Sequences, linkers comprising sequences, and uses thereof

Abstract

The invention provides sequences, linkers comprising sequences, and uses thereof. The invention provides sequences for binding and blocking a polynucleotide binding protein, the sequences comprising a first segment for binding the polynucleotide binding protein and a second segment for blocking the polynucleotide binding protein, wherein the second segment comprises a polynucleotide, the phosphate groups of which are modified, the modification reducing the net negative charge of the polynucleotide, thereby effecting blocking of the polynucleotide binding protein. The invention provides linkers for characterizing target polynucleotides. The linkers of the invention are simple to make and can be used directly in nanopore sequencing, significantly improving the efficiency of library construction and the effectiveness of sequencing data.

Description

Technical Field

[0001] This invention belongs to the field of gene sequencing and relates to a sequence for binding and blocking polynucleotide binding proteins, a linker comprising the sequence, and a method for characterizing polynucleotides using the linker. Background Technology

[0002] Nanopore sequencing technology features long read lengths, direct access to modification information, and real-time data production and parallel analysis. It offers significant advantages over next-generation sequencing or other sequencing platforms in detecting variations (including but not limited to point mutations, insertions / deletions, inversions / translocations, gene fusions, RNA splicing, RNA editing, and other nucleic acid-related variations) and modifications (including but not limited to methylation and acetylation) in long-fragment nucleic acids. This platform's ability to support parallel data production and analysis enables real-time variation / modification detection and diagnosis, and its portable design makes it promising for a wide range of applications.

[0003] When a voltage is applied across a nanopore, the current decreases as the analyte (e.g., polynucleotides, peptides) passes through it. Different analyte structures cause varying degrees of current blocking. The current changes when the analyte temporarily resides in the nanopore's barrel. Nanopore detection of nucleotides provides current changes with known characteristics and durations.

[0004] In nanopore sequencing, when no potential is applied, the retardation region of a polynucleotide typically halts polynucleotide-binding proteins (Pnucleotide-binding proteins) such as helicases, preventing them from moving further along the target polynucleotide through the spacer region. However, when the complex of a Pnucleotide-binding protein, such as a helicase, and the polynucleotide comes into contact with the nanopore and a potential is applied, one or more halted Pnucleotide-binding proteins, such as helicases, can move through the retardation region on the polynucleotide and move along the sequence of the polynucleotide to be sequenced, thus achieving the purpose of sequencing. Therefore, existing polynucleotide linkers all contain the binding region of a normal polynucleotide-binding protein and various structural retardation regions.

[0005] However, in existing nanopore sequencing technologies, the blocking regions of polynucleotides used to halt polynucleotide-binding proteins such as helicases are mostly modified on the bases and pentose sugars of the nucleotides. These modifications require the preparation of phosphorylated monomers of the modified nucleic acids through several or even dozens of reaction steps.

[0006] Therefore, there is a current need for simpler methods that can block polynucleotide-binding proteins such as helicases. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to provide a new adapter sequence. This invention also provides a method for preparing the adapter sequence and its use in nanopore sequencing.

[0008] The objective of this invention is achieved through the following technical solution: A first aspect of the invention provides a sequence for binding to and blocking a polynucleotide-binding protein, the sequence comprising a first segment and a second segment, the first segment for binding the polynucleotide-binding protein and the second segment for blocking the polynucleotide-binding protein, wherein the second segment comprises a polynucleotide whose phosphate group is modified to reduce the net negative charge of the polynucleotide, thereby achieving the blocking of the polynucleotide-binding protein.

[0009] The sequence includes, but is not limited to, polynucleotides.

[0010] According to the sequence described in this invention, the inventors discovered that the presence of a modifying group on the phosphate group of a polynucleotide can achieve the effect of helicase repression. Specifically, alkylation modification of the phosphate group of the polynucleotide reduces the charge density on the polynucleotide, which can serve as a novel repression mechanism. Since thioalkylation modification of the phosphate backbone of the polynucleotide can be performed at any position, the modification at different positions is relatively flexible, and different hydrophobic groups can be changed as needed.

[0011] According to the sequence of the present invention, the modification includes a hydrophobic modification.

[0012] The groups used for the hydrophobic modification are selected from C. 1-6 -alkyl, C 1-6 -alkoxy group, C 2-6 -Alkenyl, C 2-6 -Alynyl group, C 2-6 -One or more of alkylthio, aryl, arylthioaryl, aryloxy, and arylcarbonyl groups; and / or In the polynucleotide of the second segment, the number of modified phosphate groups is one or more; and / or The sequence comprises one or more first segments and one or more second segments; wherein the first segments and second segments are arranged alternately; and the number of polynucleotide-binding proteins that bind and stagnate in each adjacent first segment and second segment is 1.

[0013] According to the sequence described in this invention, the group used for the hydrophobic modification is an ethyl or phenylacetamide group.

[0014] According to the sequence described in this invention, the polynucleotide-binding protein is derived from a polynucleotide processing enzyme.

[0015] The polynucleotide processing enzyme is selected from one or more of polymerase, exonuclease, helicase, and topoisomerase.

[0016] The helicase is selected from one or more of Hel308 helicase, RecD helicase, Tral helicase, TrwC helicase, XPD helicase, and DDA helicase.

[0017] A second aspect of the present invention provides a method for preparing the said sequence, comprising: Synthesize a sequence comprising a first segment and a second segment, wherein one or more nucleotides in the second segment have phosphate groups that are thiophosphate groups; and The thiophosphate group is alkylated, thereby modifying the phosphate group of the polynucleotide in the second segment, and the modification reduces the net negative charge of the polynucleotide.

[0018] A third aspect of the invention provides a linker for characterizing a target polynucleotide, wherein the linker includes a third segment for guiding the linker into a nanopore and a fourth segment for connecting the target polynucleotide, wherein the aforementioned sequence is inserted between the third segment and the fourth segment.

[0019] The first segment of the sequence is close to the third segment, and the second segment of the sequence is close to the fourth segment.

[0020] A fourth aspect of the present invention provides a construct for characterizing a target polynucleotide, wherein the construct comprises a target polynucleotide and the aforementioned linker, wherein the linker is connected to either end or both ends of the target polynucleotide.

[0021] A fifth aspect of the invention provides a complex for characterizing a target polynucleotide, wherein the complex comprises the aforementioned linker or construct, and a polynucleotide-binding protein; The polynucleotide-binding protein binds to the first segment of the linker or the construct and can stop at the second segment.

[0022] A sixth aspect of the present invention provides a kit for characterizing target polynucleotides, the kit comprising: (a) one or more of the aforementioned linkers, and (b) a polynucleotide-binding protein.

[0023] A seventh aspect of the present invention provides a method for controlling the movement of a target polynucleotide through a nanopore, comprising: I. Provide the aforementioned complex; II. Contacting the composite with nanopores; and III. Apply a potential across the nanopore to cause the polynucleotide-binding protein to move through the second segment and control the movement of the target polynucleotide through the pore.

[0024] An eighth aspect of the present invention provides a method for characterizing a target polynucleotide, comprising: 1) Using the method described above, target polynucleotides are made to move through nanopores; and 2) As the polynucleotide moves relative to the pore, one or more electrical and / or optical measurements are acquired, wherein the measurements represent one or more characteristics of the polynucleotide and thereby characterize the target polynucleotide.

[0025] According to the method of the present invention, the target polynucleotide includes deoxyribonucleic acid and / or ribonucleic acid; and / or At least a portion of the polynucleotide is double-stranded; and / or The sequence for binding and blocking polynucleotide-binding proteins is included in the single-stranded region or non-hybridized region of the polynucleotide.

[0026] A ninth aspect of the present invention provides a method for controlling the loading of one or more polynucleotide-binding proteins onto a target polynucleotide, comprising: a. Provide a target polynucleotide with one or more of the aforementioned sequences inserted; and b. Contact the target polynucleotide provided in step a with the polynucleotide-binding protein such that the polynucleotide-binding protein binds to the target polynucleotide and stops at the second segment.

[0027] Compared with the prior art, the technical solution of the present invention has the following advantages: 1) The technical solution of the present invention is the first to reduce the charge of the phosphate group by modifying the phosphate group of the polynucleotide sequence, which has a good effect on blocking the movement of polynucleotide binding proteins such as helicases, and can pass through the blockage and perform normal sequencing after the application of an electric field. 2) The technical solution of the present invention is a new blocking method, which is different from the blocking schemes in the prior art and enriches the existing sequencing schemes; 3) The assembly and construction of sequencing libraries using the method of this invention can be directly used for nanopore sequencing, which significantly improves the efficiency of library construction and the validity of sequencing data. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a schematic diagram of the sequence designed to bind and block the enzyme according to Embodiment 1 of the present invention.

[0030] Figure 2 This is an electrophoresis diagram of the result obtained in Embodiment 1 of the present invention.

[0031] Figure 3 This is an electrophoresis diagram of the result obtained in Embodiment 2 of the present invention.

[0032] Figure 4 This is Embodiment 3 of the present invention, showing the results of nanopore sequencing using the adapter of the present invention. Detailed Implementation

[0033] The features and exemplary embodiments of various aspects of the present invention will now be described in detail. In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention may be practiced without requiring some of these specific details. The following description of embodiments is merely intended to provide a better understanding of the invention by illustrating examples of the invention.

[0034] Furthermore, unless otherwise expressly stated in the content, the singular forms “a,” “an,” and “described” used in this specification and the appended claims include plural references. Thus, for example, referring to “polynucleotide” includes two or more polynucleotides, referring to “an anchor” includes two or more anchors, referring to “helicase” includes two or more helicases, referring to “nanopore” includes two or more pores, etc.

[0035] The present invention provides a sequence for binding and blocking polynucleotide-binding proteins. The present invention provides a sequence for binding to and blocking a polynucleotide-binding protein, the sequence comprising a first segment and a second segment, the first segment for binding the polynucleotide-binding protein and the second segment for blocking the polynucleotide-binding protein, wherein the second segment comprises a polynucleotide whose phosphate group is modified to reduce the net negative charge of the polynucleotide, thereby achieving the blocking of the polynucleotide-binding protein.

[0036] The inventors discovered that the presence of a modifying group on the phosphate group of a polynucleotide can inhibit the action of helicases. Specifically, alkylating the phosphate group of a polynucleotide to reduce the charge density on the polynucleotide can serve as a novel inhibition mechanism. Since thioalkylation of the phosphate backbone of a polynucleotide can be performed at any position, the modification at different positions is relatively flexible, and different hydrophobic groups can be used as needed.

[0037] The groups used for the hydrophobic modification are selected from C. 1-6 -alkyl, C 1-6 -alkoxy group, C 2-6 -Alkenyl, C 2-6 -Alynyl group, C 2-6 -One or more of alkylthio, aryl, arylthioaryl, aryloxy, and arylcarbonyl groups. Those skilled in the art will understand that these hydrophobic groups, having similarly sized aromatic heterocycles, carbon chains, and other structures, can exert the same retardation effect.

[0038] The first segment preferably comprises at least a portion of a single-stranded polynucleotide for binding a polynucleotide-binding protein. The ability of the polynucleotide-binding protein (such as a helicase) to bind to the polynucleotide can be determined using any method known in the art. Suitable binding assays include, but are not limited to, polyacrylamide gel electrophoresis (PAGE), fluorescence anisotropy, calorimetry, and surface plasmon resonance (SPR, such as Biacore™). The second segment comprises at least a portion of a polynucleotide, one or more phosphate groups of which are modified such that the polynucleotide-binding protein can arrest at the second segment.

[0039] The present invention also provides a method for preparing the aforementioned sequence, comprising: i) Synthesize a sequence comprising a first segment and a second segment, wherein one or more nucleotides in the second segment have phosphate groups that are thiophosphate groups; and ii) Alkylating the thiophosphate group, thereby modifying the phosphate group of the polynucleotide in the second segment, the modification reducing the net negative charge of the polynucleotide.

[0040] In step i), one or more nucleotides in the polynucleotide of the second segment have phosphate groups of thiophosphate. In step ii), the nucleotide containing the thiophosphate group is modified by adding a phosphate group to the hydrophobic group through an alkylation reaction.

[0041] Alkylation reaction Nucleotides typically contain a nucleobase, a sugar, and at least one phosphate group. The nucleobase and sugar form a nucleoside. Nucleotides can be natural or non-natural. Each nucleotide carries a negatively charged phosphate group and a partially negatively charged base. When an electrophile attacks a negatively charged center, it often adds an alkyl group to the negatively charged center, a process known as alkylation.

[0042] The steps for modifying the phosphate groups of the sequence described in this invention via alkylation reaction in this application are as follows: In one specific embodiment, considering the poor water solubility of the 2-bromoacetamide analog, it was first dissolved in methanol, and then reacted with the thiophosphate ester on the DNA backbone in PBS buffer at 80°C for 30 min to obtain the target product. The product was then purified by HPLC to obtain a product meeting the purity requirements.

[0043] Polynucleotide binding protein Polynucleotide-binding proteins can be any protein capable of binding to a polynucleotide and controlling its movement relative to a pore, such as through the pore. In the prior art, it is possible to directly determine whether a protein is bound to a polynucleotide. The protein typically interacts with the polynucleotide and modifies at least one property of the polynucleotide. The protein can modify the polynucleotide by cleaving it to form a single nucleotide or a shorter chain of nucleotides, such as a dinucleotide or a trinucleotide.

[0044] Any number of polynucleotide proteins can be linked to the target polynucleotide. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more proteins can be linked. The one or more polynucleotide-binding proteins can be one or more single-stranded binding proteins (SSBs). The one or more single-stranded binding proteins (SSBs) may contain a region with a carboxyl terminus (C-terminus) without a net negative charge or (ii) contain one or more modified SSBs in their C-terminal region that reduce the net negative charge of the C-terminal region.

[0045] The one or more polynucleotide-binding proteins are preferably derived from polynucleotide-processing enzymes. The polynucleotide-processing enzyme is a polypeptide capable of interacting with and modifying at least one property of a polynucleotide. The enzyme can modify the polynucleotide by cleaving it to form a single nucleotide or a shorter chain of nucleotides, such as a dinucleotide or trinucleotide. The enzyme can also modify the polynucleotide by directing it or by moving it to a specific location. The polynucleotide-processing enzyme does not need to exhibit enzymatic activity as long as it can bind to the polynucleotide and control its movement relative to a pore, such as through the pore. For example, the enzyme can be modified to remove its enzymatic activity or can be used under conditions that prevent it from functioning as an enzyme.

[0046] For example, the polynucleotide-binding protein is derived from a polynucleotide-processing enzyme; the polynucleotide-processing enzyme is selected from one or more of polymerases, exonucleases, helicases and topoisomerases, and the helicase is selected from one or more of Hel308 helicase, RecD helicase, Tral helicase, TrwC helicase, XPD helicase and DDA helicase.

[0047] connector The adapter is used to connect and characterize the target polynucleotide, wherein the adapter includes a third segment for guiding the adapter into the nanopore and a fourth segment for connecting the target polynucleotide, with the aforementioned sequence inserted between the third segment and the fourth segment; The first segment of the sequence is close to the third segment, and the second segment of the sequence is close to the fourth segment.

[0048] It is understood that the third segment preferably includes a leader sequence. The leader sequence facilitates the method of the present invention. The leader sequence is designed to preferentially enter the nanopore and thereby facilitate the movement of the target polynucleotide relative to the pore, such as through the pore. The leader sequence can also be used to attach the polynucleotide to one or more anchors, as described above.

[0049] The leader sequence typically contains a polymer. The polymer is preferably negatively charged. The polymer is preferably a polynucleotide, such as DNA or RNA, a modified nucleotide (e.g., debaseled DNA), PNA, LNA, BNA, polyethylene glycol (PEG), or a polypeptide. The leader sequence preferably contains a polynucleotide, and more preferably a single-stranded polynucleotide.

[0050] The leader sequence can contain any of the polynucleotides mentioned above. The single-stranded leader sequence is preferably composed of a single strand of DNA, such as a polydT segment.

[0051] The leader sequence can be of any length, but is typically 10 to 150 nucleotides long, for example, 20 to 150 nucleotides long. The length of the leader sequence usually depends on the nanopore used in this method.

[0052] Nanopores The nanopores are selected from solid nanopores and / or biological nanopores, preferably biological nanopores, which include transmembrane pores, preferably transmembrane protein pores.

[0053] Transmembrane protein pores are polypeptides or a series of polypeptides that allow hydrated ions, such as analytes, to flow from one side of a membrane to the other. In this invention, transmembrane protein pores are capable of forming pores that allow a flow of hydrated ions driven by an applied potential to flow from one side of the membrane to the other. Transmembrane protein pores preferably allow analytes, such as nucleotides, to flow from one side of a membrane, such as a lipid bilayer, to the other. Transmembrane protein pores allow polynucleotides or nucleic acids, such as DNA or RNA, to be moved through the pores.

[0054] The transmembrane protein pore can be a monomer or an oligomer. The pore is preferably composed of several repeating subunits, such as 6, 7, 8, or 9 subunits. The pore is preferably a hexamer, heptamer, octamer, or nonamer.

[0055] Transmembrane protein pores typically consist of barrels or channels through which ions can flow. The subunits of the pore usually provide strands around a central axis to the transmembrane β-barrel or channel, or transmembrane α-helical bundle or channel.

[0056] The barrel-shaped or channel-like structure of a transmembrane protein pore typically includes amino acids that facilitate interaction with analytes such as nucleotides, polynucleotides, or nucleic acids. These amino acids are preferably located near a constriction in the barrel-shaped or channel-like structure. Transmembrane protein pores typically include one or more positively charged amino acids, such as arginine, lysine, or histidine, or aromatic amino acids such as tyrosine or tryptophan. These amino acids typically facilitate the interaction between the pore and the nucleotide, polynucleotide, or nucleic acid.

[0057] complex The present invention provides a complex comprising the linker and polynucleotide binding protein described herein, wherein the polynucleotide binding protein binds to a first segment of the linker or the construct and is capable of stopping at a second segment.

[0058] Reagent test kit The present invention also provides a kit for characterizing polynucleotides, the kit comprising the adapter or the complex described herein.

[0059] The kit includes (a) one or more adapters, and (b) one or more polynucleotide-binding proteins. The kit may include any helicases and pores discussed above.

[0060] The kit may also include membrane components, such as phospholipids or lipid bilayers required to form amphoteric molecular layers.

[0061] The kit of the present invention may additionally include one or more other reagents or instruments that enable any of the embodiments mentioned above to be carried out. Such reagents or instruments include one or more of the following: suitable buffer solutions (aqueous solutions), tools for obtaining samples from a recipient (e.g., containers or instruments containing needles), tools for amplifying and / or expressing polynucleotides, and membrane or pressure clamp or patch clamp devices as defined above. The reagents may be present in a dry state in the kit, allowing for resuspending of the reagents in a fluid sample. The kit may also, optionally, include instructions on how to use the kit in the method of the present invention, or details of which patients the method may be used for. The kit may optionally include necessary components to promote helicase movement (e.g., ATP and Mg). 2+ ).

[0062] Polynucleotides Polynucleotides, such as nucleic acids, are large molecules containing two or more nucleotides. Polynucleotides or nucleic acids can comprise any combination of any nucleotides. Nucleotides can be naturally occurring or artificially synthesized. 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 may contain pyrimidine dimers. Such dimers are commonly associated with UV-induced damage and are a leading cause of melanoma. One or more nucleotides in a polynucleotide can be modified, for example, by using markers or labels. Suitable markers are described below.

[0063] The nucleotides in polynucleotides are typically ribonucleotides or deoxyribonucleotides. The polynucleotides may contain the following nucleosides: adenosine, uridine, guanosine, and cytidine. The nucleotides are preferably deoxyribonucleotides. The polynucleotides preferably include the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU), and / or thymidine (dT), deoxyguanosine (dG), and deoxycytidine (dC).

[0064] Nucleotides typically contain monophosphate, diphosphate, or triphosphate. Phosphate can be attached to the 5' or 3' side of the nucleotide.

[0065] 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 polynucleotides preferably include the following nucleotides: dAMP, dUMP, and / or dTMP and dCMP.

[0066] The nucleotides in the polynucleotide can be linked to each other in any way. Nucleotides are typically linked by their sugar and phosphate groups, as in nucleic acids. The nucleotides can also be linked by their nucleobases, as in pyrimidine dimers.

[0067] 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), threonine nucleic acid (TNA), locked nucleic acid (LNA), or other synthetic polymers with nucleotide side chains. The PNA backbone consists of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The GNA backbone consists of repeating ethylene glycol units linked by phosphodiester bonds. The TNA backbone consists of repeating threose units linked together by phosphodiester bonds. The LNA is formed from nucleotides as discussed above, having additional bridges linking the 2'' oxygen and 4'' carbon in the ribose.

[0068] The preferred polynucleotide is either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

[0069] 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 1000 or more nucleotides, 5000 or more nucleotides in length, or 100000 or more nucleotides in length.

[0070] Helicases can move along all or only part of the target polynucleotides in the method of the present invention. All or part of the target polynucleotides can be characterized using the method of the present invention.

[0071] The target polynucleotide may 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 single-stranded regions or non-hybridized regions. The one or more helicases are capable of binding to one strand of the single-stranded or non-hybridized region. The target polynucleotide preferably includes one or more single-stranded regions or one or more non-hybridized regions.

[0072] sample The target polynucleotide is present in any suitable sample. This invention is typically carried out on samples known to contain or suspected of containing the target polynucleotide. Alternatively, the invention can be carried out on a sample to confirm the identification of one or more target polynucleotides known or expected to be present in the sample.

[0073] The sample may be a biological sample. This invention can be performed in vitro on samples obtained or extracted from any organism or microorganism. The organism or microorganism is typically archaean, prokaryotic, or eukaryotic, and generally belongs to one of the following five kingdoms: plant, animal, fungi, prokaryotes, and protists. This invention is performed in vitro on samples obtained or extracted from any virus. The sample is preferably a liquid sample. The sample typically includes bodily fluids from a patient. The sample may be urine, lymph, saliva, mucus, or amniotic fluid, but preferably blood, plasma, or serum. Typically, the sample is derived from a human, but alternatively it may be derived from other mammalian animals, such as commercially raised animals like horses, cattle, sheep, or pigs, or from pets such as cats or dogs. Alternatively, plant-derived samples are typically obtained from commercial crops such as cereals, legumes, fruits, or vegetables, such as wheat, quinoa, barley, oats, brass, corn, soybeans, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco, kidney beans, lentils, sugarcane, cocoa, and cotton.

[0074] The sample may be a non-biological sample. Non-biological samples are preferably liquid samples. Examples of non-biological samples include surgical fluids, water such as drinking water, seawater or river water, and reagents used for laboratory testing.

[0075] Samples are typically processed before testing, for example by centrifugation or membrane filtration to remove unwanted molecules or cells, such as red blood cells. Testing can be performed immediately after sample acquisition. Samples can also typically be stored prior to analysis, preferably below -70°C.

[0076] When a portion of the target polynucleotide enters the pore and moves relative to the pore along a field generated by the applied potential, such as through the pore, the one or more helicases are moved by the pore through the second segment of the sequence as the polynucleotide moves relative to the pore, such as through the pore. This is because the polynucleotide (including the one or more second segments) moves relative to the pore, such as through the pore, and the one or more helicases remain at the top of the pore.

[0077] method This invention provides a method for controlling the movement of target polynucleotides through nanopores, comprising: I. Provide the aforementioned complex; II. Contacting the composite with nanopores; and III. Apply a potential across the nanopore to cause the polynucleotide-binding protein to move through the second segment and control the movement of the target polynucleotide through the pore.

[0078] The present invention also provides a method for characterizing a target polynucleotide, comprising: 1) Using the aforementioned method, target polynucleotides are made to move through nanopores; and 2) As the polynucleotide moves relative to the pore, one or more electrical and / or optical measurements are acquired, wherein the measurements represent one or more characteristics of the polynucleotide and thereby characterize the target polynucleotide.

[0079] The method of the present invention includes measuring one or more characteristics of the target polynucleotide. The method may include 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) may be measured according to the present invention.

[0080] For (i), the length of the polynucleotide can be determined, for example, by determining the number of interactions between the target polynucleotide and the pore, and the duration of the interaction between the target polynucleotide and the pore.

[0081] 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 target polynucleotide's sequence, or not in conjunction with the determination of the target polynucleotide's sequence. The former is direct; the polynucleotide is sequenced and thus identified. The latter can be accomplished in several ways. For example, the presence of a specific motif in the polynucleotide can be determined (without determining the rest of the polynucleotide's sequence). Alternatively, specific electrical and / or optical signals determined in the method can identify a target polynucleotide from a specific source.

[0082] For (iii), the sequence of the polynucleotide can be determined as previously described. Suitable sequencing methods, especially 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 International Application WO 2000 / 28312.

[0083] For (iv), the secondary structure can be measured in a variety of ways. For example, if the method includes electrical measurements, the secondary structure can be measured using changes in residence time through the pore or variations in current. This allows for the identification of regions of single-stranded and double-stranded polynucleotides.

[0084] For (v), any modification, whether present or absent, can be determined. The method preferably includes determining whether the target polynucleotide has been modified by methylation, oxidation, damage, or by one or more proteins or one or more markers / tags. 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 identified based on the current passing through the pore during the interaction between the pore and each nucleotide.

[0085] This method is typically carried out in the presence of a buffer. In the exemplary apparatus discussed above, the buffer is present in an aqueous solution in the chamber. Any buffer can be used in the method of the present invention. Typically, the buffer is a phosphate buffer. Other suitable buffers are HEPES and Tris-HCl buffers. The method is typically carried out at pH values ​​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.

[0086] This method can be carried out at temperatures ranging from 0 to 100°C, 15°C to 95°C, 16°C to 90°C, 17°C to 85°C, 18°C ​​to 80°C, 19°C to 70°C, or 20°C to 60°C. The method is typically performed at room temperature. Optionally, the method can be carried out at temperatures that support helicase function, such as approximately 37°C.

[0087] This method can be carried out in the presence of free nucleotides or free nucleotide analogs and / or cofactors that assist helicase function. The method can also be carried out in the absence of free nucleotides or free nucleotide analogs and the cofactors of the helicase. The free nucleotide 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 (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and deoxymonophosphate. Adenosine 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). A 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 optimal cofactor for helicase is Mg. 2+ .

[0088] Example 1 This embodiment describes how alkylation modification of thiophosphate nucleotide sequences can inhibit helicase motility.

[0089] like Figure 1As shown in primers A, B, and C, nucleotide chains with thiophosphate groups are modified with ethyl or acetamide, and nucleotide chains with thiophosphate groups that are not alkylated are modified with ethyl or acetamide, respectively, to form corresponding complementary strand forms.

[0090] For primer A, Seq-10T-P_S is first synthesized, and its sequence is as follows: TTTTT TTTTT *C As shown in GGTTTCGTCCGTCG, the synthesis method involves first synthesizing a phosphate-thiocyanate primer, where the nucleotide at the * position is cytosine and its phosphate group is a phosphate-thiocyanate group, followed by an alkylation reaction to add a phenylacetamide group (e.g., ...). Figure 1 As shown in Figure A, where Ph represents phenylacetamide group, its complementary sequence is Seq-com-1.

[0091] For primer B, first synthesize Seq-10T-P_S, the sequence of which is as follows: TTTTT TTTTT *C As shown in GGTTTCGTCCGTCG, the synthesis method involves first synthesizing a phosphate-thiocyanate primer, where the nucleotide at the * position is cytosine and its phosphate group is a phosphate-thiocyanate group, followed by an alkylation reaction to add an ethane group (such as...). Figure 1 As shown in B, where C2H5 represents ethane, its complementary sequence is Seq-com-1.

[0092] For primer C, Seq-10T-P_S is first synthesized, and its sequence is as follows: TTTTT TTTTT *C As shown in GGTTTCGTCCGTCG, the synthesis method involves first synthesizing a phosphate-thiocyanate primer, where the nucleotide at the * position is cytosine, and its phosphate group is a phosphate-thiocyanate group (e.g., ...). Figure 1 (as shown in C), its complementary sequence is Seq-com-1.

[0093] Seq-10T-P_S:TTTTTTTTTT*CGGTT T CGTCCGTCG (SEQ ID NO: 1); Seq-com-1: FAM-CGACG GACGA AACCG (SEQ ID NO: 2).

[0094] The above sequence and its paired sequence were annealed at a ratio of 1:0.8, and 100 nM of annealing primer (e.g., ...) was added to a 10 μL incubation system. Figure 1As shown in A, B, and C), 30× ED1 enzyme (its sequence is shown in SEQ ID NO: 3) (dilution ratio 1:30), 2× incubation buffer (50mM KCl, 20mM HEPES, pH 7.0, 1mM EDTA), incubated at 30℃ for 1h, then 1000× TMAD was added, and incubation continued at 30℃ for another 30min, finally 10mM ATP / Mg 2+ Process for 20 minutes. For primers A, B, and C, anneal the corresponding double strands (e.g., ...). Figure 2 Lanes 1, 4, and 7), the complex formed after enzyme incubation (e.g., ... Figure 2 Lanes 2, 5, 8 and ATP / Mg 2+ The processed products (e.g.) Figure 2 Gel electrophoresis was performed on lanes 3, 6, and 9.

[0095] T4 Dda-M1G / E94C / C109A / C136A / A360C: SEQ ID NO: 3 GTFDDLTEGQKNAFNIVMKAIKEKKHHVTINGPAGTGKTTLTKFIIEALISTGETGIILAAPTHAAKKILSKLSGKEASTIHSILKINPVTYECNVLFEQKEVPDLAKA RVLICDEVSMYDRKLFKILLSTIPPWATIIGIGDNKQIRPVDPGENTAYISPFFTHKDFYQCELTEVKRSNAPIIDVATDVRNGKWIYDKVVDGHGVRGFTGDTALRDFM VNYFSIVKSLDDLFENRVMAFTNKSVDKLNSIIRKKIFETDKDFIVGEIIVMQEPLFKTYKIDGKPVSEIIFNNGQLVRIIEAEYTSTFVKARGVPGEYLIRHWDLTVET YGDDEYYREKIKIISSDEELYKFNLFLGKTCETYKNWNKGGKAPWSDFWDAKSQFSKVKALPASTFHKAQGMSVDRAFIYTPCIHYADVELAQQLLYVGVTRGRYDVFYV As shown in primer A, phenylacetamide modification of the phosphate group exhibits a significant blocking effect. Primer structure B (ethyl-modified phosphate group) also shows a significant blocking effect, while thiophosphate modification, i.e., primer structure C, has no blocking effect on ED1 enzyme.

[0096] The above results indicate that in the primer structure, the single-stranded region is equivalent to the first segment described in this invention and can be used to bind ED1 enzyme (polynucleotide binding protein), while the polynucleotide region modified with phenylacetamide or ethyl can act as an enzyme blocker.

[0097] Example 2 Based on Example 1, we further explored increasing the number of phenylacetamide groups on the phosphate group. The experiment was conducted according to the method described in Example 1.

[0098] For primer ds-2, Seq-10T-P_S_2 is first synthesized, and its sequence is as follows: TTTTT TTTTT *C* As shown in GGTTTCGTC CGTCG, the synthesis method is to first synthesize a thiophosphate primer, in which the nucleotides at * are cytosine and guanine, and their phosphate groups are thiophosphate groups, and then perform an alkylation reaction to add two phenylacetamides, whose complementary sequence is Seq-com-1.

[0099] For primer ds-3, Seq-10T-P_S_3 is first synthesized, and its sequence is as follows: TTTTT TTTTT *C* As shown in G*GTTTCGTC CGTCG, the synthesis method is to first synthesize a thiophosphate primer, in which the nucleotide at * is cytosine and two guanines, and its phosphate group is a thiophosphate group, and then perform an alkylation reaction to add three phenylacetamides, whose complementary sequence is Seq-com-1.

[0100] For primer Ds-4, Seq-10T-P_S_4 is first synthesized, and its sequence is as follows: TTTTT TTTTT *C* As shown in G*G*TTTCGTC CGTCG, the synthesis method is to first synthesize a thiophosphate primer, in which the nucleotides at * are cytosine, two guanines and thymine, and their phosphate groups are thiophosphate groups. Then, an alkylation reaction is performed to add four phenylacetamides, and its complementary sequence is Seq-com-1.

[0101] The above sequence and its paired sequence were annealed at a ratio of 1:0.8. 100 nM of annealing primer, 30× ED1 enzyme (sequence shown in SEQ ID NO: 3) (dilution ratio 1:30), and 2× incubation buffer (50 mM KCl, 20 mM HEPES, pH 7.0, 1 mM EDTA) were added to a 10 μL incubation system. After incubation at 30°C for 1 h, 1000× TMAD was added, and incubation continued at 30°C for another 30 min. Finally, 10 mM ATP / Mg was added. 2+ Process for 20 minutes. For primers A, B, and C, anneal the corresponding double strands (e.g., ...). Figure 3Lanes 1, 4, and 7), the complex formed after enzyme incubation (e.g., ... Figure 3 Lanes 2, 5, 8 and ATP / Mg 2+ The processed products (e.g.) Figure 3 Gel electrophoresis was performed on lanes 3, 6, and 9.

[0102] The results are as follows Figure 3 As shown, the results indicate that as the number of phenylacetamide groups on the phosphate group increases, the binding of multiple enzymes becomes more pronounced, thus weakening the helicase inhibition phenomenon. It is speculated that phenylacetamide modification on the phosphate group also has the ability to bind helicases.

[0103] Example 3 SEQ ID NO: 4 ATCCT TTTTA GAATT TTAGA GAT TTTTT TTTTT A GAGA TTCAGAGATT CAGAG ATTCA GAG SEQ ID NO: 5 ATCTC TAAAA TTCTA AAAAG GAT SEQ ID NO: 6 CTCTG AATCT CTGAA TCTCT GAATC TCTAG TCCAG CACCG ACC SEQ ID NO: 7 GGTCG GTGCT GGACT 1. The sequence of the joint structure is as follows: ① Y1-S: 5'-XXXXX XXXXX XXXXX XXXXX ATCCT TTTTA GAATT TTAGA GAT TTTTT TTTTT *AGAGA TTCAG AGATT CAGAG ATTCA GAG -3', where *A The phosphate group was replaced with phenylacetamide to modify the phosphate group.

[0104] The Y1-S chain contains a leader sequence, i.e., iSpC3, denoted as X, which is connected to the 5′ end of SEQ ID NO: 5.

[0105] ② Y2-S: 5'- ATCTC TAAAA TTCTA AAAAG GAT -3' ③ Y-Bottom-S:5'-P- CTCTG AATCT CTGAA TCTCT GAATC TCT AG TCCAG CACCG ACC -3' ④ Tether-S: 5'-Chol-(iSpC3)20- GGTCG GTGCT GGACT -3' Where Chol represents cholesterol, and (iSpC3)20 represents a chain composed of 20 iSpC3 molecules.

[0106] Using the above sequences, synthesize as follows: Figure 4 The adapter shown in (A) was synthesized as follows: Primers ①, ②, and ③ were annealed in a ratio of 1:2.5:2.5, with a final annealing concentration of 4 μM. The annealing program was 98℃ for 10 min; 6 s / -0.1℃, 300 × Cys; 65℃ for 5 min; 6 s / -0.1℃, 400 × Cys; 12℃, Hold.

[0107] The annealed primers were incubated according to the system shown in Table 1 below.

[0108] Table 1: Incubation system of primers after annealing Add the sample to a 1.5 mL low-adsorption centrifuge tube (wrapped in aluminum foil to protect from light), mix gently (do not use a vortex mixer), and place in a 30°C metal bath for 30 min. Finally, purify the incubated product using magnetic beads to obtain the helicase-bound complex.

[0109] 2. Sequencing using synthetic adapters: Prepare the connection reaction system according to the table below, centrifuge briefly, and let stand at room temperature for 10 minutes.

[0110] Table 2: Systems for the linkage reaction: The product was purified using magnetic beads to obtain the product to be sequenced.

[0111] Using the QNome9604 sequencing platform, 4 μL of primer ④ at a concentration of 1 μM was added to 200 μL of sequencing buffer (600 mM KCl, 10 mM HEPES pH 8.0, 3 mM MgCl2, 3 mM ATP), vortexed to mix, and briefly centrifuged. The mixture was then added to the sequencing chip and allowed to stand for 15 min. Next, 40 fmol of the prepared adapter was added to 200 μL of sequencing buffer, gently inverted to mix, and briefly centrifuged. The mixture was then added to the sequencing chip and allowed to stand for 15 min before sequencing began.

[0112] The test was conducted under the conditions of 600mM KCl, 10mM HEPES, pH 8.0, 3mM MgCl2, 3mM ATP, and 35℃. The signals captured during the signal acquisition process and some magnified images are shown below. Figure 4As shown in (B), the figure demonstrates that a complete sequencing signal was obtained. This indicates that the modified structure inhibits the movement of helicase along the target polynucleotide sequence before reaching the nanopore for sequencing. Normal movement along the target polynucleotide sequence only begins when the adapter complex contacts the nanopore and the inhibitory effect of the modified structure is released under voltage. This achieves the goal of obtaining a complete sequencing signal.

[0113] 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 these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims. sequence list <110> Beijing Qitan Technology Co., Ltd. <120> Sequences, connectors containing sequences, and their uses <130> 21NI2699 <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 25 <212> DNA <213> artificial sequence <400> 1 tttttttttt cggtttcgtc cgtcg 25 <210> 2 <211> 16 <212> DNA <213> artificial sequence <400> 2 cgacggacga aaaccg 16 <210> 3 <211> 439 <212> PRT <213> artificial sequence <400> 3 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 <210> 4 <211> 61 <212> DNA <213> artificial sequence <400> 4 atccttttta gaattttaga gatttttttt tttagagatt cagagattca gagattcaga 60 g 61 <210> 5 <211> 23 <212> DNA <213> artificial sequence <400> 5 atctctaaaa ttctaaaaag gat 23 <210> 6 <211> 43 <212> DNA <213> artificial sequence <400> 6 ctctgaatct ctgaatctct gaatctctag tccagcaccg acc 43 <210> 7 <211> 15 <212> DNA <213> artificial sequence <400> 7 ggtcggtgct ggact 15

Claims

1. A sequence for binding to and blocking polynucleotide-binding proteins, characterized in that, The sequence comprises a first segment and a second segment. The first segment is used to bind the polynucleotide-binding protein, and the second segment is used to block the polynucleotide-binding protein. The second segment comprises a polynucleotide whose phosphate group is modified. The modified phosphate group is a thiophosphate group, and the modification includes C... 1-6 -alkyl or phenylacetamide group, the modification reduces the net negative charge of the polynucleotide, thereby achieving the retardation of the polynucleotide-binding protein; The polynucleotide-binding protein includes helicase.

2. The sequence according to claim 1, wherein the number of modified phosphate groups in the polynucleotide of the second segment is one or more.

3. The sequence according to claim 1, wherein the polynucleotide-binding protein is derived from a polynucleotide processing enzyme; The helicase is selected from one or more of Hel308 helicase, RecD helicase, Tral helicase, TrwC helicase, XPD helicase, and DDA helicase.

4. A method for preparing the sequence according to any one of claims 1 to 3, comprising: Synthesize a sequence comprising a first segment and a second segment, wherein one or more nucleotides in the second segment have phosphate groups that are thiophosphate groups; and The thiophosphate group is alkylated, thereby modifying the phosphate group of the polynucleotide in the second segment.

5. A linker for characterizing a target polynucleotide, wherein the linker comprises the sequence of any one of claims 1 to 3 for binding and blocking polynucleotide-binding proteins, and the linker comprises the following sequence: ① Y1-S: 5'- (iSpC3)20-ATCCT TTTTA GAATT TTAGA GAT TTTTTTTTTT *AGAGA TTCAGAGATT CAGAG ATTCA GAG-3', where the phosphate group of *A is replaced with a phenylacetamide-modified phosphate group; ② Y2-S: 5'-ATCTC TAAAA TTCTA AAAAG GAT -3'; ③ Y-Bottom-S: 5'-P-CTCTG AATCT CTGAA TCTCT GAATC TCT AG TCCAG CACCG ACC -3'; ④ Tether-S: 5'-Chol-(iSpC3)20-GGTCG GTGCT GGACT-3'; Where Chol represents cholesterol, and (iSpC3)20 represents a chain composed of 20 iSpC3 molecules.

6. A construct for characterizing a target polynucleotide, wherein, The construct comprises a target polynucleotide and a linker as described in claim 5, wherein the linker is connected to either end or both ends of the target polynucleotide.

7. A complex for characterizing a target polynucleotide, wherein, The complex comprises the linker of claim 5 or the construct of claim 6, and a polynucleotide-binding protein; The polynucleotide-binding protein binds to the first segment of the linker or the construct and can stop at the second segment.

8. A kit for characterizing target polynucleotides, the kit comprising: (a) one or more connectors as described in claim 5, (b) a polynucleotide-binding protein.

9. A method for controlling the movement of a target polynucleotide through a nanopore, comprising: I. The complex of claim 7; II. Contact the composite with nanopores; as well as III. Apply a potential across the pore to cause the polynucleotide-binding protein to move through the second segment and control the movement of the target polynucleotide through the pore.

10. A method for characterizing a target polynucleotide, comprising: 1) Using the method of claim 9, the target polynucleotide is moved through a nanopore; as well as 2) As the polynucleotide moves relative to the pore, one or more electrical and / or optical measurements are acquired, wherein the measurements represent one or more characteristics of the polynucleotide and thereby characterize the target polynucleotide.

11. The method according to claim 9 or 10, wherein the target polynucleotide comprises deoxyribonucleic acid and / or ribonucleic acid; and / or At least a portion of the polynucleotide is double-stranded; and / or The sequence for binding and blocking polynucleotide-binding proteins is included in the single-stranded region or non-hybridized region of the polynucleotide.

12. A method for controlling the loading of one or more polynucleotide-binding proteins onto a target polynucleotide, comprising: a. Provide a target polynucleotide having one or more sequences as described in any one of claims 1 to 3; as well as b. Contact the target polynucleotide provided in step a with the polynucleotide-binding protein, such that the polynucleotide... The nucleotide-binding protein binds to the target polynucleotide and stops at the second segment.

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