Method

Abstract

To provide a method of loading a motor protein onto a polynucleotide adapter, a method for characterizing a polynucleotide using this method, and a novel polynucleotide adapter.SOLUTION: There is provided a method of loading a motor protein onto a polynucleotide adapter, the method comprising: i) providing a polynucleotide adapter comprising a spacer; ii) contacting the polynucleotide adapter with a motor protein; and iii) positioning the motor protein on the spacer, wherein a blocking moiety bound to the polynucleotide adapter prevents the motor protein from moving off the spacer.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention provides a method for loading a motor protein onto a polynucleotide adaptor, Methods for characterizing polynucleotides using the novel methods, and novel polynucleotide assays Regarding adapters. [Background technology]

[0002] Nanopore sensing relies on the individual binding or interaction between an analyte molecule and an ion-conducting channel. Nanoparticles are an approach to the detection and characterization of analytes that relies on the observation of interaction events. The sensor is a nanometer-sized single pore placed in an insulating film, which detects the pore in the presence of an analyte molecule. The nanopore can be fabricated by measuring the voltage-driven ionic current through the nanopore. In the presence of an analyte in or near the pore, the ion flow through the channel is altered, This will result in a change in the ion current or current that is measured. , its unique current signature, in particular the duration and extent of current block, as well as the pore and This is manifested through the fluctuations in the current level during the interaction time.

[0003] Polynucleotides are important analytes for sensing in this manner. Nanopore sensing of analytes reveals the identity and specificity of the sensed analytes Single molecule counting can be performed, but their composition, such as their nucleotide sequence, etc. It also provides information on the presence of features such as base modifications, oxidation, reduction, decarboxylation, and deamination. Nanopore sensing can also provide rapid and inexpensive polynucleotide sequencing. This could potentially enable single molecule sequence reads of polynucleotides that are tens to tens of thousands of bases long. to deliver.

[0004] Two of the key components of polymer characterization using nanopore sensing are: 1) Control of polymer movement through the pores, and (2) the ability to detect the movement of the polymer through the pores. The identification of the constituent building blocks for the nanopore During sensing, it is important to control the movement of the polynucleotide relative to the pore. Failure to control the movement of the polynucleotides may prevent or hinder accurate characterization of the polynucleotides. For example, if the movement of the polynucleotide relative to the pore is uncontrolled, It becomes difficult to precisely distinguish each nucleotide in a homopolymeric polynucleotide.

[0005] To address this issue, polymers such as polynucleotides have been characterized. It is known that bacteria use motor proteins to control their movement. The target proteins include polymerases such as helicases, exonucleases, and topoisomerases. The motor proteins transport polynucleotides. Thus, motor proteins are used to regulate the transport of the pore The movement of polymers such as polynucleotides can be controlled.

[0006] Use of motor proteins to generate polynucleotides for characterization by nanopore sensing Although it has become possible to achieve significant improvements in the processing of nucleotides, technical challenges remain. One problem is that the identity of the motor protein must be determined before characterization can begin. This involves ensuring that the nucleic acid is in the correct position relative to the polynucleotide sequence. For example, motor proteins may have already begun processing polynucleotides before characterization began. In this case, the portion of the polynucleotide that was processed before the start of characterization cannot be accurately determined. It's possible.

[0007] To address this, we first characterized the motor domains on the polynucleotide adapters. Motor proteins are known to "stall" the protein. In this way, the target polynucleotide is not processed until it is detected. Improved characterization is possible.

[0008] One method known in the art is to use a spacer to attach a helical molecule to a molecular adaptor. The spacer is characterized by its ability to stall motor proteins such as ribosomal enzymes. This prevents the movement of motor proteins (e.g., helicases) to the target polynucleotide. In the absence of external force, the motor protein binds to the target polynucleotide. However, the transfer of the protein (helicase) is prevented. Under the influence of an external force, which can be provided by passing a nanopore through the The target polynucleotide is then translocated to the pore, thereby inhibiting the translocation of the target polynucleotide relative to the pore. Such a method is described in WO2014 / 1 It is listed in 35838.

[0009] Although methods to stall motor proteins have provided great benefits, Production of "fuel" molecules (e.g., cofactors required for polynucleotide processing) by target proteins The efficiency of such systems could be improved if turnover could be reduced. Therefore, the polynucleotide is linked to a molecular adaptor that addresses this issue. There is a need for improved methods for loading Summary of the Invention

[0010] The present disclosure relates to a method for loading a motor protein onto a polynucleotide adaptor. The method includes providing a polynucleotide adaptor that includes a spacer. The polynucleotide adaptor contacts the motor protein and binds it to the target protein. The blocking moiety is then bound to the polynucleotide adaptor. Blocking moieties are attached to polynucleotide adaptors to inhibit motor transcription. The protein is prevented from moving away from the spacer. By preventing motor proteins from moving away from the polynucleotide The motor protein turns over fuel molecules slower than when it is bound to a This may result in beneficial effects such as

[0011] Thus, a method for loading motor proteins onto polynucleotide adaptors is So, i) providing a polynucleotide adaptor comprising a spacer; ii) contacting the polynucleotide adaptor with a motor protein; iii) disposing the motor protein on the spacer, The blocking moiety attached to the polynucleotide adaptor inhibits the motor protein from stimulating the Methods are provided herein to prevent migration away from the pacer.

[0012] In some embodiments, the method comprises: i) providing a polynucleotide adaptor, a spacer and a blocking moiety attached to the polynucleotide adaptor; Including, providing, ii) contacting the polynucleotide adaptor with a motor protein; iii) disposing the motor protein on the spacer, The blocking moiety prevents the motor protein from moving off the spacer. .

[0013] 1. A method for loading a motor protein onto a polynucleotide adaptor, comprising: i) providing a polynucleotide adaptor comprising a spacer; ii) contacting the polynucleotide adaptor with a motor protein; iii) advancing the motor protein over the spacer; and iv) attaching a blocking moiety to the polynucleotide adaptor; The blocking moiety prevents the motor protein from moving off the spacer. , a method is also provided.

[0014] In one embodiment, the method comprises: i) providing a polynucleotide adaptor comprising a loading site connected to a spacer; To provide ii) contacting the loading site with a motor protein; iii) advancing the motor protein from the loading site onto the spacer; and , iv) attaching a blocking moiety to the polynucleotide adaptor; The blocking portion prevents the motor protein from being released from the spacer and transferred to the loading site. to prevent it from moving.

[0015] In one embodiment, step (ii) of the method comprises attaching a loading site to a motor protein. wherein the motor protein binds to the loading site, Step (iv) comprises attaching a blocking moiety to the polynucleotide adaptor. The blocking portion prevents the motor protein from moving away from the spacer and from This prevents the nucleotide from rebinding to the binding site.

[0016] In one embodiment, the polynucleotide adaptor comprises a loading molecule connected to a spacer. The motor protein binds to the loading site of the polynucleotide adaptor. Combine.

[0017] In some embodiments, advancing the motor protein over the spacer comprises advancing the motor protein over the spacer. In one embodiment, the motor protein is activated by applying a physical or chemical force to the motor protein. -Moving a protein along a spacer involves directing a motor protein to one or more fuels. In one embodiment, the polynucleotide adaptor is contacted with a molecule. The motor protein comprises a loading site connected to a first motor protein. The first motor protein is advanced from the loading site onto the spacer. The loading of the second motor protein into the loading site and the second advancing the motor protein from the loading site toward the spacer; The second motor protein presses the first motor protein against the spacer. In one embodiment, the blocking moiety is attached to a polynucleotide adaptor. This presses the motor protein against the spacer.

[0018] In some embodiments, the polynucleotide adaptor is a lone nucleotide connected to a spacer. In one embodiment, the blocking moiety comprises a loading moiety and the blocking moiety binds to the loading moiety. , the loading site is adjacent to the spacer, and the blocking moiety is directly attached to the spacer. It binds to an adjacent loading site.

[0019] In one embodiment, step (iii) comprises providing a spacer at the active site of the motor protein. The method includes advancing a motor protein over the spacer so as to occupy the spacer.

[0020] In some embodiments, the polynucleotide adaptor is a lone nucleotide connected to a spacer. The loading site contains a single-stranded or unhybridized polynucleotide. In one embodiment, the loading site comprises a sequence of about 2 to about 1000 nucleotide units in length. The present invention includes single-stranded or unhybridized polynucleotides having the following structure:

[0021] In some embodiments, the blocking moiety is a physical or chemical blocking moiety. In one embodiment, the blocking moiety is attached to a polynucleotide adaptor. This sterically prevents the movement of the motor protein away from the spacer. In embodiments, the blocking moiety is attached to a polynucleotide adaptor. In this way, chemical groups are introduced that prevent the movement of the motor protein away from the spacer.

[0022] In some embodiments, (i) the loading moiety is a single-stranded or unhybridized polypeptide. the blocking moiety comprises a single stranded or unhybridized polynucleotide; (ii) binding a blocking moiety to the loading site In one embodiment, the loading portion hybridizes to the loading site. The binding portion is a single-stranded or non-hybridized nucleic acid having a length of about 2 to about 1000 nucleotide units. Contains soybean polynucleotides.

[0023] In some embodiments, the spacer is i) one or more nitroindoles, one or more inosines, one or more acridines, one or more of 2-aminopurine, one or more of 2-6-diaminopurine, one or more of 5-bromo- Deoxyuridine, one or more inverted thymidines (inverted dT), one or more inverted dideoxy- Thymidine (ddT), one or more dideoxycytidines (ddC), one or more 5-methyl cytidine, one or more 5-hydroxymethylcytidines, one or more 2'-O-methyl R NA base, one or more iso-deoxycytidines (Iso-dC), one or more iso-deoxy Iso-dG, one or more C3(OC 3 H 6 OPO 3 ) group, one or more of photocleavable (PC)[OC 3 H 6 -C(O)NHCH 2 -C 6 H 3 NO 2 -CH(C H 3 )OPO 3 ] group, one or more hexanediol groups, one or more spacers 9(iSp 9) [(OCH 2 CH 2 ) 3 OPO 3 ] group, or one or more spacers 18 (iSp1 8) [(OCH 2 CH 2 ) 6OPO 3 ] group, ii) one or more thiol connections; iii) one or more abasic nucleotides; iv) one or more nucleotides having a backbone structure different from that of the loading site; v) one or more chemical groups that stall one or more motor proteins; and / or vi) a polymer, optionally a polypeptide or polyethylene glycol (PEG).

[0024] In some embodiments, the spacer comprises one or more nucleotides, preferably , the spacer comprises one or more nucleotide islands.

[0025] In some embodiments, i) A polynucleotide adaptor comprises one or more nucleotides, preferably one or more A spacer containing a nucleotide island and a polynucleotide adapter are attached to the spacer. a blocking moiety, ii) Placing the motor protein on a spacer allows the motor protein to be attached to the spacer. Contact with the spacer and preventing the motor protein from detaching from the spacer and modifying the motor protein so as to

[0026] In some embodiments, the spacer is -SNSNSNS-, -SNNSNNSNNS-, -S -SSSSSNNSS-, -SSSSSNNSNNSSS-, -SSSNNS -NNSSNNS-, -SSSSSNNSNNSSNNS-, -SSSN -NSNNSNNS-, -NNSSNNSSSSS-, -SNNSSNNS -SSS-, -SSNNSSNNSSS-, -SSSSNNSS -NNS-, -NNSNNSSSSSSS-, -SNNSNNSSSSS-, -SSNNSNNS -SSS-, -SSSNNSNNSSS-, -SSSSNNSNNSS-, and -SSSNNS- SNNS-, where each S is a spacer unit and each N is a nucleotide.

[0027] In some embodiments, the motor protein is a helicase, polymerase, exon In one embodiment, the enzyme is a polynucleotide that is a nuclease, a topoisomerase, or a variant thereof. The motor protein on the spacer of the nucleotide adaptor is In one embodiment, the modified spacer is also modified to prevent it from being released from the spacer. Each motor protein is involved in the synthesis of Hel308 helicase, RecD helicase, and TraI helicase. helicase, TrwC helicase, XPD helicase, and Dda helicase, or The helicase is independently selected from these mutant forms.

[0028] In one embodiment, the method further comprises: (v) excess motor protein not located on a spacer. The method further comprises the step of removing the matrix molecules.

[0029] 1. A method for controlling translocation of a target polynucleotide relative to a transmembrane nanopore, comprising: i) (A) a target polynucleotide, (B) a polynucleotide adaptor comprising a spacer and (C) providing a motor protein. ii) carrying out the method according to any one of the above embodiments, thereby producing a poly Stalling a motor protein on the spacer of the nucleotide adaptor; iii) Deletions on the spacer of the target polynucleotide and the polynucleotide adaptor contacting the accelerated motor protein with a nanopore; iv) applying a voltage potential across the transmembrane nanopore, thereby activating the motor protein; The target polynucleotide is then translocated across the spacer, thereby targeting the nanopore. and controlling the movement of the target polynucleotide.

[0030] In one embodiment, after the motor protein has stalled on the polynucleotide adaptor, In another embodiment, the polynucleotide adaptor binds to the target polynucleotide. After the polynucleotide adaptor binds to the target polynucleotide, the motor protein The quality stalls on the polynucleotide adapter.

[0031] 1. A method for controlling translocation of a target polynucleotide relative to a transmembrane nanopore, comprising: i) providing a target polynucleotide; ii) A polynucleotide that contains a spacer and on which the motor protein stalls. and a polynucleotide adaptor comprising the polynucleotide adaptor disclosed herein. and providing the method according to claim 1, iii) contacting the target polynucleotide and the polynucleotide adaptor with the nanopore. And iv) applying a voltage potential across the transmembrane nanopore, thereby activating the motor protein; The target polynucleotide is then translocated across the spacer, thereby targeting the nanopore. and controlling the movement of the target polynucleotide.

[0032] In some embodiments, the method further comprises: Upon detection of the target polynucleotide, one or more measurements are made that are indicative of one or more characteristics of the target polynucleotide. This allows characterization of the target polynucleotide as it translocates relative to the nanopore. Further includes:

[0033] (i) a spacer; and (ii) a motor protein stalled on the spacer, The active site of the motor protein is occupied by a spacer. and (iii) a blocking moiety attached to the adaptor, the blocking moiety being linked to the motor protein. and a blocking portion that prevents the polymer from migrating off the spacer. Nucleotide adaptors are also provided.

[0034] In one embodiment, (i) the adaptor comprises a loading site connected to a spacer. (ii) a blocking moiety is attached to the loading site; and (iii) the blocking moiety is This prevents target proteins from binding to the loading site.

[0035] In one embodiment, the polynucleotide adaptor comprises: i) In the 5' to 3' direction, B -SD} n or {DSL B} n (In the formula, L B is a blocked loading site, S is a spacer, and D is a double-stranded polynucleotide. leutide, and n is an integer, optionally an integer from 1 to about 20; ii) one or more motor proteins stalled on a spacer (S), The or each L B The portion is a double stranded polynucleotide comprising the or each motor protein. Prevents movement off of the spacer (S) in a direction away from (D).

[0036] In one embodiment, the polynucleotide adaptor comprises: i) In the 5' to 3' direction, B -SD} n or {DSL B} n (In the formula, L B is a first double-stranded polynucleotide, S is a spacer, and D is a second double-stranded polynucleotide. nucleotides, n being an integer, optionally an integer from 1 to about 20; Polynucleotide (L B ) is adjacent to the spacer (S), and the spacer (S) is the second (D)) ii) One or more motor proteins stall on the spacer (S).

[0037] 1. A kit for modifying a target polynucleotide, comprising: i) a polynucleotide adaptor comprising a spacer; ii) a motor protein capable of controlling the movement of a target polynucleotide; iii) the motor protein is located on the spacer of the polynucleotide adaptor; When the active site of the motor protein is occupied by a spacer, the motor protein The polynucleotide adapter is inserted so that the protein is prevented from moving away from the spacer. and a blocking moiety capable of binding to a peptide.

[0038] In one embodiment, the polynucleotide adaptor comprises a loading site and a blocking site. The binding moiety is a polypeptide that prevents the motor protein from binding to the loading site. It can be bound to a oligonucleotide adaptor.

[0039] 1. A kit for modifying a target polynucleotide, comprising: i) a polynucleotide adaptor, comprising a spacer and a polynucleotide adaptor a blocking moiety attached to said polynucleotide adaptor; ii) a motor protein capable of controlling the movement of a target polynucleotide; Including, When the motor protein is bound to the adaptor, the blocking moiety binds to the motor Kits are also provided that prevent the protein from migrating off the spacer.

[0040] In some embodiments, the polynucleotide adaptor or primer described in the preceding embodiment The bits may each independently be a loader as defined in any one of the preceding embodiments. The polypeptide comprises a binding site, a blocking moiety, a spacer, and / or a motor protein. [Brief description of the drawings]

[0041] [Figure 1]Using the methods disclosed herein, wasteful turnover is reduced. Figure 1(A) shows a schematic diagram of a construct that includes double-stranded DNA, followed by a spacer (4xSp18 spacer group), followed by double-stranded DNA. Because the spacer is adjacent to the double-stranded DNA, there is no single-stranded DNA accessible at either end of the spacer. The Dda helicase stalls on the spacer. Figure 1(B) shows a similar construct where the spacer is not adjacent to the double-stranded DNA and the bottom strand is missing 10 residues. Thus, this construct includes single-stranded DNA adjacent to the 5' end of the spacer. Figure 1(C) shows a bar graph comparing the catalytic ATP turnover rates by the stalled Dda helicase enzyme on the constructs of Figure 1(A) and Figure 1(B), respectively. This data is discussed in Example 1. [Diagram 2] Loading titration results showing effective stalling of motor proteins on adaptors containing spacers as defined herein. This data is discussed in Example 3. [Diagram 3] Results showing improved efficiency when exemplary adapters according to the methods described herein were tested in a DNA sequencing system compared to adapters lacking the spacer described herein. This data is discussed in Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The present invention will now be described with respect to particular embodiments and with reference to certain drawings. The invention is not limited thereto, but rather is limited only by the scope of the claims. Of course, any reference signs should not be construed as limiting the scope. However, not all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. It should be understood that this is not necessarily the case. It is to be understood that the present invention is not limited to the disclosed embodiments, and may be modified without necessarily achieving other aspects or advantages that may be taught or suggested in the specification. The invention is embodied in a manner that achieves or optimizes one or a group of advantages taught herein. It will be appreciated that the present invention may be embodied or practiced in any manner.

[0043] The present invention, both as to its organization and method of operation, together with its features and advantages, is described in the accompanying The present invention is best understood by reference to the following detailed description when read in conjunction with the drawings. Aspects and advantages of the invention will become apparent from and will be apparent from and described hereinafter. Throughout this specification, the terms "one embodiment" and "an embodiment" will be used. Reference to an embodiment of the present invention is to be understood as meaning that a particular feature, structure, or characteristic described in connection with that embodiment is incorporated herein by reference. In at least one embodiment of the present invention, The appearance of the phrases "in one embodiment" or "in an embodiment" in various places is necessarily All of the above references are not necessarily intended to refer to the same embodiment, although they may. The description of the exemplary embodiment is provided to simplify the disclosure and to provide a clear understanding of one or more of the various inventive aspects. To aid in understanding the foregoing, various features of the present invention may be presented in the form of a single embodiment, figure, or combination thereof. However, it should be understood that the method of the present disclosure may be summarized as follows: , the claimed invention comprises more features than are expressly recited in each claim. Rather, the following patent claims should not be construed as reflecting an intention to require As the claims reflect, inventive aspects may comprise all features of a single foregoing disclosed embodiment. It doesn't meet the criteria.

[0044] Unless the context dictates otherwise, "embodiments" of the present disclosure may be specifically combined together. It is to be understood that specific combinations of all disclosed embodiments (if the context requires) may be combined. Unless otherwise implied, these are further disclosed embodiments of the claimed invention.

[0045] Additionally, as used in this specification and the appended claims, the singular forms "a," "a "n," "the," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "polynucleotide" includes two or more polynucleotides. and reference to a "motor protein" includes two or more such proteins, Reference to a "helicase" includes two or more helicases and reference to a "monomer" includes two or more monomers. and a reference to "a pore" includes two or more pores.

[0046] All publications, patents, and patent applications cited herein, either above or below, are hereby incorporated by reference. No. 6,399,433, all of which are incorporated herein by reference in their entireties.

[0047] definition Indefinite or definite articles when referring to a singular noun, e.g. "a" or "an", "th When "e" is used, this includes the plural of the noun, unless otherwise specified. When the term "comprises" is used in the present description and claims, it includes other elements. Furthermore, the first, second and third elements or steps in the description and claims are not excluded. Terms such as second, third, etc. are used to distinguish between similar elements and do not necessarily refer to the order in which they occurred. They are not necessarily used to describe the order or chronological order of events. It is understood that the terms are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are It should be understood that the steps may operate in orders other than those described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the present invention. Unless expressly defined, all terms used herein are understood by those skilled in the art of the present invention. The skilled artisan should consult, especially Sambro for definitions and technical terms. ok et al.,Molecular Cloning: A Laboratory Manual,4 th ed., Cold Spring Harbor Press ,Plainsview,New York(2012), and Ausubel et al. al.,Current Protocols in Molecular Biol ogy(Supplement 114),John Wiley & Sons,Ne See, e.g., John W. York (2016). The definitions provided herein are understood by those of skill in the art. It should not be construed to be narrower than the scope of the disclosure.

[0048] As used herein, "about" refers to measurable values, such as amounts and durations of time. , ±20% or ±10% from the specified value, more preferably ±5%, even more preferably and is intended to encompass a variation of at least ±1%, and even more preferably ±0.1%. Any of the variations described herein are suitable for carrying out the disclosed methods.

[0049] As used herein, the term "nucleotide sequence," "DNA sequence," or "nucleic acid molecule" refers to a is a nucleotide sequence of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. As used herein, the term includes double- and single-stranded DNA and RNA. The term "nucleic acid" as used herein refers to a nucleic acid in which the 3' and 5' ends of each nucleotide are phosphodiesterase. A single- or double-stranded covalently linked nucleotide sequence linked by cyclic bonds. A polynucleotide can be composed of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids can be synthetically produced in vitro or naturally occurring. The nucleic acid may be isolated from a natural source. DNA or RNA that has been modified with post-translational modifications, e.g., 7-methylguanosine 5'-capping, 3'-processing such as cleavage and polyadenylation of The nucleic acid may further include an RNA that is undergoing splicing. (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol Synthesis of GNA, locked nucleic acid (LNA), and peptide nucleic acid (PNA) The sequences of nucleic acids, also referred to herein as "polynucleotides", may also be used. The length is typically measured in terms of the number of base pairs (bp) in a double-stranded polynucleotide, or in terms of the number of base pairs (bp) in a single-stranded polynucleotide. In the case of nucleotides, it is expressed as the number of nucleotides (nt). 1000bp or nt A polynucleotide less than about 40 nucleotides in length is Typically called "oligonucleotides," they are synthesized by polymerase chain reaction (PCR) and other methods. The nucleic acid sequence may include primers for use in manipulating the DNA.

[0050] The term "amino acid" in the context of this disclosure is used in its broadest sense and refers to each amino acid. The amine (NH 2 ) and carboxyl ( In some embodiments, the term "COOH" is intended to include organic compounds that contain a COOH functional group. Amino acid refers to naturally occurring L α-amino acids or residues. Commonly used one-letter and three-letter abbreviations for the acids: A=Ala, C=Cys, D= Asp, E=Glu, F=Phe, G=Gly, H=His, I=Ile, K=Lys, L=Leu, M=Met, N=Asn, P=Pro, Q=Gln, R=Arg, S=Se As used herein, r=Thr, V=Val, W=Trp, and Y=Tyr. Lehninger, AL, (1975) Biochemistry, 2d ed. , pp.71-92, Worth Publishers, New York). The general term "amino acids" refers to D-amino acids, retro-inverso amino acids, and amino acids. Chemically modified amino acids, such as glycine analogues, which are normally incorporated into proteins, such as norleucine, Naturally occurring amino acids that are not included in the It further includes chemically synthesized compounds having properties known in the art. Phenylalanine allows for the same conformational restriction of peptide compounds as Phe or Pro. Analogs or mimetics of nin or proline are included within the definition of amino acid. The amino acids and mimetics are referred to herein as "functional equivalents" of the respective amino acids. Other examples of non-ionic acids are described in Roberts and Vel, incorporated herein by reference. laccio,The Peptides:Analysis,Synthesis,B iology,Gross and Meiehofer,eds.,Vol.5 p. 341, Academic Press, Inc., NY 1983 is.

[0051] The terms "polypeptide" and "peptide" refer to a polymer of amino acid residues, and The terms are used interchangeably herein to refer to variants and synthetic analogues thereof. These terms refer to the chemical structure of one or more amino acid residues corresponding to the corresponding naturally occurring amino acids. Amino acid polymers that are non-naturally occurring synthetic amino acids, such as amino acid analogs, as well as naturally occurring Polypeptides are polymers of amino acids that are present in the body. These may include selective cleavage, lipidation, signal peptide cleavage, propeptide cleavage, phosphorylation, etc. The peptide may also be subjected to processes such as, but not limited to, maturation or post-translational modification. Techniques can be used to produce, for example, recombinant or synthetic polynucleotides. Recombinantly produced peptides are typically substantially free of culture medium, e.g. , the culture medium accounts for less than about 20% of the volume of the protein preparation, more preferably less than about 10%; Most preferably it corresponds to less than about 5%.

[0052] The term "protein" refers to a folded polypeptide having secondary or tertiary structure. A protein can consist of a single polypeptide or A multimer may comprise a single polypeptide or a plurality of polypeptides that assemble to form a multimer. A multimer is a homo-oligomer. The protein may be a monosaccharide or a hetero-oligomer. The protein may be a mutant or wild-type protein, or may be a modified or naturally occurring protein. A protein may be, for example, a non-existent protein. The additions, substitutions, or deletions may differ from the wild-type protein.

[0053] A "mutant" form of a protein is a variant of the unmodified or wild-type protein in question. have amino acid substitutions, deletions, and / or insertions in the unmodified protein from which they are derived Peptides, oligopeptides, and polypeptides that have biological and functional activities similar to those of proteins. As used herein, "amino acid identity" includes peptides, proteins, and enzymes. The term "identical" refers to the degree to which sequences are identical amino acid-by-amino acid over a comparison window. Therefore, the "Percentage of Sequence Identity" is the best fit between the two over the comparison window. The aligned sequences are compared, and identical amino acid residues (e.g., Ala, Pro, Ser, T hr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, H is, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences. Determine the number of positions to calculate the number of matched positions, and display the number of matched positions in the Comparison window. Divide by the total number of positions (i.e., the window size) and multiply the result by 100 to obtain the sequence identity It is calculated by calculating the percentage.

[0054] For all aspects and embodiments of the present invention, a "mutant" is a mutant of the corresponding wild-type protein. At least 50%, 60%, 70%, 80%, 90%, 95% %, or 99% complete sequence identity. Sequence identity is based on the full length polynucleotide or or to a fragment or portion of a polypeptide. A sequence may have only 50% overall sequence identity to the full-length reference sequence, but may have specific regions, domains, or regions that are identical to the full-length reference sequence. The sequences of the introns or subunits are 80%, 90%, or even 99% identical to the reference sequence. They may share an identity.

[0055] The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is one that is most frequently observed in a population and therefore represents any In contrast, "modified" or "mutated" The terms "mutant" and "mutation" refer to a gene or gene product that is modified in sequence as compared to a wild-type gene or gene product. modifications (e.g., substitutions, truncations, or insertions), post-translational modifications, and / or functional properties of A naturally occurring mutation refers to a gene or gene product that exhibits a specific characteristic (e.g., an altered trait). It should be noted that variants can be isolated, which may be either wild-type or variants of the gene. It is identified by the fact that it has altered characteristics compared to the naturally occurring product. Methods for introducing or substituting amino acids are well known in the art. The amino acid (M) is a methionine at the relevant position in the polynucleotide encoding the mutant monomer. By replacing the codon for arginine (CGT) with the codon for lysine (ATG), Methods for introducing or substituting non-naturally occurring amino acids. Methods for the synthesis of non-naturally occurring amino acids are also well known in the art. Inclusion of synthetic aminoacyl-tRNA in the IVTT system used to express Alternatively, they can be introduced by synthesis of specific amino acids (i.e., naturally occurring E. that is auxotrophic for those particular amino acids in the presence of their (non-existent) analogues. They can be introduced by expressing mutant monomers in E. coli. If the monomers are produced using partial peptide synthesis, naked ligation ( Conservative substitutions can also be made by naked ligation. Replace the acid with another amino acid of similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acids have similar polarity, hydrophilicity, and phobicity properties to the amino acids they replace. It may be aqueous, basic, acidic, neutral, or charged. Alternatively, conservative substitutions may be made to replace existing In place of an aromatic or aliphatic amino acid, another amino acid that is aromatic or aliphatic is introduced. Conservative amino acid changes are well known in the art and include the 20 amino acid changes defined in Table 1 below. The amino acids may be selected according to the properties of the major amino acids. This is determined by reference to the hydropathy scale of amino acid side chains in Table 2. It is also possible. [Table 1] [Table 2]

[0056] The mutant or modified proteins, monomers, or peptides can be prepared by any method and in any The mutant or modified monomer or peptide may be preferably Preferably, the molecule is bound to one or more cysteines (cysteine ​​bond), one or more lysines conjugation of the molecule to one or more unnatural amino acids, enzymatic modification of the epitope, or The nucleic acid is chemically modified by modification of the ends of the nucleic acid or by modification of the ends of the nucleic acid. Variants of modified proteins, monomers, or peptides are well known in the art. They can be chemically modified by the attachment of any molecule, e.g., modified proteins, monomers, or or peptide variants can be chemically modified by attachment of dyes or fluorophores. do.

[0057] Motor protein stalling The present disclosure provides a method for linking a motor protein to a polynucleotide adaptor that includes a spacer. a blocking moiety that blocks the release of the motor protein from the spacer; As described in more detail below, the method includes: When the motor protein is bound to a polynucleotide, the motor protein is more easily expressed. This can slow down the rate at which proteins turn over fuel molecules.

[0058] As discussed above, the present invention provides a method for controlling the movement of a motor protein to a target polynucleotide. Methods for this purpose are known in the art. One method known in the art is The present invention relates to a method for preventing the movement of a motor protein to a target polynucleotide using a probe. The method includes ligating a motor protein to a spacer (e.g., adjacent to the spacer). In this way, the motor protein can be attached to, for example, a polynucleotide. Motor proteins can be directional (i.e. (The polynucleotide may be processed in the 5' to 3' direction or the 3' to 5' direction). The motor protein is then guided in the direction of processing by the motor protein to the target polynucleotide. By providing a spacer, the movement of the motor protein is restricted by the spacer. On the one hand, the movement of the motor protein to the target polynucleotide can be achieved by the spacer On the other hand, the movement of motor proteins is also affected by their directionality. This means that it is constrained away from the spacer in a direction away from the target polynucleotide. This means that the motor proteins cannot move in space. Move either "forward" towards the spacer or "backward" away from the spacer This restriction makes it difficult to retain the motor protein (adjacent to the spacer). can.

[0059] However, constraining motor proteins in this known manner typically The motor protein is capable of reacting with fuels that may be present under the reaction conditions (e.g., In theory, this may not prevent the consumption of cofactors necessary for polynucleotide processing. By stalling the motor protein with a spacer without being tethered, the motor It is believed that this causes the "delay" of the motor protein. The nucleic acid is loaded onto the nucleotide adaptor and advanced in the manner described above until it reaches the spacer. When the motor protein reaches the spacer, its movement stalls. The motor protein does not remain stationary at the spacer. Rather, the motor protein The spacer is periodically released from binding to the adjacent polynucleotide adaptor, and the The target protein is then diffused back along the polynucleotide adaptor away from the spacer. The motor protein then recombines with the polynucleotide adaptor. It binds and advances again until it reaches the spacer, after which the cycle is repeated. Sequential detachment of motor proteins from polynucleotide adaptors. Recombination of the motor protein with the polynucleotide adaptor and the motor protein along the polynucleotide adaptor Although the progression of the motor protein toward the spacer is not related to the overall progression of the motor protein, The motor protein stalls at the spacer and consumes the fuel present in the system. This unproductive consumption of fuel is called wasteful turnover.

[0060] Wasteful turnover is undesirable and reduces the overall efficiency of the system. If the protein stalls at the spacer for an extended period in the manner described, wasteful turnover The amount of fuel consumed by motor proteins in the mammalian genome can be significant. Such unproductive fuel use can, for example, severely limit the life of the system in storage. A reduction in fuel, for example, can slow down motor proteins to an undesirable extent. In order to take this unwanted consumption into account, wasteful turnover is It may be necessary to initially increase the amount of fuel in the reaction system to an undesirable extent. Avoiding artifacts caused by impurities during characterization of targeted polynucleotides The requirement for high purity fuel to operate the plant means that wasteful turnover can significantly increase costs. In addition, this means that motor proteins react to high concentrations of spent fuel molecules (i.e. , fuel molecules turned over by motor proteins such as ADP) are adversely affected. In some cases, motor proteins have been characterized to exert low control over target polynucleotides. and / or the speed of the motor protein may be undesirably slowed. There is a possibility that it will go down.

[0061] While searching for a solution to this problem, we were surprised to find that the motor protein on the spacer By stalling the Specifically, it was found that the motor protein is prevented from moving out of the spacer. In other words, motor proteins are able to bind polynucleotides, which reduces wasteful turnover. The motor protein turns over fuel molecules faster than when it is bound to a leution. Suitable spacers are described in more detail herein.

[0062] In developing the presently claimed method, it was surprising to discover that the blocking moiety Motor proteins bind to oligonucleotide adaptors to initiate polynucleotide synthesis. Advantageously, the head adapter can be prevented from moving out of the spacer included therein. It has been found that the blocking moiety, for example, blocks the motor protein from the spacer. It can prevent the motor from coming off and moving. The blocking part is a motor tank. It is possible to chemically prevent proteins from moving away from the spacer. The binding portion is described in more detail herein.

[0063] Thus, a method for loading motor proteins onto polynucleotide adaptors is So, i) providing a polynucleotide adaptor comprising a spacer; ii) contacting the polynucleotide adaptor with a motor protein; iii) disposing the motor protein on the spacer, A blocking moiety attached to the polynucleotide adaptor binds to the motor protein. Methods are provided herein to prevent migration away from the spacer.

[0064] In one embodiment, a method for loading a motor protein onto a polynucleotide adaptor And, i) providing a polynucleotide adaptor comprising a spacer; ii) contacting the polynucleotide adaptor with a motor protein; iii) advancing the motor protein over the spacer; and iv) attaching a blocking moiety to the polynucleotide adaptor; The blocking moiety prevents the motor protein from moving off the spacer. , methods are provided herein.

[0065] In some embodiments, the polynucleotide comprises a loading polypeptide linked to a spacer. In some embodiments, the loading site contacts the motor protein. By contacting the loading site with the motor protein, the motor protein can be The protein advances from the loading site onto the spacer. In embodiments where the adapter comprises a loading site, the blocking moiety is Typically, motor proteins are linked to adaptors to cleave the spacer. The loading site is prevented from dislodging and migrating to the loading site. It will be described in more detail.

[0066] Thus, in some embodiments, the methods provided include (i) providing a loading site contacting a motor protein with a loading site; and (ii) binding or contacting a blocking moiety with a polynucleotide adaptor. wherein the blocking moiety prevents the motor protein from being attached to the spacer. and preventing the binding of the ribozyme to the loading site and rebinding to the loading site. .

[0067] In other embodiments, the polynucleotide adaptor does not include a loading site. In such embodiments, the motor protein is typically a polynucleotide, e.g., with a spacer. The motor protein contacts the nucleotide adaptor, i.e., the motor protein contacts the spacer. The motor protein may be located on the spacer. The motor protein may be any The spacer may be disposed on the substrate by any suitable means, for example, any of the methods described in more detail herein. As described above, the motor protein can be contacted with the spacer, and then the motor protein can be attached to the spacer. The motor protein can be modified to prevent the protein from detaching from the spacer. The spacer prevents the motor protein from moving away from the spacer. The blocking moiety may be adjacent to the spacer. In some embodiments, the spacer may be a single spacer. In addition to the amino acid positions, natural nucleotides may be included, as described in more detail herein.

[0068] Polynucleotide Adapters The provided methods include loading a motor protein onto a polynucleotide adaptor. WO2015 / 110813 describes a method for targeting motor protein adaptors and the like. The loading of polynucleotides is described in detail herein and is incorporated by reference in its entirety. is incorporated into the book.

[0069] An adaptor typically comprises a polynucleotide that can be attached to the end of a target polynucleotide. The target polynucleotide typically comprises a chain of nucleotides as disclosed herein. It is intended to be characterized by law.

[0070] Polynucleotide adaptors can be added to both ends of a target polynucleotide. Alternatively, different adaptors can be added to the two ends of the target polynucleotide. An adaptor may be added to only one end of a target polynucleotide. Methods for adding oligonucleotides to the adaptors are known in the art. By ligation, by click chemistry, by tagmentation The polynucleotide may be synthesized by topoisomerase conversion, or by any other suitable method. It can bind to the

[0071] In one embodiment, the adapter is synthetic or artificial. Typically, the adapter is The adaptor comprises a polymer as described herein. The adaptor comprises a spacer as described herein. As described in more detail below, the adapter may contain a loading site, e.g., a spacer. The loading site may also include a loading site connected to the sensor. The loading site is described in more detail herein. It is described in detail.

[0072] In some embodiments, the adaptor comprises a polynucleotide. As will be appreciated, the loading site may comprise a single stranded polynucleotide strand. The nucleotide adapters are DNA, RNA, modified DNA (basic DNA, etc.), RNA, and P The adapter may comprise a single stranded NA, LNA, BNA, and / or PEG. and / or double-stranded DNA or RNA.

[0073] In one embodiment, the adaptor is a Y adaptor. The Y adaptor typically comprises: A Y adaptor is a polynucleotide adaptor. A Y adaptor is typically double-stranded and has (a) (b) a region at one end where the two strands hybridize together and (b) a region at the other end where the two The strands contain regions that are not complementary. The non-complementary portions of the strands form overhangs. The presence of a non-complementary region in the Y adaptor ensures that the two strands are distinct from the double-stranded portion. They typically do not hybridize to each other, giving the adapters their Y shape. As will be described in more detail, in one embodiment, the motor protein is a Y adaptor. In another embodiment, the motor protein can bind to the overhang of any adaptor. In other embodiments, the motor protein can bind to the double-stranded region of the adaptor. In another embodiment, the first motor may bind to the single-stranded region and / or the double-stranded region. A protein can bind to a single-stranded region of such an adaptor, and a second motor protein can bind to it. The double-stranded region of the adaptor can be bound to the double-stranded region of the adaptor.

[0074] In one embodiment, the adaptor comprises a membrane anchor or a polymeric component as described in more detail herein. In some embodiments, the anchor comprises a nucleic acid handling enzyme attached thereto. The overhang is complementary to the polynucleotide that hybridizes to it. It can be bound to leotide.

[0075] In some embodiments, the non-complementary structure of a polynucleotide adaptor, such as a Y adaptor, One of the strands is a leader sequence that can thread through the nanopore when it comes into contact with the membrane pore. In one embodiment, the leader sequence may comprise a lobe sequence, as described in more detail herein. In one embodiment, the leader sequence precedes the loading site. (i.e., the adapter may include a leader sequence attached to the loading site, The loading site is connected to a spacer, which allows the loading site to be linked to a leader sequence. (The loading site is located between the row and the spacer.) In one embodiment, the loading site is The leader sequence may precede the adapter (i.e., the adapter may be a load sequence connected to the leader sequence). The leader sequence may include a spacer region, whereby the leader sequence is connected to the spacer. is located between the loading site and the spacer).

[0076] The leader sequence is typically a polynucleotide, e.g., DNA or RNA, modified Polynucleotides (such as abasic DNA), PNA, LNA, polyethylene glycol (P EG), or a polymer such as a polypeptide. In some embodiments, the leader The sequence comprises a single stranded DNA such as a poly dT segment. The leader sequence can be of any length. However, typically, the length is 10 to 150 nucleotides, for example, 20 to 120, 30 to 100 , 40 to 80, or 50 to 70 nucleotides in length.

[0077] In one embodiment, the polynucleotide adaptor is a hairpin loop adaptor. A hairpin loop adaptor is an adaptor that contains a single polynucleotide strand and The ends of the nucleotide chain can hybridize to each other or The polynucleotide is then reordered so that the central portion of the polynucleotide forms a loop. Adaptors can be designed using methods known in the art.

[0078] As described in more detail below, polynucleotide adaptors are used to bind target polynucleotides. The target polynucleotide can be characterized by binding to the tide.

[0079] Those skilled in the art will therefore be able to easily identify and use the described adapters for use in the methods provided herein. The adapter lacks the blocking moiety described herein, as compared to the adapter lacking the blocking moiety described herein. It will be appreciated that this will reduce wasteful turnover by accelerated motor proteins. Typically, the described adapters for use in the methods provided herein are Improves efficiency of fuel usage compared to adapters lacking the blocking portion provided in .

[0080] Those skilled in the art will recognize that when the adaptor comprises a polynucleotide strand, the sequence of the adaptor is typically The type is not conclusive, and the motor protein and any polynucleotides to be characterized are It will be appreciated that the concentration of ribozyme may also be controlled or selected according to other experimental conditions, such as the amount of ribozyme present. Exemplary sequences are provided in the Examples for illustrative purposes only. For example, the adapter may be 13, or at least 20%, e.g. at least 30%, e.g. For example at least 40%, for example at least 50%, for example at least 60%, for example at least at least 70%, for example at least 80%, for example at least 90%, for example at least 9 The adaptor may comprise a polynucleotide sequence having 5% sequence similarity or identity. , sequences such as those provided in SEQ ID NOs: 14 and 15, or sequences such as those provided in SEQ ID NOs: 14 and 15 and at least 20%, for example at least 30%, for example at least 40%, for example less At least 50%, for example at least 60%, for example at least 70%, for example at least 80% %, e.g., at least 90%, e.g., at least 95% sequence similarity or identity. The polynucleotide sequence may include a spacer as described herein adjacent to the polynucleotide sequence having the Those skilled in the art will appreciate that SEQ ID NO:14 includes sequences such as SEQ ID NO:15 and therefore The target is a polynucleotide sequence such as SEQ ID NO: 15, or a sequence that is at least 20 %, for example at least 30%, for example at least 40%, for example at least 50%, for example For example at least 60%, for example at least 70%, for example at least 80%, for example at least Polynucleotides having at least 90%, for example at least 95%, sequence similarity or identity with each other. It will be understood that the order of the adapters may include the sequence of the may be modified without adversely affecting the reduction in waste turnover provided by the pacer. do.

[0081] Spacer The disclosed method comprises attaching a motor protein to a polynucleotide adaptor comprising a spacer. The method includes loading one or more spacers into a polynucleotide adaptor. For example, the polynucleotide adaptor may include from 1 to about 10 spacers, e.g. , 1 to about 5 spacers, e.g., 1, 2, 3, 4, or 5 spacers. The terms "spacer" and "stall site" may be used interchangeably. Polynucleotides that stall proteins, typically preventing wasteful turnover as described above, are used to slow down the A spacer (stall site) refers to a portion of a head adaptor. Suitable spacer units are described herein and may include abasic nucleotides. and spacer groups, e.g., iSp9, iSp18, and C3 groups (hereinafter The spacer (stall site) is a nucleotide unit in addition to the spacer unit. For example, as described in more detail herein, a spacer (stall site) may be included. ) may contain spacer units spaced by one or more nucleotides. A pacer is a sequence of one or more nucleotide units, such as one or more polynucleotides. The nucleotide sequence may include one or more spacer units adjacent to or separated by the positions. This concept is sometimes referred to as "nucleotide islands" and is discussed further below. can be.

[0082] One or more spacers are included in the polynucleotide adaptor. The nucleotide adaptor may comprise a strand of a single-stranded and / or double-stranded polynucleotide, One or more spacers may be included in the polynucleotide chain, e.g., to interrupt the chain or can separate one strand from another. Exemplary polynucleotide adapters include: This is described in more detail herein.

[0083] Each spacer in the polynucleotide adaptor impedes the movement of the motor protein Provides an energy barrier. For example, the spacer reduces the pulling force of the motor protein. This can stall the motor protein by, for example, i.e., bases are removed from one or more nucleotides in the polynucleotide adaptor. This can be achieved by using spacers that are removed. The spacers can be, for example, bulky By introducing chemical groups that physically impede the movement of motor proteins, Protein translocation can be physically blocked.

[0084] A spacer is any molecule or combination of molecules that stalls one or more proteins. The spacer may comprise a spacer that allows the motor protein to move along the target polynucleotide. The present invention may include any molecule or combination of molecules that prevents a motor protein from being stimulated. It is easy to determine if a pacer is stalling. For example, this is described in WO201 4 / 135838, the contents of which are incorporated by reference. For example, a motor that moves across a spacer to displace a complementary strand of DNA. Protein capacity can be measured by PAGE.

[0085] In some embodiments, the spacer may comprise a linear molecule, such as a polymer. Typically, such a spacer has a structure different from that of the target polynucleotide. For example, When the target polynucleotide is DNA, the or each spacer is typically Specifically, the target polynucleotide does not include deoxyribonucleic acid (DNA) or is ribonucleic acid (RNA), the or each spacer is preferably a peptide nucleic acid. Polynucleotides (PNA), Glycerol Nucleic Acid (GNA), Threose Nucleic Acid (TNA), Locked Nucleic Acid (LNA), bridged nucleic acid (BNA), or synthetic polymers with nucleotide side chains .

[0086] In some embodiments, the spacer is one or more nitroindoles, one or more indoles, nosine, one or more acridines, one or more 2-aminopurine, one or more 2-6-diazo minopurine, one or more 5-bromo-deoxyuridines, one or more inverted thymidines (inverted dT), one or more inverted dideoxy-thymine (ddT), one or more dideoxy-cytidine 5-methylcytidine (ddC), one or more 5-hydroxymethylcytidines, cytidine, one or more 2'-O-methyl RNA bases, one or more iso-deoxycytidines (I so-dC), one or more iso-deoxyguanosines (Iso-dG), one or more C3 (OC 3 H 6 OPO 3 ) groups, one or more photocleavable (PC)[OC 3 H 6 -C(O)N HCH 2 -C 6 H 3 NO 2 -CH(CH 3 )OPO 3 ] group, one or more hexanediols group, one or more spacers 9 (iSp9) [(OCH 2 CH 2 ) 3 OPO 3 ] basis, or is one or more spacers 18 (iSp18) [(OCH 2 CH 2 ) 6 OPO 3 ] basis, and may contain one or more thiol bonds. The spacer may contain any combination of these groups. Many of these groups are available from IDT® (Integrated Digital Commercially available products include C3, i The Sp9 and iSp18 spacers are all available from IDT®.

[0087] The spacer may contain any number of the above groups as spacer units. Ser is 2-aminopurine, 2-6-diaminopurine, 5-bromo-deoxyuridine, Inverted dT, ddT, ddC, 5-methylcytidine, 5-hydroxymethylcytidine, 2' -O-methyl RNA base, Iso-dC, Iso-dG, iSpC3 base, PC base, Hexa 1 to about 12 or more (e.g., 1 to about 12 or more) selected from phenyl, diol, and thiol bonds. The spacer may include from about 1 to about 8, for example, from 1 to about 6, such as from 1 to about 4, spacers. The sensor can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 It may contain groups.

[0088] The spacer may be 1 to about 12 or more (e.g., about 1 to about 8, e.g., 1 to about The spacer may contain from 1 to about 6, such as 4, C3 groups, for example, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, or 12 C groups. The C groups are typically -OC 3 H 6 OPO 3 -based.

[0089] The spacer may be from about 1 to about 8 or more (e.g., from about 1 to about 6, such as from 1 to about 4). ), for example, the spacer can include 2, 3, 4, 5, 6, 7, or 8 iSp9 groups. The iSp9 group may typically include the iSp9 group: -(OCH 2 CH 2 ) 3 OPO 3 -based.

[0090] The spacer may be from about 1 to about 8 or more (e.g., from about 1 to about 6, such as from 1 to about 4). ), for example, the spacer can include 2, 3, 4, 5, 6, 7, or 8 iSp18 groups. iSp9 groups may be included. iSp18 groups are typically -(OCH 2 CH 2 ) 6 OP O 3-groups. In one embodiment, the spacer comprises four iSp18 groups.

[0091] In some embodiments, the spacer comprises one or more iSp18 groups and / or one or more iSp9 groups and / or one or more C3 groups.

[0092] In some embodiments, the spacer comprises one or more molecules that stall the motor protein. In some embodiments, suitable chemical groups include one or more pendant The one or more chemical groups are linked to one or more nucleobases in the polynucleotide adaptor. One or more chemical groups may be attached to the backbone of the polynucleotide adaptor. Any number such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more Suitable chemical groups include fluorophores, streptavidin and and / or biotin, cholesterol, methylene blue, dinitrophenol (DNP) , digoxigenin and / or antidigoxigenin, and dibenzylcyclooctyne Groups include, but are not limited to,

[0093] In some embodiments, the spacer may comprise a polymer. The spacer is a polymer that is a polypeptide or polyethylene glycol (PEG). may include.

[0094] When the spacer comprises a polypeptide, the polypeptide may comprise any number of amino acids. For example, the polypeptide may comprise from about 1 to about 12 or more (e.g., from about 1 to about 8 For example, the spacer may comprise from 1 to about 6 amino acids, such as from 1 to about 4 amino acids. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids.

[0095] When the spacer contains a polymer such as PEG (polyethylene glycol), the A polymer can contain any number of monomer units. For example, a polymer can contain from about 1 to about 12 or or more (e.g., from about 1 to about 8, e.g., from 1 to about 6, such as from 1 to about 4) monomers. For example, the spacer can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Or it may contain 12 monomer (eg, PEG) units.

[0096] In some embodiments, the spacer comprises one or more abasic nucleotides (i.e., Nucleotides lacking nucleobases, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 The nucleobase may contain 12, 13, or more abasic nucleotides. It can be replaced by -H (idSp) or -OH in the oxidase. The nucleotide sequence of the target nucleotide is determined by removing the nucleotide from one or more adjacent nucleotides. For example, the polynucleotide may be 3-methyladenine, Contains 7-methylguanine, 1,N6-ethenoadenine inosine, or hypoxanthine The nucleobase may be modified by human alkyladenine DNA glycosylase (hA These nucleotides can be removed using polynucleotides (AG). may be modified to include uracil, and the nucleobase is uracil-DNA glycosylase (UDG). In one embodiment, the one or more spacers can be any It also does not contain abasic nucleotides.

[0097] In some embodiments, the spacer comprises one or more nucleotide islands, also referred to as nucleotide islands. The spacer may comprise the above nucleotide units. The spacer typically comprises a nucleotide unit that is essential for the activity of the motor protein. does not contain a polynucleotide chain of sufficient length to completely occupy a specific site. In embodiments where the spacer comprises one or more nucleotide units, the spacer is typically Contains 1 to 3 nucleotides, more often 1 to 2 nucleotides. In embodiments, the spacer is a nucleotide sequence that is 0.01 to 0.01 and is preferably 0.01 to 0.01. In some embodiments, the spacer comprises 1 to 3 consecutive nucleotides. 1-5 nucleotide islands, each containing 1-3 consecutive nucleotides, e.g. For example, one, two, or three nucleotide islands may be included. - The active site of a protein (polynucleotide-binding groove) is typically approximately 8 nucleotides long You will recognize that the size corresponds to the unit. Therefore, the spacer is 1 to 3 In embodiments that include one, e.g., one or two, nucleotide spacer units, The spacer unit does not completely occupy the active site of the motor protein. In embodiments, an exemplary spacer includes 1 to 3, e.g., 1 to 2, nucleotides (e.g., The fast site) is typically -[(S) x -(N) y -(S) z ] w -One to be selected and wherein each S is a spacer unit (e.g., a spacer unit as described herein). units), each N is a nucleotide, x is an integer from 1 to 7, and y is an integer from 1 to 3. Typically, z is 1 or 2, z is an integer from 1 to 7, and w is an integer from 1 to 3. In this case, each S is the same or different, and each N is the same or different. In such an embodiment, each S is selected from C3, iSp9, and iSp18 spacer units. each N is independently selected from adenine, cytosine, anine, and thymine, x is an integer from 1 to 7, and y is 1 or 2; z is an integer from 1 to 4; w is an integer from 1 to 3.

[0098] In some embodiments, the spacer may comprise one or more nucleotide islands. In the present invention, the adaptor is a protein that is bound to the motor protein before the motor protein contacts the adaptor. In such an embodiment, the blocking moiety may be The motor protein can be loaded directly into the spacer. The motor protein can be loaded into a nucleotide island contained within the spacer. is loaded into a nucleotide island contained within a spacer and then By dissociating from the pacer, the motor protein is prevented from leaving the spacer. The motor protein can be modified to inhibit its activity. Modifications of the motor protein are described in more detail herein. In some embodiments, the motor protein is loaded onto a nucleotide island. In some implementations, the spacer unit may be inserted into the spacer and then advanced to the spacer unit contained within the spacer. In this morphology, motor proteins translocate motor proteins off the islands. The nucleotide island may remain located within the spacer until a force is applied to the nucleotide island. In some embodiments, this force is provided by a nanopore as described herein. Provided.

[0099] In some embodiments, スペーサーは、-SNNSS-、-SNSN -S-、-SSNNS-、-SSNSS-、-NNSNNS -、-SSNNSS-、 -SNNSNNS-、-SNSNSNS-、-SSSS -SSS-, -SSNNSNNS-、-SSSSNNSS-、-SN -NSSNNNSS-, -SSNNNSNNS-、-SSNNSSNNS-、 -SSNNSSNNSS-, -SSSNNNNSNS-、-SSSNNSNNNS-、 -SSSSNNSNS-、-SSSSNNSNS-、 -SNNSNNSNNS-, -SSSNNSNNSS-、-SSSNNSSNN -S-、-SSSSSSNNSS-、 -NNSNNSSSSSS-、-NNSSNNSS -SSS-,-NNSSSNNSSSS-, -NNSSSSNNSSS-、-NNSSSSN -NSS-,-NNSSSSSSNNS-, -SNNSNNSSSSS-、-SNNSSNNS -SSS-、-SNNSSSNNSSS-、 -SNNSSSSSNNSS-、-SNNSSSSS -NNS-、-SSNNSSNNSSSS-、 -SSNNSSNNSSS-, -SSNNSSSN -NSS-, -SSNNSSSSNNS-, -SSSNNSNNSSS-, -SSSNNSSN -NSS-, -SSSSNNSNNSS-, -SSSSNNSS -NNS-, -SSSSSNNSNNS-, -SSSNNSNNSNNS-, -SSSNNSN -NSSSS-, -SSSNNSSNNSSS-, -SSSNNSS -SSNNS-, -SSSNNSNNSSNNS-, -SSSNNS -SNNSSSSS-, -SSSSSNNSNNSSS-, -SSSNNS -SNNSSSSS-, -SSSNNSSSSSSNNS-, -SSSSS -NNSNNSNNS-, -SSSNNSSNNSSSSSS-, -SSSS -SNNSNNSSNNS-, -SSSNNSSNNSSSSSSS-, -SSS One selected from -NNSSNNSSSSSSSS- etc. or the like, wherein each S is a spacer unit as described herein and each N is a nucleoside. It is leotide.

[0100] In some embodiments, the spacer is -SNSNSNS-, -SNNSNNSNNS-, -S -SSSSSNNSS-, -SSSSSNNSNNSSS-, -SSSNNS -NNSSNNS-, -SSSSSNNSNNSSNNS-, -SSSN -NSNNSNNS-, -NNSSNNSSSSS-, -SNNSSNNS -SSS-, -SSNNSSNNSSS-, -SSSSNNSS -NNS-, -NNSNNSSSSSSS-, -SNNSNNSSSSS-, -SSNNSNNS -SSS-, -SSSNNSNNSSS-, -SSSSNNSNNSS-, -SSSNNSSN -NS-, and the like, wherein each S is as described herein. A spacer unit, where each N is a nucleotide.

[0101] In some embodiments, the spacer is -SNNSNNSNNS-, -SSSSSNNSN -NSSS-, -NNSSNNSSSSS-, -SNNSSNNSSSS-, -SSNNSSNN -SSS-, -SSSSNNSSNNS-, -SSNNSNNSSSS-, -SSSNNSNN -SSS-, -SSSSNNSNNSS-, -SSSNNSSNNS-, etc. where each S is a spacer unit as described herein and each N is a nucleotide.

[0102] In some embodiments, the spacer is -SSSSSNNSNNSSS-, -NNSSNN -SSSSS-, -SNNSSNNSSSS-, -SSNNSSNNSSS-, -SSSSNNSS One selected from -NNS-, -SSSNNSSNNS-, etc. or the like, wherein each S is a spacer unit as described herein and each N is a nucleoside. It is leotide.

[0103] In some embodiments, the spacer is -SSNNSS-, -SNSNSNS, -SNNSN- NSNNS-, -SSSSNNSS-, -SSSSSSNNSS-, -SSSNNSNNS -NNS-, -SSSSSNNSNNSNNS-, SSSNN- SNNSSS-, -SSSSSNNSNNSSS-, -SSSNNS -NNSSNNS-, and -SSSSSNNSNNSSNNS- etc. can be selected. may include one or more moieties, where each S is a spacer unit as described herein and each N is In some embodiments, each S is the same spacer unit. In some embodiments, the S groups are different spacer units. , the spacer is a nucleotide-based spacer unit (e.g., 2'-O-methyl RN modified nucleotides such as A bases) and non-nucleotide based spacer units (iSp 18, iSp9, and C3 spacer units). In this embodiment, each S is independently selected from iSp18, iSp9, 2'-O-methyluridine, and and C3 groups. In some embodiments, each S is independently selected from iSp18 groups, i In some embodiments, each S is independently selected from the group consisting of i The uridine group is selected from the group consisting of an Spl8 group, a 2'-O-methyluridine group, and a C3 group. In some embodiments, each S is independently selected from an iSp18 group and a C3 group. In some embodiments, each N is the same nucleotide. In some embodiments, each N group is independently selected from adenine, cytosine, guanine, In some embodiments, each N group is independently selected from cytosine, ... In some embodiments, each N group is selected from thymine and thymine.

[0104] In some embodiments, the spacer is -8-TT-9-TT-8-, -8-T -8-T-8-T-8-, -8-TT-8-TT-8-TT-8-, -3-3-8 -8-TT-8-8-, -3-3-3-3-8-8-TT-8-8-, -3-3-3-3-8-TT-8-T -T-8-3-8-, -3-3-8-TT-8-TT-8-3-TT-8-, -3-3-3-3-8-TT-8-TT-8-3-TT-8-, -3-3-3-T -T-8-TT-8-TT-8-, -3-3-3-TT-8-TT-8-, -3-TT-8-TT-8-TT-8-, -TT-3-3-TT-3-3-3 -3-8-, -3-TT-3-3-TT-3-3-3-8-, -3-3-TT-3-3-TT-3-3-8-, -3-3-3-3-TT-3-3 -TT-8-, -TT-3-3-3-TT-3-3-3-8-, -TT-8-TT-3-3-3-3-3-8-, -3-TT-8-TT-3-3 -3-3-8-, -3-3-TT-8-TT-3-3-3-8-, -3-3-3-TT-8-TT-3-3-8-, -3-3-3-3-TT-8-T -T-3-8-, -3-3-3-TT-3-3-TT-9-, -3-3-3-TT-mU-mU-mU-TT-8-, -3-3-3-TT-mU -mU-TT-8-, where 3 is the first spacer sar, 8 is the second spacer, 9 is the third spacer, and mU is the fourth spacer. spacer, typically 3 is a C3 spacer as defined herein and 8 is iSp18 spacer as defined herein, and 9 is iSp9 as defined herein spacer, mU is 2'-O-methyluridine, and each N is independently an adenine , cytosine, guanine, and thymine, preferably thymine.

[0105] In some embodiments, the spacer is -8-8-NN-8-8-, -8-N-8 -N-8-N-8, -8-NN-8-NN-8-NN-8-, -3-3-8-8- NN-8-8-, -3-3-3-3-8-8-NN-8-8-, -3-3-8-NN-8-NN-8 -NN-8-, -3-3-3-3-8-NN-8-NN-8-NN-8-, 3- 3-8-NN-8-NN-8-3-8-, -3-3-3-3-8-NN-8-N- N-8-3-8-, -3-3-8-NN-8-NN-8-3-NN-8-, and -One selected from 3-3-3-3-8-NN-8-NN-8-3-NN-8- or the moiety, wherein 3 is a first spacer and 8 is a second spacer. Typically, 3 is a C3 spacer as defined herein and 8 is a C4 spacer as defined herein. iSp18 or iSp9 spacer, typically 8 as defined herein. iSp18 spacer, where each N is independently adenine, cytosine, guanine, and and thymine, preferably thymine.

[0106] In some embodiments, the spacer is 88TTT88, 88mUmUmU88, 8 8 / iBNA-T / / iBNA-T / / iBNA-T / 88, 88T88, 8TT88, 88TT8, 8T8T8, 99TT9TT99, 8TT9TT8, 8TT3TT8, 8T 8T8T8, 8TT8TT8TT8, 3388TT88, 333388TT88, 338 TT8TT8TT8, 33338TT8TT8TT8, 338TT8TT838, 333 38TT8TT838, 338TT8TT83TT8, 33338TT8TT83TT8 , 8TT8TT8, TT8TT8, 3TT8TT8, 333TT8TT8TT8, 333 TT8TT8, 3TT8TT8TT8, 33TT8TT8, 333TT8TT838, 3 <h2 style=";text-align:left;direction:ltr">3338TT8TT8, 333TT33333TT8, 333TT99TT8, 333<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TT9TT8, 333TT3TT8, 9TT8TT8, 99TT8TT8, 3333T8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TT8, 3333TT8T8, 333T8TTT8, 333TTT8T8, 33TTT8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TT8, 33TT8TTT8, 9TT99TT99, 333TT8TT9999, 333<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TT8TT999, 333TT8TT99, 333TT8TT9, TT3TT33333<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8, 3TT3TT3338, 33TT3TT3338, 333TT3TT338, 33<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 33TT3TT38, 33333TT3TT8, TT33TT3338, 3TT33T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T3338, 33TT33TT338, 333TT33TT38, 3333TT33TT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8, TT333TT3338, 3TT333TT338, 33TT333TT38, 33<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3TT333TT8, TT3333TT338, 3TT3333TT38, 33TT33<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 33TT8, TT33333TT38, 3TT33333TT8, TT333333TT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8, TT8TT333338, 3TT8TT33338, 33TT8TT3338, 33<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3TT8TT338, 3333TT8TT38, 33333TT8TT8, 333TT3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3TT8, 333TT33TT9, 333TTmUmUmUTT8, 333TTSpSp<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TT8, 333TTSpTT8, 9TT33TT99, 333TTmUmUTT8, 33<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3TTrUTT8, 333TTrUrUTT8, 333TTrUrUrUTT8, 333<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TTrUrUrUrUTT8, 333TT33TTC12, 333TT33TTC6, m<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> UmUmUTTmUmUTT8,mUmUmUTT33TT8,333TT33TT88<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 333TT33TT888, 333TT33TT8888, 333TT33TT99<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 333TT33TT999、333TT33TT9999、333TT33TT333、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 333TT33TT3333、333TT33TT3333、333TT33TT33<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3333, 333TT33TT333333, 333TT33TT333333<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 33<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0107] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 88mUmUmU88 / 88 / iBNA-T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> / / iBNA-T / / iBNA-T / 88、88T88、88TT88、8TT88、8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8TT8、8T8T8、8TT9TT8、8TT3TT8、8T8T8T8、8TT8T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T8TT8, 3388TT88, 333388TT88, 33338TT8TT838<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 338TT8TT83TT8,33338TT8TT83TT8,8TT8TT8,TT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8TT8, 3TT8TT8, 333TT8TT8TT8, 333TT8TT8, 3TT8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> TT8TT8, TT3TT333338, 3TT3TT33338, 33TT3TT3333<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 38, 333TT3TT338, 3333TT3TT38, 33333TT3TT8, T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T33TT33338, 3TT33TT3338, 33TT33TT338, 3333T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T33TT8, TT333TT3338, 3TT333TT338, 33TT333TT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 38, TT3333TT338, 3TT3333TT38, 33TT333TT8, T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> T33333TT38, 3TT3333TT8, TT33333TT8, TT8TT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 333338, 3TT8TT33338, 33TT8TT3338, 333TT8TT3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 38, 3333TT8TT38, 33333TT8TT8, 333TT33TT9, 33<h2 style=";text-align:left;direction:ltr"> 3TTmUmUmUTT8, 333TTSpSpTT8, 333TTSpTT8, 333 TTmUmUTT8, and 333TT33TT8. obtain.

[0108] In some embodiments, the spacer is -8-8-TT-8-8-, -8-T-8 -T-8-T-8, -8-TT-8-TT-8-TT-8-, -3-3-8-8- TT-8-8-, -3-3-3-3-8-8-TT-8-8-, -3-3-8-TT-8-TT-8 -TT-8-, -3-3-3-3-8-TT-8-TT-8-TT-8-, 3-3-8-TT-8-TT-8-3-8-, -3-3-3-3-8-TT-8- TT-8-3-8-, -3-3-8-TT-8-TT-8-3-TT-8-, and -3-3-3-3-8-TT-8-TT-8-3-TT-8- may contain one or more moieties, where 3 is a first spacer and 8 is a second spacer. Typically, 3 is a C3 spacer as defined herein and 8 is a C4 spacer as defined herein. iSp18 or iSp9 spacer as defined herein, typically 8 being as defined herein. is an iSp18 spacer in which each T is a thymine.

[0109] In some embodiments, the polynucleotide adaptor comprises two or more spacers. In such embodiments, the two or more spacers are the same or different. For example, , one spacer may comprise one of the linear molecules discussed herein; Another spacer may be one or more chemical molecules that physically stall one or more motor proteins. The spacer may comprise any of the linear molecules discussed herein and one or more one or more chemical groups that physically stall one or more motor proteins, e.g., one or more and a fluorophore.

[0110] Suitable spacers depend on the nature of the polynucleotide adaptor, the motor protein, and The method may be designed or selected depending on the conditions under which the method is to be carried out. motor proteins process DNA in vivo, and such motor proteins are typically Essentially, you could stall using anything that isn't DNA.

[0111] Characterization of target polynucleotides is often performed using free nucleotides and / or burn-in nucleotides. The disclosed method is carried out in the presence of a feed molecule (a cofactor for the motor protein). Motor proteins typically transport free nucleotides and / or fuel molecules (motor In the presence of the protein cofactor, the polynuclear ligand is prevented from migrating off the spacer. Methods in which the nucleotide adaptor does not involve the presence of free nucleotides and / or fuel molecules When the motor protein is to be used in a ribozyme, the motor protein typically does not react with free nucleotides or and / or are prevented from migrating off the spacer in the absence of fuel molecules. In this embodiment, different spacers may be used. For example, free nucleotides and / or In embodiments where free nucleotides and / or fuel molecules are present, free nucleotides and / or fuel molecules are present. A longer spacer may be advantageously used compared to the embodiment in which no spacer is present.

[0112] In some embodiments, the disclosed methods are based on the salt concentration present under reaction conditions. It is beneficial to design or select a suitable spacer for use. The present invention includes characterization of a target polynucleotide strand by translocating the target strand relative to a nanopore. Methods to achieve this often involve the use of relatively high salt concentrations, which can disrupt one or more motor This may affect the ability of one or more spacers to stall the protein. The higher the salt concentration used in the method, the shorter the spacer or spaces typically used. and vice versa.

[0113] Some specific combinations of features are shown in Table 1 below. [Table 3]

[0114] In embodiments involving the use of multiple motor proteins, longer spacers may be beneficial. For example, in embodiments involving the use of two motor proteins as described herein Suitable spacers include from about 1 to about 25 of the above-mentioned spacers (e.g., iSp18 group, i The aryl group may include from about 1 to about 25 groups selected from the group consisting of C1, C2, C3, and Sp9 groups.

[0115] Blocking part In some embodiments, the disclosed methods include binding a blocking moiety to a polynucleotide. The blocking moiety is a blocker that blocks the binding of the motor protein to a polynucleotide. This prevents the catheter from moving off the spacer of the catheter adapter.

[0116] The blocking moiety typically inhibits the synthesis of a polynucleotide that is normally processed by a motor protein. A portion of the motor protein that prevents the movement of the motor protein in the opposite direction to the direction of the motor protein. For example, If the motor protein processes the polynucleotide chain in the native 5' to 3' direction, A suitable blocking moiety, in some embodiments, is a 3' to 5' amino acid sequence of the motor protein. Similarly, motor proteins are the parts that prevent the polynucleotide from moving in the opposite direction. When the tide strand is engineered in the native 3' to 5' direction, suitable blocking moieties include: In one embodiment, the moiety prevents the motor protein from moving in the 5' to 3' direction. It is.

[0117] In the disclosed methods, the blocking moiety is removed from the spacer of the motor protein. The motor protein is bound to a polynucleotide adaptor to prevent translocation. Preventing movement out of the spacer provides a solid block to the motor tamper This can be achieved by physically preventing the movement of the motor protein. Preventing migration of the molecule off the spacer is achieved by the use of chemical blocking moieties. Chemical blockade, on or across which target proteins cannot migrate This can be achieved by using a blocking moiety. The binding moiety may include one or more of the spacer groups discussed herein. In some embodiments, the blocking moiety may comprise a polynucleotide strand, as described in more detail below. It is discussed in detail.

[0118] In the disclosed methods, the blocking moiety prevents the motor protein from being dissociated from the spacer. In one embodiment, this is achieved by attaching a blocking moiety to a spacer. This is achieved by ligating to adjacent polynucleotide adaptors.

[0119] For example, in some embodiments, the polynucleotide adaptor is flanked by a spacer. In such an embodiment, the blocking moiety comprises a polynucleotide strand that is spaced apart from the For example, the blocking moiety can be a spacer. The blocking moiety may bind to one or more of the 20 nucleotides adjacent to the sar. It can bind to one or more of the 10 nucleotides adjacent to the spacer. The blocking moiety may be attached to one or more of the five nucleotides adjacent to the spacer. The binding portion may be located at either 2, 3, or 4 nucleotides adjacent to the spacer, or at both sites. In one embodiment, the blocking moiety is a terminal nucleoside adjacent to the spacer. In such an embodiment, the position of the blocking group relative to the spacer is The target protein is released from the spacer and the blocking moiety is attached to the polynucleotide. In some embodiments, the position of the tip adapter is such that the tip adapter cannot be moved to a position other than the tip adapter. In some embodiments, the blocking moiety binds to a loading site as described herein.

[0120] In some embodiments, the blocking moiety is an E. coli single-stranded binding protein. This protein binds to the polynucleotides in the ribosomal DNA and is involved in the synthesis of the single-stranded binding protein (SSB). Binding to a nucleotide adaptor and translocation of the motor protein away from the spacer Prevent.

[0121] In some embodiments, the blocking moiety is an intercalator. Any suitable intercalator can be used. The tethered adaptor polynucleotide chain is then intercalated, thus forming a motor tank. In some embodiments, the intercalator prevents the transfer of the protein through the intercalator. By placing the calculator close to the spacer, the motor protein's spacer In one embodiment, the intercalator is a spacer. In one embodiment, the interleukin-1 (IL-1) may be introduced between two of the 20 nucleotides adjacent to the interleukin-1 (IL-1). The chromatinator can be introduced between two of the 10 nucleotides adjacent to the spacer. In one embodiment, the intercalator is a 5-nucleotide nucleotide sequence adjacent to the spacer. In one embodiment, the intercalator is adjacent to the spacer. It can be introduced between the two adjacent terminal nucleotides.

[0122] In some embodiments, the blocking moiety comprises a secondary structure of the polynucleotide strand. For example, a blocking moiety can prevent the movement of a motor protein out of its spacer. The nucleic acid may contain secondary structures such as pseudoknots for the purpose of integrating the nucleic acid.

[0123] In some embodiments, the blocking moiety may comprise a chemical tag. The target tag is a polynucleotide, such as a polynucleotide, that can bind to a polynucleotide adaptor. Examples of suitable tags include biotin, a selectable polynucleotide sequence, an antibody, antibodies, antibody fragments, e.g., Fab and ScSv, antigens, polynucleotide binding proteins , polyhistidine tails, as well as GST tags. Biotin specifically binds to avidin, such as streptavidin. Biotin groups bound to avidin, such as riboflavin, would prevent the translocation of motor proteins. In some embodiments, a biotin group bound to an avidin, such as streptavidin. would prevent translocation of the motor protein across the biotin group.

[0124] In some embodiments, the blocking moiety is from about 2 to about 1000 nucleotide units in length. The blocking moiety comprises a single-stranded or unhybridized polynucleotide having the following structure: The polynucleotide adaptor is designed to prevent the motor protein from moving out of the spacer. The nucleic acid sequence can be hybridized to a peptide.

[0125] In some embodiments, the blocking moiety is from about 2 to about 500 nucleotide units in length. For example, about 2 to about 100 nucleotide units in length, for example, about 10 to about 100 nucleotide units in length nucleotide unit length, about 20 to about 80 nucleotide unit length, for example, about 30 to about 50 nucleotide unit length In some embodiments, the polynucleotide comprises a single stranded or unhybridized polynucleotide having the formula: In general, the blocking moiety can be about 1 to about 100 nucleotide units in length, for example, about 1 to about 90 nucleotides. Nucleotide unit length, for example, about 1 to about 80 nucleotide unit length, for example, about 1 to about 70 nucleotide unit length. Nucleotide unit length, for example, about 1 to about 60 nucleotide unit length, for example, about 1 to about 50 nucleotide unit length. Nucleotide unit length, for example, about 1 to about 40 nucleotide unit length, for example, about 1 to about 30 nucleotide unit length. Nucleotide unit length, for example, about 1 to about 20 nucleotide unit length, for example, about 1 to about 10 nucleotide unit length It includes single-stranded or unhybridized polynucleotides having a nucleotide unit length. In some embodiments, the blocking moiety is from about 2 to about 100 nucleotide units in length, e.g. , about 2 to about 90 nucleotide units in length, for example, about 2 to about 80 nucleotide units in length, for example , about 2 to about 70 nucleotide units in length, for example, about 2 to about 60 nucleotide units in length, for example , about 2 to about 50 nucleotide units in length, for example, about 2 to about 40 nucleotide units in length, for example , about 2 to about 30 nucleotide units in length, for example, about 2 to about 20 nucleotide units in length, for example , a single-stranded or unhybridized polynucleotide having a length of about 2 to about 10 nucleotide units. Includes chid.

[0126] Those skilled in the art will appreciate that a blocking moiety may be a single-stranded or non-hybridizing polynucleotide. In cases where the sequence of the blocking moiety polynucleotide is not typically critical, The proteins can be controlled or selected according to the adapter and motor proteins and other experimental conditions. It will be understood that exemplary sequences are provided in the Examples for illustrative purposes only. For example, the blocking moiety may be a polynucleotide sequence such as SEQ ID NO:19, or SEQ ID NO:1 9 and at least 20%, for example at least 30%, for example at least 40%, for example at least at least 50%, for example at least 60%, for example at least 70%, for example at least 8 0%, for example at least 90%, for example at least 95% sequence similarity or identity. For example, the blocking moiety may comprise a polynucleotide sequence that encodes a or a polynucleotide sequence having at least 20%, for example at least 3 0%, e.g. at least 40%, e.g. at least 50%, e.g. at least 60%, e.g. For example at least 70%, for example at least 80%, for example at least 90%, for example at least The polynucleotide sequence may include a polynucleotide sequence having at least 95% sequence similarity or identity. The binding portion corresponds to SEQ ID NO: 10 or SEQ ID NO: 19 and has at least one At least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least A polynucleotide containing at least 8, at least 9, or at least 10 nucleotide substitutions. Without being bound by theory, one of skill in the art can The blocking moiety prevents the DNA from moving out of the spacer and is compatible with the adapter sequence. It will be appreciated that this can be accomplished by a blocking moiety selected to be compatible with the The blocking moiety is attached (e.g., by hybridization) to the adapter. Blocking the motor protein from moving out of the spacer The structure of the king moiety may be modified.

[0127] The blocking moiety adaptor is a polynucleotide adaptor within the spacer region. It may also have a sequence complementary to the sequence (e.g., within about 20 nucleotides of the spacer). For example, within about 10 nucleotides of the spacer, for example, within about 5 nucleotides of the spacer. within 2, 3, or 4 nucleotides of the spacer), which allows The binding portion polynucleotide is attached to the polynucleotide adaptor within the spacer region. The motor proteins are then hybridized to the spacer and translocated. It will be possible to prevent this.

[0128] For example, a single strand capable of binding to a polynucleotide adaptor by hybridization. The blocking portion comprising the non-hybridizing polynucleotide or strand is also called the "capture strand." The capture strand captures the motor protein on the spacer. Binding to a nucleotide adaptor can be achieved by, for example, capture by hybridization. The strand is then ligated to the sequence of the polynucleotide adaptor, e.g., the spacing of the polynucleotide adaptor. This may involve binding to a sequence adjacent or adjacent to the sequence.

[0129] Typically, the blocking moiety or capture strand is bound to the target protein, e.g., by hybridization. The binding sequence is such that the resulting adapter containing the blocking moiety is a double-stranded polynucleotide. Thus, those skilled in the art can easily understand that an adapter is a single-stranded sequence that can be a nucleotide. may comprise a region of double stranded polynucleotide linked to a spacer, in such embodiments, The double-stranded polynucleotide blocks the movement of the motor protein away from the spacer, A single strand of a single-stranded polynucleotide or a portion of a single strand of a double-stranded polynucleotide is captured. It will be understood that this includes the inclusion of a chain, which is discussed in more detail in the Examples.

[0130] As described in more detail herein, in some embodiments, a blocking moiety If present, when the motor protein is placed on the spacer portion, the adapter In some embodiments, the blocking moiety, if present, is coupled to a motor The protein is bound to an adaptor before being placed on the spacer unit. In embodiments, the blocking moiety is a molecule that binds to one or more of the nucleases described herein. Binding to adaptors prior to placement on spacer units comprising nucleotide islands .

[0131] Loading site As discussed above, in some embodiments, the polynucleotide adaptor is It contains a loading site connected to the pacer. The loading site is a motor protein. This is the site for loading a molecule onto a polynucleotide adaptor.

[0132] In one embodiment, the loading moiety is a linear molecule, such as a polymer. In embodiments, the loading site is a deoxyribonucleic acid (DNA), ribonucleic acid (RNA) , peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), It may include locked nucleic acids (LNA), or synthetic polymers with nucleotide side chains. The loading moiety may comprise a polymer such as PEG.

[0133] In some embodiments, the motor protein is a polynucleotide adaptor. The motor protein binds to the polynucleotide adaptor (e.g., , the loading site of the polynucleotide adaptor) to When associated with a polynucleotide adaptor (e.g., polynucleotide adaptor For example, motor proteins can bind to the polynucleotide loading site of a By binding to a polynucleotide adaptor or by not binding to a polynucleotide adaptor. forms a complex around the portion of the polynucleotide adaptor to which it binds. The polynucleotide may be attached to a polynucleotide adaptor (e.g., a loading site) by Thus, for example, a motor protein may be a DNA processing enzyme and a rhodi This includes linear molecules in which the loading site is not DNA (e.g., RNA, A, GNA, TNA, LNA, or PEG, etc., the motor protein is It can associate (bind) with the loading site without binding to the loading site. If the loading site contains DNA, the motor protein binds to the loading site. Similarly, motor proteins can bind to the loading site by binding to the RNA. A processing enzyme, and the loading site is a linear molecule that is not RNA ( For example, the loading moiety may be DNA, PNA, GNA, TNA, LNA, or PEG. In some cases, such as those involving the ribosomal protein, the motor protein is loaded without binding to the loading site. On the other hand, if the loading site contains RNA, the mode The target protein binds to the loading site by binding to the loading site. Needless to say, in some embodiments, the motor protein is a motor protein. can bind to loading sites that do not contain the natural substrate of a protein (e.g., a DNA-binding enzyme In some embodiments, the nucleotide sequence may bind to a loading site that contains a linear molecule other than DNA. (And so on.)

[0134] Thus, in some embodiments, the polynucleotide adaptor is attached to a spacer. The motor protein contains a loading site linked to the polynucleotide adaptor. Binds to the loading site.

[0135] In some embodiments of the disclosed methods, the polynucleotide adaptor comprises a spacer and a blocking moiety as described herein comprises a loading site connected to a loading site As described in more detail herein, the blocking moiety binds to the loading site. When binding to the binding site, the blocking moiety prevents the motor protein from In some embodiments, the blocking portion prevents the motor from moving out of place. This may prevent the protein from detaching from the spacer and migrating to the loading site.

[0136] In some embodiments, the loading site is adjacent to the spacer. For example, the loading moiety may be directly attached to the spacer. The blocking moiety is adjacent to the spacer and the blocking moiety is directly adjacent to the spacer. As will be apparent, in some embodiments, the blotting The binding moiety is attached to the loading site directly adjacent to the spacer. The target protein is prevented from being removed from the spacer and transferred to the polynucleotide adaptor. In some embodiments, the blocking moiety is located immediately adjacent to the spacer. By binding to the loading site, the motor protein is released from the spacer and loaded. This prevents the cells from migrating to the target site.

[0137] For example, the polynucleotide adaptor may comprise a loading site connected to a spacer. The loading site may comprise a single-stranded or non-hybridizing polynucleotide. In some embodiments, the motor protein binds the polynucleotide at the loading site. The blocking moiety can bind to the adapter and advance from the loading site onto the spacer. The loading moiety can be linked to a loading site (such as those described above) and a blocking moiety can be linked to the loading moiety. By binding to the binding site, the motor protein is bound to a single-stranded or unhybridized polymerase. Transfer back to the ribonucleotide may be prevented.

[0138] In some embodiments, the loading site is a single-stranded or unhybridized polynucleotide. A single-stranded polynucleotide chain is an overhanging double-stranded polynucleotide chain. The non-hybridizing polynucleotide may, for example, comprise a hanging region, as described in more detail above. It may comprise the Y-shaped polynucleotides described.

[0139] In some preferred embodiments, the loading site is single-stranded or non-hybridizing. The blocking moiety comprises a single stranded or unhybridized polynucleotide. and (ii) attaching a blocking moiety to the loading site The method comprises hybridizing a binding moiety to a loading site.

[0140] In some embodiments, the loading site is about 2 to about 1000 nucleotide units in length. In some embodiments, the polynucleotide comprises a single stranded or unhybridized polynucleotide having the formula: In the present invention, the loading site is about 2 to about 500 nucleotide units in length, for example, about 2 to about 100 Nucleotide unit length, for example, about 10 to about 100 nucleotide unit length, about 20 to about 80 nucleotide unit length A single-stranded or non-stranded nucleic acid having a nucleotide unit length, for example, about 30 to about 50 nucleotide units. In some embodiments, the loading site comprises a hybridizing polynucleotide. , about 1 to about 100 nucleotide units in length, for example, about 1 to about 90 nucleotide units in length, for example For example, about 1 to about 80 nucleotide unit lengths, for example, about 1 to about 70 nucleotide unit lengths, e.g. For example, about 1 to about 60 nucleotide unit lengths, for example, about 1 to about 50 nucleotide unit lengths, e.g. For example, about 1 to about 40 nucleotide unit lengths, for example, about 1 to about 30 nucleotide unit lengths, e.g. For example, it has a length of about 1 to about 20 nucleotide units, for example, a length of about 1 to about 10 nucleotide units. In some embodiments, the nucleic acid sequence includes a single stranded or unhybridized polynucleotide. The loading site is about 2 to about 100 nucleotide units in length, for example, about 2 to about 90 nucleotides. nucleotide unit length, for example, about 2 to about 80 nucleotide unit length, for example, about 2 to about 70 nucleotide unit length nucleotide unit length, for example, about 2 to about 60 nucleotide unit length, for example, about 2 to about 50 nucleotide unit length nucleotide unit length, for example, about 2 to about 40 nucleotide unit length, for example, about 2 to about 30 nucleotide unit length nucleotide unit length, for example, about 2 to about 20 nucleotide unit length, for example, about 2 to about 10 nucleotide unit length A single stranded or unhybridized polynucleotide having a length of several tid units. In an embodiment, the blocking moiety is a single strand having a length of about 2 to about 1000 nucleotide units. In some embodiments, the blockade comprises a non-hybridizing polynucleotide. The binding portion may be about 2 to about 500 nucleotide units in length, for example, about 2 to about 100 nucleotides. Unit length, for example, about 10 to about 100 nucleotide unit length, about 20 to about 80 nucleotide unit length A single-stranded or non-hybridized nucleic acid having a length of, for example, about 30 to about 50 nucleotide units. In some embodiments, the blocking moiety comprises about 1 to about 1 00 nucleotide unit length, for example, about 1 to about 90 nucleotide unit length, for example, about 1 to about 80 nucleotide units in length, for example, about 1 to about 70 nucleotide units in length, for example, about 1 to about 60 nucleotide units in length, for example, about 1 to about 50 nucleotide units in length, for example, about 1 to about 40 nucleotide units in length, for example, about 1 to about 30 nucleotide units in length, for example, about 1 to about A single strand having a length of 20 nucleotide units, for example, a length of about 1 to about 10 nucleotide units. comprises a non-hybridizing polynucleotide. The length of the nucleic acid may be from about 2 to about 100 nucleotide units, for example, from about 2 to about 90 nucleotide units. For example, about 2 to about 80 nucleotide unit lengths, for example, about 2 to about 70 nucleotide unit lengths, For example, about 2 to about 60 nucleotide unit lengths, for example, about 2 to about 50 nucleotide unit lengths, For example, about 2 to about 40 nucleotide unit lengths, for example, about 2 to about 30 nucleotide unit lengths, For example, about 2 to about 20 nucleotide unit lengths, for example, about 2 to about 10 nucleotide unit lengths The term includes single-stranded or unhybridized polynucleotides having the structure

[0141] In some embodiments, the loading site comprises a sequence complementary to the sequence of the blocking moiety. It has columns.

[0142] Those skilled in the art will appreciate that the sequence of the loading site is typically not critical and may vary depending on the adapter and and motor proteins, as well as other experimental conditions, can be controlled or selected according to the It will be understood that exemplary sequences are provided in the Examples for illustrative purposes only. Without the need for a molecular weight reagent, one skilled in the art can easily determine the molecular weight of the motor protein by facilitating loading of the motor protein into the spacer. The function of the loading site in the adapter and motor proteins used in the method It will be appreciated that this can be achieved by a loading site selected to be compatible with Thus, as described herein, the motor protein may be a loading The sequence of the loading site is provided that the loading site can proceed from the loading site onto the spacer. may be modified.

[0143] In some embodiments, the backbone of the loading moiety and the blocking moiety are the same. (e.g., both the loading moiety and the blocking moiety have a DNA backbone, In another embodiment, the loading site and The backbones of the loading moiety and the blocking moiety are different (e.g., the loading site can have a DNA backbone, The blocking moiety has an RNA backbone or the loading moiety has an RNA backbone. the blocking moiety has a DNA backbone, etc.

[0144] In some embodiments, hybridization to the loading site of the blocking moiety This creates a region of double-stranded polynucleotide flanked by spacers. Typically, the region where the motor protein is released from the spacer and attached to the blocking portion This prevents migration into the region of the hybridized loading site. The blocking moiety may comprise a single stranded polynucleotide, and the blocking moiety may be adjacent to a spacer. the ends being flanked by spacers to create a region of double stranded polynucleotide It can be bound by nucleotides.

[0145] Loading Chain In some embodiments, the motor protein comprises a polynucleotide comprising a loading strand. In some embodiments, the loading strand is specifically loaded onto one It is desirable to load only one motor protein onto a single adaptor. Embodiments of the methods provided improve the efficiency of motor protein loading. In some embodiments, the loading strand comprises a spacer of the polynucleotide adaptor. Adjacent to (or nearby, e.g., within 1, 2, 3, 4, or 5 nucleotides of) In some embodiments, the loading strand comprises a portion that is complementary to the sequence of the primer. The polynucleotide further comprises a portion that is not complementary to the sequence of the polynucleotide adapter. In this form, the loading strand is complementary to the sequence of the adapter and thus is a spacer a portion that hybridizes to the adapter near the spacer (e.g., adjacent to the spacer); A portion that is not complementary to the adapter sequence and therefore does not hybridize to the adapter In some embodiments, the portion that does not hybridize to the adapter comprises a motor In some embodiments, the adapter facilitates loading of the polypeptide protein to the adapter. The adapter protein processes the hybridized loading strand / adaptor complex. , thereby allowing it to be processed on the spacer. The motor protein on the spacer then processes the loading strand into the polynucleotide. In some embodiments, the loading strand is displaced from the adapter. Displacement prevents subsequent loading of the motor protein onto the adaptor.

[0146] In some embodiments, the loading strand is from about 2 to about 500 nucleotide units in length, e.g. For example, about 5 to about 100 nucleotide unit lengths, for example, about 10 to about 60 nucleotide unit lengths. For example, it has a length of about 20 to about 40 nucleotide units.

[0147] Those skilled in the art will appreciate that the sequence of the loading strand is typically not critical and that the adapter and It is understood that the expression of the β-amino acid sequence can be controlled or selected according to the motor protein and other experimental conditions. Exemplary sequences are provided in the Examples for illustrative purposes only. The linker may have a polynucleotide sequence such as SEQ ID NO:18, or at least a sequence similar to SEQ ID NO:18. 20%, for example at least 30%, for example at least 40%, for example at least 50%, For example at least 60%, for example at least 70%, for example at least 80%, for example at least Polynucleotides having at least 90%, for example at least 95%, sequence similarity or identity. Without being bound by theory, one of skill in the art can easily determine whether a motor protein is a nucleotide sequence. The function of the loading strand is to facilitate loading of the target nucleic acid into the spacer used in the method. The loading strand is selected to be compatible with the adaptor and motor proteins. It will be appreciated that this can be achieved by the following: , that the motor protein can advance from the loading strand onto the spacer. Under certain conditions, the sequence of the loading strand may be modified.

[0148] In some embodiments, the blocking moiety is, in place of the displaced loading strand, Binds to an adaptor near (e.g., adjacent to) the spacer. The binding moiety may be, for example, a single-stranded or non-hybridizing polynucleotide (i.e., a blotting The displaced locus may be a binding strand, e.g., a binding strand that is a binding site for a displaced locus by hybridization. The nucleic acid sequence may be linked to a polynucleotide adaptor instead of the backing strand.

[0149] A loading strand is used to advance the motor protein over the spacer and block the The king strand is used to prevent the motor protein from moving away from the spacer. In some embodiments, the blocking strand has a higher polynucleotide affinity than the loading strand. For example, the blocking strand may be longer than the loading strand. In some embodiments, the loading strand may hybridize more strongly than the loading strand. In this embodiment, the blocking strand is provided in a concentration greater than the loading strand. By providing a loading strand, reannealing of the displaced loading strand is reduced or prevented. This is because the number of motor proteins on the polynucleotide adaptor is controlled by the presence of the motor protein. For example, the blocking strand should be at least twice as long as the loading strand, e.g. For example, at least 5 times, for example, at least 10 times, for example, at least 20 times, for example, at least Both may be present in a concentration of 50-fold, or at least 100-fold.

[0150] Move the motor protein forward along the spacer As discussed in more detail herein, the methods provided herein include The method includes placing a protein onto a spacer contained in the adapter.

[0151] As discussed in more detail herein, in some embodiments, a polynucleotide By contacting the adaptor with the motor protein, the motor protein binds to the polynucleotide It will advance onto the spacer of the nucleotide adapter.

[0152] In some embodiments, the motor protein is attached to a spacer from the loading site. Propulsion requires the motor protein to move from the loading site to the spacer. Any suitable propulsion may be used.

[0153] Typically, the motor protein is cleaved from a polynucleotide adaptor (e.g., There is an energy barrier to proceeding on the spacer (from the binding site). Barriers can arise from changes in the chemical composition of the linear molecule along which the motor protein moves. For example, in some embodiments, the change is from the polynucleotide backbone of the loading site. In other embodiments, the energy The G-barrier is a change in the DNA or RNA backbone at the loading site to an abasic spacer. The typical existence of an energy barrier can result from the binding of motor proteins to polynucleotides. Push the adapter (e.g., from the loading site) forward onto the spacer. This means that thrust is often required.

[0154] For example, in some embodiments, motor proteins can be advanced over spacers. The induction of motor activity involves applying a physical or chemical force to the motor protein. In the form, moving the motor protein forward on the spacer is In some embodiments, the motor tank comprises contacting the motor tank with one or more fuel molecules. Force is applied by a second motor protein that "pushes" the protein against the spacer. In other words, in some embodiments, the polynucleotide adaptor is attached to the spacer. The motor protein is the first motor protein and contains a connected loading site. and advancing the first motor protein from the loading site onto the spacer. loading a second motor protein into the loading site and and advancing the protein from the loading site toward the spacer, The second motor protein presses the first motor protein against the spacer.

[0155] In another embodiment, the blocking moiety is attached to a polynucleotide adaptor. The motor protein is pressed against the spacer. Binding to a nucleotide adaptor (e.g., a loading site) binds the motor protein to This may be advantageous enough to overcome the energy barrier to pressing against the spacer.

[0156] In another embodiment, the motor protein has a blocking moiety attached to an adaptor. For example, a motor protein may be attached to the spacer before or after it is attached to the spacer. A blocking moiety can be disposed on the pacer and attached to the polynucleotide adaptor. This may prevent the motor protein from moving away from the spacer. In this embodiment, the motor protein is a blocking molecule linked to a spacer and an adaptor. The motor protein may be attached to an adaptor that includes a binding moiety. For example, a motor protein can be placed on the adaptor in a spacer. For example, in some embodiments, the motor protein may preferentially localize to a specific location. The protein can bind to a nucleotide island within the spacer. In embodiments, contacting the polynucleotide adaptor with the motor protein comprises: Contacting the motor protein with the nucleotide island contained in the spacer It may include.

[0157] In such embodiments, the motor protein is typically free of, e.g., a spacer. The motor is modified to prevent it from separating from the spacer by moving along the The protein may be modified before or after it is placed on the spacer. Typically, the motor protein is modified after being placed on the spacer. Protein modifications are described in more detail herein.

[0158] From the above discussion, it is understood that some embodiments of the disclosed method include: It will be apparent that this includes proceeding from the nucleotide adaptor onto the spacer. .

[0159] In some embodiments, when the motor protein is present on a spacer, the spacer Sir occupies the active site of the motor protein. In some embodiments, the active site is partially occupied by a spacer. In other embodiments, the active site is The active site is fully occupied by the motor protein. Typically, as used herein, In this case, the active site of the motor protein is the polynucleotide binding site of the motor protein. Those skilled in the art will recognize that the ATP binding site of a motor protein is a polynucleotide analog. It will be appreciated that the adapter may be separate from the In an embodiment, the polynucleotide binding groove of the motor protein is occupied by a spacer. The ATP binding site of the motor protein is not occupied by the spacer. In this morphology, both the polynucleotide-binding groove and the ATP-binding site of the motor protein are The spacer is occupied by the polynucleotide binding site of the motor protein. The groove can be occupied by a spacer, and the ATP binding site of the motor protein is occupied by the spacer. The theory is that the ATP-dependent ATP pathway may be blocked or blocked by ATP receptors or may be accessible to ATP. Without being tethered, the motor protein still stalls on the spacer and binds ATP. Even when the binding site is accessible, it may not be involved in wasteful turnover. For example, In some embodiments, ATP hydrolysis is mediated by the polynucleotide Conformational changes in motor proteins induced by polynucleotide binding to the binding groove The polynucleotide binding groove is stalled on the spacer and cannot be attached to the polynucleotide. If the protein is not accessible to the target protein, such conformational changes will not occur, reducing wasteful metabolic turnover or Be excluded.

[0160] In some embodiments, the spacer comprises the entire footprint of the motor protein. In some embodiments, the spacer spans a region of the motor protein. It spans the entire footprint and occupies the active site of the motor protein. For example, in some embodiments, the motor protein advances over the spacer, thereby , the motor protein is now entirely present on the spacer.

[0161] Those skilled in the art will recognize that a motor protein that stalls on a spacer as defined herein , motor proteins that simply stall near the spacer (e.g., adjacent to the spacer) It will be seen that there is a significant difference between the quality of the motor protein and the spacer protein. If it is adjacent to or only slightly spans the spacer, wasteful metabolism Rotation may not necessarily be prevented. Allowing the device to move to this extent is within the scope of the present claims. This does not correspond to preventing the transfer of the target protein away from the spacer. There is a slight overlap between the target protein and the spacer, but Prevents fuel molecules from turning over at substantially the same rate as when bound to a polynucleotide. Unstopped motor proteins are not present on the spacer used herein. .

[0162] Thus, in some embodiments, the spacer separates the active site of the motor protein. When the motor protein is occupied, the turnover of fuel molecules by the motor protein is reduced. In embodiments, turnover of fuel molecules by motor proteins is eliminated.

[0163] Motor proteins As one of skill in the art would understand, any suitable motor protein is provided herein. In one embodiment, the motor protein can be used in methods and products comprising the steps of: , capable of binding to a polynucleotide and its relative position to, for example, the nanopore through the pore. It can be any protein capable of controlling migration.

[0164] In one embodiment, the motor protein is a polynucleotide handling enzyme. Polynucleotide handling enzymes are those that interact with polynucleotides. It is a polypeptide capable of interacting with and modifying at least one property of the polypeptide. This enzyme cleaves polynucleotides into individual nucleotides or shorter chains of nucleotides. Polynucleotides can be formed by forming nucleotides, e.g., dinucleotides or trinucleotides. This enzyme can modify the nucleotides in a polynucleotide or to modify the specific The polynucleotide may be modified by moving the nucleic acid to a specific position.

[0165] In one embodiment, the motor protein is more preferably Enzyme Classification (EC) group 3.1 .11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21, 3 .1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30, and 3.1 It is derived from any member of .31.

[0166] Typically, motor proteins are helicases, polymerases, exonucleases, etc. , topoisomerase, or a variant thereof.

[0167] In some embodiments, the motor domain on the spacer of the polynucleotide adaptor The protein is transported by the motor protein (other than by the movement of the motor protein from the end of the spacer). The motor protein is modified to prevent it from being released from the spacer. For example, the motor protein can be attached to the adaptor in a suitable manner. It can be loaded and then modified to prevent it from being released from the spacer. Alternatively, the motor protein may detach from the spacer before it is loaded onto the adaptor. The motor protein may be modified to prevent it from detaching from the spacer. Modification of motor proteins to prevent this can be accomplished by methods known in the art, e.g., Discussed in WO2014 / 013260, which is incorporated herein by reference in its entirety. and preventing the motor protein from disengaging from the polynucleotide chain. See in particular the section describing the modification of motor proteins such as helicases to For example, the motor protein can be synthesized by the method of It can be modified by treatment with thiamidamide (TMAD).

[0168] For example, motor proteins are cleaved from a polynucleotide chain and a polynucleotide-free opening, e.g., a cavity, through which the strand can pass when the In some embodiments, the polynucleotide non-binding opening may have a groove, a groove, or a gap. When the motor protein is released from the spacer, the spacer can pass through. In some embodiments, the polynucleotide sequence of a given motor protein is The non-bonded openings are identified by reference to their structure, e.g., by reference to their X-ray crystal structure. X-ray crystal structures can be determined in the presence of a polynucleotide substrate. In some embodiments, a given motor tank may be obtained with and / or without The location of the polynucleotide-free opening in the protein is determined by standard protein binding methods known in the art. The cage may be used to predict or confirm the effect of molecular modeling. In the present invention, the polynucleotide-free opening is a region of one or more parts of the motor protein, e.g. , can be generated transiently by the movement of one or more domains.

[0169] Motor proteins are modified by closing the polynucleotide-free opening Therefore, by closing the polynucleotide-free opening, the motor For example, the motor protein can be prevented from detaching from the spacer. The polynucleotide-free openings may be modified by covalently closing them. In some embodiments, the preferred motor protein to address in this manner is a helical motor protein. It is Ze.

[0170] In one embodiment, the motor protein is an exonuclease. Suitable enzymes include , Exonuclease I from E. coli (SEQ ID NO: 1), Exonuclease I from E. coli Nuclease III enzyme (SEQ ID NO:2), RecJ from T. thermophilus (SEQ ID NO: 3) and bacteriophage lambda exonuclease (SEQ ID NO: 4), T atD exonuclease, and variants thereof. Three subunits containing the sequence shown in SEQ ID NO:3 or a variant thereof interact with each other. This results in the formation of a trimeric exonuclease.

[0171] In one embodiment, the motor protein is a polymerase. roPhage® 3173 DNA polymerase (Lucigen® (commercially available from Bioron Corporation), SD polymerase (commercially available from Bioron Corporation), (registered trademark) manufactured by NEB, Klenow, or variants thereof. In one embodiment, the enzyme is Phi29 DNA polymerase (SEQ ID NO:5) or Modified versions of Phi29 polymerase that can be used in the present invention A similar technique is disclosed in U.S. Pat. No. 5,576,204.

[0172] In one embodiment, the motor protein is a topoisomerase. The topoisomerase is preferably of the subclassification (EC) groups 5.99.1.2 and 5.99. Topoisomerases are members of any of the three families of topoisomerases that catalyze the synthesis of cDNA from an RNA template. They can be reverse transcriptases, enzymes that can catalyze the formation of DNA. For example, New England Biolabs® and Invitrogen (registered trademark).

[0173] In one embodiment, the motor protein is a helicase. Any suitable helicase can be used in accordance with the methods provided herein. For example, The or each motor protein used independently is Hel308 helicase, Rec D helicase, TraI helicase, TrwC helicase, XPD helicase, and D The monomeric helicase may be selected from a da helicase, a da helicase, or a variant thereof. For example, the TraI helicase and the TraI subunit may comprise several domains linked together. The B group helicases consist of two RecD helicase domains, a relaxase domain, and and C-terminal domains. These domains typically form oligomers. The monomeric helicase can function without the need for a cleavage reaction. Specific examples include Hel308, NS3, Dda, UvrD, Rep, PcrA, and Pif1. These helicases typically act on single-stranded DNA. Examples of helicases that can translocate along both strands of double-stranded DNA include F tfK and hexameric enzyme complexes, or multisubunit complexes such as RecBCD Some examples include:

[0174] Hel308 helicase is disclosed in WO2013 / 0574, the entire contents of which are incorporated by reference. 95, the entire contents of which are incorporated by reference. XPD helicase is described in publications such as WO2013 / 098562, which is incorporated herein by reference. and WO2013 / 098561, the entire contents of which are incorporated by reference. Dda helicase is disclosed in WO2015 / 013362, the entire contents of each of which are incorporated by reference. No. 55981 and WO2016 / 055777.

[0175] In one embodiment, the helicase has the sequence shown in SEQ ID NO:6 (Trwc Cba) or or a variant thereof, the sequence shown in SEQ ID NO: 7 (Hel308 Mbu) or a variant thereof or the sequence shown in SEQ ID NO: 8 (Dda) or a variant thereof. It may differ from the native sequence in any of the ways discussed herein. Exemplary mutations include E94C / A360C. Further exemplary mutations of SEQ ID NO:8 are , E94C / A360C, then (ΔM1)G1G2 (i.e., deletion of M1, then G 1 and the addition of G2).

[0176] In some embodiments, the motor protein (e.g., a helicase) comprises at least Two active modes of operation (all the components a motor protein needs to facilitate movement) , e.g., ATP and Mg as discussed herein 2+ Equipped with fuel and cofactors such as one inactive mode of operation (when the motor protein is active to facilitate movement) and one inactive mode of operation (when the motor protein is active to facilitate movement) (if the necessary components are not present) can be used to control the movement of polynucleotides.

[0177] When it has all the components necessary to facilitate movement (i.e., in active mode) The motor protein (egelicase) moves the polynucleotide from 5' to 3' or The motor proteins move in a 3' to 5' direction (depending on the motor protein). In embodiments in which a protein is used to control the translocation of a polynucleotide strand relative to a nanopore, Motor proteins can be used to move polynucleotides (e.g., against an applied field) ) to translocate the polynucleotide away from (e.g., out of) the pore or For example, by moving the pore toward (e.g., into) the pore (according to an applied field). For example, the motor protein can move toward a polynucleotide that is a target of translation. When the end of the oxodil is captured by the pore, the motor protein is pulled away from the applied potential. It acts against the direction of the generated field and drives the threaded polynucleotide into the pore. However, the motor protein is pulled away from the cytoplasm (e.g., into the cis chamber). When the moving end is trapped within the pore, the motor protein is activated by the applied electrical potential The field acts in a directional manner to drive the threaded polynucleotide into the pore (e.g., the trans-channel Push it into the compartment.

[0178] Motor proteins (e.g., helicases) provide the components necessary to facilitate movement. When not present (i.e., in the inactive mode), the motor protein binds to the polynucleotide and are drawn into the pore by, for example, the field resulting from an applied potential. a brake that slows the movement of the polynucleotide as it moves relative to the nanopore In the inactive mode, either end of the polynucleotide can be captured. It does not matter whether the polynucleotide is captured or not, but rather the applied voltage determines the translocation of the polynucleotide relative to the pore. The field is important, and the motor protein acts as a brake. In the inactive mode, The control of polynucleotide movement by motor proteins is a process known as ratcheting and slicing. This can be described in a number of ways, including ding, and grey-king.

[0179] As discussed above, the methods disclosed herein reduce the concept of wasteful turnover. Turnover is the chemical (enzymatic) conversion of fuel molecules by motor proteins. .

[0180] The fuel is typically a free nucleotide or a free nucleotide analog. Nucleotides are adenosine monophosphate (AMP), adenosine diphosphate (ADP), 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 (CTP), phosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP) , cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), Deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), Oxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), Deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), Deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), Uridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine diphosphate (dUDP) deoxycytidine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine deoxycytidine diphosphate (dCDP), and deoxycytidine triphosphate (dCTP) The free nucleotides are usually, but not limited to, AMP, TMP, G Selected from MP, CMP, UMP, dAMP, dTMP, dGMP, or dCMP The free nucleotide is typically adenosine triphosphate (ATP).

[0181] Motor protein cofactors are factors that enable motor proteins to function. The cofactor is preferably a divalent metal cation. The divalent metal cation is preferably For example, Mg 2+ , Mn 2+ , Ca 2+ , or Co 2+ The cofactor is most preferably is Mg 2+ It is.

[0182] Removal of excess motor protein In some embodiments, the methods provided include providing an excess motor that is not located on a spacer. The method further comprises the step of removing excess motor protein molecules. The stress step may be referred to as a stress step. This allows the removal of misbound motor proteins from the promoter. Modifying the motor protein to prevent the protein from detaching from the spacer In embodiments of the provided methods, including: In one embodiment, the motor protein is removed.

[0183] The removal of excess motor protein can be achieved by the addition of the nucleotide sequence to the polynucleotide adapter described herein. It is beneficial to reduce or eliminate the presence of unbound motor proteins that do not stall at the same time. Motor proteins that do not move can bind to polynucleotide chains and / or are present Excessive motor tanks can consume fuel molecules that are not needed, i.e., they can participate in wasteful metabolic turnover. By removing the protein, this wasteful turnover is eliminated from the motor protein that does not stall. The non-productive consumption of fuel molecules is reduced or prevented, thus reducing the non-productive consumption of fuel molecules.

[0184] Unbound motor protein can be removed in any suitable manner (e.g., by removing the motor protein from the For example, unbound motor proteins can be removed by washing or filtration. Unbound motor proteins can be removed using a high salt wash buffer. Unbound motor proteins can be removed by specific pH or pH-dependent cleavage, which facilitates their removal. can be removed using temperature conditions. For example, stress conditions can be, for example, Such applications may include high salt concentrations in the presence of fuels such as

[0185] Remove the motor protein from the spacer and move it forward As discussed in more detail herein, in some embodiments, the adapter to control the movement of the target polynucleotide by stalling the motor protein prior to use of the For example, in some embodiments, the disclosed adapters can be used to A motor protein is provided to control the movement of the nucleic acid relative to the nanopore. In some embodiments, the movement of the target polynucleotide relative to the nanopore comprises It is useful for characterizing octides.

[0186] As discussed in more detail herein, the disclosed method comprises: The blocking moiety prevents the motor protein from stalling. The nucleic acid sequence is transferred from the spacer to the polynucleotide adaptor, e.g., the spacer is transferred to the polynucleotide adaptor. This prevents migration of the nucleotide adaptor to the loading site.

[0187] Typically, a motor protein advances from the spacer to the target polynucleotide to target the target. There is an energy barrier that controls the movement of the target polynucleotide. can result from changes in the chemical composition of the linear molecule along which the motor protein moves. For example, In some embodiments, the change is from the non-polynucleotide backbone of the spacer to the target polynucleotide. In other embodiments, the change may be to the polynucleotide backbone. The glycine barrier is the distance from the basic spacer to the DNA or RNA backbone of the target polynucleotide. The typical existence of an energy barrier can lead to the motor protein being bound to a polynucleotide. To advance from the nucleotide adaptor (e.g., from the spacer) to the target polynucleotide, This means that propulsion is often required to

[0188] Thus, in some embodiments, the motor protein is attached to the target polypeptide from the spacer. The motor protein advances the nucleotide from the spacer to the target polynucleotide. The method includes providing a driving force for the transfer of the leotide to the leotide. Any suitable driving force may be used. It is possible.

[0189] For example, in some embodiments, the driving force drives the polynucleotide adaptor through the nanopore. For example, in some embodiments, Nopore binds to a polynucleotide adaptor and directs the motor protein to the target polynucleotide. Press it onto the punch line.

[0190] More specifically, in some embodiments, the motor protein is stalled on a spacer. The polynucleotide adapter is a target polynucleotide adapter. The target polynucleotide is contacted under conditions that allow the target polynucleotide to bind to the target polypeptide. For example, As described in more detail below, the polynucleotide adaptor is a target polynucleotide adaptor. The polynucleotide adapter can be linked, for example, to the end of the target polynucleotide. The polynucleotide adaptor-target polynucleotide is then coupled to the nanopore. The polynucleotide adduct can be contacted with the nanopore. The adapter can pass through the nanopore. Translocation (e.g., through a nanopore) of the polynucleotide adaptor on the spacer. For example, a force can be provided to a stalled motor protein at a polynucleotide adaptor. In the embodiment in which the nanopore is passed under the influence of an applied potential, The force exerted by the nanopore on the motor protein when it contacts or moves against the motor This is sufficient to force the target protein onto the target polynucleotide. In this case, the nanopore releases the motor protein from the spacer and delivers it to the target polynucleotide. Such an embodiment can be thought of as "pushing" the polynucleotide onto the The tethered adaptor comprises (i) a loading site and (ii) a target polynucleotide. and a spacer disposed between the polynucleotide strand and the polynucleotide strand that may be attached or otherwise linked. , which are particularly suited (but not limited to) to embodiments of the present disclosure.

[0191] In another embodiment, the motor protein advances from the spacer to the target polynucleotide. The force required to induce the movement can be generated by applying physical or chemical forces to the motor proteins. In some embodiments, the motor protein is attached to the target polypeptide from the spacer. Moving a nucleotide forward brings the motor protein into contact with one or more fuel molecules. In some embodiments, the motor protein is removed from the spacer to form a target protein. Force is applied by a second motor protein that "pushes" the target polynucleotide In other words, in some embodiments, the polynucleotide adaptor comprises a spacer. and the motor protein comprises a first motor protein, the first motor protein being capable of binding to a target polynucleotide. and drives the first motor protein from the spacer to the target polynucleotide. This involves loading a second motor protein onto a polynucleotide adaptor; and propelling a second motor protein toward the target polynucleotide. The second protein presses the first motor protein against the spacer. In this embodiment, the polynucleotide adaptor is typically spaced apart from the target polynucleotide. (a polynucleotide that is or will bind to a target polynucleotide) It contains a loading site connected to the end of a spacer (away from the end of the adaptor).

[0192] In such embodiments, the motor protein is typically removed from the spacer to form the target polypeptide. It has been shown that it is not necessary to remove the blocking moiety in order to advance to the nucleotide. It will be appreciated that in some embodiments, however, the method may include blocking. For example, contacting a polynucleotide adaptor with a nanopore can include displacing a binding portion. The blocking moiety can be removed or displaced by In some embodiments, removal of the blocking moiety from the polynucleotide adaptor results in The motor protein is released from the spacer to form the polynucleotide adaptor and / or It is then allowed to migrate towards the target polynucleotide.

[0193] In some embodiments, the blocking moiety is spaced (or separated) from the target polynucleotide. That is, a polynucleotide that is or will bind to a target polynucleotide. The spacer adaptor is attached to the polynucleotide adaptor on the spacer side (away from the end of the spacer adaptor). In such embodiments, the polynucleotide adaptor typically Apart from the nucleotide (which is bound or will bind to the target polynucleotide), The loading sequence is connected to the end of the spacer (away from the end of the oligonucleotide adapter). The blocking moiety is a polynucleotide between the loading site and the spacer. The nucleotide sequence of the ...

[0194] In other embodiments, the blocking moiety is or towards the end of the polynucleotide adaptor to which it will be attached) In such an embodiment, the target polynucleotide is bound to a polynucleotide adaptor. In the present invention, the polynucleotide adaptor is typically spaced apart from the target polynucleotide (target A polynucleotide adaptor that is or will bind to a target polynucleotide. a loading site connected to the end of a spacer (away from the end of the block) The binding portion is a polynucleotide that is or will bind to a target polynucleotide. The polynucleotide adaptor is ligated towards the end of the nucleotide adaptor.

[0195] Movement Control and Characterization 1. A method for controlling translocation of a target polynucleotide relative to a transmembrane nanopore, comprising: i) (A) a target polynucleotide, (B) a polynucleotide adaptor comprising a spacer and (C) providing a motor protein. ii) carrying out the methods disclosed herein, thereby obtaining polynucleotide adaptors; and stalling the motor protein on the spacer of iii) Deletions on the spacer of the target polynucleotide and the polynucleotide adaptor contacting the accelerated motor protein with a nanopore; iv) applying a voltage potential across the transmembrane nanopore, thereby activating the motor protein; The target polynucleotide is then translocated across the spacer, thereby targeting the nanopore. and controlling the movement of a target polynucleotide.

[0196] Any of the methods of the embodiments disclosed herein may further comprise a polynucleotide adaptor. can be used to stall the motor protein on the spacer.

[0197] In one embodiment, the target polynucleotide is attached to a polynucleotide adaptor. In one embodiment, step (iii) comprises ligating a polynucleotide adaptor to a target polynucleotide. The method includes conjugating the compound to a leotide.

[0198] In one embodiment, after the motor protein has stalled on the polynucleotide adaptor, In another embodiment, the polynucleotide adaptor binds to the target polynucleotide. After the polynucleotide adaptor binds to the target polynucleotide, the motor protein The quality stalls on the polynucleotide adapter.

[0199] 1. A method for controlling translocation of a target polynucleotide relative to a transmembrane nanopore, comprising: i) providing a target polynucleotide; ii) A polynucleotide that contains a spacer and on which the motor protein stalls. and a polynucleotide adaptor comprising the polynucleotide adaptor disclosed herein. providing a method for producing a composition comprising the steps of: iii) contacting the target polynucleotide and the polynucleotide adaptor with the nanopore. And iv) applying a voltage potential across the transmembrane nanopore, thereby activating the motor protein; The target polynucleotide is then translocated across the spacer, thereby targeting the nanopore. and controlling the movement of a target polynucleotide.

[0200] Any of the methods of the embodiments disclosed herein may further comprise a polynucleotide adaptor. can be used to stall the motor protein on the spacer.

[0201] In one embodiment, the target polynucleotide is attached to a polynucleotide adaptor. In one embodiment, step (iii) comprises ligating a polynucleotide adaptor to a target polynucleotide. The method includes conjugating the compound to a leotide.

[0202] In the disclosed methods, the polynucleotide adaptor can be attached to the target polynucleotide in any manner. The adaptor is preferably capable of binding to a target polynucleotide. The adaptor can be ligated to a target polynucleotide. Either end of the oligonucleotide, i.e., the 5' end or the 3' end, or the target polypeptide Adaptation can be performed at both ends of a oligonucleotide, i.e., at the 5' end and the 3' end. The primer is ligated to the target polynucleotide using any method known in the art. The adapter reacts in the absence of ATP or with gamma-S-ATP instead of ATP ( ATPγS) can be used to link to a target polynucleotide. When the adapter binds to the target polynucleotide, the motor protein binds to the polynucleotide. When stalled on the ATP adapter, the adapter reacts with the target polynucleotide in the absence of ATP. The adapter is ligated to the nucleotide sequence of ... , Taq DNA ligase, Tma DNA ligase, and 9 o N DNA ligase Any ligase may be used to ligate the fragments. The ligase may be tested prior to step (iii) of the method. The adaptors may be attached using a topoisomerase. The merases are, for example, those of the EC groups 5.99.1.2 and 5.99.1.3. The member may be any of the following:

[0203] 1. A method for characterizing a target polynucleotide, comprising: i) carrying out one of the methods disclosed above; ii) detecting the target polynucleotide as it is translocated relative to the nanopore; and performing one or more measurements that indicate one or more characteristics of the target polynucleotide. and characterizing it as it translocates relative to the nanopore.

[0204] Polynucleotide Adapters A polynucleoside that includes a spacer and stalls a motor protein on the spacer. Also provided are polynucleotide adapters. It will be appreciated that this is also applicable to the method embodiments discussed herein and above. It will be possible.

[0205] In one embodiment, the present invention comprises: (i) a spacer; and (ii) a motor tank stalled on the spacer. a protein in which the active site of the motor protein is occupied by a spacer; a motor protein; and (iii) a blocking moiety attached to an adaptor, A blocking moiety that prevents the motor protein from moving away from the spacer.

[0023] Provided herein is a polynucleotide adapter comprising:

[0206] In some embodiments, the spacer, the motor protein, and the blocking moiety is as described in detail herein.

[0207] In one embodiment, (i) the adaptor comprises a loading site connected to a spacer. (ii) a blocking moiety is attached to the loading site; and (iii) the blocking moiety is In some embodiments, the target protein is prevented from binding to the loading site. The loading sites are as described herein.

[0208] In one embodiment, i) In the 5' to 3' direction, B -SD} n or {DSL B} n (In the formula, L B is a blocked loading site, S is a spacer, and D is a double-stranded polynucleotide. leutide, and n is an integer, optionally an integer from 1 to about 20; ii) one or more motor proteins stalled on a spacer (S), The or each L B The portion is a double-stranded polynucleotide ( D) A polynucleotide that prevents the polynucleotide from moving away from the spacer (S) in a direction away from the A tip adapter is provided.

[0209] In some embodiments, the loading site, the spacer, and the motor protein is as described herein.

[0210] In some embodiments, n is from 1 to about 10, e.g., from 1 to about 5, e.g., 1, 2, 3 , or 4, and often an integer 1 or 2. In some embodiments, n is 1 or and multiple motor proteins can stall on multiple spacer Ss. .

[0211] In some embodiments, the double-stranded polynucleotide is from about 1 to about 1000 nucleotides. In some embodiments, the double-stranded polynucleotide has a length of about 5 to about 500 nucleotides. The length of the nucleic acid fragment is, for example, about 10 to about 100 nucleotides, for example, about 20 to about 80 nucleotides. The length of the nucleic acid is, for example, about 30 to about 50 nucleotides.

[0212] In some embodiments, L B is a polynucleotide that is the same type as D L may be a nucleotide or may be a different type of polynucleotide than D. B and / or D is a polynucleotide of the same type as the target polynucleotide to which the adaptor can bind. The target polynucleotide may be a polynucleotide of a different type. For example, L B can be double stranded DNA, D can be double stranded DNA, The adaptor may be suitable for binding to a target polynucleotide that is double-stranded DNA.

[0213] In one embodiment, i) In the 5' to 3' direction, B -SD} n or {DSL B} n (In the formula, L B is a first double-stranded polynucleotide, S is a spacer, and D is a second double-stranded polynucleotide. nucleotides, n being an integer, optionally an integer from 1 to about 20; Polynucleotide (L B ) is adjacent to the spacer (S), and the spacer (S) is the second (D)) ii) One or more motor proteins stall on the spacer (S), a polynucleotide sequence that A head adapter is provided.

[0214] In some embodiments, the spacer and the motor protein are As can be seen.

[0215] In some embodiments, n is from 1 to about 10, e.g., from 1 to about 5, e.g., 1, 2, 3 , or 4, often an integer between 1 or 2.

[0216] In some embodiments, L B is a polynucleotide that is the same type as D L may be a nucleotide or may be a different type of polynucleotide than D. B and / or D is a polynucleotide of the same type as the target polynucleotide to which the adaptor can bind. The target polynucleotide may be a polynucleotide of a different type. For example, L B can be double stranded DNA, D can be double stranded DNA, The adaptor may be suitable for binding to a target polynucleotide that is double-stranded DNA.

[0217] kit Kits containing the polynucleotide adaptor and the motor protein are also provided. Any of the polynucleotide adaptors disclosed herein may be used as described herein and above. It will be understood that the present invention is applicable to any kit embodiment.

[0218] In one embodiment, there is provided a kit for modifying a target polynucleotide, comprising: i) a polynucleotide adaptor comprising a spacer; ii) a motor protein capable of controlling the movement of a target polynucleotide; iii) the motor protein is located on the spacer of the polynucleotide adaptor; When the active site of the motor protein is occupied by a spacer, the motor protein The polynucleotide adapter is inserted so that the protein is prevented from moving away from the spacer. and a blocking moiety capable of binding to a peptide.

[0219] In one embodiment, the polynucleotide adaptor is a polynucleotide adaptor as described in more detail herein. In one embodiment, the motor protein is a nucleotide adaptor as described herein. In one embodiment, the blocking moiety is a motor protein as described herein. This is the blocking portion of the above.

[0220] In one embodiment, the polynucleotide adaptor comprises a loading site. The loading site can be any of the loading sites described herein. In this state, the blocking moiety prevents the motor protein from binding to the loading site. The polynucleotide can be bound to a polynucleotide adaptor such that the nucleic acid is blocked.

[0221] 1. A kit for modifying a target polynucleotide, comprising: i) a polynucleotide adaptor, comprising a spacer and a polynucleotide adaptor a blocking moiety attached to said polynucleotide adaptor; ii) a motor protein capable of controlling the movement of a target polynucleotide; Including, When the motor protein is bound to the adaptor, the blocking moiety binds to the motor Kits are also provided that prevent the protein from migrating off the spacer.

[0222] Polynucleotide adaptors, spacers, blocking moieties, and motor proteins The substance is typically as defined in more detail herein.

[0223] system Also provided is a system comprising a polynucleotide adaptor, a motor protein, and a nanopore. Any of the polynucleotide adaptors disclosed herein may be used as described herein and above. It will be understood that this is applicable to the system embodiments discussed.

[0224] In one embodiment, there is provided a system for characterizing a target polynucleotide, comprising: a polynucleotide adaptor comprising a spacer; A motor protein, wherein the active site of the motor protein is occupied by a spacer. A motor protein, A blocking part coupled to the adapter, the blocking part being connected to the motor a blocking portion that prevents the protein from moving away from the spacer; - characterizing the target polynucleotide as it translocates relative to the nanopore; and a nanopore for attaching the nanopore to the target.

[0225] In one embodiment, the polynucleotide adaptor is a polynucleotide adaptor as described in more detail herein. In one embodiment, the motor protein is a nucleotide adaptor as described herein. In one embodiment, the blocking moiety is a motor protein as described herein. In one embodiment, the nanopore is a nanopore as described herein. and the system may further comprise a membrane, a control device, etc. as defined herein.

[0226] In one embodiment, the polynucleotide adaptor comprises a loading site. The loading site can be any of the loading sites described herein. In this state, the blocking moiety prevents the motor protein from binding to the loading site. The polynucleotide can be bound to a polynucleotide adaptor such that the nucleic acid is blocked.

[0227] In one embodiment, the system further comprises a target polynucleotide. The polynucleotide is a target polynucleotide as described herein.

[0228] Target Polynucleotide In embodiments of the present disclosure relating to characterization of a target polynucleotide, the target analyte is As described in more detail herein, the transmembrane nanopore may be coupled to, for example, the interior of, or moves through it.

[0229] In one embodiment, the presence, absence, or one or more characteristics of a target polynucleotide is determined. The method comprises detecting the presence, absence, or one or more abnormalities of at least one target polynucleotide. The method can be for determining a characteristic of two or more target polynucleotides. The method may involve determining the presence, absence, or one or more characteristics of any number of targets. Target polynucleotides, e.g., 2, 5, 10, 15, 20, 30, 40, 50, 100, Determine the presence, absence, or one or more characteristics of one or more target polynucleotides. Any number of features of one or more target polynucleotides, e.g., one, two, Three, four, five, ten or more features may be determined.

[0230] Binding of a molecule (e.g., a target polynucleotide) within the channel of the pore induces its transport through the pore. The pore channel affects ion flow, which is the basis of the book "Molecular Sensing" of pore channels. The open channel ion current fluctuations are measured using a suitable measurement technique based on changes in electrical current. (See, for example, WO2000 / 28312 and D. Stodda rt et al.,Proc.Natl.Acad.Sci.,2010,106,7 702-7 or WO2009 / 077734). Ions measured by a decrease in current The degree of flow reduction is related to the size of the obstruction in or near the pore. Thus, For binding of a molecule of interest (e.g., a target polynucleotide) within or near the pore This provides a detectable and measurable event, thereby forming the basis of a "biological sensor." Detecting the presence of biomolecules can be used to advance personalized drug development, medicine, and diagnostics. diagnostics, life science research, environmental monitoring, and security and / or defense industries Uses for the following are found.

[0231] The target polynucleotide may be secreted from the cell. Alternatively, the target analyte may be intracellularly The analyte may be present in the cell and therefore may need to be extracted from the cell before the method can be carried out. It must be done.

[0232] In one embodiment, the target polynucleotide is a nucleic acid. It is defined as a macromolecule that contains the above nucleotides. It occurs naturally in DNA and RNA. Nucleic acid bases can be distinguished by their physical size. A nucleic acid molecule or an individual base is When passing through the nanopore channel, the size difference between the bases allows ions to pass through the channel. A directly correlated decrease in current results. Fluctuations in ion current can be recorded. Fluctuations in ion current Suitable electrical measurement techniques for recording are described, for example, in WO2000 / 28312 and D. Stoddart et al.,Proc.Natl.Acad.Sci.,2010 ,106,pp7702-7 (single channel recording device), as well as, for example, WO2009 / With suitable calibration, the ion The characteristic decrease in current is used to identify specific nucleotides and related bases crossing the channel. In a typical nanopore nucleic acid sequencing, the nucleotide Due to partial blocking of the channel by the nucleotide, individual nucleotides of the nucleotide sequence of interest can be blocked. When the ions pass through the nanopore channel in succession, the ion current in the open channel decreases. It is this reduction in ion current that is measured using the preferred recording technique described above. The decrease in ion flux corresponds to the decrease in ion flux measured for a given nucleotide through the channel. This allows for calibration and therefore the determination of which nucleotides will pass through the channel. This provides a means for quantification of nucleic acids passing through the nanopore, if carried out sequentially. This provides a method for determining the sequence of a nucleic acid fragment. Typically, a reduction in ion flow through a channel is called a constriction (or "leading head") It is necessary to directly correlate the size of the individual nucleotides passing through the sequence. The determination is, for example, by the action of a polymerase or an associated motor protein such as a helicase. It will be understood that this can be performed on an intact nucleic acid polymer that is "threaded" into the pore. Alternatively, the sequence may be a sequence of nucleotide triphosphates successively removed from the target nucleic acid near the pore. The acid-base transition can be determined by the acid-base transition (see, for example, WO2014 / 187924). stomach).

[0233] A polynucleotide or nucleic acid can contain any combination of any nucleotides. The nucleotides may be naturally occurring or artificial. One or more of the nucleotides in a polynucleotide The nucleotides in the polynucleotide may be oxidized or methylated. The nucleotides in the polynucleotide may be damaged. For example, the polynucleotide may contain pyrimidine Such dimers are typically associated with UV damage. , the main cause of cutaneous melanoma. One or more nucleotides in the polynucleotide may For example, it may be modified with a label or tag, suitable examples of which are known to those skilled in the art. A nucleotide may contain one or more spacers. A nucleotide is typically a nucleobase. , a sugar, and at least one phosphate group. The nucleobase and sugar form a nucleoside. Nucleobases are typically heterocyclic. Nucleobases include purines and pyrimidines. More specifically, adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). The sugars are typically pentoses. Nucleotide sugars include ribose and deoxyribose. The sugar is preferably deoxyribose. The polynucleotide is preferably or the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and / or thymidine (dT), deoxyguanosine (dG), and deoxycytidine (dT). Nucleotides are typically ribonucleotides or deoxyribonucleic acids. Nucleotides are typically monophosphate, diphosphate, or triphosphate. Nucleotides contain more than three phosphates, e.g., four or five phosphates. The phosphate may be attached to the 5' or 3' side of the nucleotide. The nucleotides in a nucleotide can be linked to each other in any manner. The nucleotides are typically Nucleotides are linked by their sugar and phosphate groups, just like nucleic acids. Similar to the dimers, polynucleotides can be linked through their nucleobases. At least a portion of the polynucleotide may be single-stranded or double-stranded. The polynucleotide is most preferably a ribonucleic acid (RNA) or The nucleic acid is deoxyribonucleic acid (DNA). Specifically, polynucleotides are used as analytes. Alternatively, the method may include: (i) the length of the polynucleotide; (iii) the sequence of the polynucleotide; (iv) the secondary structure of the polynucleotide; and (v) determining one or more characteristics selected from whether the polynucleotide is modified. This includes:

[0234] A polynucleotide can be of any length (i). For example, a polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 20 0, at least 250, at least 300, at least 400, or at least 500 A polynucleotide can be 1000 nucleotides or nucleotide pairs long. nucleotide length or nucleotide pair length, 5000 nucleotides or nucleotide pairs or 100,000 nucleotides or nucleotide pairs long. Any number of polynucleotides can be interrogated. For example, the method can involve the interrogation of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more polynu The present invention relates to characterizing two or more polynucleotides. In this case, they may be different polynucleotides or two copies of the same polynucleotide. Polynucleotides may be naturally occurring or artificial. For example, This method can be used to verify the sequence of the oligonucleotides produced. The methods are typically carried out in vitro.

[0235] The nucleotides may have any identity (ii), and the nucleotides may include adenosine monophosphate (ATP), AMP, guanosine monophosphate (GMP), thymidine monophosphate (TMP), Uridine monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethyl Cytidine monophosphate, cytidine monophosphate (CMP), cyclic adenosine monophosphate (cA MP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dA MP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (d TMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate (d These include, but are not limited to, deoxymethylcytidine monophosphate (CMP), and deoxymethylcytidine monophosphate. The nucleotide is preferably AMP, TMP, GMP, CMP, UMP, dAMP. , dTMP, dGMP, dCMP, and dUMP. A nucleotide can be a base (i.e., it can lack a nucleobase). A nucleotide is a nucleobase and a sugar. The sequence of nucleotides (iii) may also be absent (i.e., a C3 spacer). The following nucleotides are linked together across the polynucleotide strand in the 5' to 3' direction: It is determined by the consecutive identity of the

[0236] Target polynucleotides can be products of PCR reactions, genomic DNA, or endonuclease digests. The target polynucleotide may comprise any of the products of The target polynucleotide may be obtained or extracted from an organism or microorganism. Tides are often extracted from humans or animals, for example from urine, lymph, saliva, mucus, semen, or from amniotic fluid, or from whole blood, plasma, or serum. The target polypeptide may be obtained from a plant, for example, a cereal, a legume, a fruit, or a vegetable. The nucleotides may include genomic DNA. The genomic DNA may be fragmented. The DNA may be any The fragmentation may be carried out by any suitable method. For example, methods for fragmenting DNA are known in the art. Such methods are known in the art and involve the use of transposases such as MuA transposase. Preferably, the genomic DNA is not fragmented. In some embodiments, The target polynucleotide may be DNA, RNA, and / or DNA / RNA hybrids. It can be a good idea to use

[0237] Nanopore In embodiments of the invention relating to nanopores, any suitable nanopore may be used. In one embodiment, the nanopore is a transmembrane pore.

[0238] A transmembrane pore is a structure that spans the membrane to some extent, allowing it to be driven by an applied potential. This allows the hydrated ions that are released into the membrane to flow across or into the membrane. A typically crosses the entire membrane, allowing hydrated ions to pass from one side of the membrane to the other side of the membrane. However, the transmembrane pore does not have to traverse the membrane. One end may be closed. For example, a pore may represent a well, gap, channel, or other structure in a membrane. It may be a groove, or a slit, along or through which hydrated ions can flow. Cut.

[0239] Any transmembrane pore may be used in the methods provided herein. The pore may be biological. Suitable pores include protein pores, polynucleotide pores, and solid pores. The pore is a DNA origami pore. (Langecker et al., Science, 2012;338:932 -936). A suitable DNA origami pore is disclosed in WO2013 / 083983. .

[0240] In one embodiment, the nanopore is a transmembrane protein pore. It allows hydrated ions, such as polynucleotides, to flow from one side of the membrane to the other side of the membrane. In the methods provided herein, the membrane is a polypeptide or a collection of polypeptides. Transmembrane protein pores allow hydrated ions to pass from one side of the membrane to the other, driven by an applied potential. The membrane-spanning protein pore is preferably capable of forming a pore that allows the flow of the For example, polynucleotides flow from one side of a membrane, such as a triblock copolymer membrane, to the other side. The transmembrane protein pore allows the polynucleotide to move through the pore. This makes it possible to

[0241] In one embodiment, the nanopore is a transmembrane protein pore that is monomeric or oligomeric. The pores are preferably at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 1 Several repeat subunits, such as 4, at least 15, or at least 16 subunits. The pore is preferably composed of hexamers, heptamers, octamers or It is a nonameric pore. The pore may be homo- or hetero-oligomeric. .

[0242] In one embodiment, the transmembrane protein pore is a barrel through which ions can flow. The subunits of the pore typically surround a central axis and organize the strands into a membrane. into a transmembrane β-barrel or channel or a transmembrane α-helical bundle or channel Contribute.

[0243] Typically, the barrel or channel of a transmembrane protein pore is adapted to bind a target polynucleotide. These include amino acids that facilitate interactions with analytes, such as those described herein. The amino acid of is preferably located near the constriction of the barrel or channel. Protein pores typically contain one or more amino acids, such as arginine, lysine, or histidine. It contains a positively charged amino acid or an aromatic amino acid such as tyrosine or tryptophan. These amino acids are typically linked to the pore and to a nucleotide, polynucleotide, or nucleobase. Promotes interaction between the acid and

[0244] In one embodiment, the nanopore is derived from a β-barrel pore or an α-helical bundle pore. β-barrel pores are transmembrane protein pores that are formed by a barrel or chelator made of β-strands. Suitable β-barrel pores include β-toxins, such as α-hemolysin, anthrax toxin, and leukocidins, as well as bacterial outer membrane proteins / porins, e.g., Mycobacterium M. smegmatis porins (Msp), e.g., MspA, MspB, M spC, or MspD, CsgG, outer membrane porin F (OmpF), outer membrane porin G (Omp G), outer membrane phospholipase A, and Neisseria autotransporter lipoprotein These include, but are not limited to, NaIP, as well as other pores such as lysenin. The α-helical bundle pore is a barrel or pore formed from α-helices. Suitable α-helical bundle pores include inner membrane proteins and α-external membrane proteins. Membrane proteins, including but not limited to WZA and ClyA toxins .

[0245] In one embodiment, the nanopore is capable of detecting Msp, α-hemolysin (α-HL), lysenin, CsgG , ClyA, Sp1, or from the hemolytic protein Fragaceatoxin C (FraC) or a transmembrane pore based thereon.

[0246] In one embodiment, the nanopore is a CsgG vector, e.g., E. coli strain K-12 substrain MC4 100. Such pores are oligomeric and typically contain Csg The pore contains 7, 8, 9, or 10 monomers derived from G. Alternatively, the pore may be a homo-oligomeric pore derived from CsgG. It may be a hetero-oligomeric pore derived from CsgG comprising at least one monomer. An example of a suitable pore derived from CsgG is disclosed in WO2016 / 034591.

[0247] In one embodiment, the nanopore is a transmembrane pore derived from lysenin. Examples of suitable pores are disclosed in WO2013 / 153359.

[0248] In one embodiment, the nanopore is derived from or based on α-hemolysin (α-HL). The wild-type α-hemolysin pore is composed of seven identical monomers or subunits. The α-hemolysin pore is formed from α-hemolysin The mutation may be -NN or a variant thereof. The mutation preferably has positions E111 and K147 Contains an N residue at position 1.

[0249] In one embodiment, the nanopore is derived from an Msp, e.g., a transmembrane domain derived from MspA. A suitable example of a pore derived from MspA is described in WO2012 / 1077 This is disclosed at 78.

[0250] In one embodiment, the nanopore is a transmembrane pore derived from or based on ClyA. It is.

[0251] film In embodiments of the invention involving the use of a transmembrane nanopore, the transmembrane nanopore will typically comprise: Any suitable membrane can be used in the system.

[0252] The membrane is preferably an amphiphilic layer. An amphiphilic layer has both hydrophilic and lipophilic properties. The layer is made of amphiphilic molecules such as phospholipids having the structure Non-naturally occurring amphiphiles and those that form monolayers may be used. Amphiphiles that form cyclic amines are known in the art and include, for example, block copolymers. Rimmer (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Block copolymers are polymers made of two or more monomers. It is a polymeric material in which subunits are polymerized together to create a single polymer chain. A copolymer typically has properties contributed by each monomer subunit. However, block copolymers are polymers formed from individual subunits. Block copolymers can have unique properties that are not available from monomer subunits. One of the subunits is hydrophobic (i.e., lipophilic) while the other subunit is lipophilic in aqueous media. It can be engineered to be water-based, in which case the block copolymer can have amphiphilic properties. Block copolymers can form structures that mimic biological membranes. It can be constructed from three or more monomer subunits. The copolymers can form more complex arrangements in which the tribromo groups are bonded together and behave as amphiphiles. The membrane may be a block, tetrablock, or pentablock copolymer. , a triblock copolymer membrane.

[0253] Archaeal bipolar tetraether lipids are naturally organized structures in which lipids are organized to form monolayer membranes. These lipids are generally extremophiles that can survive in harsh biological environments. They are found in philic bacteria, thermophiles, halophiles, and eosinophils. Their stability is determined by the final bilayer structure. This is believed to result from the fusion properties of the layers. We aimed to mimic these biological entities by creating triblock polymers that It is easy to construct a lipid copolymer that behaves similarly to lipid bilayers. They form monomeric membranes that can encompass a wide range of phase behaviors, from vesicles to lamellar membranes. Membranes formed from these triblock copolymers offer several advantages over biological lipid membranes. Since the triblock copolymers are synthetic, their exact structures need to be carefully determined. The correct structure required to control and form the membrane and interact with the pore and other proteins. Chain length and properties can be provided.

[0254] Block copolymers are constructed from subunits that are not classified as lipid submaterials. For example, hydrophobic polymers may be made from siloxanes or other non-hydrocarbon monomers. The hydrophilic subsection of the block copolymer has low protein binding properties. This allows for the creation of membranes that are highly resistant when exposed to live biological samples. The head group unit may also be derived from a non-classified lipid head group.

[0255] Triblock copolymer membranes exhibit increased mechanical and environmental stability compared to biological lipid membranes. They also have properties such as a much higher operating temperature or pH range. The properties provide a basis for customizing polymer-based membranes for a wide range of applications.

[0256] In some embodiments, the membrane is a membrane as described in International Application No. WO2014 / 064443 or This is one of the membranes disclosed in WO2014 / 064444.

[0257] The amphiphilic molecule is chemically modified to facilitate coupling of the polynucleotide. The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic layer is typically planar. The amphiphilic layer may also be curved. The conductive layer may be supported.

[0258] Amphiphilic membranes typically have a molecular weight of approximately 10 -8 cm s -1 with a lipid diffusion rate of It is naturally mobile, essentially acting as a parent fluid. This is due to the pores and the coupled This means that the polynucleotides that are bound to the nucleobase are typically capable of moving within amphiphilic membranes. .

[0259] The membrane may be a lipid bilayer. Lipid bilayers are a model for cell membranes and have been used extensively in experiments. For example, lipid bilayers are ideal for single channel recordings. Alternatively, it can be used for the in vitro investigation of membrane proteins by lipid bisection. The layer can be used as a biosensor to detect the presence of a wide range of substances. The lipid bilayer can be any lipid bilayer. Suitable lipid bilayers include planar lipids. These include, but are not limited to, bilayers, supported bilayers, or liposomes. The bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers include those described in WO200 8 / 102121, WO2009 / 077734, and WO2006 / 100484 It has been disclosed.

[0260] Methods for forming lipid bilayers are known in the art. Generally Montal and Mueller(Proc.Natl.Acad.Sci .USA., 1972;69:3561-3566), In this study, a lipid monolayer is grown on both sides of an opening perpendicular to an aqueous solution / air interface. The lipid is usually first dissolved in an organic solvent and then added to a small amount of the solvent. The solvent is evaporated on the interface of the aqueous solution on both sides of the opening, and the surface of the aqueous electrolyte solution is When the organic solvent evaporates, the solution / air interface on either side of the opening forms a bilayer. The planar lipid bilayer physically moves up and down across the aperture until it is free to move across the aperture. The recess may be formed within the recess or across the opening.

[0261] The Montal & Mueller method provides a good selection of lipids suitable for protein pore insertion. It has gained popularity because it is a cost-effective and relatively simple method for forming bilayers. Other common methods of bilayer formation include tip-dipping, bilayer Painting bilayers, and patches of liposome bilayers Clamps included.

[0262] Tip-dipping bilayer formation involves placing an open surface (e.g., This involves contacting the lipid monolayer with a thin film of water (e.g., a pipette tip). Again, the lipid monolayer is then First, a small amount of lipid dissolved in an organic solvent is evaporated at the solution surface to form a solution / air interface. The bilayer is then formed by the Langmuir-Schäfer technique. Mechanical automation is required to move the aperture relative to the solution surface.

[0263] In the case of bilayer coating, a small amount of lipid dissolved in an organic solvent is applied to an aperture immersed in the test aqueous solution. The lipid solution is applied directly to the opening using a paint brush or equivalent. Thinning the solvent leads to the formation of a lipid bilayer. However, it is difficult to completely remove the solvent from the bilayer. The bilayer formed by the method is less stable and is prone to noise during electrochemical measurements. stomach.

[0264] Patch clamping is commonly used to study biological cell membranes. The end of the pet is then fastened so that the patch of membrane is bonded across the opening. , liposomes are pinned and then ruptured to seal the lipid bilayer across the pipette opening This method has been adapted to produce lipid bilayers by the addition of stable giant It is necessary to create small openings in the material with a large monolayer liposome and a glass surface. It is necessary.

[0265] Liposomes can be synthesized by sonication, extrusion, or the Mozafari method (Colas et al. l. (2007) Micron 38:841-847) .

[0266] In some embodiments, the lipid bilayer is a lipid bilayer as described in International Application No. WO2009 / 077734. In this method, advantageously, the lipid bilayer is formed from dry lipids. In a most preferred embodiment, the lipid bilayer is The opening is formed across the opening so that the substrate can be mounted.

[0267] A lipid bilayer is formed from two opposing layers of lipids. These two lipid layers are Their hydrophobic tail groups are oriented toward each other to form a hydrophobic interior. The hydrophilic head groups of the molecules face outward toward the aqueous environment on either side of the bilayer. The layers are classified into liquid disordered phase (fluid lamellar), liquid ordered phase, and solid ordered phase (lamellar gel phase, comb-like gel phase). , and planar bilayer crystals (lamellar subgel phase, lamellar crystalline phase), but are not limited to these. It may be present in several lipid phases, not just in the lipid phase.

[0268] Any lipid composition that forms a lipid bilayer can be used. Required properties, e.g., surface charge, ability to support membrane proteins, packing density, or mechanical The lipid composition is selected to form a lipid bilayer having one or more distinct properties. For example, the lipid composition may include up to 100 lipids. The lipid composition preferably contains 1 to 10 lipids. The lipid composition may be a naturally occurring lipid. The lipid composition may include natural lipids and / or artificial lipids.

[0269] Lipids typically consist of a head group, an interface portion, and two interfacial portions that may be the same or different. Suitable head groups include neutral head groups, such as diacylglycerol. lysine (DG) and ceramide (CM), zwitterionic head groups, e.g., phosphatidylcholine (Pd) and ceramide (CM), Phosphingomyelin (PC), phosphatidylethanolamine (PE), and sphingomyelin ( SM), negatively charged head groups, e.g., phosphatidylglycerol (PG), phosphatidylglycerol (PGP), Phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (P A), and cardiolipin (CA), and positively charged head groups, e.g., trimer Suitable examples of suitable ammonium salts include, but are not limited to, ethylammonium-propane (TAP). The surface moiety may include naturally occurring interfacial moieties, such as glycerol-based moieties or ceramide-based moieties. Suitable hydrophobic tail groups include, but are not limited to, saturated hydrocarbon chains, For example, lauric acid (n-Dodecanolic acid) , myristic acid (n-tetradecononic acid d)), palmitic acid (n-hexadecanoic acid), stearic acid (n-octadecanoic acid), and arachidic acid (n-eicosanoic acid), unsaturated hydrocarbon chains, e.g., oleic acid (cyano). octadecanoic acid), as well as branched hydrocarbon chains, e.g., phytanoyl However, they are not limited to the above. The length of the unsaturated hydrocarbon chain and the position and structure of the double bond are also important. The length of the chain and the position and number of branching groups, such as methyl groups in branched hydrocarbon chains, may vary. The hydrophobic tail groups are linked to the interface moieties as ethers or esters. The lipid may be a mycolic acid.

[0270] The lipids can also be chemically modified. The head or tail groups of the lipids can be chemically modified. Suitable lipids whose head groups are chemically modified include PEG-modified lipids, e.g. For example, 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000], functionalized PEG lipids, e.g., 1,2-distearate Aroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene [glycol) 2000], as well as lipids modified for conjugation, e.g. For example, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(sucrose) cinyl) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine The tail group is chemically modified, including, but not limited to, -N-(biotinyl). Suitable lipids include polymerizable lipids, such as 1,2-bis(10,12-tricosapentaerythritol) and 1,2-bis(10,12-tricosapentaerythritol). Trichosodium glycero-3-phosphocholine, Fluorinated lipids, such as 1-palmitoyl-2-(16-fluoropalmitoyl)-sn-glycol Lysero-3-phosphocholine, deuterated lipids, e.g., 1,2-dipalmitoyl-D62- sn-glycero-3-phosphocholine, and ether-linked lipids, such as 1,2-di-O -Phytanyl-sn-glycero-3-phosphocholine, but not limited to Lipids may be chemically modified to facilitate coupling of polynucleotides. It may be functionalized.

[0271] The composition of the amphiphilic layer, e.g., lipids, will typically affect the properties of the layer. Suitable additives include fatty acids, such as palmitic acid, myristic acid, Acid, oleic acid, fatty alcohols, such as palmitic alcohol, myristic alcohol oleic alcohol, sterols, e.g., cholesterol, ergosterol esters, lanosterol, sitosterol, and stigmasterol, lysophospholipids, e.g. For example, 1-acyl-2-hydroxy-sn-glycero-3-phosphocholine, and ceramide These include, but are not limited to:

[0272] In another embodiment, the membrane comprises a solid layer. The solid layer may be a microelectronic material, an insulating material, e.g. For example, Si 3 N 4 , A1 2 O 3 , and SiO, organic and inorganic polymers, e.g., poly amides, plastics such as Teflon®, or elastomers such as Examples include, but are not limited to, organic, two-component, addition-cured silicone rubbers, and glass. The solid layer can be made of both inorganic and non-organic materials. Suitable graphene layers are disclosed in WO2009 / 035647. When the solid layer includes a solid layer, the pores are typically defined as holes, wells, gaps, etc., in the solid layer. The membrane or layer is contained within a channel, groove, or slit. Suitable solid / amphiphilic hybrid systems can be prepared if: As disclosed in WO2009 / 020682 and WO2012 / 005857. Any of the amphiphilic membranes or layers discussed can be used.

[0273] The methods disclosed herein typically include the steps of: (i) forming an artificial amphiphilic layer comprising pores; (ii) forming a pore-containing artificial amphiphilic layer; (iii) an isolated naturally occurring lipid bilayer containing a pore; or (iv) a lipid bilayer containing a pore inserted therein. The method is typically carried out using artificial triblock copolymers. This is carried out using artificial amphiphilic layers, such as polymer layers, which, in addition to the pore, also contain other transmembrane tethers. The proteins may include proteins and / or intramembrane proteins, as well as other molecules. The methods and conditions are discussed below. The methods of the invention are typically carried out in vitro.

[0274] anchor In one embodiment, the polynucleotide adaptor comprises a membrane anchor or a polypeptide linked to the adaptor. or a transmembrane pore anchor. In one embodiment, the targeting method according to the methods disclosed herein Characterization of the anchor of a polynucleotide, e.g., to attach a target polynucleotide to a transmembrane pore. In the method comprising contacting the membrane anchor or transmembrane pore anchor with the surrounding of the transmembrane pore, This can facilitate localization of a selected polynucleotide at the site of interest.

[0275] The membrane anchor may be a polypeptide anchor and / or a hydrophobic anchor that can be inserted into a membrane. In one embodiment, the hydrophobic anchor can be a lipid, a fatty acid, a sterol, a carbohydrate, Nanotubes, polypeptides, proteins, or amino acids, e.g., cholesterol , palmitic acid, or tocopherol. The anchor can be a thiol, biotin, or may contain a surfactant. In one embodiment, the anchor is biotin (for binding to streptavidin), maltodextrin (for binding to maltodextrin), or maltodextrin (for binding to maltodextrin). amylose (for binding to a histidine-binding protein or fusion protein), Ni-NTA for binding to histidine- or poly-histidine-tagged proteins, or or a peptide (such as an antigen).

[0276] In one embodiment, the anchor comprises one linker, or two, three, four, or more Preferred linkers include polymers, e.g., polynucleotides, These include polyethylene glycol (PEG), polysaccharides, and polypeptides. These linkers can be linear, branched, or cyclic. For example, The linker can be a circular polynucleotide. The adaptor can be a circular polynucleotide linker. One or more anchors or one or more linkers can hybridize to complementary sequences on the anchors. The anchor may contain a moiety that can be cleaved or degraded, such as a restriction site or a photolabile group. The linker is functionalized with a maleimide group to couple to a cysteine ​​residue of the protein. Suitable linkers are described in WO2010 / 086602.

[0277] In one embodiment, the anchor is cholesterol or a fatty acyl chain. Any fatty acyl chain having a length of 6 to 30 carbon atoms, such as xadecanoic acid, can be used. Examples of suitable anchors and methods for attaching the anchors to the adaptors are described in WO2012 / 1 64270 and WO2015 / 150786.

[0278] Characterization In embodiments of the present disclosure relating to characterization of a target polynucleotide, the target analyte is As described in more detail herein, the transmembrane nanopore may be coupled to, for example, the interior of, or moves through it.

[0279] The characterization method can use any device suitable for investigating membrane / pore systems where the pore is inserted into the membrane. The characterization method can be carried out using any device suitable for transmembrane pore sensing. For example, the device may include a chamber containing an aqueous solution and a chamber separating the chamber into two compartments. The barrier may have an opening in which the membrane containing the transmembrane pore is formed. The pores are described herein.

[0280] Characterization methods are described in WO2008 / 102120, WO2010 / 122293, or This may be carried out using the apparatus described in WO00 / 28312.

[0281] Characterization methods typically involve measuring the flow of ionic current through the pores. Alternatively, the flow of ions through the pore can include the method described by Heron et al. Disclosed by J. Am. Chem. Soc. 9 Vol. 131, No. 5, 2009 Thus, the device applies a potential and detects the membrane and An electrical circuit may also be included that can measure an electrical signal across the pore. The characterization method may be performed using patch clamp or voltage clamp. involves the use of a voltage clamp.

[0282] The characterization method is as follows: each array is 128, 256, 512, 1024, 2000, 3000 , 4000, 6000, 10000, 12000, 15000, or more wells The method may be implemented in a silicon-based well array comprising:

[0283] The characterization method may include measuring the current through the pore. This method typically comprises The procedure is carried out with a voltage applied across the membrane and the pores. The voltages used are typically Typically, it is +2V to -2V, typically -400mV to +400mV. The voltage is preferably -400mV, -300mV, -200mV, -150mV, -10 The lower limit can be selected from 0mV, -50mV, -20mV, and 0mV, and the lower limit can be selected from +10mV, + 20mV, +50mV, +100mV, +150mV, +200mV, +300mV, and and an upper limit independently selected from +400 mV. More preferably, the range is 100 mV to 240 mV, and most preferably, the range is 120 mV to 22 By using increased applied potentials, different nuclei were obtained for each pore. It is possible to increase discrimination between leucotides.

[0284] Characterization methods typically involve the use of metal salts, e.g., alkali metal salts, halide salts, e.g. For example, the reaction may be carried out in the presence of any charge carrier, such as a chloride salt, e.g., an alkali metal chloride salt. The charge carriers include ionic liquids or organic salts, such as tetramethylammonium chloride. ammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, or can include 1-ethyl-3-methylimidazolium chloride. In this device, salts are present in the aqueous solution in the chamber. Thorium chloride (NaCl), or cesium chloride (CsCl) are typically used. is preferred. The salt may be an alkaline earth metal salt, such as calcium chloride (CaCl2). The salt concentration may be at saturation. The salt concentration may be up to 3M, typically 0. 1~2.5M, 0.3~1.9M, 0.5~1.8M, 0.7~1.7M, 0.9~1. The salt concentration is 6 M, or 1 M to 1.4 M. The salt concentration is preferably 150 mM to 1 M. The characterization method preferably involves detecting at least 0.3M, for example at least 0.4M, At least 0.5M, at least 0.6M, at least 0.8M, at least 1.0M, at least at least 1.5M, at least 2.0M, at least 2.5M, or at least 3.0M Higher salt concentrations provide a higher signal-to-noise ratio and are normally This allows identification of currents indicative of binding / no binding against a background of random current fluctuations.

[0285] The characterization method is typically carried out in the presence of a buffer. In the device, the buffer is present in an aqueous solution within the chamber. Any suitable buffer may be used. Typically, the buffer is HEPES. Another suitable buffer is Tris-HC The method is typically carried out in the range of 4.0 to 12.0, 4.5 to 10.0, 5.0 ~9.0, 5.5~8.8, 6.0~8.7, or 7.0~8.8, or 7.5~8 The pH used is preferably about 7.5.

[0286] Characterization methods are 0℃~100℃, 15℃~95℃, 16℃~90℃, 17℃~85℃ , 18°C ​​to 80°C, 19°C to 70°C, or 20°C to 60°C. The method is typically carried out at room temperature. The characterization method optionally supports enzyme function. The reaction is carried out at a temperature suitable for carrying out the reaction, for example, about 37°C.

[0287] Certain embodiments, particular configurations, and materials and / or molecules are contemplated for the method according to the invention. Although discussed herein, various changes or modifications in form and detail may be made to the present invention. It should be understood that the following may be implemented without departing from the scope and spirit of the present invention. The examples are provided to better illustrate certain embodiments and are not intended to limit the present application. This application is limited only by the claims. EXAMPLES

[0288] Example 1 This example shows that the motor protein stalls adjacent to a spacer, compared to when the motor protein stalls adjacent to a spacer. When the motor protein is stalled on the spacer according to the methods disclosed herein, This indicates that wasteful metabolic turnover is reduced.

[0289] A single-stranded polynucleotide strand of SEQ ID NO: 9 and a spacer (four iSp18 units) A first DNA construct containing a complementary single-stranded DNA strand (SEQ ID NO: 10 and and 11). SEQ ID NO:10 is a sequence that hybridizes to the 5' end of SEQ ID NO:9 and a spacer 9 between the 5' end of SEQ ID NO: 9 and the 5' end of SEQ ID NO: 9, which is referred to as the "test" strand. The motor protein (closed T4 Dda helicase) was stalled on the spacer. The construct is shown diagrammatically in FIG. 1A.

[0290] A second DNA construct is generated by loading the closing T4 Dda helicase onto the spacer. The second DNA construct consisted of the same sequence as in SEQ ID NO:12, except that SEQ ID NO:10 was used instead of SEQ ID NO:12. SEQ ID NO:12 is identical to the first DNA construct at the 5' end of SEQ ID NO:10. It is identical to SEQ ID NO: 10, except that it lacks the terminal 10 nucleotides. Sequence number 12 hybridizes to SEQ ID NO:9 and has 10 nucleotides at the 5' end of the spacer. Thus, SEQ ID NO:12 is referred to as the "Test-10" strand. The construct is shown diagrammatically in FIG.

[0291] Using a spectrophotometric assay, the first DNA construct and the second DNA construct were respectively The stalled motor protein (in this example, the closed T4 Dda helicase) The rate of fuel utilization (ATP turnover) was determined. Briefly, the spectrophotometric assay was performed using a coupled enzyme system Monitor ATP usage by motor proteins using pyruvate kinase (P K, rabbit muscle, 600-1000U / mL, Sigma) is a stimulatory protein that stimulates the motor protein This reaction converts the ADP formed by the reaction into ATP. (PEP, phospho(enol)pyruvate monopotassium salt, Sigma) 1 molecule of pyruvate Pyruvate is converted to acid by the enzyme lactate dehydrogenase (LDH, rabbit muscle meat, 900-1400 U / mL, Sigma) to convert it to lactic acid, resulting in NA The NADH molecule is oxidized. The oxidation of NADH is indicated by a decrease in absorbance at 340-380 nm. , followed spectrophotometrically (extinction coefficient is 1210 M at 380 nm). -1 cm -1 and 6220M at 340nm -1 cm -1 One ADP molecule is converted to ATP. Since one molecule of NADH is oxidized for each reaction, the decrease in absorbance signal is due to the motor protein is proportional to the steady-state rate of ATP hydrolysis.

[0292] FIG. 1C shows the results of stalling on the first and second DNA constructs, respectively. Figure 1 shows the calculated ATP turnover rate by the closed T4 Dda helicase. Catalytic ATP turnover rate when Dda helicase stalls on the spacer of the first construct degree(k cat ) is about 115s -1 In contrast, the closed T4 Dda helicase When stalled on a second construct adjacent to the pacer, the ATP turnover rate was It was twice as much as the other cat Approximately 235s -1 ). Therefore, the closing of the T4 Dda helicopter The rate of wasteful metabolic turnover by Ze was roughly halved.

[0293] Example 2 This example compares the adapters with a variety of different spacer designs and provides When the motor protein stalls on the spacer, ATP metabolism is wasted according to the method Demonstrate reduced rotation.

[0294] As shown in the results table below, multiple different adapters were inserted using the spacer design. The adapters tested were (1) top strand, (2) bottom strand, and (3) block strand. The top strand contained polynucleotide strands on both sides. The sequences included the spacer designs to be tested. The blocking moiety was a single-stranded polynucleotide as described in

[0295] Adapter sequences Top strand:333333333333333333333333333333CCTTT TTTTTGGCGTCTGCTTGGGTGTTTAACC / [spacer sequence] / C CACAACTTCGTTCAGTTACGTATTGCT (i.e., SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 16 are composed of the spacer sequence. SEQ ID NO: 14 is SEQ ID NO: 1 Configure 5) Bottom chain: 5phos / GCAATACGTAACTGAACGAAGTTGTGG( i.e., SEQ ID NO:17) Loading strand: GGTTAAACACCCAAGCAGACGCCTTT (i.e. SEQ ID NO:18) Blocking strand: GGTTAAACACCCAAGCAGACGCCAAAAAAAG G / 3Cy5Sp / (i.e., SEQ ID NO: 19)

[0296] Adapter preparation and loading The motor protein was first divided into a top strand, a bottom strand, and a loading strand. The use of a loading strand allows multiple motor proteins to be loaded onto a single adapter. If it is desired to reduce the ratio of adapters loaded to the number of adapters, This is an optional feature that can improve the efficiency of target protein loading. According to the method provided in the document, the motor protein is advanced onto the spacer and By this, the motor protein displaces the loading strand from the adaptor. The blocking strand is then annealed to the adapter to allow the motor protein to bind to the spacer. The completed adapter is fast and prevented from moving off by the blocking chain. Since the blocking strand is longer, it is more efficient than the loading strand. The preferential binding of the adapter strand to the loading strand is significantly greater than that of the loading strand. This is further enhanced by the blocking strand being provided in high concentration.

[0297] Adapter Preparation Protocol The adaptor polynucleotide strands were diluted in potassium acetate buffer (400 mM H The strands were annealed in 100 mM EPES pH 8.0, 400 mM potassium acetate. Built it. [Table 4]

[0298] Motor protein (Dda helicase) at a final concentration of 50–500 nM DNA 10 with annealed adaptors and at least 7-fold molar excess of protein under ambient conditions Combine for ~20 min. TMAD (100 μM) in potassium acetate buffer (as above) and incubated at 35°C for 1 hour. Salt / ATP buffer (final concentration: 0.5M NaCl, 1 mM ATP, 10 mM MgCl2) together with the blocking strand 20x The top strand concentration was added and the mixture was incubated at room temperature for 25-30 min.

[0299] The helicase-bound adapters were purified using the SPRI bead purification system and Adaptor production was verified using TBE gels.

[0300] ATP turnover and stability studies Each adaptor was tested for ATP turnover and stability.

[0301] ATP turnover occurs when the adapter is not involved in any sequencing reaction. It provides a measure of wasteful ATP turnover, with lower ATP turnover indicating less wasteful ATP use. The smaller the dose, the more fuel efficient the adapter will be.

[0302] The stability of the adapters was tested under conditions typically found in nanopore sequencing reactions. , which indicates how long a given adapter is expected to function in such a setting. Sequences run under different conditions, e.g., lower salt concentration or reduced temperature. Note that the synthesizing reaction may provide increased stability of the adapter.

[0303] The adapters tested were named B1-B52 (see the results table below). These results were compared with a commercially available Oxford nanopore sequencing assay that contains motor proteins. The adapter was compared to a 3-aminobutyric acid (herein referred to as "AA" adapter).

[0304] ATPase Assay Protocol The adapter samples for testing were dissolved in SPRI elution buffer (50 mM Tris pH 8, 20 mM NaCl, 1 ... The solution was diluted to a concentration of 10 nM in 10 M NaCl. 10 μL of each 10 nM adapter sample was added to a 384-well plate. 2.2 μL of 40 U / mL lactate dehydrogenase / pyruvate kinase, 33.3 μL of 1.33x sequencing buffer, 3.3 µL 3.33 mM NADH, 0.7 µL NADH matrix containing 5.33 mM pyruvate, and 10.4 µL of nuclease-free water Star mix was prepared. Incubate the NADH master mix at room temperature for 10 min to remove free ADP in solution. There was metabolic turnover. Add 30 μL of NADH master mix to each well of a 384-well plate and 10 μL of The adapter was added to the sample and mixed. The plate is added to a UV absorbance plate reader and the absorbance is recorded at 380 nm and 120 °C at 34 °C. The cycle was measured. Analyze the generated data and cat and ATP turnover values ​​were determined.

[0305] Stability Assay Protocol 7.8 µL nuclease-free water, 10 µL 2x sequencing buffer, and 0 An assay master mix was prepared containing 0.2 μL of B-OTG. The test sample was added to 18 μL of the assay in a PCR tube with 2 μL of 125 nM adapter. The cells were prepared by adding to the Imasta mix + / - ATP. Incubate each sample in a thermal cycler at 34°C with a cover temperature of 45°C for 24 hours. I did. After incubation, samples were run on a 5% TBE gel (160 mV, 30 min) or 4–2 Electrophoresis was carried out on a 0% TBE gel (180 mV, 45 min). The gel was stained with SYBR Gold for 10 min and then stained with Amersham T using Cy3 settings. Images were taken on a yphoon scanner. Densitometric analysis was performed to determine the percentage of bound and unbound adapters. It has been decided.

[0306] result Results of ATP turnover are shown with the comparison adaptor AA serving as a baseline. The ATP turnover percentage is expressed as a percentage of the total ATP metabolism. Anything below 100% indicates a reduction in wasteful ATP turnover and All adapters tested shown in the results table show improvements in adapter -had reduced ATP turnover and therefore improved fuel efficiency compared to AA.

[0307] We also tested the stability of the adaptor and the results were compared with those of the motor protein over a 24-hour period. These results are shown as an increase in the percentage of untranslated adaptor polynucleotides. The results indicate that stable motor protein-loaded adapters were generated. [Table 5-1] [Table 5-2]

[0308] Example 3 This embodiment assumes that the blocking moiety ("blocking chain") is already in place. By directly loading the motor protein onto the spacer of the polynucleotide adaptor, This demonstrates that it is possible to

[0309] Top strand, bottom strand, and blocking strand sequences as described in Example 2 above. A polynucleotide adapter was prepared having the following structure: The DNA contained a spacer sequence containing multiple nucleotide islands such as:

[0310] The adaptor-containing polynucleotide strands were diluted with potassium acetate buffer (400 mM) at the following concentrations: The strands were annealed in 10 mM HEPES pH 8.0, 400 mM potassium acetate. And so we built it. [Table 6]

[0311] Motor protein (Dda helicase) at a final concentration of 50–500 nM DNA Annealed adapters and various molar excesses of protein at ambient conditions were used to identify 10–20 Add TMAD (100 μM) in potassium acetate buffer (as above) The mixture was then incubated at 35°C for 1 hour. Cl, 1 mM ATP, 10 mM MgCl2) was added and the mixture was incubated at room temperature for 25-30 min. The mixture was incubated for 1 h.

[0312] The helicase-bound adapter was purified by HPLC, and the adapter production was analyzed by TBE gel electrophoresis. The results were verified using the with 1x, 2x, 5x, 10x, 15x, 20x, and 30x excess of motor protein The prepared samples were run on a gel, and the results are shown in Figure 2. The samples were electrophoresed in the presence of salt / ATP buffer. The results were obtained both before ("Closed") and after ("Salt / ATP") the closing step.

[0313] The lanes show that the following was obtained: free DNA (loaded with motor proteins) (unmodified adaptor), and a 1:1 ratio of motor protein and adaptor DNA (positive (indicating properly loaded motor proteins), and low-abundance motor proteins at a ratio of >1:1. Motor proteins and adapter DNA (multiple motor proteins first bound to a single adapter The greater the excess of motor protein relative to the DNA adaptor, the higher the A high ratio (1:1 protein:DNA) of loaded adapters was obtained.

[0314] Thus, the results presented in FIG. 2 show that motor proteins bind to the DNA described herein. 1 shows that it is possible to directly load into the spacer section containing the islands.

[0315] Example 4 This example illustrates the use of exemplary adapters prepared according to the methods described herein in DNA sequencing. The results demonstrate the increased efficiency achieved when tested in a sequencing system.

[0316] Double-stranded 440mer libraries were ligated with standard commercially available sequencing adapters (AMX or any of the test adapters (adapters designated herein as B14, B21, and C1) We used either the commercially available Oxford Nanopore Sequencing Kit (SQK-LSK1 09).

[0317] The test adapters were as described in Example 2 above and prepared accordingly. The spacer sequences were as follows: B14:33338TT8TT838 B21:333TT8TT8 C1:333TT33TT8

[0318] The 440mer library was purified using Oxford Nanopore MinION Mk1 b Sequencing was performed on a MinION flow cell (FLO-MIN106) using the device. Data collection was performed using the baseline LSK109 sequencing protocol at Min. This was done with KNOW Software.

[0319] Analyzing bulk Fast5 data generated by the MiniKNOW software Individual strand data were extracted and saved in subsequent text files. Strand data was analyzed by dividing the data into subgroups based on events within a strand. The number of strands and the duration of the strand were used to estimate the rate of DNA translocation.

[0320] The results presented in Figure 3 show that the fuel Improved efficiency allows for faster and longer sequencing times than commercially available adapter AMX. These results demonstrate that the nanopore sequencing system can be used to This also confirms the stability of the adapter, which has been tested in a laboratory. Description of sequence listing SEQ ID NO:1 is the (hexahistidine tagged) exonuclease I from E. coli The amino acid sequence of (EcoExo I) is shown. SEQ ID NO:2 shows the amino acid sequence of the exonuclease III enzyme from E. coli . SEQ ID NO: 3 is the RecJ enzyme from T. thermophilus (TthRecJ-c d) The amino acid sequence is shown. SEQ ID NO: 4 shows the amino acid sequence of bacteriophage lambda exonuclease. The sequence of htt is one of three identical subunits that make up the trimer. p: / / www.neb.com / nebecomm / products / produc tM0262.asp). SEQ ID NO:5 is the Phi2 sequence from Bacillus subtilis phage Phi29. 9 The amino acid sequence of DNA polymerase is shown. SEQ ID NO:6 is the sequence identified as Trwc Cba (Citromicrobium bathyomar The amino acid sequence of the helicase is shown. SEQ ID NO: 7 is Hel308 Mbu (Methanococcoides burto nii) The amino acid sequence of the helicase is shown. SEQ ID NO:8 represents the amino acid sequence of Dda helicase 1993 from enterobacteriaceae phage T4. show. SEQ ID NO:9 shows the nucleotide sequence of the polynucleotide strand discussed in Example 1. SEQ ID NO: 10 is the nucleotide sequence of the polynucleotide strand (the "test" strand) discussed in Example 1. The code sequence is shown. SEQ ID NO:11 shows the nucleotide sequence of the polynucleotide strand discussed in Example 1. SEQ ID NO: 12 is the nucleic acid sequence of the polynucleotide strand discussed in Example 1 (the "Test-10" strand). The peptide sequence is shown. SEQ ID NO:13 is the sequence of the polynucleotide adaptor ("top strand") discussed in Example 2. The nucleotide sequence is shown. SEQ ID NO:14 is the polynucleotide adaptor ("top strand", strand) discussed in Example 2. The nucleotide sequence of the nucleotide sequence of the nucleotide sequence preceding the pacer unit is shown. SEQ ID NO:15 is the polynucleotide adaptor ("top strand", strand) discussed in Example 2. The nucleotide sequence of the portion preceding the pacer unit (the nucleotide region) is shown. SEQ ID NO:16 is the polynucleotide adaptor ("top strand", strand) discussed in Example 2. The nucleotide sequence of the nucleotide sequence of the nucleotide sequence following the pacer unit is shown. SEQ ID NO:17 is the sequence of the polynucleotide adaptor ("bottom strand") discussed in Example 2. The nucleotide sequence is shown. SEQ ID NO:18 is the polynucleotide adaptor ("loading strand") discussed in Example 2. " ) is shown. SEQ ID NO:19 is the polynucleotide adaptor ("blocking strand") discussed in Example 2. " ) is shown. Sequence Listing SEQ ID NO:1 - Exonuclease I from E. coli MMNDGKQQSTFLFHDYETFGTHPALDRPAQFAAIRTDSEF NVIGEPEVFYCKPADDYLPQ PGAVLITGITPQEARAKGENEAAFAARIHSLFTVPKTCIL GYNNVRFDDEVTRNIFYRNF YDPYAWSWQHDNSRWDLLLDVMRACYALRPEGINWPENDDG LPSFRLEHLTKANGIEHSNA HDAMADVYATIAMAKLVKTRQPRLFDYLFTHRNKHKLMAL IDVPQMKPLVHVSGMFGAWR GNTSWVAPLAWHPENRNAVIMVDLAGDISPLLELDSDTLR ERLYTAKTDLGDNAAVPVKL VHINKCPVLAQANTLRPEDADRLGINRQHCLDNLKILREN PQVREKVVAIFAEAEPFTPS DNVDAQLYNGFFSDADRAAMKIVLETEPRRNLPALDITFVD KRIEKLLFNYRARNFPGTLD YAEQQRWLEHRRQVFTPEFLQGYADELQMLVQQYADDKEK VALLKALWQYAEEIVSGSGH HHHHH SEQ ID NO:2 - Exonuclease III enzyme from E. coli MKFVSFNINGLRARPHQLEAIVEKHQPDVIGLQETKVHDD MFPLEEVAKLGYNVFYHGQK GHYGVALLTKETPIAVRRGFPGDDEEAQRRIIMAEIPSLL GNVTVINGYFPQGESRDHPI KFPAKAQFYQNLQNYLETELKRDNPVLIMGDMNISPTDLD IGIGEENRKRWLRTGKCSFL PEEREWMDRLMSWGLVDTFRHANPQTADRFSWFDYRSKGF DDNRGLRIDLLLASQPLAEC CVETGIDYEIRSMEKPSDHAPVWATFRR SEQ ID NO:3 - RecJ enzyme from T. thermophilus MFRRKEDLDPPLALLPLKGLREAAALLEEALRQGKRIRVH GDYDADGLTGTAILVRGLAA LGADVHPFIPHRLEEGYGVLMERVPEHLEASDLFLTTVDCG ITNHAELRELLENGVEVIVT DHHTPGKTPPPGLVVHPALTPDLKEKPTGAGVAFLLLWAL HERLGLPPPLEYADLAAVGT IADVAPLWGWNRALVKEGLARIPASSWVGLRLLAEAVGYT GKAVEVAFRIAPRINAASRL GEAEKALRLLLTDDAAEAQALVGELHRLNARRQTLEEAML RKLLPQADPEAKAIVLLDPE GHPGVMGIVASRILEATLRPVFLVAQGKGTVRSLAPISAV EALRSAEDLLLRYGGHKEAA GFAMDEALFPAFKARVEAYAARFPDPVREVALLLDLLPEPG LLPQVFRELALLEPYGEGNP EPLFL SEQ ID NO:4 - Bacteriophage lambda exonuclease MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNV IAKPRSGKKWPDMKMSYFHT LLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNV TESPIIYRDESMRTACSPDG LCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQY SMWVTRKNAWYFANYDPRMK REGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFV FGEQWR SEQ ID NO:5 - Phi29 DNA polymerase MKHMPRKMYSCAFETTTKVEDCRVWAYGYMNIEDHSEYKI GNSLDEFMAWVLKVQADLYF HNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQW YMIDICLGYKGKRKIHTVIY DSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITP EEYAYIKNDIQIIAEALLIQ FKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDK EVRYAYRGGFTWLNDRFKEK EIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDED YPLHIQHIRCEFELKEGYIP TIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHY DLYNVEYISGLKFKATTGLF KDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVT GKVPYLKENGALGFRLGEEE TKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSI HLTGTEIPDVIKDIVDPKKL GYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDD YTDIKFSVKCAGMTDKIKKE VTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKSGGSA WSHPQFEKGGGSGGGSGGSA WSHPQFEK SEQ ID NO: 6 - Trwc Cba Helicase MLSVANVRSPSAAASYFASDNYYASADADRSGQWIGDGAK RLGLEGKVEARAFDALLRGE LPDGSSVGNPGQAHRPGTDLTFSVPKSWSLLALVGKDERI IAAYREAVVEALHWAEKNAA ETRVVEKGMVVTQATGNLAIGLFQHDTNRNQEPNLHFHAV IANVTQGKDGKWRTLKNDRL WQLNTTLNSIAMARFRVAVEKLGYEPGPVLKHGNFEARGI SREQVMAFSTRRKEVLEARR GPGLDAGRIAALDTRASKEGIEDRATLSKQWSEAAQSIGL DLKPLVDRARTKALGQGMEA TRIGSLVERGRAWLSRFAAHVRGDPADPLVPPSVLKQDRQ TIAAAQAVASAVRHLSQREA AFERTALYKAALDFGLPTTIADVEKRTRALVRSGDLIAGK GEHKGWLASRDAVVTEQRIL SEVAAGKGDSSPAITPQKAAASVQAAALTGQGFRLNEGQL AAARLILISKDRTIAVQGIA GAGKSSVLKPVAEVLRDEGHPVIGLAIQNTLVQMLERDTG IGSQTLARFLGGWNKLLDDP GNVALRAEAQASLKDHVLVLDEASMVSNEDKEKLVRLANL AGVHRLVLIGDRKQLGAVDA GKPFALLQRAGIARAEMATNLRARDPVVREAQAAAQAGDV RKALRHLKSHTVEARGDGAQ VAAETWLALDKETRARTSIYASGRAIRSAVNAAVQQGLLA SREIGPAKMKLEVLDRVNTT REELRHLPAYRAGRVLEVSRKQQALGLFIGEYRVIGQDRK GKLVEVEDKRGKRFRFDPAR IRAGKGDDNLTLLEPRKLEIHEGDRIRWTRNDHRRGLFNA DQARVVEIANGKVTFETSKG DLVELKKDDPMLKRIDLAYALNVHMAQGLTSDRGIAVMDS RERNLSNQKTFLVTVTRLRD HLTLVVDSADKLGAAVARNKGEKASAIEVTGSVKPTATKG SGVDQPKSVEANKAEKELTR SKSKTLDFGI SEQ ID NO:7 - Hel308 Mbu helicase MMIRELDIPRDIIGFYEDSGIKELYPPQAEAIEMGLLEKK NLLAAIPTASGKTLLAELAM IKAIREGGKALYIVPLRALASEKFERFKELAPFGIKVGIS TGDLDSRADWLGVNDIIVAT SEKTDSLLRNGTSWMDEITTVVVDEIHLLDSKNRGPTLEV TITKLMRLNPDVQVVALSAT VGNAREMADWLGAALVLSEWRPTDLHEGVLFGDAINFPGS QKKIDRLEKDDAVNLVLDTI KAEGQCLVFESSRRNCAGFAKTASSKVAKILDNDIMIKLA GIAEEVESTGETDTAIVLAN CIRKGVAFHHAGLNSNHRKLVENGFRQNLIKVISSTPTLA AGLNLPARRVIIRSYRRFDS NFGMQPIPVLEYKQMAGRAGRPHLDPYGESVLLAKTYDEF AQLMENYVEADAEDIWSKLG TENALRTHVLSTIVNGFASTRQELFDFFGATFFAYQQDKW MLEEVINDCLEFLIDKAMVS ETEDIEDASKLFLRGTRLGSLVSMLYIDPLSGSKIVDGFK DIGKSTGGNMGSLEDDKGDD ITVTDMTLLHLVCSTPDMRQLYLRNTDYTIVNEYIVAHSD EFHEIPDKLKETDYEWFMGE VKTAMLLEEWVTEVSAEDITRHFNVGEGDIHALADTSEWL MHAAAKLAELLGVEYSSHAY SLEKRIRYGSGLDLMELVGIRGVGRVRARKLYNAGFVSVA KLKGADISVLSKLVGPKVAY NILSGIGVRVNDKHFNSAPISSNTLDTLLDKNQKTFNDFQ Sequence number 8 - Dda helicase MTFDDLTEGQKNAFNIVMKAIKEKKHHVTINGPAGTGKTT LTKFIIEALISTGETGIILA APTHAAKKILSKLSGKEASTIHSILKINPVTYEENVLFEQ KEVPDLAKCRVLICDEVSMY DRKLFKILLSTIPPWCTIIGIGDNKQIRPVDPGENTAYIS PFFTHKDFYQCELTEVKRSN APIIDVATDVRNGKWIYDKVVDGHGVRGFTGDTALRDFMV NYFSIVKSLDDLFENRVMAF TNKSVDKLNSIIRKKIFETDKDFIVGEIIVMQEPLFKTYK IDGKPVSEIIFNNGQLVRII EAEYTSTFVKARGVPGEYLIRHWDLTVETYGDDEYYREKI KIISSDEELYKFNLFLGKTA ETYKNWNKGGKAPWSDFWDAKSQFSKVKALPASTFHKAQG MSVDRAFIYTPCIHYADVEL AQQLLYVGVTRGRYDVFYV Sequence number 9 / 5Biosg / CCTTTTTTTTGGCGTCTGCTTGGGTGTTTAAC C / iSp18 / / iSp18 / / iSp18 / / iSp18 / / iBNA-C / / iB NA-C / / iBNA-A / / iBNA-C / / iBNA-A / ACTTCGTTCAG TTACGTATTGC / 3Bio / Sequence number 10 GGTTAAACACCCAAGCAGACGCCAAAAAAAAGG Sequence number 11 / 5phos / GCAATACGTAACTGAACGAAGT / iBNA -T / / iBNA-G / / iBNA-T / / iBNA-G / / iBNA-G / Sequence number 12 CCAAGCAGACGCCAAAAAAAAGG SEQ ID NO:13 333333333333333333333333333333CCTTTTTTTT GGCGTCTGCTTGGGTGTTTAACC / [spacer sequence] / CCACAA CTTCGTTCAGTTACGTATTGCT SEQ ID NO:14 333333333333333333333333333333CCTTTTTTTT GGCGTCTGCTTGGGTGTTTAACC SEQ ID NO:15 CCTTTTTTTTGGCGTCTGCTTGGGTGTTTAACC SEQ ID NO:16 CCACAACTTCGTTCAGTTACGTATTGCT SEQ ID NO:17 5phos / GCAATACGTAACTGAACGAAGTTGTGG SEQ ID NO:18 GGTTAAACACCCAAGCAGACGCCTTT SEQ ID NO:19 GGTTAAACACCCAAGCAGACGCCAAAAAAAAGG / 3Cy5Sp /

Claims

1. 1. A method for loading a motor protein onto a polynucleotide adaptor, comprising: i) providing a polynucleotide adaptor comprising a spacer; ii) contacting the polynucleotide adaptor with a motor protein; iii) disposing said motor protein on said spacer, A blocking moiety attached to the polynucleotide adaptor inhibits the motor protein. and preventing migration of the substance away from the spacer.

2. i) providing a polynucleotide adaptor, said polynucleotide adaptor comprising: The adapter is connected to a spacer and a blocking portion attached to the polynucleotide adapter. minutes, ii) contacting the polynucleotide adaptor with a motor protein; iii) disposing said motor protein on said spacer, The blocking moiety prevents the motor protein from moving away from the spacer. The method of claim 1 , wherein

3. 1. A method for loading a motor protein onto a polynucleotide adaptor, comprising: i) providing a polynucleotide adaptor comprising a spacer; ii) contacting the polynucleotide adaptor with a motor protein; iii) advancing the motor protein over the spacer; and iv) attaching a blocking moiety to the polynucleotide adaptor. The blocking portion prevents the motor protein from moving away from the spacer. A method for preventing the

4. i) providing a polynucleotide adaptor comprising a loading site connected to a spacer; To provide ii) contacting the loading site with a motor protein; iii) advancing the motor protein from the loading site onto the spacer; And iv) attaching a blocking moiety to the polynucleotide adaptor. and the blocking moiety prevents the motor protein from being disengaged from the spacer and from The method of claim 3, wherein the method prevents migration to the loading site.

5. Step (ii) comprises contacting the loading site with a motor protein. wherein the motor protein binds to the loading site; Step (iv) attaching a blocking moiety to said polynucleotide adaptor. wherein the blocking moiety prevents the motor protein from being released from the spacer.

5. The method of claim 4, wherein said binding site is prevented from migrating and rebinding to said loading site.

6. By preventing the motor protein from moving off the spacer when the motor protein is bound to a polynucleotide, - a compound according to any one of claims 1 to 5, which reduces the rate at which proteins turn over fuel molecules; The method described.

7. the polynucleotide adaptor comprises a loading site connected to a spacer; The motor protein binds to the loading site of the polynucleotide adapter. The method according to any one of claims 4 to 6, wherein

8. The motor protein is advanced over the spacer by a physical or chemical The method according to any one of claims 1 to 7, comprising applying a force to the motor protein. method.

9. Propelling the motor protein onto the spacer The method according to any one of claims 1 to 8, comprising contacting the protein with one or more fuel molecules. How to.

10. the polynucleotide adaptor comprises a loading site connected to a spacer; The motor protein is a first motor protein, and the first motor protein Advancing the protein from the loading site onto the spacer is performed by a second motor. loading a protein into said loading site; and from the loading site toward the spacer, A second motor protein presses the first motor protein against the spacer. The method according to any one of claims 1 or 3 to 9,

11. The blocking moiety is attached to the polynucleotide adaptor. The motor protein is pressed against the spacer. The method according to any one of claims 1 to 5.

12. the polynucleotide adaptor comprises a loading site connected to a spacer; Any one of claims 1 to 11, wherein the blocking moiety binds to the loading site. The method according to claim 5.

13. The loading moiety is adjacent to the spacer and the blocking moiety is adjacent to the The method of claim 12, wherein the loading site is directly adjacent to a spacer.

14. Step (iii) comprises determining whether the spacer occupies an active site of the motor protein. and advancing the motor protein over the spacer so as to 14. The method according to any one of 1 to 13.

15. the polynucleotide adaptor comprises a loading site connected to a spacer; The loading site comprises a single-stranded or non-hybridizing polynucleotide.

15. The method according to any one of 1 to 14.

16. The loading site is a single-stranded or non-stranded nucleic acid having a length of about 2 to about 1000 nucleotide units. The method of claim 15 comprising a hybridizing polynucleotide.

17. 16. The blocking moiety of claim 1, wherein the blocking moiety is a physical or chemical blocking moiety.

2. The method according to claim 1 .

18. The blocking moiety is attached to the polynucleotide adaptor.

10. The method of claim 1, wherein the movement of the motor protein away from the spacer is sterically prevented.

18. The method according to any one of claims 1 to 17.

19. The blocking moiety is attached to the polynucleotide adaptor. A chemical group is introduced that prevents the motor protein from moving away from the spacer.

19. The method according to any one of claims 1 to 18.

20. (i) the loading portion comprises a single-stranded or non-hybridizing polynucleotide; wherein the blocking moiety comprises a single-stranded or non-hybridizing polynucleotide; i) binding the blocking moiety to the loading site The method according to any one of claims 1 to 19, further comprising hybridizing a loading moiety to said loading site. The method according to any one of claims 1 to 5.

21. The blocking moiety is a single-stranded or non-stranded molecule having a length of about 2 to about 1000 nucleotide units. The method of any one of claims 1 to 20, comprising a hybridizing polynucleotide.

22. The spacer is i) one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurine, one or more 2-6-diaminopurine, one or more 5-bromo- deoxyuridine, one or more inverted thymidines (inverted dT), one or more inverted dideoxy- Thymidine (ddT), one or more dideoxy-cytidines (ddC), one or more 5-methyl cytidine, one or more 5-hydroxymethylcytidines, one or more 2'-O-methyl R NA base, one or more iso-deoxycytidines (Iso-dC), one or more iso-deoxy Iso-dG, one or more C3 (OC ３ H ６ O.P.O. ３ ) group, one or more of photocleavable (PC)[OC ３ H ６ -C(O)NHCH ２ -C ６ H ３ NO ２ -CH(C H ３ ) O.P.O. ３ ] group, one or more hexanediol groups, one or more spacers 9 (iSp 9) [(OCH ２ CH ２ ) ３ O.P.O. ３ ] group, or one or more spacers 18 (iSp 18) [(OCH ２ CH ２ ) ６ OPO ３ group, ii) one or more thiol attachments; iii) one or more abasic nucleotides; iv) one or more nucleotides having a backbone structure different from that of the loading site; v) one or more chemical groups that stall the one or more motor proteins; and / or teeth vi) a polymer, optionally a polypeptide or a polyethylene glycol 22. The method of claim 1, comprising administering to the patient a polymer that is a PEG-coated polyether ether (PEG).

23. The spacer comprises one or more nucleotides, and preferably the spacer comprises one The method according to any one of claims 1 to 22, comprising a nucleotide island or islands.

24. i) the polynucleotide adaptor comprises one or more nucleotides, preferably one or more A spacer containing the above nucleotide island and a polynucleotide adaptor are attached to the spacer. and a blocking moiety associated therewith, ii) placing the motor protein on the spacer contacting a motor protein with the spacer; and and modifying the motor protein to prevent it from escaping from the target protein.

24. The method of any one of claims 1, 2, 6-9, or 12-23.

25. The spacer is -S-N-S-N-S-N-S-, -S-N-N-S-N-N-S-N-N-S-, -S -S-S-S-S-S-N-N-S-S-, -S-S-S-S-S-N-N-S-N-S-S-S-, -S-S-S-N-N-S -N-N-S-S-N-N-S-, -S-S-S-S-S-N-N-S-N-N-S-S-N-N-S-, -S-S-S-N -N-S-N-N-S-N-N-S-, -N-N-S-S-N-N-S-S-S-S-, -S-N-N-S-S-N-N-S -SSSS-, -S-S-N-N-S-S-N-N-S-S-S-, -S-S-S-S-N-N-S-S -N-N-S-, -N-N-S-N-N-S-S-S-S-S-, -S-N-N-S-N-N-S-S-S-S-, -S-S-N-N-S-N-N-S -S-S-S-, -S-S-S-N-N-S-N-N-S-S-S-, --S-S-S-S-N-N-S-N-N-S-S-, and --S-S-S-N-N-S- comprising one or more moieties selected from S-N-N-S-; wherein each S is a spacer unit and each N is a nucleotide. The method according to claim 24.

26. The motor protein is a helicase, a polymerase, an exonuclease, a topoisomerase, The method according to any one of claims 1 to 25, which is a somerase or a variant thereof. 。

27. The motor protein on the spacer of the polynucleotide adaptor The motor protein is modified to prevent it from being detached from the spacer. The method according to any one of claims 1 to 26.

28. The or each motor protein is Hel308 helicase, RecD helicase, TraI helicase, TrwC helicase, XPD helicase, and Dda helicase or a variant thereof. The method according to any one of claims 1 to 5.

29. v) removing excess motor protein molecules that are not located on the spacer The method of any one of claims 1 to 28, further comprising:

30. 1. A method for controlling translocation of a target polynucleotide relative to a transmembrane nanopore, comprising: i) (A) a target polynucleotide, (B) a polynucleotide adaptor comprising a spacer and (C) providing a motor protein; ii) carrying out the method according to any one of claims 1 to 29, whereby said polynucleotide Stalling the motor protein on the spacer of a nucleotide adaptor. 、 iii) determining the spacing between the target polynucleotide and the polynucleotide adaptor; contacting the stalled motor protein on a sensor with the nanopore; iv) applying a voltage potential across the transmembrane nanopore, thereby The protein is transferred across the spacer to the target polynucleotide, thereby and controlling the movement of the target polynucleotide relative to the nanopore.

31. After the motor protein stalls on the polynucleotide adaptor, The method of claim 30, wherein a oligonucleotide adaptor binds to the target polynucleotide. method.

32. After the polynucleotide adaptor binds to the target polynucleotide, the moiety 31. The method of claim 30, wherein the adapter protein is stalled on the polynucleotide adapter. method.

33. 1. A method for controlling translocation of a target polynucleotide relative to a transmembrane nanopore, comprising: i) providing a target polynucleotide; ii) A polynucleotide that includes a spacer and on which the motor protein stalls The present invention provides a polynucleotide adaptor, the polynucleotide adaptor comprising:

29. A method for producing a medicament for the treatment of a cancer, comprising: iii) coupling the target polynucleotide and the polynucleotide adaptor to the nanoparticles. contacting the pore; iv) applying a voltage potential across the transmembrane nanopore, thereby The protein is transferred across the spacer to the target polynucleotide, thereby and controlling the movement of the target polynucleotide relative to the nanopore.

34. 1. A method for characterizing a target polynucleotide, comprising: i) carrying out a method according to any one of claims 30 to 33; ii) detecting the presence of the target polynucleotide as it moves relative to the nanopore; one or more measurements are made that indicate one or more characteristics of the nucleotides, thereby identifying the target polynucleotides. and characterizing the nucleotide as it translocates relative to the nanopore. Law.

35. (i) a spacer; and (ii) a motor protein stalled on the spacer. and wherein the active site of the motor protein is occupied by the spacer. (iii) a blocking moiety attached to an adaptor, A blocking portion that prevents the motor protein from moving away from the spacer. and a polynucleotide adaptor.

36. (i) the adapter comprises a loading site connected to the spacer, (ii) a locking moiety is attached to the loading site, and (iii) the blocking moiety is 36. The method of claim 35, which prevents the motor protein from binding to the loading site. The polynucleotide adaptor described herein.

37. i) in the 5' to 3' direction {L Ｂ -S-D} ｎ Or {D-S-L Ｂ } ｎ (In the formula, L Ｂ is a blocked loading site, S is a spacer, and D is a double-stranded polynucleotide. leutide, where n is an integer, optionally an integer from 1 to about 20; ii) one or more motor proteins stalled on said spacer (S), The or each L Ｂ a portion of the or each motor protein comprising the double-stranded polynucleotide and preventing the spacer (S) from moving away from the lead (D).

37. The polynucleotide adaptor of claim 36.

38. i) in the 5' to 3' direction {L Ｂ -S-D} ｎ Or {D-S-L Ｂ } ｎ (In the formula, L Ｂ is a first double-stranded polynucleotide, S is a spacer, and D is a second double-stranded polynucleotide. nucleotide, n being an integer, optionally an integer from 1 to about 20; Single-stranded polynucleotide (L Ｂ ) is adjacent to the spacer (S), and the spacer (S) is adjacent to the second double-stranded polynucleotide (D), ii) one or more motor proteins stall on said spacer (S), 6 or 37. A polynucleotide adaptor according to claim 37.

39. 1. A kit for modifying a target polynucleotide, comprising: i) a polynucleotide adaptor comprising a spacer; ii) a motor protein capable of controlling the movement of the target polynucleotide; 、 iii) the motor protein is coupled to the spacer of the polynucleotide adaptor. and the active site of the motor protein is occupied by the spacer. When the motor protein is released from the spacer, the motor protein is prevented from moving away from the spacer. a blocking moiety capable of binding to the polynucleotide adaptor such that Including, Optionally, the polynucleotide adaptor comprises a loading site, and the protease The binding moiety prevents the motor protein from binding to the loading site. The kit can be used to bind to the polynucleotide adaptor.

40. 1. A kit for modifying a target polynucleotide, comprising: i) a polynucleotide adaptor, comprising a spacer and a blocking moiety attached to said adaptor; ii) a motor protein capable of controlling the movement of the target polynucleotide; , including When the motor protein is bound to the adaptor, the blocking moiety prevents the motor protein from moving away from the spacer, 。

41. The loading moiety, the blocking moiety, and / or the spacer each and / or The motor protein is as defined in any one of claims 26 to 28. The polynucleotide adaptor or kit according to any one of claims 35 to 40, 。

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