably, the method comprises

[0041] S101: allowing the annealing product comprising the Y1 chain containing D1'-L'-S-D2', the Y2 chain containing D1", and the YB chain containing D2" to bind to the motor protein, wherein the binding region is located at the L' chain of the annealing product;

[0042] S102: Adding a PNA-R chain containing a complementary chain L" to obtain the complex, wherein the motor protein is driven to the blocking chain by the PNA-R chain.

[0043] The present invention also provides a method for characterizing a target polynucleotide, wherein the method uses the adapter or the complex;

[0044] Preferably, the method comprises:

[0045] (a) moving the target polynucleotide through the transmembrane pore,

[0046] wherein the target polynucleotide is linked to the adaptor or the complex; and

[0047] (b) obtaining one or more electrical and / or optical measurements as the polynucleotide moves relative to the pore, wherein the measurements represent one or more characteristics of the polynucleotide and thereby characterize the target polynucleotide.

[0048] In another aspect, the present invention also provides a kit for characterizing polynucleotides.

[0049] The kit is composed of any one of the following 1)-4):

[0050] 1) comprising the independently packaged adaptor, preferably, further comprising the independently packaged motor protein;

[0051] 2) comprising the complex;

[0052] 3) a complex obtained by the preparation method;

[0053] 4) Contains the following components, each packaged separately:

[0054] The polynucleotide chain L' for connecting to S in the adapter, the blocking chain S, and the complementary chain L' of the nucleotide chain L', preferably, further comprise the independently packaged motor protein. In nanopore sequencing, the blocking chain has a serious ATP consumption phenomenon in existing sequencing adapters, that is, the adapters that have not been sequenced through the pore will also consume a large amount of ATP. In sequencing, the reduction in ATP concentration will lead to a decrease in sequencing speed and too short sequencing time, which will affect the output value of sequencing data. The inventors of the present invention have attempted to provide a method that does not require the consumption of ATP, and drives the motor protein loaded on the adapter to the blocking chain, thereby avoiding the consumption of ATP. The technical concept of the present invention is combined with Figure 1 The description is as follows, Figure 1 Schematic diagram of the principle of using the Y-shaped adapter of the present invention to expel the helicase to the blocking strand; wherein, the Y-Top-1 strand (Y1 strand) contains D1'-L'-S-D2', the Y-Top-2 strand (Y2 strand) contains D1", and the YB strand contains D2". First, the Y1 strand, Y2 strand, and YB strand are annealed, and then the helicase is added. The helicase binds to the L' strand, and the Y-Top-2 strand contains a chemical click group. A PNA-R strand is added. The PNA-R strand can better bind to the L' in the Y-Top-1 strand (Y1 strand), and the chemical click reaction between the PNA-R strand and the Y-Top-2 strand further stabilizes the binding, thereby expelling the helicase bound to the L' strand to the S region along the 5' to 3' end direction. This expulsion relies on the driving force of the binding force between the double strands and does not require any ATP consumption, thereby reducing ATP consumption during the actual sequencing process.

[0055] Among them, Figure 1 In the figure, the star-shaped signal of the Y-Top-2 chain is a click reaction group, specifically DBCO modification in a specific embodiment; the triangle signal in the PNA-R chain is a click reaction group, specifically N3 in a specific embodiment.

[0056] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0057] The present invention provides a novel adapter, which can greatly avoid ATP consumption in nanopore sequencing and significantly improve sequencing efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0059] Figure 1 Schematic diagram of the principle of using the Y-shaped adapter of the present invention to expel the helicase to the blocking strand; wherein, the Y-Top-1 strand (Y1 strand) contains D1'-L'-S-D2', the Y-Top-2 strand (Y2 strand) contains D1", and the YB strand contains D2". First, the Y1 strand, Y2 strand, and YB strand are annealed, and then the helicase is added. The helicase binds to the L' strand, and the Y-Top-2 strand contains a chemical click group. A PNA-R strand is added. The PNA-R strand can better bind to the L' in the Y-Top-1 strand (Y1 strand), and the chemical click reaction between the PNA-R strand and the Y-Top-2 strand further stabilizes the binding, thereby expelling the helicase bound to the L' strand to the S region along the 5' to 3' end direction. This expulsion relies on the driving force of the binding force between the double strands and does not require any ATP consumption, thereby reducing ATP consumption during the actual sequencing process.

[0060] Among them, Figure 1 In the figure, the star-shaped signal of the Y-Top-2 chain is a click reaction group, specifically DBCO modification in a specific embodiment; the triangle signal in the PNA-R chain is a click reaction group, specifically N3 in a specific embodiment.

[0061] Figure 2 This is a quality control graph of the adapter-junction complex before and after loading the fourth-strand PNA according to Example 1 of the present invention.

[0062] Figure 3 This is a graph showing ATP / NADH consumption measured after the enzyme is driven to blocking chains of different structures and sequences according to Example 2 of the present invention.

[0063] Figure 4 This is the rate reduction of different adapters in actual sequencing according to Example 3 of the present invention. DETAILED DESCRIPTION

[0064] The features and exemplary embodiments of various aspects of the present invention will be described in detail below. In the detailed description below, many specific details are proposed in order to provide a comprehensive understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention can be implemented without the need for some of these specific details. The following description of the embodiments is merely intended to provide a better understanding of the present invention by illustrating examples of the present invention.

[0065] In addition, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes two or more polynucleotides, reference to "an anchor" includes two or more anchors, reference to "a helicase" includes two or more helicases, reference to "a transmembrane pore" includes two or more pores, etc.

[0066] Connector

[0067] The present invention provides an adapter for characterizing a polynucleotide, wherein the adapter comprises {LS} n or {SL} n , wherein L is a modified double-stranded polynucleotide, S is a blocking strand, and n is an integer; and the L double strand comprises a polynucleotide chain L' connected to S and a complementary chain L'' of the L', wherein the L' comprises a first segment away from the blocking strand S and a second segment close to the blocking strand S, the first segment comprises a modified portion, and the second segment comprises a motor protein binding active region, which is blocked by the complementary chain L''; the complementary chain L'' comprises a polymer capable of competing with the motor protein for binding to the L' chain.

[0068] According to the adapter of the present invention, the adapter comprises {D1-LS} n or {SL-D1} n , D1 is a first double-stranded polynucleotide; and / or, the adapter comprises {LS-D2} in the 5' to 3' direction n or {D2-SL} n , D2 is a second double-stranded polynucleotide; preferably, n is an integer of 1-20, for example, n can be 1, 2, 3, 4, 5, 6, 7, 8, or more.

[0069] According to the adaptor of the present invention, the modified portion of chain L' causes the first segment to have weaker binding ability to the motor protein than the second segment, or causes the first segment to not bind to the motor protein; and / or

[0070] The modified portion in the chain L' is a ribonucleotide and / or a nucleic acid analog.

[0071] Preferably, the ribonucleotide includes a 2'-modified ribonucleotide, preferably a 2'-alkoxy-modified ribonucleotide, more preferably a 2'-methoxy-modified ribonucleotide; or preferentially, the nucleic acid analog includes any one or any combination of two or more of peptide nucleic acid, glycerol nucleic acid, threose nucleic acid, locked nucleic acid, and bridge nucleic acid; and / or the motor protein binding active region in the chain L' is a deoxyribonucleotide; and / or the number of the ribonucleotides and / or nucleic acid analogs is 1-20, 1-15 or 1-10; preferably 2-6, for example, 2, 3, 4, 5 or 6.

[0072] Among them, modification is provided at one end of the polynucleotide chain L' away from the blocking chain S, and the binding ability of the modified end to the motor protein is weaker, so that during the preparation of the adapter, the motor protein is more likely to bind to the end of the polynucleotide chain L' close to the blocking chain S, which is more conducive to the subsequent expulsion of the motor protein. It will be understood by those skilled in the art that the modified portion can be an optional modification, as long as the modification can weaken the binding of the modified portion to the motor protein. The length of the modified portion can be determined according to the length of the polynucleotide chain L'. Without being limited to any particular theory, after ensuring that there are a sufficient number of polynucleotides binding to the motor protein at one end of the polynucleotide chain L' close to the blocking chain S, the remaining parts can be selectively modified without affecting the technical effects of the present application.

[0073] According to the adapter of the present invention, the end of the complementary chain L" close to the blocking chain S comprises a portion with stronger binding force to the polynucleotide chain L'; preferably, the portion comprises PNA or LNA.

[0074] The main function of the complementary chain L" is to expel the motor protein by complementing the polynucleotide chain L'. Those skilled in the art will understand that, without being limited to any particular theory, as long as the complementary chain L" has a stronger binding force. In the preparation of the adapter, the motor protein can be expelled to the blocking chain by complementing the polynucleotide chain L'.

[0075] According to the adapter of the present invention, the end of the complementary chain L″ away from the blocking chain S comprises a first part of click chemistry, preferably a click reaction group;

[0076] And / or, the D1 double strand comprises a polynucleotide chain D1' connected to L' and its complementary chain D1", wherein one end of the complementary chain D1" close to the blocking chain S comprises a second part of click chemistry, preferably a click reaction group;

[0077] And / or, the D2 double strand comprises a polynucleotide chain D2' connected to the blocking chain S and its complementary chain D2", wherein the complementary chain D2" comprises a portion that does not hybridize with the adapter; preferably, the portion that does not hybridize with the adapter is located at one end of the complementary chain D2" adjacent to the blocking chain S.

[0078] By introducing click chemistry groups, the binding of this part can be made stronger.

[0079] According to the adapter of the present invention, the blocking strand has a structure different from that of the polynucleotide.

[0080] Complex

[0081] The present invention provides a complex comprising the adaptor of the present invention and a motor protein, wherein the motor protein is located on a blocking strand;

[0082] Preferably, the motor protein is a protein capable of binding to a polynucleotide and controlling its movement through the pore; preferably, an enzyme. For example, the enzyme is selected from one or more of a polymerase, an exonuclease, a helicase, and a topoisomerase. For example, the helicase is selected from one or more of a Hel308 helicase, a RecD helicase, a Tral helicase, a TrwC helicase, an XPD helicase, and a DDA helicase.

[0083] The present invention provides a method for preparing the composite, comprising:

[0084] S1: enables the Y1 chain containing L'-S to bind to the motor protein, and the binding region is located in the L' chain;

[0085] S2: adding a PNA-R chain containing a complementary chain L″ to obtain the complex, wherein the motor protein is driven to the blocking chain by the PNA-R chain;

[0086] Preferably, the method comprises

[0087] S101: allowing the annealing product comprising the Y1 chain containing D1'-L'-S-D2', the Y2 chain containing D1", and the YB chain containing D2" to bind to the motor protein, wherein the binding region is located at the L' chain of the annealing product;

[0088] S102: Adding a PNA-R chain containing a complementary chain L" to obtain the complex, wherein the motor protein is driven to the blocking chain by the PNA-R chain.

[0089] polynucleotides

[0090] Polynucleotides, such as nucleic acids, are macromolecules containing two or more nucleotides. A polynucleotide or nucleic acid can include any combination of nucleotides. Nucleotides can be naturally occurring or synthetic. One or more nucleotides in a polynucleotide can be oxidized or methylated. One or more nucleotides in a polynucleotide can be damaged. For example, a polynucleotide can contain pyrimidine dimers. Such dimers are often associated with damage caused by ultraviolet light and are a leading cause of cutaneous melanoma. One or more nucleotides in a polynucleotide can be modified, for example, with a marker or label. Suitable markers are described below.

[0091] The nucleotides in a polynucleotide are typically ribonucleotides or deoxyribonucleotides. The polynucleotide may contain the following nucleosides: adenosine, uridine, guanosine, and cytidine. The nucleotides are preferably deoxyribonucleotides. The polynucleotide preferably includes the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and / or thymidine (dT), deoxyguanosine (dG), and deoxycytidine (dC).

[0092] Nucleotides typically contain monophosphate, diphosphate, or triphosphate. The phosphate can be attached to the 5" or 3" side of the nucleotide.

[0093] Suitable nucleotides include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP) and deoxycytidine monophosphate (dCMP). The nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP and dUMP. The nucleotides are most preferably selected from dAMP, dTMP, dGMP, dCMP and dUMP. The polynucleotide preferably comprises the following nucleotides: dAMP, dUMP and / or dTMP and dCMP.

[0094] The nucleotides in the polynucleotide can be linked to each other in any manner. Nucleotides are typically linked by their sugar and phosphate groups, as in nucleic acids. The nucleotides can be linked by their nucleobases, as in pyrimidine dimers.

[0095] The polynucleotide can be a nucleic acid. The polynucleotide can be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), or other synthetic polymers with nucleotide side chains. The PNA backbone is composed of repeated N-(2-aminoethyl)-glycine units connected by peptide bonds. The GNA backbone is composed of repeated ethylene glycol units connected by phosphodiester bonds. The TNA backbone is composed of repeated threose linked together by phosphodiester bonds. The LNA is formed by nucleotides having an extra bridge connecting 2 "oxygen and 4 " carbon in ribose as discussed above.

[0096] The polynucleotide is most preferably ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

[0097] 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 nucleotides in length. The polynucleotide can be 1,000 or more nucleotides in length, 5,000 or more nucleotides in length, or 100,000 or more nucleotides in length.

[0098] The helicase may move along all or only part of the target polynucleotide in the method of the invention.All or part of the target polynucleotide may be characterized using the method of the invention.

[0099] The target polynucleotide can be single-stranded. At least a portion of the target polynucleotide is preferably double-stranded. Helicases typically bind to single-stranded polynucleotides. If at least a portion of the target polynucleotide is double-stranded, the target polynucleotide preferably includes a single-stranded region or a non-hybridized region. The one or more helicases can bind to a chain in the single-stranded region or the non-hybridized region. The target polynucleotide preferably includes one or more single-stranded regions or one or more non-hybridized regions.

[0100] sample

[0101] The target polynucleotide is present in any suitable sample. The present invention is generally implemented on a sample known to contain or suspected of containing the target polynucleotide. Alternatively, the present invention can be implemented on a sample to confirm one or more target polynucleotides identified, which are known or expected to be present in the sample.

[0102] The sample can be a biological sample. The present invention can be implemented in vitro for samples obtained or extracted from any organism or microorganism. The organism or microorganism is typically archaean, prokaryotic, or eukaryotic, and typically belongs to one of the following five kingdoms: Plantae, Animalia, Fungi, Prokaryotes, and Protists. The present invention can be implemented in vitro for samples obtained or extracted from any virus. The sample is preferably a liquid sample. The sample typically includes a patient's body fluid. The sample can be urine, lymph, saliva, mucus, or amniotic fluid, but is preferably blood, plasma, or serum. Typically, the sample is human, but alternatively can be from other mammalian animals, such as commercially raised animals such as horses, cattle, sheep, or pigs, or pets such as cats or dogs. Alternatively, plant-derived samples are typically obtained from commercial crops, such as cereals, beans, fruits, or vegetables, for example, wheat, quinoa, barley, oats, rapeseed, corn, soybeans, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugarcane, cocoa, and cotton.

[0103] The sample may be a non-biological sample. The non-biological sample is preferably a liquid sample. Examples of non-biological samples include surgical fluids, water such as drinking water, sea water or river water, and reagents for laboratory tests.

[0104] The sample is typically processed prior to testing, for example by centrifugation or by filtering through a membrane to remove unwanted molecules or cells, such as red blood cells. Testing can be performed immediately after obtaining the sample. The sample is also typically stored, preferably below -70°C, prior to analysis.

[0105] Blocking Chain

[0106] The one or more blocking strands are included in the target polynucleotide. The one or more blocking strands are preferably part of the target polynucleotide, for example, they interrupt the polynucleotide sequence. The one or more blocking strands are preferably not part of one or more block molecules, such as speed bumps that hybridize to the target polynucleotide.

[0107] There can be any number of blocking strands in a target polynucleotide, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more blocking strands. Preferably, there are 2, 4 or 6 blocking strands in a target polynucleotide. Blocking strands can be present in different regions of a target polynucleotide, such as a blocking strand in a leader sequence and a blocking strand in a hairpin loop.

[0108] The one or more blocking strands each provide an energy barrier that the one or more helicases cannot overcome even in active mode. The one or more blocking strands can stall the one or more helicases by reducing the pull of the helicase (e.g., by removing bases of nucleotides in the target polynucleotide) or physically blocking the movement of the one or more helicases (e.g., using bulky chemical groups).

[0109] The one or more blocking chains can comprise any molecule or the combination of any molecule that stagnates one or more helicases. The one or more blocking chains can comprise any molecule or the combination of any molecule that stops the one or more helicases from moving along the target polynucleotide. It directly determines under the condition of lacking transmembrane pore and the electric potential applied, whether one or more helicases rest on one or more blocking chains. For example, this can be tested as shown in the Examples, and for example helicase passes the complementary chain of blocking chain and displacement DNA and can be measured by PAGE.

[0110] One or more blocking chains generally include straight chain molecules such as polymers. The one or more blocking chains generally have a structure different from the target polynucleotide. For example, if the target polynucleotide is DNA, one or more blocking chains are not deoxyribonucleic acid (DN A) or deoxyribonucleic acid (RNA). In particular, if the target polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), the one or more blocking chains preferably include peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA) or a synthetic polymer with nucleotide side chains.

[0111] One or more blocking chains preferably include one or more nitroindoles, such as one or more 5-nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuracils, one or more reversed thymidines (reverse dTs), one or more reversed deoxythymidines (ddTs), one or more dideoxycytidines (ddCs), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2'alkoxy-modified ribonucleotides (preferably 2'methoxy-modified ribonucleotides), one or more isodeoxycytidines (iso-dCs), one or more isodeoxyguanosines (iso-dGs), one or more iSpC3 groups (i.e., lacking nucleotides of sugar and base), one or more photocleavage (PC) groups, one or more hexanediol groups, one or more blocking chains 9 (iSp9) groups, one or more blocking chains 18 (iSp18) groups, polymers or one or more thiols are connected. The one or more blocking chains may include any combination of these groups. Many of these groups can be purchased from (Integrated DNA).

[0112] The one or more blocking chains can include any number of these groups. For example, for 2-aminopurine, 2-6-diaminopurine, 5-bromodeoxyuridine, reverse dTs, ddTs, ddCs, 5-methylcytidine, 5-hydroxymethylcytidine, 2'alkoxy modified ribonucleotides (preferably 2'methoxy modified ribonucleotides), different dCs, different dGs, iSpC3 groups, PC groups, hexanediol groups and thiol connections, one or more blocking chains preferably include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. One or more blocking chains preferably include 2, 3, 4, 5, 6, 7, 8 or more iSp9 groups. One or more blocking chains preferably include 2, 3, 4, 5 or 6 or more iSp18 groups. The most preferred blocking chain groups are 4 iSp18 groups.

[0113] The polymer is preferably a polypeptide or polyethylene glycol (PEG). The polypeptide preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acids. The PEG preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more monomeric units.

[0114] One or more blocking strands preferably include one or more abasic nucleotides (i.e., nucleotides lacking a core base), such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more abasic nucleotides. The core base can be replaced by -H (idSp) or -OH in the abasic nucleotide. The abasic blocking strand can be inserted into the target polynucleotide by removing the core base from one or more adjacent nucleotides.

[0115] The one or more blocking strands preferably comprise one or more chemical groups that physically cause one or more helicases to stall. The one or more chemical groups are preferably one or more pendant chemical groups. The one or more chemical groups can be attached to one or more nucleobases in the target polynucleotide. The one or more chemical groups can be attached to the backbone of the target polynucleotide. There can be any number of these chemical groups, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. Suitable groups include, but are not limited to, fluorophores, streptavidin and / or biotin, cholesterol, methylene blue, dinitrophenols (DNPs), digoxigenin and / or anti-digoxigenin and diphenylcyclooctyne groups.

[0116] Different blocking strands in the target polynucleotide can comprise different stagnation molecules. For example, one blocking strand can comprise a linear molecule as discussed above, and another blocking strand can comprise one or more chemical groups that physically cause one or more helicases to stagnate. A blocking strand can comprise any linear molecule as discussed above and one or more chemical groups that physically cause one or more helicases to stagnate, such as one or more abasic groups and fluorophores.

[0117] Suitable blocking strands can be designed based on the type of target polynucleotide and implemented under the conditions of the method of the present invention. Most helicases bind to DNA and move along DNA, so they can be stopped by any substance that is not DNA. Suitable molecules are as described above.

[0118] In a specific embodiment, the number of the 2'-methoxy modified ribonucleotides is 1-10, more preferably 2-6; and / or the 2'-methoxy modified ribonucleotides are uniformly distributed on the blocking strand.

[0119] Helicase

[0120] Any helicase can be used in the present invention. The helicase can be or be derived from a Hel308 helicase, a RecD helicase, such as a TraI helicase or a TrwC helicase, an XPD helicase, or a Dda helicase. The helicase can be the helicase provided in Example 1, or the helicase disclosed in Chinese Patent CN201880095718.X, or other helicases.

[0121] Click Chemistry

[0122] The polynucleotides of the present application can be covalently connected. For example, free copper click chemistry or copper-catalyzed click chemistry can be used. Due to the satisfactory properties of click chemistry and its scope for generating covalent connections between a variety of building blocks (building blocks), click chemistry is used in these applications. For example, it is fast, clean and non-toxic, and only produces harmless by-products. Click chemistry is a term first introduced by Kolb et al. in 2001, for describing a wider series of powerful, selective and modular building blocks, which are reliably used in small-scale and large-scale applications (Kolb HC Finn, MG, Sharp less KB, click chemistry:diverse chemical function from a few good reactions, Angew. Chem. Int. Ed. 40 (2001) 2004-2021). They defined the following stringent criteria for click chemistry: "The reactions must be modular, broad in scope, give very high yields, produce only harmless by-products that can be removed by non-chromatographic methods, and be stereospecific (but not necessarily enantioselective). Desired process features include simple reaction conditions (ideally the process should be insensitive to oxygen and water), readily available starting materials and reagents, absence of solvents or use of solvents that are either mild (e.g., water) or easily removed, and simple product isolation. Purification, if necessary, must be by non-chromatographic methods, such as crystallization or distillation, and the products must be stable under physiological conditions."

[0123] Suitable examples of click chemistry include, but are not limited to, the following:

[0124] (a) Modified 1,3-aryl cycloaddition of free copper, where the azide reacts with the alkyne in a strained, for example, cyclooctane ring;

[0125] (b) reaction of an oxygen nucleophile on one linker with an epoxide or aziridine reactive moiety on the other linker; and

[0126] (c) Staudinger ligation, in which the alkyne moiety can be replaced by an aryl phosphine, leading to a specific reaction with an azide to give an amide bond.

[0127] The click chemistry reaction is preferably a Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction between an alkyne and an azide. In a preferred embodiment, the first group is an azide group and the second group is an alkyne group. Nucleic acid bases have been synthesized, and azide and alkyne groups are inserted in preferred positions (e.g., Kocalka P, El-Sagheer AH, Brown T, Rapid and efficient DNA strand cross-linking by click chemistry, Chembiochem. 2008.9(8): 1280-5). Alkyne groups are commercially available from Berry Associates (Michigan, USA), and azide groups are synthesized by ATDBio or IDT bio.

[0128] In a specific embodiment of the present application, preferred reactive groups are azide and hexyl groups, such as azide N3 and DBCO.

[0129] method

[0130] The present invention also provides a method for characterizing a target polynucleotide, wherein the method uses the adapter or the complex;

[0131] Preferably, the method comprises:

[0132] (a) moving the target polynucleotide through the transmembrane pore,

[0133] wherein the target polynucleotide is linked to the adaptor or the complex; and

[0134] (b) obtaining one or more electrical and / or optical measurements as the polynucleotide moves relative to the pore, wherein the measurements represent one or more characteristics of the polynucleotide and thereby characterize the target polynucleotide.

[0135] The method of the present invention comprises measuring one or more characteristics of the target polynucleotide. The method may comprise measuring two, three, four, five or more characteristics of the target polynucleotide. The one or more characteristics are preferably selected from (i) the length of the target polynucleotide, (ii) the identity of the target polynucleotide, (iii) the sequence of the target polynucleotide, (iv) the secondary structure of the target polynucleotide; and (v) whether the target polynucleotide is modified. Any combination of (i) to (v) can be measured according to the present invention.

[0136] For (i), the length of the polynucleotide may, for example, be determined by determining the number of interactions between the target polynucleotide and the pore, and the duration of each interaction between the target polynucleotide and the pore.

[0137] For (ii), the identity of the polynucleotide can be determined in a variety of ways. The identity of the polynucleotide can be determined in conjunction with the determination of the sequence of the target polynucleotide, or it can be determined without the determination of the sequence of the target polynucleotide. The former is direct; the polynucleotide is sequenced and identified thereby. The latter can be accomplished in several ways. For example, the presence of a specific motif in a polynucleotide can be determined (without determining the rest of the sequence of the polynucleotide). Alternatively, a specific electrical and / or optical signal determined in the method can identify a target polynucleotide from a specific source.

[0138] For (iii), the sequence of the polynucleotide can be determined as described above. Suitable sequencing methods, particularly those using electrical measurements, are described in Stoddart D et al., Proc Natl Acad Sci, 12; 106(19): 7702-7, Lieberman KR et al, J Am Chem Soc. 2010; 132(50): 17961-72, and in International Application WO 2000 / 28312.

[0139] With respect to (iv), the secondary structure can be measured in a variety of ways. For example, if the method comprises electrical measurement, the secondary structure can be measured using changes in the residence time or current flow through the pore. This allows regions of single-stranded and double-stranded polynucleotides to be identified.

[0140] For (v), the presence or absence of any modification can be determined. The method preferably includes determining whether the target polynucleotide has been modified by methylation, oxidation, damage, modification with one or more proteins, or modification with one or more markers, tags, or blocking strands. Specific modifications will result in specific interactions with the pore, which can be determined using the methods described below. For example, cytosine and methylated cytosine can be distinguished based on the current passing through the pore during the interaction of the pore with each nucleotide.

[0141] The method is typically carried out in the presence of a buffer. In the exemplary apparatus discussed above, the buffer is present in the aqueous solution of the chamber. The method of the present invention can be used with any buffer. Typically, the buffer is a phosphate buffer. Other suitable buffers are HEPES and Tris-HCl buffers. The method is typically carried out at a pH of 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7, 7.0 to 8.8, or 7.5 to 8.5. The pH used is preferably about 7.5.

[0142] The method can be performed at 0 to 100° C., 15 to 95° C., 16 to 90° C., 17 to 85° C., 18 to 80° C., 19 to 70° C., or 20 to 60° C. The method is typically performed at room temperature. The method is optionally performed at a temperature that supports helicase function, such as about 37° C.

[0143] The method can be implemented in the presence of free nucleotides or free nucleotide analogs and / or auxiliary factors that assist the helicase function. The method can also be implemented in the absence of free nucleotides or free nucleotide analogs and in the absence of auxiliary factors for the helicase. The free nucleotides can be any one or more of the single nucleotides discussed above. 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

[0144] (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate

[0145] (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (DADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine diphosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), and 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). The helicase cofactor is a factor that enables the helicase or construct to function. The helicase cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg 2+ , Mn 2+ , Ca 2+ or Co 2+ The most preferred helicase cofactor is Mg. 2+ .

[0146] Reagent test kit

[0147] In yet another aspect, the present invention also provides a kit for characterizing a polynucleotide, the kit comprising the adapter or the complex.

[0148] The kit comprises (a) one or more adapters, (b) one or more helicases. The kit may comprise any of the helicases and pores discussed above.

[0149] The kit may also include membrane components, such as phospholipids required to form an amphiphilic layer such as a lipid bilayer.

[0150] The kit of the present invention may further comprise one or more other reagents or instruments that enable any of the embodiments mentioned above to be implemented. Such reagents or instruments include one or more of the following reagents or instruments: a suitable buffer (aqueous solution), a means for obtaining a sample from a subject (e.g. a container or instrument comprising a needle), a means for amplifying and / or expressing a polynucleotide, a membrane as defined above or a pressure clamp or patch clamp device. The reagents may be present in the kit in a dry state so that a fluid sample resuspends the reagents. The kit may also, optionally, comprise instructions for using the kit in the method of the present invention, or detailed information on which patients the method may be used for. The kit may optionally comprise the necessary components (e.g. ATP and Mg) to facilitate the movement of the helicase. 2+ ).

[0151] The following examples illustrate the invention.

[0152] Example 1: Preparation of a Y-adapter-enzyme complex that reduces ATP consumption

[0153] SEQ ID NO:1GCGGAGTCAAACGGTAGAAGTCG

[0154] SEQ ID NO:2TAACGTATTC

[0155] SEQ ID NO:3ACTGCTCATTCGGTCCTGCTGACT

[0156] SEQ ID NO:4CGACTTCTACCGTTTGACTCCGC

[0157] SEQ ID NO:5GTCAGCAGGACCGAATGA

[0158] SEQ ID NO: 6GAATACGTTAGCGG, wherein SEQ ID NO: 6 consists of PNA

[0159] SEQ ID NO:7GCAGTAGTCCAGCACCGACC

[0160] SEQ ID NO:8

[0161] GTFDDLTEGQKNAFNIVMKAIKEKKHHVTINGPAGTGKTTLTKFIIEALISTGETGIILAAPTHAAKKILSKLSGKEASTIHSILKINPVTYECNVLFEQKEVPDLAKA RVLICDEVSMYDRKLFKILLSTIPPWATIIGIGDNKQIRPVDPGENTAYISPFFTHKDFYQCELTEVKRSNAPIIDVATDVRNGKWIYDKVVDGHGVRGFTGDTALRDFM VNYFSIVKSLDDLFENRVMAFTNKSVDKLNSIIRKKIFETDKDFIVGEIIVMQEPLFKTYKIDGKPVSEIIFNNGQLVRIIEAEYTSTFVKARGVPGEYLIRHWDLTVET YGDDEYYREKIKIISSDEELYKFNLFLGKTCETYKNWNKGGKAPWSDFWDAKSQFSKVKALPASTFHKAQGMSVDRAFIYTPCIHYADVELAQQLLYVGVTRGRYDVFYV

[0162] The complex is composed of four different chains hybridized together;

[0163] The first strand (Y-Top-1) sequentially comprises a leader sequence, i.e., an iSpC3 blocking strand, indicated as 3, which is connected to the 5′ end of SEQ ID NO: 1, and its 3′ end is sequentially connected to four i2OMeCs and SEQ ID NO: 2, and the 3′ end of SEQ ID NO: 2 is connected to a blocking strand (R0-R6 as shown in Table 2) and SEQ ID NO: 3, wherein i2OmeC and i2OmeG are 2″-O-methyl RNA, i.e., 2′ methoxy-modified RNA.

[0164] The second strand (Y-Top-2), DBCO is linked to the 5' end of SEQ ID NO:4.

[0165] The third strand (Y-Bottom), the 3′ end of SEQ ID NO: 4 is linked to SEQ ID NO: 7.

[0166] The fourth chain (PNA-R), [GAATACGTTAGCGG]pna-OO-azide(N3), where O is O-liker (also known as AEEA or eg1), is used to increase the solubility of PNA-R.

[0167] Y-Top-1:

[0168]

[0169] Y-Top-2:DBCO- CGACTTCTACCGTTTGACTCCGC;

[0170] Y-Bottom:

[0171] PNA-R:

[0172] The three synthetic single strands Y1, Y2, and YB were annealed at a ratio of 1:1.1:1.1 (slowly cooled from 95°C to 25°C, with the cooling rate not exceeding 0.1°C / s). The final annealing system included 160mM HEPES 7.0; 200mM NaCl, and the final concentration of Y1 was 4-8μM, ultimately forming a Y-shaped adapter. The Y-shaped adapter (500nM) was mixed with 6 times the amount of the enzyme T4 Dda-M1G / E94C / C109A / C136A / A360C (3μM) (sequence shown in SEQ ID NO: 8) in a buffer (100mM NaAc (pH 7); 1.5mM TMAD) and incubated at room temperature for 30 minutes. This mixture is referred to as sample 1.

[0173] 1 μM of PNA-R chain was added to sample 1 and incubated at room temperature for 30 minutes to obtain sample 2.

[0174] TBE (native) PAGE gel was used to examine the migration rates of Sample 1 and Sample 2 under the same conditions. After the PNA-R chain was loaded onto the linker, the migration rate of Sample 2 decreased, and it migrated slower than the control (Sample 1) without the addition of the PNA-R chain. The comparison results using the blocking chain as R0 are shown in Figure 2. Figure 2 shown.

[0175] Sample 2 was purified using a DNAPac PA200 column using the following elution buffers (Buffer A: 20 mM Na-CHES, 250 mM NaCl, 4% (W / V) glycerol, pH 8.6; Buffer B: 20 ​​mM Na-CHES, 1 M NaCl, 4% (W / V) glycerol, pH 8.6). Sample 1 was loaded onto the column, and enzyme not bound to DNA was eluted from the column using Buffer A. The enzyme-bound Y-adapter complex was then eluted using 10 column volumes of 0-100% Buffer E. The main elution peak was then pooled and its concentration was measured for use in the assay described in Example 2.

[0176] Example 2: ATPase activity detection

[0177] First, prepare the NADH reaction mixture according to Table 1 below. After the preparation is completed, turn it horizontally at room temperature and incubate it for 10 minutes.

[0178] Table 1 Preparation of NADH reaction mixture

[0179]

[0180] Then, 112.5 μL of NADH reaction mixture and 37.5 μL (20 nM) of Y adapter-enzyme complex thing (Right now Any one of the purified samples 1 and 2 in Example 1 ) and then placed it in a UV-Vis spectrophotometer to measure absorbance at 380 nm at 34°C. 200 cycles of 5 minutes each were performed. The collected data were used to plot a standard curve, and the slope of the standard curve was used to determine ATP consumption.

[0181] The results are as follows Figure 3 and Table 2 (10h after adding the complex). The control without adding PNA-R (the blocking chain used is the same as R1) is set as the baseline (100%). If the ATP consumption percentage is less than 100%, it means that the adapter has the potential to reduce ATP consumption compared to the control adapter. The blocking chain sequences of all tested adapter adapters are shown in Table 2 below. After driving the enzyme to the blocking chain with different structures and sequences, the ATP / NADH consumption was measured. See Figure 3 shown.

[0182] Depend on Figure 3 The results showed that compared with the control R0, the ATP consumption rates of adapters R1-R6 decreased significantly. Compared with R1, the blocking chains of R2-R6 were different, and the ATP consumption was different. Among them, when the blocking chain added one 2'methoxy-modified ribonucleotide, such as R2, the ATP consumption did not change significantly. When the blocking chain added four 2'methoxy-modified ribonucleotides, such as R3-R6, the ATP consumption was significantly reduced. In addition, Figure 3 A control (CK) of an enzyme without an adapter (the same as in Example 1) was also set up. The enzyme consumes ATP depending on the substrate. When the adapter is not present, the enzyme does not bind to the substrate, so its ATP consumption is lower.

[0183] Table 2

[0184]

[0185] Example 3: On-machine testing of the Y-adapter-enzyme complex to reduce ATP consumption

[0186] A 10 kb library was prepared by end repair, and the Y adapter-enzyme complex (linker R4) prepared in Example 1 for reducing ATP consumption was used to connect the library, i.e., the target polynucleotide. The target polynucleotide connection position was Figure 1 The right end of the adapter is shown. A control without the addition of PNA-R chain (denoted as RC) was used.

[0187] Sequencing was performed using the QNome-9604 nanopore sequencer from Qitan Technology Co., Ltd. The sequencing buffer used was: final concentration 10 mM HEPEs, 100 mM MgCl2, 375 mM KCl, ATP 100 mM, pH 7.1, and sequencing temperature: 30-40°C.

[0188] Results: As Figure 4 As shown in the figure, the control RC decreased by approximately 80 bp / s over 16 hours of sequencing; the adapter R4 decreased by approximately 10 bp / s over the same period. Sequencing rate is positively correlated with ATP concentration; if ATP concentration decreases significantly during sequencing, the sequencing rate will also decrease.

[0189] In this document, the term "and / or" simply describes a relationship between related objects, indicating that three possible relationships exist. For example, "A and / or B" can represent: A exists alone, A and B exist simultaneously, or B exists alone. Furthermore, the character " / " in this document generally indicates that the related objects are in an "or" relationship.

[0190] It should be understood that in the embodiments of the present invention, "B corresponding to A" means that B is associated with A and B can be determined based on A. However, it should also be understood that determining B based on A does not mean determining B based solely on A; B can also be determined based on A and / or other information.

[0191] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and such modifications or substitutions are intended to be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims. Sequence Listing <110> Chengdu Qitan Technology Co., Ltd. <120> Adapters for characterizing polynucleotides and uses thereof <130> 21NI2349 <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty three <212> DNA <213> Artificial Sequence <400> 1 gcggagtcaa acggtagaag tcg 23 <210> 2 <211> 10 <212> DNA <213> Artificial Sequence <400> 2 taacgtattc 10 <210> 3 <211> twenty four <212> DNA <213> Artificial Sequence <400> 3 actgctcatt cggtcctgct gact 24 <210> 4 <211> twenty three <212> DNA <213> Artificial Sequence <400> 4 cgacttctac cgtttgactc cgc 23 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <400> 5 gtcagcagga ccgaatga 18 <210> 6 <211> 14 <212> DNA <213> Artificial Sequence <400> 6 gaatacgtta gcgg 14 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 gcagtagtcc agcaccgacc 20 <210> 8 <211> 439 <212> PRT <213> Artificial Sequence <400> 8 Gly Thr Phe Asp Asp Leu Thr Glu Gly Gln Lys Asn Ala Phe Asn Ile 1 5 10 15 Val Met Lys Ala Ile Lys Glu Lys Lys His His Val Thr Ile Asn Gly 20 25 30 Pro Ala Gly Thr Gly Lys Thr Thr Leu Thr Lys Phe Ile Ile Glu Ala 35 40 45 Leu Ile Ser Thr Gly Glu Thr Gly Ile Ile Leu Ala Ala Pro Thr His 50 55 60 Ala Ala Lys Lys Ile Leu Ser Lys Leu Ser Gly Lys Glu Ala Ser Thr 65 70 75 80 Ile His Ser Ile Leu Lys Ile Asn Pro Val Thr Tyr Glu Cys Asn Val 85 90 95 Leu Phe Glu Gln Lys Glu Val Pro Asp Leu Ala Lys Ala Arg Val Leu 100 105 110 Ile Cys Asp Glu Val Ser Met Tyr Asp Arg Lys Leu Phe Lys Ile Leu 115 120 125 Leu Ser Thr Ile Pro Pro Trp Ala Thr Ile Ile Gly Ile Gly Asp Asn 130 135 140 Lys Gln Ile Arg Pro Val Asp Pro Gly Glu Asn Thr Ala Tyr Ile Ser 145 150 155 160 Pro Phe Phe Thr His Lys Asp Phe Tyr Gln Cys Glu Leu Thr Glu Val 165 170 175 Lys Arg Ser Asn Ala Pro Ile Ile Asp Val Ala Thr Asp Val Arg Asn 180 185 190 Gly Lys Trp Ile Tyr Asp Lys Val Val Asp Gly His Gly Val Arg Gly 195 200 205 Phe Thr Gly Asp Thr Ala Leu Arg Asp Phe Met Val Asn Tyr Phe Ser 210 215 220 Ile Val Lys Ser Leu Asp Asp Leu Phe Glu Asn Arg Val Met Ala Phe 225 230 235 240 Thr Asn Lys Ser Val Asp Lys Leu Asn Ser Ile Ile Arg Lys Lys Ile 245 250 255 Phe Glu Thr Asp Lys Asp Phe Ile Val Gly Glu Ile Ile Val Met Gln 260 265 270 Glu Pro Leu Phe Lys Thr Tyr Lys Ile Asp Gly Lys Pro Val Ser Glu 275 280 285 Ile Ile Phe Asn Asn Gly Gln Leu Val Arg Ile Ile Glu Ala Glu Tyr 290 295 300 Thr Ser Thr Phe Val Lys Ala Arg Gly Val Pro Gly Glu Tyr Leu Ile 305 310 315 320 Arg His Trp Asp Leu Thr Val Glu Thr Tyr Gly Asp Asp Glu Tyr Tyr 325 330 335 Arg Glu Lys Ile Lys Ile Ile Ser Ser Asp Glu Glu Leu Tyr Lys Phe 340 345 350 Asn Leu Phe Leu Gly Lys Thr Cys Glu Thr Tyr Lys Asn Trp Asn Lys 355 360 365 Gly Gly Lys Ala Pro Trp Ser Asp Phe Trp Asp Ala Lys Ser Gln Phe 370 375 380 Ser Lys Val Lys Ala Leu Pro Ala Ser Thr Phe His Lys Ala Gln Gly 385 390 395 400 Met Ser Val Asp Arg Ala Phe Ile Tyr Thr Pro Cys Ile His Tyr Ala 405 410 415 Asp Val Glu Leu Ala Gln Gln Leu Leu Tyr Val Gly Val Thr Arg Gly 420 425 430 Arg Tyr Asp Val Phe Tyr Val 435

Claims

1. An adapter for characterizing a target polynucleotide, characterized in that The adapter comprises {D1-LS} in the 5' to 3' direction n or {SL-D1} n , D1 is the first double-stranded polynucleotide; Wherein, L is a modified double-stranded polynucleotide, S is a blocking strand, and n is a positive integer; Furthermore, the L double strand comprises a polynucleotide chain L' connected to S and a complementary chain L'' of the L', wherein the L' comprises a first segment away from the blocking chain S and a second segment close to the blocking chain S, wherein the first segment comprises a modified portion, and the second segment comprises a motor protein binding active region, which is blocked by the complementary chain L''; The complementary chain L'' comprises a polymer capable of competing with the motor protein for binding to the L' chain; The modified portion of the chain L' causes the first segment to have weaker binding ability to the motor protein than the second segment, or causes the first segment to not bind to the motor protein; the modified portion in the chain L' is a ribonucleotide and / or a nucleic acid analog, and the nucleic acid analog is any one or any combination of two or more of a peptide nucleic acid, a glycerol nucleic acid, a threose nucleic acid, a locked nucleic acid, and a bridge nucleic acid; The end of the complementary chain L'' close to the blocking chain S comprises a portion with stronger binding force to the polynucleotide chain L' or the second segment, and the portion is PNA or LNA; The end of the complementary chain L'' away from the blocking chain S comprises the first part of click chemistry; The D1 double-stranded chain comprises a polynucleotide chain D1' connected to L' and its complementary chain D1'', wherein one end of the complementary chain D1'' close to the blocking chain S comprises the second part of click chemistry.

2. The adapter according to claim 1, wherein: The adapter comprises a double strand of D2, and the adapter comprises {D1-LS} in the 5' to 3' direction. n or {SL-D1} n , the adapter comprises {LS-D2} in the 5' to 3' direction n or {D2-SL} n , D2 is a second double-stranded polynucleotide.

3. The adapter according to claim 2, wherein: Said n is an integer of 1-20.

4. The adapter according to claim 1, wherein: The ribonucleotides include 2'-modified ribonucleotides; and / or The motor protein binding active region in the chain L' is a deoxyribonucleotide; and / or The number of the ribonucleotides and / or nucleic acid analogs is 1-20.

5. The adapter according to claim 4, characterized in that: The ribonucleotides include 2' alkoxy modified ribonucleotides.

6. The adapter according to claim 4, characterized in that: The ribonucleotides include 2'-methoxy-modified ribonucleotides.

7. The adapter according to claim 4, characterized in that: The number of the ribonucleotides and / or nucleic acid analogs is 1-15.

8. The adapter according to claim 4, characterized in that: The number of the ribonucleotides and / or nucleic acid analogs is 1-10.

9. The adapter according to claim 4, characterized in that: The number of the ribonucleotides and / or nucleic acid analogs is 2-6.

10. The adapter according to claim 2, wherein: The D2 double strand comprises a polynucleotide chain D2′ connected to a blocking chain S and a complementary chain D2″ thereof, wherein the complementary chain D2″ comprises a portion that does not hybridize with the adapter. The adapter according to claim 1 , wherein: The first part of the click chemistry is the click reaction group.

12. The adapter according to claim 1, wherein: The second part of the click chemistry is the click reaction group.

13. The adapter according to claim 10, wherein: The portion that does not hybridize with the adapter is located at one end of the complementary strand D2″ adjacent to the blocking strand S.

14. The adapter according to claim 1, wherein: The blocking strand has a structure different from that of the polynucleotide and is used to block the motor protein.

15. The adapter according to claim 14, wherein: The blocking chain comprises one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuracils, one or more inverted thymidines, one or more inverted dideoxythymidines, one or more dideoxycytidines, one or more 5-methylcytidylic acid, one or more 5-hydroxymethylcytidines, one or more 2'alkoxy-modified ribonucleotides, one or more isodeoxycytidines, one or more isodeoxyguanosines, one or more C3 groups, one or more photocleavable (PC) groups, one or more hexanediols, one or more iSp9 groups, one or more iSp18 groups, a polymer or one or more thiol groups. The adapter according to claim 15 , wherein: The 2'alkoxy modified ribonucleotide is a 2'methoxy modified ribonucleotide.

17. The adapter according to claim 1, wherein: The end of S away from L' is used to connect to the target polynucleotide; The motor protein is a protein that is able to bind to a polynucleotide and control its movement through the pore.

18. The adapter according to claim 17, wherein: The motor protein is selected from one or more of polymerase, exonuclease, helicase and topoisomerase.

19. The adapter according to claim 18, wherein: The helicase is selected from one or more of Hel308 helicase, RecD helicase, Tral helicase, TrwC helicase, XPD helicase and DDA helicase. 20 . A complex comprising the adaptor according to claim 1 , and the motor protein and / or the target polynucleotide. The complex according to claim 20 , wherein the motor protein is located on the blocking strand.

22. The method for preparing the composite according to claim 20 or 21, comprising: S1: enables the Y1 chain containing L'-S to bind to the motor protein, and the binding region is located in the L' chain; S2: Adding a PNA-R chain containing a complementary chain L″ to obtain the complex, wherein the motor protein is driven to the blocking chain by the PNA-R chain.

23. The method for preparing the composite according to claim 22, comprising: S101: allowing the annealing product comprising the Y1 chain containing D1'-L'-S-D2', the Y2 chain containing D1'' and the YB chain containing D2'' to bind to the motor protein, wherein the binding region is located at the L' chain of the annealing product; S102: Adding a PNA-R chain containing a complementary chain L″ to obtain the complex, wherein the motor protein is driven to the blocking chain by the PNA-R chain. 24 . A method for characterizing a target polynucleotide, the method using the adaptor according to claim 1 or the complex according to claim 20 or 21 , wherein the method is a method for non-disease diagnosis and treatment.

25. The method according to claim 24, comprising: (a) moving the target polynucleotide through the transmembrane pore, wherein the target polynucleotide is linked to the adapter of any one of claims 1 to 19 or the complex of claim 20 or 21; and (b) obtaining one or more electrical and / or optical measurements as the polynucleotide moves relative to the pore, wherein the measurements represent one or more characteristics of the polynucleotide and thereby characterize the target polynucleotide.

26. A kit for characterizing a polynucleotide, the kit comprising any one of the following 1) to 4): 1) comprising an independently packaged adapter according to any one of claims 1 to 19; 2) comprising the complex according to claim 20 or 21; 3) A composite obtained by the preparation method according to claim 22 or 23; 4) Contains the following components in separate packages: The double-stranded polynucleotide chains L' and D1 for connection to S, the blocking chain S, and the complementary chain L'' of the nucleotide chain L' in the adapter according to any one of claims 1 to 19.

27. The kit according to claim 26, wherein 1) further comprises the motor protein according to any one of claims 1 to 19 in an independently packaged form.

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