method

Abstract

Provided herein is a method for characterizing a target polypeptide as it moves relative to a nanopore. Also provided are related kits, systems, and devices for performing such methods.

Description

Technical Field

[0001] The present disclosure relates to a method for characterizing a target polypeptide by forming a construct comprising a first strand comprising the target polypeptide and a second strand such as a polynucleotide strand, moving the construct relative to a nanopore under conditions such that the first and second strands of the construct move through the nanopore, and obtaining a measurement characteristic of the polypeptide during such movement. The present disclosure also relates to kits, systems, and devices for performing such methods.

Background Art

[0002] The characterization of biological molecules is becoming increasingly important in biomedical and biotechnological applications. For example, nucleic acid sequencing enables the study of genomes and the proteins they encode, and for example, enables the association of observable phenomena such as nucleic acid mutations with disease symptoms. Nucleic acid sequencing can be used in evolutionary biology to study relationships between organisms. Metagenomics involves identifying the organisms present in a sample, such as the microorganisms in a microbiome, and nucleic acid sequencing enables the identification of such organisms. Techniques for characterizing polynucleotides (e.g., sequencing) have been widely developed, but techniques for characterizing polypeptides have not advanced as much, despite their great biotechnological importance. For example, knowledge of protein sequences can establish structure-activity correlations and has influenced rational drug development strategies for developing ligands for specific receptors. Identification of post-translational modifications is also key to understanding the functional properties of many proteins. For example, in eukaryotes, typically 30-50% of protein species are phosphorylated. Some proteins can have multiple phosphorylation sites that serve to activate or inactivate the protein, promote its degradation, or regulate its interaction with protein partners.

Summary of the Invention

[0003] Known methods for characterizing polypeptides include mass spectrometry and Edman degradation.

[0004] Protein mass spectrometry involves characterizing whole proteins or fragments thereof in their ionized form. Known methods of protein mass spectrometry include electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI). While mass spectrometry has several advantages, the results obtained can be affected by the presence of contaminants, and processing fragile molecules without fragmentation can be difficult. Furthermore, mass spectrometry is not a single-molecule technique and only provides holistic information about the sample being analyzed. Mass spectrometry is not suitable for characterizing differences within a population of polypeptide samples and is cumbersome when attempting to distinguish between adjacent residues.

[0005] Edman sequencing is an alternative to mass spectrometry that enables residue-wise sequencing of polypeptides. Edman sequencing sequences polypeptides by sequentially cleaving the N-terminal amino acids and then characterizing the individually cleaved residues using chromatography or electrophoresis. However, Edman sequencing is slow, requires expensive reagents, and is not a single-molecule technique like mass spectrometry.

[0006] Therefore, there remains an urgent need for new technologies for characterizing polypeptides, particularly at the single-molecule level. Single-molecule technologies for characterizing biomolecules such as polynucleotides have proven particularly attractive due to their high fidelity and avoidance of amplification bias.

[0007] One attractive method for single-molecule characterization of biomolecules such as polypeptides is nanopore sensing. Nanopore sensing is an approach to analytes detection and characterization that relies on observing individual binding or interaction events between the analyte molecule and ion conduction channels. Nanopore sensors can be made by placing a single nanometer-sized pore in an electrical insulating film and measuring the voltage-driven ion current through the pore in the presence of the analyte molecule. When the analyte is present inside or near the nanopore, the ion flow through the pore changes, resulting in an ion current or current change measured on the channel. Identification of the analyte is revealed through its distinctive current signature, particularly the duration and degree of current interruption, as well as the variation in current levels during its interaction with the pore. Nanopore sensing has the potential to enable rapid and inexpensive polypeptide characterization.

[0008] Nanopore sensing and characterization of polypeptides have been proposed in the art. For example, WO2013 / 123379 discloses the use of an NTP-driven protein processing unfoldase enzyme to process proteins and rearrange them through nanopores. WO2021 / 111125 discloses a method in which a target polypeptide is conjugated to a polynucleotide to form a single-strand polypeptide-polynucleotide conjugate, and the conjugate is moved through nanopores using a polynucleotide handling protein. However, alternative and / or improved methods for characterizing polypeptides are still needed.

[0009] The methods disclosed herein can also be applied to the characterization of polynucleotides described below.

[0010] This disclosure relates to a method for characterizing a target polypeptide.

[0011] In one aspect, the method involves a conjugate chain comprising a target polypeptide. The target polypeptide is conjugated at each end of the polypeptide to one or more polynucleotide adjacent chains. The conjugate is contacted with a polynucleotide carrier chain, thereby forming a polynucleotide-polypeptide construct. The construct is contacted with a nanopore. The contact is carried out under conditions such that both the polynucleotide-polypeptide conjugate chain and the polynucleotide carrier chain co-translocate through the nanopore. As the conjugate moves relative to the nanopore, one or more measurements characteristic of the polypeptide are obtained. In this way, the target polypeptide contained in the conjugate is characterized.

[0012] In another aspect, the method involves a conjugate chain comprising a target polypeptide. The polypeptide attaches to a polynucleotide adjacent chain, thereby forming a conjugate chain. The conjugate chain is contacted with a polynucleotide handling protein. The conjugate chain is contacted with a nanopore. The polynucleotide handling protein controls the movement of the conjugate relative to the nanopore. As the conjugate moves relative to the nanopore, one or more measurements characteristic of the polypeptide are obtained. In this way, the target polypeptide contained in the conjugate is characterized.

[0013] Thus, provided herein is a method for characterizing a target polypeptide, comprising: -(i) contacting a polynucleotide-polypeptide conjugate chain comprising the target polypeptide conjugated at each end of the polypeptide to one or more polynucleotide adjacent chains with (ii) a polynucleotide carrier chain, thereby forming a polynucleotide-polypeptide construct; -contacting the construct and the nanopore under conditions such that both the polynucleotide-polypeptide conjugate chain and the polynucleotide carrier chain co-translocate through the nanopore; - obtaining one or more measurements characteristic of the polypeptide as the construct moves relative to the nanopore, thereby characterizing the target polypeptide.

[0014] In some embodiments, each of the one or more polynucleotide flanking strands is independently complementary to a region of the polynucleotide carrier strand. In some embodiments, each of the one or more polynucleotide flanking strands is independently at least partially hybridized to the polynucleotide carrier strand.

[0015] Also provided is a method of characterizing a target polypeptide, comprising: - contacting (i) a polynucleotide-polypeptide conjugate strand comprising a target polypeptide attached to a polynucleotide flanking strand with (ii) a polynucleotide handling protein that can control the movement of the polynucleotide flanking strand relative to the nanopore; - contacting the polynucleotide-polypeptide conjugate strand and the nanopore under conditions such that the polynucleotide handling protein controls the movement of the polynucleotide-polypeptide conjugate strand relative to the nanopore; - obtaining one or more measurements characteristic of the polypeptide as the polynucleotide flanking strand and the target polypeptide co-translocate through the nanopore, thereby characterizing the target polypeptide.

[0016] In some embodiments, prior to the methods described above, the polynucleotide flanking strand at least partially hybridizes to the polynucleotide carrier strand, thereby forming a polynucleotide-polypeptide construct.

[0017] In some embodiments of the methods described above, the polynucleotide-polypeptide conjugate strand comprises a plurality of target polypeptides.

[0018] In some embodiments, during the methods described above, the polypeptide or each polypeptide is independently maintained in a linearized form.

[0019] In some embodiments, the target polypeptide or each target polypeptide independently has a length of from about 5 to about 1000 peptide units.

[0020] In some embodiments, the method includes mechanically manipulating the construct, the polynucleotide - polypeptide conjugate chain, and / or the polynucleotide carrier chain, thereby moving the construct, the polynucleotide - polypeptide conjugate chain, and / or the polynucleotide carrier chain relative to the nanopore. In some embodiments, the construct, the polynucleotide - polypeptide conjugate chain, and / or the polynucleotide carrier chain are moved by a mechanical manipulation in a direction opposite to the potential applied across the nanopore. In some embodiments, the potential is a voltage potential applied across the nanopore.

[0021] In some embodiments, the method includes contacting the construct with a polynucleotide - handling protein that can control the movement of one or more polynucleotide - adjacent chains and / or polynucleotide carrier chains, wherein the polynucleotide - handling protein controls the movement of the target polypeptide relative to the nanopore.

[0022] In some embodiments, the method includes contacting both the polynucleotide - polypeptide conjugate chain and the polynucleotide carrier chain of the construct with a polynucleotide - handling protein that can control the movement of the polynucleotide - polypeptide conjugate chain and / or the polynucleotide carrier chain, wherein the polynucleotide - handling protein controls the movement of the construct relative to the nanopore.

[0023] In some embodiments, the method involves contacting a polynucleotide-polypeptide conjugate chain with a polynucleotide handling protein capable of controlling the movement of the polynucleotide-polypeptide conjugate chain, the polynucleotide handling protein controlling the movement of a target polypeptide into a nanopore.

[0024] In some embodiments, the method involves contacting a polynucleotide carrier chain with a polynucleotide handling protein that can control the movement of the polynucleotide carrier chain, the polynucleotide handling protein controlling the movement of a target polypeptide into a nanopore.

[0025] In some embodiments, i) Polynucleotide handling proteins are located on the cis side of the nanopore, and the polynucleotide handling proteins control the movement of constructs, polynucleotide-polypeptide conjugate chains and / or polynucleotide carrier chains from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of target polypeptides from the cis side of the nanopore to the trans side of the nanopore, or ii) The polynucleotide handling protein is located on the trans side of the nanopore and controls the movement of constructs, polynucleotide-polypeptide conjugate chains and / or polynucleotide carrier chains from the trans side to the cis side of the nanopore, thereby controlling the movement of target polypeptides from the trans side to the cis side of the nanopore.

[0026] In some embodiments, the polynucleotide handling protein is located on the cis side of the nanopore and controls the movement of the polynucleotide carrier chain from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of the target polypeptide through the nanopore. In some embodiments, the polynucleotide handling protein is located on the trans side of the nanopore and controls the movement of the polynucleotide carrier chain from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of the target polypeptide through the nanopore.

[0027] In some embodiments, i) The polynucleotide handling protein is located on the cis side of the nanopore, and the polynucleotide handling protein controls the movement of constructs, polynucleotide-polypeptide conjugate chains and / or polynucleotide carrier chains from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of target polypeptides from the trans side of the nanopore to the cis side of the nanopore, or ii) A polynucleotide handling protein is located on the trans side of the nanopore, and the polynucleotide handling protein controls the movement of constructs, polynucleotide-polypeptide conjugate chains and / or polynucleotide carrier chains from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of target polypeptides from the cis side of the nanopore to the trans side of the nanopore.

[0028] In some embodiments, the polynucleotide handling protein is located on the cis side of the nanopore, and the polynucleotide handling protein controls the movement of the polynucleotide carrier strand from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of the target polypeptide through the nanopore. In some embodiments, the polynucleotide handling protein is located on the trans side of the nanopore, and the polynucleotide handling protein controls the movement of the polynucleotide carrier strand from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of the target polypeptide through the nanopore.

[0029] In some embodiments, before contacting the construct with the nanopore, the polynucleotide handling protein is bound to the polynucleotide carrier strand within the region of the polynucleotide carrier strand that extends into the non-hybridized region of the polynucleotide adjacent strand.

[0030] In some embodiments, when a portion of the polynucleotide-polypeptide conjugate strand that contacts the active site of the polynucleotide handling protein contains the target polypeptide, the polynucleotide handling protein can maintain its binding to the polynucleotide-polypeptide conjugate strand. In some embodiments, when the polynucleotide handling protein contacts the target polypeptide, the polynucleotide handling protein is modified to prevent dissociation from the construct, the polynucleotide-polypeptide conjugate strand, and / or the polynucleotide carrier strand. In some embodiments, the polynucleotide handling protein is modified to fully or partially close an opening present in at least one conformational state of the unmodified protein that can unbind the polynucleotide strand. In some embodiments, the polynucleotide handling protein is a helicase, translocase, or helicase-nuclease complex, or includes them.

[0031] In some embodiments, the construct includes a stall portion, and prior to translocation of the target polypeptide through the nanopore, the polynucleotide handling protein is arranged such that the stall portion is positioned between the polynucleotide handling protein and the target polypeptide.

[0032] In some embodiments, one or more adapters and / or one or more tethers and / or one or more anchors are attached to the construct, the polynucleotide-polypeptide conjugate chain, and / or the polynucleotide carrier chain. In some embodiments, the construct, the polynucleotide-polypeptide conjugate chain, and / or the polynucleotide carrier chain includes a blocking portion attached via an optional linker, and the blocking portion is unable to translocate through the nanopore.

[0033] In some embodiments, the method comprises i) performing the method described herein such that the target polypeptide translocates the nanopore in a first direction relative to the nanopore; ii) enabling the construct, the polynucleotide-polypeptide conjugate chain, and / or the polynucleotide carrier chain to be moved in a direction opposite to the direction of movement relative to the nanopore in step (i) such that the target polypeptide translocates the nanopore in a second direction opposite to the first direction; iii) optionally, enabling the construct, the polynucleotide-polypeptide conjugate chain, and / or the polynucleotide carrier chain to be moved in the first direction such that the target polypeptide re-translocates the nanopore in the first direction; iv) optionally, repeating steps (ii) and (iii) to oscillate the polypeptide through the nanopore.

[0034] In some embodiments, one or more of the measured values are characteristic of one or more features of the target polypeptide selected from: (i) the length of the target polypeptide, (ii) the identity of the target polypeptide, (iii) the sequence of the target polypeptide, (iv) the secondary structure of the target polypeptide, and (v) whether the target polypeptide is modified.

[0035] In some embodiments, the nanopore is a protein nanopore, preferably a β-barrel protein nanopore.

[0036] Also provided by the present invention is a system comprising: -(i) a polynucleotide-polypeptide conjugate chain comprising the target polypeptide conjugated to one or more polynucleotide flanking chains at each end of the target polypeptide, and (ii) a polynucleotide carrier chain, a construct; -a nanopore capable of cotranslocating the polynucleotide-polypeptide conjugate chain and the polynucleotide flanking chain of the construct; -a polynucleotide handling protein, a system.

[0037] Also provided by the present invention is a kit comprising: -a nanopore; -a first polynucleotide comprising a reactive functional group for conjugating to the first end of the target polypeptide; -a second polynucleotide comprising a reactive functional group for conjugating to the second end of the target polypeptide; -a polynucleotide handling protein, a kit.

[0038] In some embodiments of the systems and kits provided herein, the nanopore, construct, and / or polynucleotide handling protein are as defined herein.

[0039] Also provided are methods for characterizing target polynucleotides. Unless otherwise implied by the context, the methods herein relating to polypeptide characterization may be applied similarly to the characterization of target polynucleotide sequences, as described in more detail herein. [Brief explanation of the drawing]

[0040] [Figure 1]A schematic diagram illustrating a non-limiting example of an embodiment of the disclosed method, in which a polynucleotide handling protein moving along ssDNA on the cis side of a nanopore controls the movement of a construct comprising a polynucleotide-polypeptide conjugate chain described herein, hybridized to a polynucleotide carrier chain from the cis side of the nanopore to the trans side of the nanopore, thus enabling characterization of the polypeptide as it moves toward the nanopore. As depicted, the polynucleotide handling protein is first loaded onto the ssDNA opposite an optional bubble region (F). Both ends of the polynucleotide may be trapped in the nanopore, for example from the cis side of the membrane, by applying a positive voltage, for example (i) to the range of the polynucleotide handling enzyme, for example (ii), and (iii) unstalling the polynucleotide handling protein (if optional stalling chemistry is used) and transposing it on the ssDNA. The movement of the polynucleotide handling protein along the polynucleotide section of the carrier chain feeds the construct into the pore, and as the polynucleotide handling protein moves along the polynucleotide (e.g., in a step driven by a single nucleotide fuel), it feeds the construct into the nanopore, and the peptide section passes through the nanopore, making it possible to characterize it. Both ssDNA strands re-anneal behind the enzyme and transpose within the migrating bubble corresponding to the polymer strand spreading into the polynucleotide handling protein, as depicted. A: peptide conjugated in a dsDNA context, B: optional motor protein stall chemistry (e.g., BNA, LNA, or RNA), C: optional motor protein stall chemistry (e.g., Spacer 18 or similar), D: polynucleotide handling enzyme, E: nanopore inserted into the membrane, F: optional bubble-conjugated ssDNA, ssRNA, or the opposite enzyme of the Spacer chemistry. [Figure 2]A schematic diagram illustrating a non-limiting example of an embodiment of the disclosed method, in which a polynucleotide handling protein moving along ssDNA on the cis side of a nanopore controls the movement of a construct comprising a polynucleotide-polypeptide conjugate chain hybridized to a polynucleotide carrier chain from the trans side of a nanopore to the cis side of a nanopore, thus enabling characterization of the polypeptide as it moves toward the nanopore. As depicted, the polynucleotide handling protein is first loaded onto the ssDNA opposite an optional bubble region (F). Both ends of the polynucleotide may be captured within the nanopore, for example from the cis side of the membrane, by applying a positive voltage, for example (i) to the range of the polynucleotide handling enzyme, for example (ii), for example (i) to the trans side of the membrane, and (iii) to allow the removal of an optional enzyme stall blocking portion (if an optional blocking portion is used) and its repositioning on the ssDNA. The movement of the polynucleotide handling protein along the polynucleotide section of the carrier chain feeds the construct into the pore and pulls it out of the pore (e.g., from the trans side of the pore to the cis side of the pore), and as the polynucleotide handling protein moves along the polynucleotide (e.g., in a step driven by a single nucleotide fuel), it pulls the construct out of the nanopore, allowing the peptide section to pass through the nanopore and become available for characterization. Both ssDNA strands re-anneal behind the enzyme and rearrange within the moving bubble as described, thereby controlling both strands of the conjugate as they exit the nanopore. All steps can be performed with a positive bias on the trans side of the membrane, except in case (ii), and can be performed with zero potential or a low negative (trans) bias.A: A selectively bubbled ssDNA, ssRNA, or spacer chemistry opposite enzyme; B: A polynucleotide handling enzyme; C: A selectively enzyme stall blocking moiety (e.g., LNA, BNA, or RNA); D: A selectively enzyme stall chemistry (e.g., Spacer 18 or similar); E: A peptide conjugated in a dsDNA context; F: A nanopore inserted into a membrane. [Figure 3]A schematic diagram illustrating a non-limiting example of an embodiment of the disclosed method, in which a polynucleotide handling protein on the cis side of a nanopore controls the movement of a construct comprising a polynucleotide-polypeptide conjugate chain described herein, hybridized to a polynucleotide carrier chain from the trans side of the nanopore to the cis side of the nanopore, thus enabling characterization of the polypeptide as it moves toward the nanopore. As depicted, the polynucleotide handling protein is first loaded and optionally loaded onto the ssDNA opposite an optional bubble (D). An optional leader (A) may be present on the construct to facilitate threading of the construct through the nanopore. Both ends of the polynucleotide may be captured within the nanopore, for example from the cis side of the membrane, by applying a positive voltage, for example (i) to the range of the polynucleotide handling enzyme, for example (ii), and then (iv) the enzyme is pushed backward toward an optional blocking portion (F), and then transposed toward the ssDNA. The movement of the polynucleotide handling protein involves supplying the construct into the pore and pulling it out of the pore (e.g., from the trans side of the pore to the cis side of the pore), and as the polynucleotide handling protein moves along the polynucleotide (e.g., in a step driven by a single nucleotide fuel), it pulls the construct out of the nanopore, allowing the peptide section to pass through the nanopore and become available for characterization. Both ssDNA strands re-anneal behind the enzyme and rearrange within the moving bubble as described, thereby controlling both strands of the conjugate exiting the nanopore. The enzyme may be pushed back to its previous position at any point via a force acting on the DNA, thereby resetting the cycle. The polynucleotide handling protein can then again control the movement of the construct into the nanopore, allowing the target polypeptide to be repeatedly "floored" through the nanopore.A: Optionally selected leader section, optionally selected C3 section to stall the enzyme, C: Polynucleotide handling enzyme, D: Optionally selected bubble-tipped ssDNA:ssRNA: or spacer chemistry opposite enzyme, E: Peptide conjugated in a dsDNA context, F: Optionally selected backblocker portion to prevent enzyme dissociation, G: Nanopores inserted into the membrane. [Figure 4] This schematic diagram illustrates a non-limiting example of an embodiment of the disclosed method, in which a polynucleotide handling protein on the cis side of a nanopore controls the movement of a construct comprising a polynucleotide-polypeptide conjugate chain hybridized to a polynucleotide carrier chain from the cis side of the nanopore to the trans side of the nanopore, thus enabling characterization of the polypeptide as it moves toward the nanopore. The polynucleotide handling protein can engage with both chains of the construct (e.g., the polynucleotide-polypeptide conjugate chain and the carrier chain) and actively control the movement of either or both chains. The movement of the construct and characterization of the target polypeptide are discussed in Figure 1. [Figure 5] This schematic diagram illustrates a non-limiting example of an embodiment of the disclosed method, in which a polynucleotide handling protein on the cis side of a nanopore controls the movement of a construct comprising a polynucleotide-polypeptide conjugate chain hybridized to a polynucleotide carrier chain from the trans side of the nanopore to the cis side of the nanopore, thus enabling characterization of the polypeptide as it moves toward the nanopore. The polynucleotide handling protein can engage with both chains of the construct (e.g., the polynucleotide-polypeptide conjugate chain and the carrier chain) and actively control the movement of either or both chains. The movement of the construct and characterization of the target polypeptide are discussed in Figure 2. [Figure 6]A schematic diagram illustrating further non-limiting examples of embodiments of the disclosed method. A: The target polypeptide is contained in a polynucleotide-polypeptide conjugate chain by attachment to a polynucleotide adjoint chain. The polynucleotide-polypeptide conjugate chain is brought into contact with a polynucleotide handling protein, thereby enabling the feature of the polypeptide as it moves into nanopores. As shown, the movement scheme is similar to that in Figure 1, but those skilled in the art will understand that the exact movement scheme is not limiting, and that movement schemes such as those described herein, particularly in each of Figures 1 to 5, are also compatible with this embodiment and are specifically disclosed herein. B: The polynucleotide-polypeptide conjugate chain in panel A may optionally be brought into contact with a polynucleotide carrier chain to form a construct as depicted. The construct is brought into contact with a polynucleotide handling protein, thereby enabling the feature of the polypeptide as it moves into nanopores. As shown, the transfer scheme is similar to that in Figure 4, but those skilled in the art will understand that the exact transfer scheme is not limited, and that transfer schemes such as those described herein, in particular in each of Figures 1 to 5, are also compatible with this embodiment and are specifically disclosed herein. C: The polynucleotide-polypeptide conjugate chain of panel A may optionally be in contact with a polynucleotide carrier chain to form a construct as depicted, and the carrier chain is optionally excluded from the nanopore during the rearrangement of the construct through the nanopore. As shown, the transfer scheme is similar to that in Figure 1, but those skilled in the art will understand that the exact transfer scheme is not limited, and that transfer schemes such as those described herein, in particular in each of Figures 1 to 5, are also compatible with this embodiment and are specifically disclosed herein. [Figure 7]This schematic diagram illustrates a further non-limiting example of a method for the repetitive and controlled transfer of a trans-to-cis polynucleotide-polypeptide conjugate, where the transfer is controlled via a polynucleotide handling enzyme moving along ssDNA. The enzyme is first loaded and stalls on the ssDNA overhang. This method is performed under conditions where multiple enzymes can be loaded onto the overhang. Both ends of the polynucleotide are (i) trapped in the nanopore to the polynucleotide handling enzyme (ii), after which the enzyme controls the transfer of the conjugate out of the nanopore to the peptide (iii). The enzyme may then dissociate from the polynucleotide, at which point the position of the conjugate in the nanopore descends to a second enzyme loaded at its previous position on the DNA, thereby resetting the cycle. A: Polynucleotide handling enzyme loaded onto an ssDNA overhang; B: Peptide conjugated in a dsDNA context; C: Nanopore inserted into the membrane; D: Second polynucleotide handling enzyme binding to the ssDNA overhang while initially transposing. [Figure 8] This schematic diagram illustrates a further non-limiting example of a method for the repetitive and controlled transfer of trans-to-cis polynucleotide-polypeptide conjugates, where the transfer is controlled via a polynucleotide handling enzyme moving along ssDNA. The scheme is identical to that of Figure 7, except that the enzyme is loaded onto a strand that is successively ssDNA, with the exception of a spacer portion (A) that restricts the movement of the enzyme and causes the enzyme to deassociate from the DNA. [Figure 9]This is a schematic diagram illustrating a further non-limiting example of a scheme in which polypeptides are characterized using nanopores. In each example, the polypeptide is conjugated between two internal groups on a polynucleotide. A: A polynucleotide handling enzyme controls the polynucleotide-polypeptide conjugate exiting the nanopore. The polypeptide attaches to two internal points on the ssDNA oligonucleotide, and either (i) ssDNA and peptide, or (ii) dsDNA and peptide, cotranspose through the nanopore. B: A polynucleotide handling enzyme controls the polynucleotide-polypeptide conjugate entering the nanopore. The polypeptide attaches to a single point on the ssDNA oligonucleotide, and either (i) ssDNA and peptide, or (ii) dsDNA and peptide, cotranspose through the nanopore. C: A polynucleotide handling enzyme controls the polynucleotide-polypeptide conjugate entering the nanopore. The polypeptide attaches to two internal points on the ssDNA oligonucleotide, and either (i) ssDNA and the peptide, or (ii) dsDNA and the peptide, co-transpose through the nanopore. In case (ii) of each scheme shown, the polynucleotide handling enzyme can transpose on either DNA strand. [Figure 10] Method for assembling a polynucleotide-polypeptide construct. In the first click reaction (step (i)), a hairpin DNA (DNA1) having a 3'TCO group is reacted with a polypeptide having an N-terminal azide and a C-terminal methyltetrazine group. Then, a second click reaction (step (ii)) is performed on DNA2, which has a 5'BCN group and a 3'biotin group. Monovalent traptabidine is added to produce the final construct (A) (step (iii)). Distance x is defined as the distance in the base pair between monovalent traptabidine and the peptide (excluding intervening linker chemistry) and varies in the experiment described in Example 1. In this example, the “carrier” chain (opposite side of the peptide) has a length of 6 nucleotides. The hairpin ends are trapped in the nanopores shown in (A). [Figure 11]This is an example of a current-time trace demonstrating the capture of a polynucleotide-polypeptide conjugate, as described in Example 1. The peptide sequence used throughout is: [ka] In trace (A), XX=DD; in (B), XX=RR; and in (C), XX=YY. Each trace shows an initial stage of open-pore current (i), followed by a decrease to a lower level (ii) due to conjugate capture. The normalized current (I / I0, Figure 12) is then scored for each capture by dividing the median current at level (ii) by the median current at level (i). [Figure 12] 3 peptides (sequence) [ka] (A) shows the normalized current histogram for XX=DD, (B) for XX=RR, and (C) for XX=YY. The normalized current is defined according to Figure 11. [Figure 13] Polypeptide sequence [ka] This is a plot of normalized current against distance for a series of polynucleotide-polypeptide conjugates having the central two residues mutated to DD, RR, or YY. The normalized current (Figure 11) is plotted as a function of the base pair distance between monovalent traptabidine and the peptide. The dsDNA level, shown as a dashed line, was determined from a double-stranded DNA control. [Figure 14] This is an example of a current-time trace demonstrating the capture of a polynucleotide-polypeptide conjugate, accompanied by the movement of the conjugate out of a nanopore controlled by a helicase, as described in Example 2. The peptide sequence used throughout is: [ka] In trace (A), XX=DD; in (B), XX=RR; and in (C), XX=YY. Each trace shows an initial stage of open-pore current (i), followed by a decrease to a lower level (ii) due to conjugate capture. Each example shows a repeating pattern indicating the repeated movement of the conjugate through the nanopore. In (iii), the helicase controlling the movement dissociates from the conjugate and the second helicase is no longer bound, so the current trace returns to the open-pore level (i). [Figure 15] This is an example of a current-time trace showing the capture of a polynucleotide-polypeptide conjugate in the absence of ATP, using a helicase-bound conjugate as described in Example 2. The peptide sequence used is N-GGSGDDSGSG-C. The trace shows an initial stage of open-pore current (i), followed by a decrease to a lower level marked by (ii) due to the capture of the conjugate. Unlike Figure 14, no repetitive motion is observed, indicating that the helicase movement is ATP-dependent. In (iii), the helicase controlling the movement dissociates from the conjugate, and since the second helicase is not bound, the current trace returns to the open-pore level (i). [Figure 16] This is the polynucleotide-polypeptide construct used in Example 3. a. Leader, b. Tether, c. Hairpin, d. DNA1 oligo; e. DNA2 oligo; f. Peptide; g. DNA3 oligo; x. ssDNA overhang for enzyme loading; y. Helicase (arrows indicate the direction of helicase movement); circular = click chemical group, vertical line = ligation site. [Figure 17-1]Figure 17 shows current-time traces illustrating the capture of a polynucleotide-polypeptide conjugate, accompanied by helicase-controlled conjugate movement out of a nanopore, as described in Example 3. The peptide sequences tested were RSDSGQQARY (Figures 17A, 2D), GGSGSSSGSG (Figures 17B, 17E), and EAIYAAPFAKKK (Figures 17C, 17F). Traces A, B, and C show an initial stage of open-pore current (i) followed by a decrease to a lower level (ii) due to conjugate capture. Traces A, B, and C further show a repeating pattern indicating repeated movement of the conjugate through the nanopore. In (iii), the helicase controlling the movement dissociates from the conjugate, and since no further helicase is bound, the current trace returns to the open-pore current level. Examples of single reads for each peptide are shown in D, E, and F. A single read shows a single-stranded DNA stage (iv), followed by a decrease in current caused by the peptide (v). The length of block (iv) depends on where the enzyme binds as the conjugate enters the nanopore, along the single-stranded overhang. A current level of 100 pA is shown as a dashed line to highlight the difference in peptide dip amplitude between different sequences tested. [Figure 17-2](Continued) Figure 17 is a current-time trace showing the capture of a polynucleotide-polypeptide conjugate, accompanied by the movement of the conjugate out of a nanopore controlled by a helicase, as described in Example 3. The peptide sequences tested were RSDSGQQARY (Figures 17A, 2D), GGSGSSSGSG (Figures 17B, 17E), and EAIYAAPFAKKK (Figures 17C, 17F). Traces A, B, and C show an initial stage of open-pore current (i) followed by a decrease to a lower level (ii) due to the capture of the conjugate. Traces A, B, and C further show a repeating pattern indicating the repeated movement of the conjugate through the nanopore. In (iii), the helicase controlling the movement dissociates from the conjugate, and since no more helicase is bound, the current trace returns to the open-pore current level. Examples of single reads for each peptide are shown in D, E, and F. A single read shows a single-stranded DNA stage (iv), followed by a decrease in current caused by the peptide (v). The length of block (iv) depends on where the enzyme binds as the conjugate enters the nanopore, along the single-stranded overhang. A current level of 100 pA is shown as a dashed line to highlight the difference in peptide dip amplitude between different sequences tested. [Figure 18] The current-time traces from Example 3 show single reads of polynucleotide-polypeptide conjugates containing peptides of varying charges. The movement of the conjugates out of the nanopores is controlled by helicase, as described in Example 3. The peptide sequences are SRRRRRRRRS(A), HDSGYEVHHQK(B), and SEEEEEEEES(C), with charges of +8, -2, and -8, respectively. Each single read shows a single-stranded DNA stage (iv), followed by a decrease in current caused by the peptide (v). The length of block (iv) depends on where the enzyme binds as the conjugate enters the nanopore, along the single-stranded overhang. Current levels of 0 pA are shown as dashed lines to highlight the differences in peptide block amplitudes between the different sequences tested. [Figure 19]This is the polynucleotide-polypeptide construct used in Example 4. a. Leader, b. Tether, c. Hairpin adapter, d. DNA1 oligo, e. DNA2 oligo, f. Peptide, g. DNA3 oligo, x. Stall, y. Helicase (arrows indicate the direction of helicase movement), z. Bubble, Circle = click chemical group, Vertical line = ligation site. [Figure 20] This is a current-time trace from Example 4, showing the capture of a polynucleotide-polypeptide conjugate, involving the movement of the conjugate into a nanopore controlled by a Dda helicase, as described in Example 4. The peptide sequence is HDSGDEVHHQK, a fragment of amyloid-beta protein. Traces from three examples are shown, demonstrating the reproducibility of the signal. Each trace shows an initial stage of open-pore current (i), followed by a decrease to a lower level (ii) due to the capture of the conjugate and stalling of the helicase. Once the enzyme is released from stall by electrophoretic force, the signal transitions to the dsDNA level (iii), followed by a decrease in the signal caused by the peptide (iv). As the helicase moves the peptide across the constriction, the signal returns to the dsDNA level (iii). [Figure 21] This is the polynucleotide-polypeptide construct used in Example 5. a. DNA1 oligo, b. DNA2 oligo, c. peptide, d. DNA3 oligo, e. DNA4 oligo, f. sequencing adapter, x. stall, y. helicase (arrows indicate the direction of helicase movement), circle = click chemical group (located within DNA2 and DNA4 oligos), vertical line = ligation site. [Figure 22-1]A1: A current-time trace of the enzyme-controlled transfer of a polypeptide-polynucleotide conjugate, including a DNA-peptide cotransposition, as described in Example 5. A2: A magnified view of the highlighted section of the A1 trace. Each trace shows the initial stage of open-pore current (i), followed by a decrease to a section of the adapter sequence upon capture of the analyte (ii), and then a current spike caused by the peptide (iii). The peptide is adjacent to an oligo containing 40 dT bases, thereby creating a section with flat signals on both sides of the peptide (iv). [Figure 22-2] (Continued) B1: Current-time trace for a control experiment in which the peptide displaces only in nanopores. B2: Enlarged view of the highlighted section of trace B1. Each trace shows the initial stage of open-pore current (i), followed by a decrease to the adapter sequence section (ii) as the analyte is captured, and then a current spike caused by the peptide (iii). The peptide is adjacent to an oligo containing 40 dT bases, which creates a flat section in the signal on both sides of the peptide (iv). [Figure 23] This is the polynucleotide-polypeptide construct used in Example 6. a. DNA1 oligo, b. DNA2 oligo (containing an internal click chemical group and showing a DNA "flap" alongside the peptide), c. peptide, d. DNA3 oligo, e. DNA4 oligo, f. sequencing adapter, x. stall, y. helicase (arrows indicate the direction of helicase movement), circle = click chemical group, vertical line = ligation site. [Figure 24]A1: A current-time trace of the enzyme-controlled transfer of a polypeptide-polynucleotide conjugate, including the cotransposition of a DNA flap and a peptide, as described in Example 6. A2: A magnified view of the highlighted section of the A1 trace. Each trace shows the initial stage of open-pore current (i), followed by a decrease to a section of the adapter sequence upon capture of the analyte (ii), and then a current spike caused by the peptide (iii). The peptide is adjacent to an oligo containing 40 dT bases, thereby creating a section with flat signals on both sides of the peptide (iv). [Modes for carrying out the invention]

[0041] The present invention is described with respect to specific embodiments and with reference to certain drawings, but is not limited thereto and is limited only by the claims. None of the reference numerals in the claims should be construed as limiting the scope. Needless to say, it should be understood that not all aspects or advantages can necessarily be achieved according to any particular embodiment of the present invention. Therefore, for example, a person skilled in the art will recognize that the present invention can be embodied or practiced in a manner that achieves or optimizes one advantage or group of advantages taught herein without necessarily achieving other aspects or advantages that can be taught or suggested herein.

[0042] The present invention, with regard to both its organization and methods of operation, along with its characteristics and advantages, can be best understood by referring to the embodiments for carrying out the invention as described below, when read in conjunction with the accompanying drawings. The aspects and advantages of the present invention will become apparent and clarified by referring to the embodiments(s) described below. Throughout this specification, any reference to “one embodiment” or “a certain embodiment” means that a particular characteristic, structure, or feature described in relation to that embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrase “in one embodiment” or “in a certain embodiment” in various places throughout this specification does not necessarily all refer to the same embodiment, although it may. Similarly, in the description of exemplary embodiments of the present invention, it should be understood that various features of the invention may be grouped together in a single embodiment, figure, or description for the purpose of simplifying this disclosure and aiding in the understanding of one or more of the various aspects of the present invention. However, the manner of this disclosure should not be interpreted as reflecting an intention that the claimed invention requires more characteristics than are explicitly enumerated in each claim. Rather, as reflected in the following claims, the embodiments of the invention are fewer than all the characteristics of a single, aforementioned disclosed embodiment.

[0043] Unless otherwise specified in the context, the “embodiments” of this disclosure should be understood to be specific combinations. Any specific combination of all disclosed embodiments (unless otherwise implied in the context) constitutes a further disclosed embodiment of the claimed invention.

[0044] In addition, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless it is clearly evident from the context. Thus, for example, a reference to “polynucleotide” includes two or more polynucleotides, a reference to “motor protein” includes two or more such proteins, a reference to “helicase” includes two or more helicases, a reference to “monomer” refers to two or more monomers, and a reference to “pore” includes two or more pores.

[0045] All publications, patents, and patent applications cited herein, whether in whole or in part, are incorporated herein by reference.

[0046] definition When an indefinite or definite article, such as "a" or "an" or "the," is used to refer to a singular noun, unless otherwise specified, this includes the plural form of that noun. When the term "includes" is used in this description and claims, it does not exclude other elements or steps. Furthermore, terms such as first, second, third, etc., in this description and claims are used to distinguish similar elements and are not necessarily used to describe the order in which they occur or the order in which they occur. It should be understood that the terms used in this manner are interchangeable under appropriate circumstances and that embodiments of the invention described herein may operate in an order other than that described or illustrated herein. The following terms or definitions are provided solely to aid in understanding the invention. Unless specifically defined herein, all terms used herein have the same meaning as they have to those skilled in the art. Practitioners should refer to definitions and terms in the art, in particular, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 th See ed., Cold Spring Harbor Press, Plainsview, New York (2012), and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016). The definitions provided herein should not be construed as narrower than those understood by those skilled in the art.

[0047] As used herein, "about" when referring to measurable values ​​such as quantity or temporal duration means that it includes a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, such variation being appropriate for carrying out the disclosed method.

[0048] As used herein, “nucleotide sequence,” “DNA sequence,” or “nucleic acid molecule” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Therefore, this term includes double-stranded and single-stranded DNA and RNA. As used herein, “nucleic acid” refers to a single-stranded or double-stranded covalent nucleotide sequence in which the 3' and 5' ends of each nucleotide are linked by phosphodiester bonds. Polynucleotides may consist of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids may be produced synthetically in vitro or isolated from natural sources. Nucleic acids may further include modified DNA or RNA, e.g., methylated DNA or RNA, or RNA subjected to post-translational modifications, e.g., 3'-processing such as 5'-capping, cleavage and polyadenylation with 7-methylguanosine, and splicing. Nucleic acids may also include synthetic nucleic acids (XNAs) such as hexitol nucleic acids (HNAs), cyclohexene nucleic acids (CeNAs), threose nucleic acids (TNAs), glycerol nucleic acids (GNAs), locked nucleic acids (LNAs), and peptide nucleic acids (PNAs). The size of nucleic acids, also referred to herein as “polynucleotides,” is typically expressed in terms of the number of base pairs (bp) for double-stranded polynucleotides, or the number of nucleotides (nts) for single-stranded polynucleotides. 1000 bp or nts corresponds to kilobases (kb). Polynucleotides with a length of less than approximately 40 nucleotides are typically referred to as “oligonucleotides” and may include primers used for manipulating DNA, such as via polymerase chain reaction (PCR).

[0049] In the context of this disclosure, the term “amino acid” is used in its broadest sense and means organic compounds containing amine (NH2) and carboxyl (COOH) functional groups, along with a side chain specific to each amino acid (e.g., an R group). In some embodiments, an amino acid refers to a naturally occurring L α-amino acid or residue. One- and three-letter abbreviations commonly used for naturally occurring amino acids are used herein: 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=Ser, T=Thr, V=Val, W=Trp, and Y=Tyr (Lehninger, AL, (1975) Biochemistry, 2nd ed., pp. 71-92, Worth Publishers, New York). The general term “amino acid” further includes chemically modified amino acids such as D-amino acids, retro-inversoamino acids, and amino acid analogs, naturally occurring amino acids not typically incorporated into proteins such as norleucine, and chemically synthesized compounds that exhibit amino acid-like characteristics known in the art, such as β-amino acids. For example, analogs or mimics of Phe or Pro that allow for the same stereochemical constraints on peptide compounds as natural phenylalanine or proline are included within the definition of an amino acid. Such analogs and mimics are referred to herein as “functional equivalents” of their respective amino acids. Other examples of amino acids are listed by reference in Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5 p. 341, Academic Press, Inc., NY 1983, which is incorporated herein by reference.

[0050] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to polymers of amino acid residues, as well as their variants and synthetic analogs. Therefore, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic amino acids that do not exist naturally, such as chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. Polypeptides may undergo maturation or post-translational modification processes, including but not limited to glycosylation, proteolytic cleavage, lipidation, signal peptide cleavage, propeptide cleavage, and phosphorylation. Peptides can be prepared using recombination techniques, for example, by the expression of recombinant or synthetic polynucleotides. Peptides produced by recombination typically contain substantially no culture medium, for example, the culture medium constitutes less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0051] The term "protein" is used to describe folded polypeptides that have a secondary or tertiary structure. A protein may consist of a single polypeptide or may contain multiple polypeptides that assemble to form a multimer. A multimer may be a homooligomer or a heterooligomer. A protein may be a naturally occurring protein or a wild-type protein, or it may be a modified protein or a protein that does not exist in nature. A protein may differ from a wild-type protein, for example, by the addition, substitution, or deletion of one or more amino acids.

[0052] A protein "variant" includes peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions compared to the unmodified or wild-type protein in question, and that have similar biological and functional activity to the unmodified protein from which they are derived. As used herein, the term "amino acid identity" refers to the degree to which sequences are identical amino acid-wise across a comparison window. Thus, the "percentage of sequence identity" is calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions in which identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences, calculating the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to calculate the percentage of sequence identity.

[0053] In all aspects and embodiments of the present invention, the "variant" has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% complete sequence identity with respect to the amino acid sequence of the corresponding wild-type protein. Sequence identity may also be to a full-length polynucleotide or a fragment or portion of a polypeptide. Thus, a sequence may have only 50% sequence identity overall with a full-length reference sequence, while the sequence of a particular region, domain, or subunit may share as much as 80%, 90%, or 99% sequence identity with the reference sequence.

[0054] The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is the most frequently observed and therefore arbitrarily designed "normal" or "wild-type" form of that gene in a given population. In contrast, the terms "modified," "mutant," or "variant" refer to a gene or gene product that exhibits sequence modifications (e.g., substitution, cleavage, or insertion), post-translational modifications, and / or functional characteristics (e.g., altered features) compared to a wild-type gene or gene product. It should be noted that naturally occurring mutants can be isolated, and these are identified by the fact that they have altered features compared to a wild-type gene or gene product. Methods for introducing or substituting naturally occurring amino acids are well known in the art. For example, methionine (M) can be substituted with arginine (R) by replacing the methionine codon (ATG) with the arginine codon (CGT) at the relevant position in the polynucleotide encoding the mutant monomer. Methods for introducing or substituting amino acids that do not exist in nature are also well known in the art. For example, amino acids that do not exist in nature can be introduced by including synthetic aminoacyl-tRNA in the IVTT system used to express mutant monomers. Alternatively, they can be introduced by expressing mutant monomers in E. coli that are nutrientally required for those specific amino acids, in the presence of synthetic (i.e., non-naturally occurring) analogs of those specific amino acids. They may also be produced by naked ligation when the mutant monomers are produced using partial peptide synthesis. Conservative substitutions replace an amino acid with another amino acid having a similar chemical structure, similar chemical properties, or similar side-chain volume. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acid they replace. Alternatively, conservative substitutions can introduce another amino acid that is aromatic or aliphatic in place of an existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected according to the properties of 20 major amino acids defined in Table 1 below.If amino acids have similar polarity, this can also be determined by referring to the hydrophobicity scale of the amino acid side chains in Table 2. [Table 1] [Table 2]

[0055] Mutant or modified proteins, monomers, or peptides can be chemically modified in any manner and at any site. Preferably, mutant or modified monomers or peptides are chemically modified by attachment of molecules to one or more cysteine ​​molecules (cysteine ​​bonding), attachment of molecules to one or more lysines, attachment of molecules to one or more non-natural amino acids, enzymatic modification of epitopes, or terminal modification. Suitable methods for carrying out such modifications are well known in the art. Mutants of modified proteins, monomers, or peptides can be chemically modified by attachment of any molecule. For example, mutants of modified proteins, monomers, or peptides can be chemically modified by attachment of dyes or fluorophores.

[0056] Method of disclosure This disclosure relates to a method for characterizing polypeptides. Polypeptides are characterized when they co-transform through nanopores with one or more polynucleotide chains, for example, one or more polynucleotide adjacent chains and / or carrier chains as described in detail herein. Polypeptides and polynucleotide chains together may be referred to as constructs.

[0057] This method utilizes the ability of numerous nanopores to accommodate multiple polymer chains simultaneously. Co-transposition of polypeptide chains and one or more polynucleotide chains has advantages that will be discussed in more detail herein.

[0058] In contrast to methods that attempt to control polypeptide movement into nanopores using polypeptide handling enzymes, certain embodiments of the present disclosure relate to methods that involve controlling polypeptide movement into nanopores using polynucleotide handling enzymes. Other embodiments of the present disclosure do not require the use of enzymes to control the movement of constructs.

[0059] Therefore, in one embodiment, what is provided herein is a method for characterizing a target polypeptide, -(i) a polynucleotide-polypeptide conjugate chain containing the target polypeptide, which is conjugated to one or more adjacent polynucleotide chains at each end of the target polypeptide, (ii) contact with a polynucleotide carrier chain, thereby forming a polynucleotide-polypeptide construct, - The construct and the nanopores are brought into contact under conditions such that both the polynucleotide-polypeptide conjugate chain and the polynucleotide carrier chain co-transform through the nanopores, - This includes obtaining one or more characteristic measurements of the polypeptide as the construct moves into the nanopores, This method is used to characterize target polypeptides.

[0060] In a related aspect, what is provided is a method for characterizing a target polypeptide, -(i) a polynucleotide-polypeptide conjugate chain containing the target polypeptide, which is conjugated to one or more adjacent polynucleotide chains at each end of the target polypeptide, (ii) contact with a polynucleotide carrier chain, thereby forming a polynucleotide-polypeptide construct, - Controlling the movement of the construct through the nanopores so that both the polynucleotide-polypeptide conjugate chain and the polynucleotide carrier chain co-transition through the nanopores, - This includes obtaining one or more characteristic measurements of the polypeptide as the construct moves into the nanopores, This method is used to characterize target polypeptides.

[0061] In another relevant aspect of this disclosure, a method for characterizing a target polypeptide is provided. -(i) a polynucleotide-polypeptide conjugate chain containing a target polypeptide attached to a polynucleotide flanking chain, and (ii) contacting a polynucleotide handling protein capable of controlling the movement of the polynucleotide flanking chain into a nanopore, - The polynucleotide-polypeptide conjugate chain and the nanopore are brought into contact under conditions such that a polynucleotide handling protein controls the movement of the polynucleotide-polypeptide conjugate chain to and from the nanopore. - Obtaining one or more characteristic measurements of the polypeptide as the polynucleotide adjacent chain and the target polypeptide co-transition through the nanopore, This method is used to characterize target polypeptides.

[0062] In a related aspect, what is provided is a method for characterizing a target polypeptide, -(i) a polynucleotide-polypeptide conjugate chain containing a target polypeptide attached to a polynucleotide adjacent chain, and (ii) contact with a polynucleotide handling protein. - Using polynucleotide handling proteins to control the movement of adjacent polynucleotide chains into nanopores, - The polynucleotide-polypeptide conjugate chain and the nanopore are brought into contact under conditions such that a polynucleotide handling protein controls the movement of the polynucleotide-polypeptide conjugate chain to and from the nanopore. - Obtaining one or more characteristic measurements of the polypeptide as the polynucleotide adjacent chain and the target polypeptide co-transition through the nanopore, This method is used to characterize target polypeptides.

[0063] In some preferred embodiments, polynucleotide handling proteins are used to control the movement of the constructs, polynucleotide-polypeptide conjugate chains and / or carrier chains described herein, but it will be apparent to those skilled in the art that polynucleotide handling proteins are not essential in the methods provided herein. Therefore, in some embodiments, for example, a method for characterizing a target polypeptide is provided herein, which does not necessarily involve polynucleotide handling proteins. -(i) bringing a polynucleotide-polypeptide conjugate chain containing a target polypeptide attached to an adjacent polynucleotide chain, and (ii) bringing it into contact with nanopores, - Controlling the movement of polynucleotide-polypeptide conjugate chains into nanopores, - Obtaining one or more characteristic measurements of the polypeptide as the polynucleotide adjacent chain and the target polypeptide co-transition through the nanopore, This method is used to characterize target polypeptides.

[0064] The methods described above may be referred to herein as the disclosed methods. To avoid misunderstanding, embodiments of the disclosure are described herein in relation to the disclosed methods for the sake of brevity. Unless otherwise required by context, such embodiments are expressly disclosed in relation to and as those preferred characteristics of each of the disclosed methods described above.

[0065] Any suitable polypeptide can be characterized using the methods disclosed herein. In some embodiments, the target polypeptide is a protein or a naturally occurring polypeptide. In some embodiments, the target polypeptide is a portion of a protein or a naturally occurring polypeptide, such as one that can be obtained by nuclease digestion of the protein or the naturally occurring polypeptide. In some embodiments, the polypeptide is a synthetic polypeptide. Polypeptides that can be characterized according to the disclosed methods are described in more detail herein.

[0066] Any suitable polynucleotide can be used when forming polynucleotide-polypeptide conjugate chains for use in the methods disclosed herein. Polynucleotide chains attached to the polypeptide chain in a polynucleotide-polypeptide conjugate chain may be referred to as adjacent chains. Adjacent chains are described in more detail herein.

[0067] In some embodiments, the polynucleotide flanking chain or each polynucleotide flanking chain has at least the same length as the portion of the target polypeptide being characterized. In some embodiments, the polynucleotide flanking chain or each polynucleotide flanking chain has a length longer than the portion of the target polypeptide being characterized. In some embodiments, the polynucleotide flanking chain or each polynucleotide flanking chain has a length shorter than the portion of the target polypeptide being characterized. Polynucleotides suitable for use in the disclosed methods are disclosed in more detail herein.

[0068] In some embodiments, a polynucleotide-polypeptide conjugate chain or each polynucleotide-polypeptide conjugate chain is complexed with a polynucleotide carrier chain. In some embodiments, the polynucleotide flanking chains of the polynucleotide-polypeptide conjugate chain are independently complementary to the region of the polynucleotide carrier chain. In some embodiments, the polynucleotide flanking chains of the polynucleotide-polypeptide conjugate chain are independently complementary to the corresponding region of the polynucleotide carrier chain by at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%.

[0069] In some embodiments, one or more polynucleotide flanking chains are independently at least partially hybridized to a polynucleotide carrier chain. Each flanking chain may independently be at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% hybridized to a complementary chain over the length of the flanking chain. For example, in some embodiments, one or more polynucleotide flanking chains are independently hybridized to a polynucleotide carrier chain over a length of at least 5, e.g., at least 10, e.g., at least 20, e.g., at least 30, e.g., at least 40, e.g., at least 50, e.g., at least 60, e.g., at least 80, e.g., at least 100, e.g., at least 150, e.g., at least 200, e.g., at least 500, or longer. The complementary chains described herein may associate, for example, via hydrogen bonds (e.g., base pairing) and thus hybridize together. Those skilled in the art will understand that the intensity of hybridization between the polynucleotide carrier chain and the polynucleotide neighbor chain of a polynucleotide-polypeptide conjugate chain can be controlled, i.e., by controlling the length of the hybridized region and the degree of complementarity. Using shorter neighbor chains can decrease the hybridization force between the neighbor chain and the carrier chain of a polynucleotide-polypeptide conjugate chain. Using longer neighbor chains can increase the hybridization force between the neighbor chain and the carrier chain of a polynucleotide-polypeptide conjugate chain. Increasing the degree of complementarity can also increase the hybridization force between the neighbor chain and the carrier chain of a polynucleotide-polypeptide conjugate chain.

[0070] In some embodiments, a polynucleotide-polypeptide conjugate chain or carrier chain may overhang other chains of the construct. For example, in some embodiments, a polynucleotide-polypeptide conjugate chain overhangs a carrier chain at one end. For example, the 3' end of a polynucleotide-polypeptide conjugate chain may overhang the 5' end of a carrier chain. In other embodiments, a carrier chain may overhang a polynucleotide-polypeptide conjugate chain at one end. For example, the 3' end of a carrier chain may overhang the 5' end of a polynucleotide-polypeptide conjugate chain. Of course, the overhang may alternatively be located at another end of the construct; for example, the 5' end of a polynucleotide-polypeptide conjugate chain may overhang the 3' end of a carrier chain, or the 5' end of a carrier chain may overhang the 3' end of a polynucleotide-polypeptide conjugate chain. In some embodiments, the overhang may be located at each end of the construct. In some embodiments, the overhang provides a loading site for the polynucleotide handling protein.

[0071] In the disclosed method, the target polypeptide can be conjugated to a polynucleotide using any preferred means. Several exemplary means are described in more detail herein.

[0072] In some embodiments, the conjugate formed in the manner disclosed is brought into contact with a polynucleotide handling protein, as will be discussed in more detail herein. When present in nanopores, the polynucleotide handling protein can typically control the movement of the adjacent polynucleotide chains and / or the polynucleotide carrier chain. Exemplary polynucleotide handling proteins are described in more detail herein.

[0073] If present, polynucleotide handling proteins control the movement of polynucleotide chains into nanopores. As will be discussed in more detail herein, polynucleotide handling proteins may control the movement of polynucleotide adjacent chains into nanopores. Polynucleotide handling proteins may control the movement of polynucleotide carrier chains into nanopores. Polynucleotide handling proteins may control the movement of both polynucleotide adjacent chains and polynucleotide carrier chains into nanopores. Thus, polynucleotide handling proteins control the movement of constructs into nanopores. Any suitable nanopore can be used in the disclosed method. Nanopores suitable for use in the disclosed method are described in more detail herein.

[0074] The disclosed method involves obtaining one or more characteristic measurements of a polypeptide as the polypeptide portion of a polynucleotide-polypeptide conjugate chain or each polypeptide portion moves toward a nanopore. The one or more measurements may be any preferred measurements. Typically, the one or more measurements are electrical measurements, e.g., current measurements, and / or one or more optical measurements. Apparatus for recording preferred measurements and information that can provide such measurements are described in more detail herein.

[0075] Certain disclosed methods can also be used to characterize target polynucleotides, and the properties of the disclosed methods can be generally applied to such methods unless otherwise implied by context. Specific methods for characterizing target polynucleotides are described in more detail herein.

[0076] Characterize the target polypeptide. This specification describes a method for characterizing a target polypeptide, comprising: forming a construct comprising a first chain containing the target polypeptide and a second chain containing a polynucleotide; moving the construct toward nanopores under conditions such that the first and second chains of the construct co-transition through nanopores; and obtaining one or more characteristic measurements of the polypeptide as the construct moves toward the nanopores. The method described herein may include a step of controlling the movement of the construct toward nanopores.

[0077] As disclosed herein, polynucleotides can be used to control the movement of polypeptides into nanopores. Since the polynucleotide is conjugated to the polypeptide in the conjugate, the movement of the polynucleotide drives the movement of the polypeptide. The polypeptide portion of the polynucleotide-polypeptide conjugate chain is flanked by one or more adjacent polynucleotide chains. The polynucleotide-polypeptide conjugate chain can be hybridized to a polynucleotide carrier chain.

[0078] Accordingly, the methods provided herein involve cotransition through nanopores of (i) a target polypeptide and (ii) a polynucleotide chain. In some embodiments, the polynucleotide chain cotransitioning through nanopores is a polynucleotide adjacent chain as described herein. In some embodiments, the polynucleotide chain cotransitioning through nanopores is a polynucleotide carrier chain as described herein.

[0079] As used herein, the term “cotransition” means to displace together and simultaneously. Therefore, with respect to the methods described herein, cotransition through nanopores means to displace together and simultaneously through nanopores.

[0080] In the methods described herein, the target polypeptide and polynucleotide chain co-transplant through nanopores. Thus, the target polypeptide and polynucleotide chain transplant together and simultaneously through nanopores. Such co-transplantation of two chains necessarily requires that the two chains pass through the nanopores in a double-strand configuration, aligned with each other (side by side). Therefore, as a further example, in some embodiments of the methods described herein, the target polypeptide and polynucleotide chain co-transplanting through nanopores may transplant in the form of a double-stranded polypeptide-polynucleotide chimera.

[0081] The above may be in contrast to prior art methods such as those described in WO2021 / 111125 and WO2021 / 133168. In such prior art methods, single-stranded polypeptide-polynucleotide conjugates are moved through nanopores using polynucleotide handling enzymes, resulting in linear succession (i.e., rearrangement) of the polypeptide and polynucleotide portions of the conjugate through the nanopores, while double-stranded polypeptide-polynucleotides do not move through the pores. Such linear succession of polypeptides and polynucleotides is in contrast to the "parallel" movement of target polypeptides and polynucleotide chains provided by the methods of the present invention disclosed herein.

[0082] The simultaneous cotransposition of the target polypeptide and the polynucleotide chain means that, at the time of measurement, the analyte (e.g., construct) moving through the pore is necessarily double-stranded. One chain contains the polynucleotide; the other chain contains the target polypeptide. Thus, as used herein, the term “double-stranded” encompasses a polynucleotide chain parallel to the polypeptide chain. This offers advantages compared to methods for characterizing polypeptides known in the art.

[0083] For example, target polypeptides are typically substantially uncharged, have a low net charge and / or charge density, and / or are irregularly charged. In other words, the charge distribution in a target polypeptide is typically irregularly distributed along the length of the target polypeptide. As shown in Table 1 above, some amino acids in the target polypeptide are polar, and some are nonpolar. Some are positively or negatively charged under physiological conditions, others are uncharged under physiological conditions but may be charged under conditions in which methods such as those disclosed herein are performed, and still others are uncharged under all relevant conditions. The distribution of amino acids in the target polypeptide depends on the exact analyte characterized by the disclosed method and is therefore not known in advance by the user.

[0084] In known polypeptide analysis methods that rely on the electrophoretic migration of polypeptides through nanopores, this irregular charge along the target polypeptide can create difficulties because it alters the electrophoretic force acting on the polypeptide as the polypeptide chain moves through the nanopore. As a result, the migration rate of polypeptides through nanopores can be unpredictable, hindering accurate characterization. For example, it can be difficult to distinguish between two identical amino acids moving quickly through the pore and one amino acid moving more slowly. The low average charge density of the target polypeptide is one reason why methods that may include the detection or characterization of polynucleotides and their analogues (PNAs, peptide nucleic acids) are typically unsuitable for accurate characterization of the target polypeptide. Therefore, a method that does not rely on the changing charge of the polypeptide is needed to determine the migration of polypeptides through the pore.

[0085] A further problem arising in known methods of polypeptide analysis is that target polypeptides typically possess irregular 3D structures. For example, proteins are known to fold to conform to 3D structures that may be associated with their biological function. The presence of 3D structures (e.g., secondary or tertiary structures) within a target polypeptide can hinder its characterization using nanopores in known methods that rely on single-strand rearrangement of the target polypeptide through the pore. This is because different parts of the folded target polypeptide require different degrees of force to unfold in order to rearrange in this manner. As a result, the movement of the polypeptide through the pore can be irregular, for example, some parts moving faster through the pore than others. This can hinder accurate characterization. Furthermore, it may not always be possible to unfold a protein using nanopores in this manner. Therefore, methods that simply decorate a double-stranded polynucleotide with a folded protein and involve rearranging the construct through nanopores (e.g., solid-state nanopores) typically cannot provide detailed information about the protein, such as its sequence.

[0086] A further problem that can arise with some known methods of characterizing polypeptides using nanopores is that motor proteins that can be used to control the movement of such polypeptides may be inefficient in precisely controlling the movement of long polypeptide chains, even though they can effectively rearrange on such chains. For example, when used to control the movement of very long polypeptide chains (e.g., in the form of polynucleotide-polypeptide conjugates), motor proteins may slip on the polypeptide portion of the chain as they move through the nanopores. Slipping is problematic because it can lead to inaccurate characterization of the polypeptide.

[0087] This method addresses some or all of these problems. As shown above, the simultaneous cotransposition of the target polypeptide and polynucleotide chain means that, at the time of measurement, the analyte moving through the pore is necessarily double-stranded. In some embodiments, this results in some or all of the following advantages.

[0088] Firstly, polynucleotide chains co-transitioning with target polypeptides through nanopores typically possess a regular charge density and therefore exert regular electrophoretic forces on the construct as they move through the nanopores. The forces exerted by the movement of polynucleotides through nanopores are well understood and can be accurately modeled. Thus, co-transitioning polynucleotides can drive the predictable and consistent movement of target polypeptides through the pores, facilitating polypeptide characterization.

[0089] Secondly, the polynucleotide chains co-transposition the nanopores with the target polypeptide can be selected or configured to effectively linearize the target polypeptide. For example, the target polypeptide may be attached to polynucleotide fringe chains at each end of the analyte, and the polynucleotide fringe chains may be hybridized to a polynucleotide carrier chain. Hybridization of the fringe chains prevents polypeptide folding. The fringe chains can be designed to extend the target polypeptide to a desired degree according to the parameters of the method operated by the user. Since folding of the target polypeptide can be reduced or eliminated, the movement of the target polypeptide through the nanopores is typically more regular.

[0090] Thirdly, a polynucleotide handling protein may be brought into contact with a polynucleotide-polypeptide conjugate chain and / or a polynucleotide carrier chain, and this may be used to control the construct with respect to the nanopore. In embodiments in which a polynucleotide handling protein is used to control the movement of a polynucleotide chain in order to control the movement of a construct, the length of the target polypeptide is not particularly limited, as the polynucleotide handling protein progressively processes the polynucleotide chain co-transferring the pore with the polypeptide chain even while the polynucleotide chain is moving through the nanopore.

[0091] Therefore, the methods provided herein enable some or all of the following: improved ability to detect target polypeptides, improved accuracy of polypeptide feature evaluation, improved reproducibility of polypeptide data, improved (increased) polypeptide read length, and reduced slip.

[0092] The method of the present invention can be understood by referring to Figure 1, which shows one non-limiting example of the disclosed method.

[0093] The construct may comprise a polynucleotide-polypeptide conjugate chain containing a target polypeptide flanked by one or more adjacent polynucleotide chains. In some embodiments, the construct may be brought into contact with a polynucleotide handling protein so that the construct threads through nanopores. Optionally, further polymers, such as a leader (as described herein), may be used to facilitate the threading of polypeptides through nanopores. Such use is within the scope of the disclosed methods, but is not essential. Optionally, spacers and / or stalls may be used to inhibit the movement of polynucleotide handling proteins before the user operates the method (as shown in Figure 1). This is within the scope of the methods described and disclosed herein, but is not essential.

[0094] In some embodiments, a polynucleotide handling protein can process a polynucleotide carrier chain. When a polynucleotide handling protein processes a polynucleotide carrier chain, the carrier chain hybridizes to a polynucleotide-polypeptide conjugate chain (e.g., the adjacent chain portion of the polynucleotide-polypeptide conjugate chain), so that the construct passes through the nanopore. Thus, the polypeptide passes through the nanopore. As the polypeptide passes through the nanopore, it is characterized.

[0095] In some embodiments, the polynucleotide handling protein may process a polynucleotide-polypeptide conjugate chain (for example, by processing polynucleotide adjacent chains). When the polynucleotide handling protein processes polynucleotide adjacent chains, the construct passes through the nanopores because the adjacent chains hybridize to the polynucleotide carrier chain. Thus, the polypeptide passes through the nanopores. As the polypeptide passes through the nanopores, it is characterized.

[0096] The method of the present invention can also be understood by referring to Figure 6, which illustrates another non-limiting example of the disclosed method.

[0097] A polynucleotide-polypeptide conjugate chain containing one or more adjacent polynucleotide chains and a target polypeptide can be threaded through nanopores.

[0098] In some embodiments, the polynucleotide-polypeptide conjugate chain moves relative to the pore. In some embodiments, the target polypeptide is spread onto the polynucleotide adjacent chain. Therefore, when the target polypeptide rearranges through the nanopore, the target polypeptide is spread onto the polynucleotide adjacent chain, and thus the polynucleotide adjacent chain and the target polypeptide co-rearrange through the pore. In some embodiments, the target polypeptide is spread onto the polynucleotide adjacent chain and attached to the polynucleotide adjacent chain at one end of the target polypeptide. In some embodiments, the target polypeptide is spread onto the polynucleotide adjacent chain and attached to the polynucleotide adjacent chain at each end of the target polypeptide. In some embodiments, each end of the target polypeptide is conjugated to a polynucleotide adjacent chain, and one or both of the polynucleotide adjacent chains are further spread onto the target polypeptide. In some embodiments, one end of the target polypeptide is conjugated to a first polynucleotide adjacent chain, and the other end of the target polypeptide is conjugated to a second polynucleotide adjacent chain, and one or both of the first and second polynucleotide adjacent chains are spread onto the target polypeptide. In some embodiments, the adjacent chain portion extending into the target polypeptide is of the same type of polynucleotide as the rest of the adjacent chain. In some embodiments, the adjacent chain portion extending into the target polypeptide is of a different type of polynucleotide than the rest of the adjacent chain.

[0099] In some embodiments, a polynucleotide-polypeptide conjugate chain is hybridized to a polynucleotide carrier chain as described herein. In some embodiments, the resulting construct undergoes rearrangement through nanopores.

[0100] In some embodiments, the movement of the construct through the nanopore results in the cotransition of three chains: the target polypeptide, the polynucleotide adjacent chains extending to the target polypeptide, and the polynucleotide carrier chain. This is shown in Figure 6B. However, in some embodiments, the polynucleotide carrier chain is removed by the nanopore, for example (e.g., dehybridized and thereby opened). In such embodiments, when the target polypeptide transposes through the nanopore, the target polypeptide extends to the polynucleotide adjacent chains, and thus the polynucleotide adjacent chains and the target polypeptide cotransition through the pore, while the polynucleotide carrier chain is excluded from the pore. This is shown in Figure 6C.

[0101] In some embodiments, a polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain are brought into contact with a polynucleotide handling protein so that the construct threads through the nanopores. However, this is not essential. Optionally, further polymers, such as a leader (as described herein), may be used to facilitate the threading of polypeptides through the nanopores. Such use is within the scope of the disclosed methods, but is not essential. Optionally, spacers and / or stalls may be used to inhibit the movement of polynucleotide handling proteins (if present) before the user operates the method. This is within the scope of the methods described and disclosed herein, but is not essential.

[0102] In the examples shown in Figures 1 and 6, the polynucleotide handling protein moves the construct "into" the pore from the "perspective" of the polynucleotide handling protein. For example, as shown, the polynucleotide handling protein can be positioned on the cis side of the nanopore and move the construct into the pore, i.e., from the cis side to the trans side. The opposite setup can also be used.

[0103] In other words, in some embodiments, the polynucleotide handling protein may be located on the cis side of the nanopore, and the polynucleotide handling protein controls the movement of constructs, polynucleotide-polypeptide conjugate chains and / or polynucleotide carrier chains from the cis side of the nanopore to the trans side of the nanopore. In some embodiments, the polynucleotide handling protein is located on the cis side of the nanopore, and the polynucleotide handling protein controls the movement of polynucleotide carrier chains from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of target polypeptides through the nanopore. Thus, in some embodiments, the polynucleotide handling protein is located on the cis side of the nanopore, and the polynucleotide handling protein controls the movement of polypeptides from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of polypeptides through the nanopore.

[0104] In other embodiments, the polynucleotide handling protein is located on the trans side of the nanopore and controls the movement of constructs, polynucleotide-polypeptide conjugate chains, and / or polynucleotide carrier chains from the trans side to the cis side of the nanopore. In some embodiments, the polynucleotide handling protein is located on the trans side of the nanopore and controls the movement of polynucleotide carrier chains from the trans side to the cis side of the nanopore, thereby controlling the movement of target polypeptides through the nanopore. Thus, in some embodiments, the polynucleotide handling protein is located on the trans side of the nanopore and controls the movement of polypeptides from the trans side to the cis side of the nanopore, thereby controlling the movement of polypeptides through the nanopore.

[0105] As will be described in more detail herein, the construct may include a leader. The leader may be located on the polynucleotide-polypeptide conjugate chain. If present, the leader may be located on the carrier chain.

[0106] The leader, if present, may be located on the same chain processed by the polynucleotide handling protein. For example, in some embodiments, the polynucleotide-polypeptide conjugate chain includes a leader, and the polynucleotide handling protein controls the movement of the polynucleotide-polypeptide conjugate chain into the nanopore. In other embodiments, the carrier chain includes a leader, and the polynucleotide handling protein controls the movement of the carrier chain into the nanopore.

[0107] The leader may be located on one chain of the construct, and a polynucleotide handling protein may control the movement of the other chain of the construct. For example, in some embodiments, the polynucleotide-polypeptide conjugate chain includes a leader, and the polynucleotide handling protein controls the movement of the carrier chain to the nanopore. In other embodiments, the carrier chain includes a leader, and the polynucleotide handling protein controls the movement of the polynucleotide-polypeptide conjugate chain to the nanopore.

[0108] If a carrier chain is present, the leader can attach to both the polynucleotide-polypeptide conjugate chain and the carrier chain. For example, the polynucleotide-polypeptide conjugate chain and the carrier chain can be attached using the Y adapter described herein. The stem of the Y adapter may provide a leader sequence, such as a single-stranded or double-stranded polynucleotide portion.

[0109] If present, the leader may include nucleotide units, spacer units, and / or other monomer units that can be attached together to form the leader. For example, the leader may include one or more nucleotide units and / or one or more spacer units. Preferred spacer units are described in more detail herein and include, for example, one or more C3, iSp9, and / or iSp18 spacers as described herein.

[0110] Any suitable leader can be used as described herein. Optionally, the leader may be a polynucleotide. The leader may be the same as or different from the polynucleotide in the conjugate. As described above, the leader may facilitate threading of the conjugate through the nanopores.

[0111] In other words, in some embodiments, the polynucleotide-polypeptide conjugate chain is L-{PN}-P m It includes one or more structures of the form, -L is the leader, and L is optionally part N. -P is a polypeptide, -N contains a polynucleotide, -m is 0 or 1, The method may include threading a leader (L) through nanopores, thereby bringing a polypeptide (P) into contact with the nanopores.

[0112] A polynucleotide-polypeptide conjugate chain can be hybridized to a carrier chain. The N portion(s) of a polynucleotide-polypeptide conjugate chain can be hybridized to one or more polynucleotide carrier chains. In some embodiments, each N portion of the polynucleotide-polypeptide conjugate chain is hybridized to a polynucleotide carrier chain.

[0113] In some such embodiments, the polynucleotide handling protein is located on the cis side of the nanopore, and the method includes enabling the polynucleotide handling protein to control the movement of a polynucleotide moiety (N) from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of polypeptides (P) through the nanopore. In other embodiments, the polynucleotide handling protein is located on the trans side of the nanopore, and the method includes enabling the polynucleotide handling protein to control the movement of a polynucleotide moiety (N) from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of polypeptides (P) through the nanopore.

[0114] As will be described in more detail herein, in some embodiments the construct comprises multiple polynucleotides and polypeptides. In such embodiments, a polynucleotide handling protein sequentially controls the movement of the polynucleotide-polypeptide conjugate chain and / or carrier chain into the nanopores, and thus sequentially moves each polypeptide into the nanopores. In this way, each polypeptide within the conjugate can be sequentially characterized in the disclosed manner.

[0115] In some embodiments, the polynucleotide-polypeptide conjugate chain comprises multiple target polypeptides. For example, the polynucleotide-polypeptide conjugate chain may comprise one or more portions of the form ...NPNPN..., where each N may be the same or different polypeptide and each N may be the same or different polynucleotide. Some or all of the N portions of the polynucleotide-polypeptide conjugate chain can be hybridized to one or more polynucleotide carrier chains. In some embodiments, each N portion of the polynucleotide-polypeptide conjugate chain is hybridized to a polynucleotide carrier chain.

[0116] For example, a polynucleotide-polypeptide conjugate chain is L-P1-N-{PN} n -P m It may include one or more structures of the form, -n is a positive integer, -L is the leader, and L is optionally part N. -Each P may be the same or different, and is a polypeptide. -Each N may be the same or different, and it may contain polynucleotides. -m is 0 or 1, The method may include threading a leader (L) through nanopores, thereby bringing polypeptide (P1) into contact with the nanopores.

[0117] Typically, in such embodiments, n is 1 to about 1000, for example 2 to about 100, for example 3 to about 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0118] In some such embodiments, the polynucleotide handling protein is located on the cis side of the nanopore, and the method includes enabling the polynucleotide handling protein to sequentially control the movement of each polynucleotide (N) from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the sequential movement of each polypeptide (P) through the nanopore. In other such embodiments, the polynucleotide handling protein is located on the trans side of the nanopore, and the method includes enabling the polynucleotide handling protein to sequentially control the movement of each polynucleotide (N) from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the sequential movement of each polypeptide (P) through the nanopore.

[0119] Those skilled in the art will understand that when the conjugate contains more than one polypeptide, it may be advantageous for the polynucleotide handling protein to remain bound to the conjugate without dissociating upon contact with the polypeptide (as described in more detail herein). For example, as shown in Figures 4 and 5, this allows the polynucleotide handling protein to pass over the polypeptide moieties in the conjugate upon contact with them in order to sequentially move to the polynucleotide moieties in order to control the movement of the conjugate into the nanopores.

[0120] Another non-limiting embodiment of the disclosed method is schematically shown in Figure 2. In this example, the polynucleotide handling protein moves the conjugate "outside" the pore from the "point of view" of the polynucleotide handling protein. For example, as shown, the polynucleotide handling protein is positioned on the cis side of the nanopore and moves the conjugate into the pore, i.e., from the trans side to the cis side. The opposite setting may also be used.

[0121] In other words, in some embodiments, the polynucleotide handling protein may be located on the cis side of the nanopore, and the polynucleotide handling protein controls the movement of constructs, polynucleotide-polypeptide conjugate chains and / or polynucleotide carrier chains from the trans side of the nanopore to the cis side of the nanopore. In some embodiments, the polynucleotide handling protein is located on the cis side of the nanopore, and the polynucleotide handling protein controls the movement of polynucleotide carrier chains from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of target polypeptides through the nanopore. Thus, in some embodiments, the polynucleotide handling protein is located on the cis side of the nanopore, and the polynucleotide handling protein controls the movement of polypeptides from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of polypeptides through the nanopore.

[0122] In other embodiments, the polynucleotide handling protein is located on the trans side of the nanopore and controls the movement of constructs, polynucleotide-polypeptide conjugate chains, and / or polynucleotide carrier chains from the cis side of the nanopore to the trans side of the nanopore. In some embodiments, the polynucleotide handling protein is located on the trans side of the nanopore and controls the movement of polynucleotide carrier chains from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of target polypeptides through the nanopore. Thus, in some embodiments, the polynucleotide handling protein is located on the trans side of the nanopore and controls the movement of polypeptides from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of polypeptides through the nanopore.

[0123] Using the same nomenclature as above, in some embodiments, a polynucleotide-polypeptide conjugate chain may comprise one or more portions of the form …NPNPN…, where each N may be the same or different polypeptide, and each N may be the same or different polynucleotide. Some or all of the N portions of the polynucleotide-polypeptide conjugate chain may be hybridized to one or more polynucleotide carrier chains. In some embodiments, each N portion of the polynucleotide-polypeptide conjugate chain is hybridized to a polynucleotide carrier chain.

[0124] In some embodiments, the polynucleotide-polypeptide conjugate chain is L-{PN}-P m Or L-P1-N-{PN} n -P m It includes one or more structures of the form, -n is a positive integer, -L is the leader, and L is optionally part N. -Each P may be the same or different, and is a polypeptide. -Each N may be the same or different, and it may contain polynucleotides. -m is 0 or 1, The method may include threading a leader (L) through nanopores, thereby bringing a polypeptide (P) into contact with the nanopores.

[0125] A polynucleotide-polypeptide conjugate chain can be hybridized to a carrier chain. The N portion(s) of a polynucleotide-polypeptide conjugate chain can be hybridized to one or more polynucleotide carrier chains. In some embodiments, each N portion of the polynucleotide-polypeptide conjugate chain is hybridized to a polynucleotide carrier chain.

[0126] In some such embodiments, the polynucleotide handling protein is located on the cis side of the nanopore, and the method includes enabling the polynucleotide handling protein to control the movement of the polynucleotide moiety (N) from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of the polypeptide moiety (P) through the nanopore. In other such embodiments, the polynucleotide handling protein is located on the trans side of the nanopore, and the method includes enabling the polynucleotide handling protein to control the movement of the polynucleotide moiety (N) from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of the polypeptide moiety (P) through the nanopore.

[0127] In some embodiments, the methods provided herein involve enabling a construct, a polynucleotide-polypeptide conjugate chain, and / or a polynucleotide carrier chain to move back and forth within a nanopore. This process can be repeated multiple times. In this way, the polypeptide can vibrate through the pore (i.e., be “flossed” through the nanopore). This “flossing” allows the polypeptide portion of the conjugate to be repeatedly characterized by the nanopore. In some embodiments, this can improve the accuracy of the characteristic information. This “flossing” is also referred to herein as rereading. An example of a rereading method is shown in Figure 3.

[0128] In some embodiments, this method i) Performing the method herein such that the target polypeptide displaces the nanopore in a first direction relative to the nanopore, ii) To enable the construct, polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain to move in a direction opposite to the direction of movement to the nanopore in step (i) such that the target polypeptide displaces the nanopore in a second direction opposite to the first direction, iii) To enable the construct, polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain to move in the first direction such that the target polypeptide re-transposes the nanopore in the first direction, iv) optionally repeating steps (ii) and (iii) to vibrate the polypeptide through the nanopores.

[0129] In some embodiments, particularly in some embodiments involving the rereading described above, the conjugate may include a blocking moiety attached to the construct (e.g., a polynucleotide-polypeptide conjugate chain and / or carrier chain) via an optional linker.

[0130] The blocking portion is typically too large to pass through the nanopore; therefore, as the construct moves toward the nanopore, the blocking portion comes into contact with the nanopore, preventing further movement of the conjugate through the nanopore. In embodiments of the disclosed method, where the conjugate moves toward the nanopore under an applied force (e.g., electric potential or chemical potential), the conjugate can "return" through the pore in the opposite direction to the movement controlled by the polynucleotide handling protein. This return movement of the conjugate through the pore allows the polypeptide portion of the conjugate to be re-characterized according to the again disclosed method. This is schematically shown in Figure 3, where the circle at the top of the chain (shown here as a non-limiting example at the top of the carrier chain) represents the blocking portion.

[0131] In such embodiments, any suitable blocking portion can be used. For example, the conjugate can be modified with biotin, and the blocking portion may be streptavidin, avidin, or neutraavidin, for example. The blocking portion may be a large chemical group such as a dendrimer. The blocking portion may be nanoparticles or beads. Other suitable blocking portions will be obvious to those skilled in the art.

[0132] Accordingly, in some embodiments of the disclosed method, the construct, polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain includes a blocking moiety attached via an optional linker, the blocking moiety being unable to displace through nanopores.

[0133] In some embodiments, the disclosed method is i) Bringing a polynucleotide-polypeptide conjugate chain or a construct containing a polynucleotide-polypeptide conjugate chain to contact a nanopore such that the blocking portion is on the same side as the polynucleotide handling protein of the nanopore, ii) Contacting the polynucleotide region of the construct with a polynucleotide handling protein, iii) Polynucleotide handling proteins enable the movement of polynucleotides into nanopores to be controlled in the opposite direction to a force applied across the nanopore (e.g., voltage potential), thereby controlling the movement of polypeptides through the nanopores. iv) To enable the construct to move in a certain direction with respect to a force applied across the nanopores (e.g., voltage potential), i.e., in the opposite direction to the direction of movement controlled by the polynucleotide handling protein, v) If the blocking portion comes into contact with the nanopore, thereby preventing further movement of the conjugate through the nanopore, the polynucleotide handling protein can control the movement of the construct through the nanopore. vi) optionally repeating steps (iv) to (iv) to vibrate the polypeptide through the nanopores, and

[0134] polypeptide Any suitable polypeptide can be characterized in the manner disclosed.

[0135] As described herein, in some embodiments, multiple polypeptides are characterized. In some embodiments, the polynucleotide-polypeptide conjugate chain comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, or at least 100 polypeptide moieties.

[0136] In some embodiments, the target polypeptide or each target polypeptide is an unmodified protein or a portion thereof, or a naturally occurring polypeptide or a portion thereof.

[0137] In some embodiments, the target polypeptide or each target polypeptide is secreted from the cell. Alternatively, the target polypeptide or each target polypeptide may be produced intracellularly so that it must be extracted from the cell for characterization by the disclosed method. The polypeptide or each polypeptide is a plasmid, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 th This may include the product of cellular expression of plasmids used for protein cloning according to the methods described in ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016).

[0138] Polypeptides or each polypeptide may be obtained from or extracted from any organism or microorganism. Polypeptides or each polypeptide may be obtained from humans or animals, for example from urine, lymph, saliva, mucus, semen, or amniotic fluid, or from whole blood, plasma, or serum. Polypeptides or each polypeptide may be obtained from plants, for example from cereals, legumes, fruits, or vegetables.

[0139] A target polypeptide, or each target polypeptide, may be provided as an impure mixture of one or more polypeptides and one or more impurities. The impurities may include truncated forms of target polypeptides that are different from the “target polypeptide” used for characterization in the disclosed method. For example, a target polypeptide, or each target polypeptide, may be a full-length protein, and the impurities may include protein fractions. The impurities may also include proteins other than the target protein, which can be co-purified, for example, from cell cultures or obtained from samples.

[0140] Polypeptides can contain any combination of any amino acid, amino acid analogs, and modified amino acids (i.e., amino acid derivatives). Amino acids (and their derivatives, analogs, etc.) in a polypeptide can be distinguished by their physical size and charge.

[0141] The amino acids / derivatives / analogs may be naturally occurring or artificial. In some embodiments, the polypeptide does not contain or consists of peptide nucleic acids (PNA).

[0142] In some embodiments, polypeptides may contain any naturally occurring amino acids. Twenty amino acids are encoded by the universal genetic code. These are alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamic acid / glutamate (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). Other naturally occurring amino acids include selenocysteine ​​and pyrrolicin.

[0143] In some embodiments, the polypeptide or each polypeptide is unmodified, for example, not chemically modified as described in more detail herein. In some embodiments, the polypeptide or each polypeptide is not modified for detection (e.g., not chemically modified). In some embodiments, the polypeptide or each polypeptide does not contain modified (e.g., chemically modified) amino acids. In some embodiments, the polypeptide contains unmodified amino acids. In some embodiments, the polypeptide is a synthetic polypeptide. Thus, the polypeptide may be, for example, a naturally occurring polypeptide, or a synthetic polypeptide synthesized using unmodified (e.g., canonical) amino acids as shown in Table 1 above.

[0144] In some embodiments, the polypeptide or each polypeptide is modified. In some embodiments, the polypeptide or each polypeptide is modified for detection using the disclosed method. In some embodiments, the disclosed method is for characterizing the modification in the target polypeptide(s).

[0145] In some embodiments, one or more of the polypeptides or amino acids / derivatives / analogs in each polypeptide are modified. In some embodiments, one or more of the polypeptides or amino acids / derivatives / analogs in each polypeptide are modified post-translationally. Thus, the methods disclosed herein can be used to detect the presence, absence, and number of positions of post-translational modifications in polypeptides. The disclosed methods can be used to characterize the degree to which a polypeptide is post-translationally modified.

[0146] Any one or more post-translational modifications can be present in a polypeptide or each polypeptide. Typical post-translational modifications include modification by hydrophobic groups, modification by cofactors, addition of chemical groups, glycosylation (non-enzymatic binding of sugars), biotinylation, and pegylation. Post-translational modifications can be unnatural, such as chemical modifications performed in the laboratory for biotechnological or biomedical purposes. This can enable monitoring of the levels of peptides, polypeptides, or proteins produced in the laboratory, in contrast to their natural counterparts.

[0147] Examples of post-translational modifications by hydrophobic groups include myristoylation, attachment of myristic acid, C 14 attachment of saturated acids; palmitoylation, attachment of palmitic acid, C 16 attachment of saturated acids; isoprenylation or prenylation, attachment of isoprenoid groups; farnesylation, attachment of farnesol groups; geranylgeranylation, attachment of geranylgeraniol groups; and glypiation, and formation of glycosylphosphatidylinositol (GPI) anchors via amide bonds.

[0148] Examples of post-translational modifications by cofactors include lipoylation, attachment of lipoate (C8) functional groups; flavinylation, attachment of flavin moieties (e.g., flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD)); attachment of heme C, e.g., via a thioether bond with cysteine; phosphopantetheinylation, attachment of 4'-phosphopantetheinyl groups; and formation of retinylidene Schiff bases.

[0149] Examples of post-translational modifications by the addition of chemical groups include: acylation, e.g., O-acylation (ester), N-acylation (amide), or S-acylation (thioester); acetylation, e.g., attachment of an acetyl group to the N-terminus or lysine; formylation; alkylation, addition of alkyl groups such as methyl or ethyl; methylation, e.g., addition of a methyl group to lysine or arginine; amidation; butyrication; gammacarboxylation; glycosylation, e.g., enzymatic attachment of a glycosyl group to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, or tryptophan; polysialylation, attachment of polysialic acid; malonylation; hydroxylation; iodization; bromination; and citrulline. This includes nucleotide addition, attachment of any nucleotide such as any of those considered above, ADP-ribosylation; oxidation; phosphorylation, e.g., attachment of a phosphate group to serine, threonine, or tyrosine (O-linked) or histidine (N-linked); adenylylation, e.g., attachment of an adenylyl moiety to tyrosine (O-linked) or histidine or lysine (N-linked); propionation, formation of pyroglutamic acid; S-glutathione; smoylation; S-nitrosylation; succinylation, e.g., attachment of a succinyl group to lysine; selenoylation, incorporation of selenium; and ubiquitination, addition of a ubiquitin subunit (N-linked).

[0150] Labeling of polypeptides or individual polypeptides with molecular labels is within the scope of the methods provided herein. Molecular labeling may be modifications to polypeptides that facilitate the detection of polypeptides in the methods provided herein. For example, labeling may be modifications to polypeptides that alter the signal obtained when the conjugate is characterized. For example, labeling may interfere with the flow of ions through nanopores. In this way, labeling may improve the sensitivity of the methods.

[0151] In some embodiments, the polypeptide or each polypeptide comprises one or more crosslinked sections, e.g., CC crosslinks. In some embodiments, the polypeptide is not crosslinked before being characterized using the disclosed method.

[0152] In some embodiments, the polypeptide, or each polypeptide, contains sulfide-containing amino acids and therefore has the potential to form disulfide bonds. Typically, in such embodiments, the polypeptide is reduced using a reagent such as DTT (dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine) before being characterized using the disclosed method.

[0153] In some embodiments, the polypeptide or each polypeptide is a full-length protein or a naturally occurring polypeptide. In some embodiments, the protein or naturally occurring polypeptide is fragmented before conjugate to a polynucleotide. In some embodiments, the protein or polypeptide is fragmented chemically or enzymatically. In some embodiments, the polypeptide or polypeptide fragments can be conjugated to form a longer target polypeptide.

[0154] A polypeptide or each polypeptide may be a polypeptide of any preferred length. In some embodiments, a polypeptide or each polypeptide independently has a length of about 5 to about 5000 peptide units. In some embodiments, a polypeptide has a length of about 5 to about 1000 peptide units, for example, about 5 to about 500 peptide units, for example, about 5 to about 250 peptide units, for example, about 5 to about 100 peptide units, for example, about 5 to about 50 peptide units, for example, about 5 to about 25 peptide units. In some embodiments, a polypeptide or each polypeptide independently has a length of about 10 to about 5000 peptide units. In some embodiments, a polypeptide or each polypeptide has a length of about 10 to about 1000 peptide units, for example, about 10 to about 500 peptide units, for example, about 10 to about 250 peptide units, for example, about 10 to about 100 peptide units, for example, about 10 to about 50 peptide units, for example, about 10 to about 25 peptide units. In some embodiments, the polypeptide or each polypeptide independently has a length of about 25 to about 1000 peptide units, for example, about 50 to about 500 peptide units, for example, about 100 to about 250 peptide units.

[0155] Any number of polypeptides can be characterized in the disclosed manner. For example, the method may relate to the characterization of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, or more polynucleotides. If two or more polypeptides are used, they may be different polypeptides or two or more examples of the same polypeptide.

[0156] Therefore, it is clear that the measurements obtained by the disclosed method are typically characterized by one or more features of the polypeptide, selected from (i) polypeptide length, (ii) polypeptide identity, (iii) polypeptide sequence, (iv) polypeptide secondary structure, and (v) whether the polypeptide is modified. In typical embodiments, the measurements are characterized by the polypeptide sequence, or whether the polypeptide is modified, for example, by one or more post-translational modifications. In some embodiments, the measurements are characteristic of the polypeptide sequence.

[0157] In some embodiments, the polypeptide is in a relaxed form. In some embodiments, the polypeptide is held in a linearized form. Holding the polypeptide in a linearized form can facilitate the characteristic feature of the polypeptide residue by residue because it prevents "bunching" of the polypeptide within the nanopores.

[0158] The polypeptide can be held in a linearized form using any preferred means.

[0159] In some embodiments, the polypeptide is maintained in a linearized form because the polypeptide is attached to polynucleotide flanking chains at each end of the polypeptide, and the flanking chains are hybridized to complementary or substantially complementary portions of one or more polynucleotide carrier chains. The sequences of the flanking chains and / or carrier chains may be selected or designed such that the distance between the regions of the carrier chains that hybridize to the flanking chains is similar to the length of the polypeptide in its linearized form. The length of the polypeptide in its linearized form can be determined from the number of amino acids in the polypeptide, if the number of amino acids in the polypeptide is known; for example, peptide units in a polypeptide are generally considered to have a length of about 0.35 nm (3.5 Å). Thus, designing flanking and carrier chains for any desired target polypeptide, for example, based on the mass of the polypeptide, is within the capabilities of those skilled in the art, and this can be readily determined by methods known to those skilled in the art, such as denaturation (e.g., SDS-) or native PAGE, or by mass spectrometry, or based on the sequence of the polypeptide, if known.

[0160] Other methods are known for maintaining target polypeptides in a linearized form.

[0161] For example, if a polypeptide is charged, applying a voltage can hold the polypeptide in a linearized form.

[0162] If the polypeptide is uncharged or only slightly charged, the charge can be altered or controlled by adjusting the pH. For example, by using a high pH to increase the relative negative charge of the polypeptide, the polypeptide can be retained in a linearized form. Increasing the negative charge of the polypeptide allows it to be retained in a linearized form, for example, under a positive voltage. Alternatively, by using a low pH to increase the relative positive charge of the polypeptide, the polypeptide can be retained in a linearized form. Increasing the positive charge of the polypeptide allows it to be retained in a linearized form, for example, under a negative voltage. In the disclosed methods, polynucleotide handling proteins are often used to control the movement of polynucleotides into nanopores. Since polynucleotides are typically negatively charged, it is generally most preferable to increase the linearization of the polypeptide by raising the pH, thereby making the polypeptide more negatively charged, as is the case with the polynucleotides. In this way, the polynucleotide-polynucleotide conjugate chain retains an overall negative charge and is therefore readily movable, for example, under an applied voltage.

[0163] Polypeptides can be retained in a linearized form by using suitable denaturation conditions. Suitable denaturation conditions include, for example, the presence of a suitable concentration of a denaturing agent such as guanidine HCl and / or urea. The concentration of such a denaturing agent used in the disclosed method depends on the target polypeptide characterized by the method and can be readily selected by those skilled in the art.

[0164] Polypeptides can be retained in a linearized form by using a suitable detergent. Suitable detergents for use in the disclosed method include SDS (sodium dodecyl sulfate).

[0165] Polypeptides can be retained in a linearized form by carrying out the disclosed method at high temperatures. Increasing the temperature overcomes intrachain bonds, allowing the polypeptide to take on a linearized form.

[0166] Polypeptides can be retained in a linearized form by performing the disclosed method under strong electroosmotic forces. Such forces can be provided by using asymmetric salt conditions and / or by providing a suitable charge to the channels of the nanopores. The charge of the channels of protein nanopores can be modified, for example, by mutagenesis. Changing the charge of nanopores is within the capabilities of those skilled in the art. By changing the charge of nanopores, a strong electroosmotic force is generated from the unbalanced flow of cations and anions through the nanopores when a potential is applied across the nanopores.

[0167] Conjugate chain formation When forming a polynucleotide-polypeptide conjugate chain, the target polypeptide or each target polypeptide can be conjugated to a polynucleotide adjacent chain or each polynucleotide adjacent chain at any suitable position. For example, the polypeptide or each polypeptide can be conjugated to a polynucleotide adjacent chain(s) at the N-terminus or C-terminus of the polypeptide. The polypeptide can be conjugated to the polynucleotide via side chain groups of residues (e.g., amino acid residues) within the polypeptide.

[0168] In some embodiments, the target polypeptide has naturally occurring reactive functional groups that can be used to facilitate conjugate to polynucleotide adjacent chains. For example, cysteine ​​residues can be used to form disulfide bonds to polynucleotide adjacent chains or modifying groups thereon.

[0169] In some embodiments, the target polypeptide is modified to facilitate its conjugate to a polynucleotide adjacent chain(s). For example, in some embodiments, the polypeptide is modified by attaching a moiety containing a reactive functional group for attachment to a polynucleotide adjacent chain(s). For example, in some embodiments, the polypeptide may be extended at the N-terminus or C-terminus by one or more residues (e.g., amino acid residues) containing one or more reactive functional groups for reacting with corresponding reactive functional groups on the polynucleotide adjacent chain(s). For example, in some embodiments, the polypeptide may be extended at the N-terminus and / or C-terminus by one or more cysteine ​​residues. Such residues may be used for attachment to the polynucleotide moiety of the polynucleotide-polypeptide conjugate chain, for example by maleimide chemistry (e.g., reaction of cysteine ​​with an azido-maleimide compound such as azido-[Pol]-maleimide, where [Pol] is typically a short-chain polymer such as PEG, e.g., PEG2, PEG3, or PEG4, followed by coupling to a appropriately functionalized polynucleotide, e.g., a polynucleotide possessing a BCN group for reaction with azide). To avoid misunderstanding, if a polypeptide contains suitable naturally occurring residues at its N-terminus and / or C-terminus (e.g., naturally occurring cysteine ​​residues at the N-terminus and / or C-terminus), such residues may be used for attachment to the polynucleotide.

[0170] In some embodiments, residues in the target polypeptide are modified to facilitate the attachment of the target polypeptide to the polynucleotide adjacent chain(s). In some embodiments, residues in the polypeptide (e.g., amino acid residues) are chemically modified for attachment to the polynucleotide adjacent chain(s). In some embodiments, residues in the polypeptide (e.g., amino acid residues) are enzymatically modified for attachment to the polynucleotide adjacent chain(s).

[0171] The conjugation chemistry between a polynucleotide adjacent chain(s) and the polypeptide portion of a polynucleotide-polypeptide conjugate chain is not particularly limited. Any suitable combination of reactive functional groups can be used. Many suitable reactive groups and their chemical targets are known in the art. Some exemplary reactive groups and their corresponding targets include aryl azides that can react with amines, carbodiimides that can react with amines and carboxyl groups, hydrazides that can react with carbohydrates, hydroxymethylphosphines that can react with amines, imide esters that can react with amines, isocyanates that can react with hydroxyl groups, carbonyls that can react with hydrazines, maleimides that can react with sulfhydryl groups, NHS esters that can react with amines, PFP esters that can react with amines, psoralens that can react with thymine, pyridyl disulfide that can react with sulfhydryl groups, vinyl sulfones and vinyl sulfonamides that can react with sulfhydrylamines and hydroxyl groups.

[0172] Other suitable chemistry for conjugating polypeptides into polynucleotides includes click chemistry. Many suitable click chemistry reagents are known in the art. Suitable examples of click chemistry include, but are not limited to, the following: (a) Azide-alkyne cycloaddition catalyzed by copper(I) (azide-alkyne hysgene cycloaddition), (b) Strain-enhanced azide-alkyne cycloaddition; [3+2] cycloaddition of alkenes and azides; reverse demand Diels-Alder reaction of alkenes and tetrazines; and photoclic reaction of alkenes and tetrazoles, (c) A copper-free variant of a 1,3-dipolar cycloaddition reaction in which an azide reacts with a strained alkyne, for example in a cyclooctane ring, for example in bicyclic [6.1.0]nonine (BCN), (d) Reaction of an oxygen nucleophile on one linker with the epoxide or aziridine reactive moiety on the other, (e) Staudinger ligation, in which the alkyne moiety is replaced with an arylphosphine to induce a specific reaction with the azide and confer an amide bond.

[0173] Any reactive group(s) may be used to form the conjugate. Some suitable reactive groups include [1,4-bis[3-(2-pyridyldithio)propionamide]butane; 1,11-bis-maleimidetriethylene glycol; 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide); ethylene glycol-bis(succinate N-hydroxysuccinimide); 4,4'-diisothiocyanatostilbene-2,2'-disodium disodium disulfonate; bis[2-(4-azidosalicylamide)ethyl]disulfide; 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide; 4-maleimide butyrate N-hydroxysuccinimide; iodoacetate N-hydroxysuccinimide; S-acetylthioglycolate N-hydroxysuccinimide; azido-PEG-maleimide; and alkyne-PEG-maleimide. The reactive group may be any of those disclosed in WO2010 / 086602, particularly in Table 3 of that application.

[0174] In some embodiments, the reactive functional group is contained within the polynucleotide and the target functional group is contained within the polypeptide prior to the conjugation step. In other embodiments, the reactive functional group is contained within the polypeptide and the target functional group is contained within the polynucleotide prior to the conjugation step. In some embodiments, the reactive functional group is directly attached to the polypeptide. In some embodiments, the reactive functional group is attached to the polypeptide via a spacer. Any suitable spacer can be used. Suitable spacers include, for example, alkyldiamines such as ethyldiamine.

[0175] As described above, in some embodiments, the conjugate comprises multiple polypeptide sections and / or multiple polynucleotide sections. For example, the conjugate may include a structure of form …-PNPNPN… where P is a polypeptide and N is a polynucleotide. In such embodiments, the multiple polynucleotides and polypeptides may be conjugated together by the same or different chemicals.

[0176] As described herein, the construct may include a leader. The leader may reside on the polynucleotide-polypeptide conjugate chain (e.g., on an adjacent strand) or on the carrier strand, if present. Any suitable leader may be used as described herein. In some embodiments, the leader is or contains a polynucleotide. In embodiments where the leader is a polynucleotide, the leader may be the same type of polynucleotide as the polynucleotide used in the conjugate, or the leader may be a different type of polynucleotide. For example, the polynucleotide in the conjugate may be DNA and the leader may be RNA, or vice versa.

[0177] In some embodiments, the leader may be about 10 to 150 nucleotides in length (e.g., DNA and / or RNA nucleotides), for example 20 to 120, for example 30 to 100, for example 40 to 80, for example 50 to 70 nucleotides, or about 10 to about 60 nucleotides in length, for example about 20 to about 50, for example about 20 to about 40, for example about 30 nucleotides.

[0178] In some embodiments, the leader is a charged polymer, for example, a negatively charged polymer. In some embodiments, the leader comprises a polymer such as PEG or a polysaccharide. In such embodiments, the leader may have a length of 10 to 150 monomer units (e.g., ethylene glycol or sugar units), for example, 20 to 120, for example, 30 to 100, for example, 40 to 80, or for example, 50 to 70 monomer units (e.g., ethylene glycol or sugar units).

[0179] Polynucleotides As will be described in more detail herein, the methods provided herein include conjugating one or more polypeptides to one or more adjacent polynucleotide chains. The resulting polynucleotide-polypeptide conjugate chain may be hybridized or otherwise attached to a polynucleotide carrier chain.

[0180] In the disclosed method, any suitable polynucleotide can be used as adjacent chains and / or carrier chains. Selecting suitable polynucleotides for use as adjacent chains and carrier chains, depending on the target polypeptide characterized by the disclosed method, is within the capabilities of those skilled in the art. For example, in some embodiments, the adjacent chains or each adjacent chain is at least the same length as the target polypeptide. In some embodiments, the adjacent chains or each adjacent chain is shorter than the target polypeptide. In some embodiments, the adjacent chains extend over the target polypeptide.

[0181] In some embodiments, the polynucleotides used, or each polynucleotide, are secreted from the cell. Alternatively, the polynucleotides may be produced intracellularly, so that they must be extracted from the cell for use in the disclosed manner.

[0182] A polynucleotide may be provided as an impure mixture of one or more polynucleotides and one or more impurities. The impurities may include partially excised polynucleotides that are different from the polynucleotides intended for use in conjugate formation. For example, the polynucleotide intended for use as an adjacent strand or carrier strand may be genomic DNA, and the impurities may include fractions of genomic DNA, plasmids, etc. The target polynucleotide may be a coding region of genomic DNA, and the undesirable polynucleotide may include a non-coding region of DNA.

[0183] Examples of polynucleotides include DNA and RNA. Bases in DNA and RNA can sometimes be identified by their physical size.

[0184] A polynucleotide or nucleic acid suitable for use as an adjacent chain or carrier chain may contain any combination of any nucleotides. The nucleotides may be naturally occurring or artificial. One or more nucleotides in a polynucleotide may be oxidized or methylated. One or more nucleotides in a polynucleotide may be damaged. For example, a polynucleotide may contain pyrimidine dimers, which are typically associated with UV damage and are a major cause of cutaneous melanoma.

[0185] One or more nucleotides in a polynucleotide may be modified, for example, by labels or tags, suitable examples of which are known to those skilled in the art. Those suitable for use as adjacent-chain or carrier-chain polynucleotides may include one or more spacers. Adapters, such as sequencing adapters, may be included in the polynucleotide. Adapters, tags, and spacers are described in more detail herein.

[0186] Examples of modified bases are disclosed herein and can be incorporated into polynucleotides by means known in the art, for example, by polymerase incorporation of modified nucleotide triphosphates in a strand copy (e.g., PCR) or by polymerase packing. In some embodiments, one or more bases may be modified by chemical means using reagents known in the art.

[0187] Nucleotides typically consist of a nucleic acid base, a sugar, and at least one phosphate group. The nucleic acid base and sugar form a nucleoside. Nucleic acid bases are typically heterocyclic. Nucleic acid bases include, but are not limited to, purines and pyrimidines, more specifically adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). Sugars are typically pentose sugars. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar is preferably deoxyribose. Polynucleotides preferably consist of the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and / or thymidine (dT), deoxyguanosine (dG), and deoxycytidine (dC). Nucleotides are typically ribonucleotides or deoxyribonucleotides. Nucleotides typically contain monophosphate, diphosphate, or triphosphate. Nucleotides may contain more than three phosphate groups, for example, four or five. The phosphate groups may be attached to the 5' or 3' end of the nucleotide. Nucleotides in a polynucleotide may be attached to each other in any manner. Nucleotides are typically attached by their sugar and phosphate groups, as is the case with nucleic acids. Nucleotides may be linked via nucleic acid bases, as is the case with pyrimidine dimers.

[0188] Generally, polynucleotides can be double-stranded or single-stranded. In the disclosed method, the adjacent strand(s) are typically single-stranded. The carrier strand(s) are typically single-stranded. Therefore, the construct formed by adhesion (e.g., hybridization) between the adjacent strand(s) and the carrier strand(s) is thus double-stranded.

[0189] A single-stranded polynucleotide suitable for use as an adjacent strand or carrier strand is typically single-stranded DNA. Single-stranded RNA may also be used. In some embodiments, a single-stranded DNA-RNA hybrid is suitable for use as an adjacent strand or carrier strand. DNA-RNA hybrids can be prepared by ligating single-stranded DNA to RNA or vice versa. Polynucleotides are most typically single-stranded deoxyribonucleic acid (DNA) or single-stranded ribonucleic acid (RNA), usually DNA.

[0190] In some embodiments, the constructs and carrier strand constructs comprising polynucleotide-polypeptide conjugate strands include double-stranded DNA. In some embodiments, they include double-stranded RNA. In some embodiments, they are double-stranded DNA-RNA hybrids. Double-stranded DNA-RNA hybrids can be prepared from single-stranded RNA by reverse transcription of cDNA complement.

[0191] A polynucleotide suitable for use as an adjacent chain or carrier chain can be of any length. For example, a polynucleotide may be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides or nucleotide pairs in length. A polynucleotide may be at least 1000 nucleotides or nucleotide pairs in length, at least 5000 nucleotides or nucleotide pairs in length, or at least 100000 nucleotides or nucleotide pairs in length.

[0192] More typically, polynucleotides suitable for use as adjacent chains or carrier chains have lengths of about 1 to about 10,000 nucleotides or nucleotide pairs, for example, about 1 to about 1,000 nucleotides or nucleotide pairs (for example, about 10 to about 1,000 nucleotides or nucleotide pairs), for example, about 5 to about 500 nucleotides or nucleotide pairs, for example, about 10 to about 100 nucleotides or nucleotide pairs, for example, about 20 to about 80 nucleotides or nucleotide pairs, for example, about 30 to about 50 nucleotides or nucleotide pairs.

[0193] Typically, polynucleotides suitable for use as carrier chains are longer than those used as adjacent chains. Therefore, typically, multiple adjacent chains and polypeptide sections can be associated with a single polynucleotide carrier chain.

[0194] For example, in some embodiments, - The target polypeptide or each target polypeptide has a length of approximately 5 to approximately 1000 peptide units, for example, approximately 5 to approximately 500 peptide units, for example, approximately 5 to approximately 250 peptide units, for example, approximately 5 to approximately 100 peptide units, for example, approximately 5 to approximately 50 peptide units. -Each adjacent strand has a length of approximately 5 to approximately 1000 nucleotides, for example, approximately 5 to approximately 500 nucleotides, for example, approximately 5 to approximately 250 nucleotides, for example, approximately 5 to approximately 100 nucleotides, for example, approximately 5 to approximately 50 nucleotides. Each carrier chain has a length of approximately 50 to 50,000 nucleotides, for example, approximately 100 to 10,000 nucleotides, for example, approximately 5,000 nucleotides. Longer carrier chains, for example, approximately 1,000 to 10M bases, for example, approximately 10,000 to 1,000,000 nucleotides, for example, approximately 100,000 nucleotides, can also be used.

[0195] In some embodiments, before contact between the construct and the nanopores, the polynucleotide handling protein is bound to the polynucleotide carrier chain within a region of the polynucleotide carrier chain that extends into the non-hybridization region of the polynucleotide adjacent chain. Thus, the adjacent chain can form a bubble region around the polynucleotide handling protein. The bubble region is the portion of the construct that is not hybridized because the polynucleotide handling protein blocks the association of the adjacent chain and the carrier chain. This is shown as a non-limiting example of schematic form in Figures 1 and 2.

[0196] If present, the bubble region extending across the polynucleotide handling protein can have any appropriate length. The length is typically a function of the size of the polynucleotide handling protein used. If present, the bubble region extending across the polynucleotide handling protein may have a length of, for example, about 2 to about 50 nucleotides, for example, about 5 to about 20 nucleotides, or for example, about 19 nucleotides. In some embodiments, the bubble region may have a length of, for example, about 2 to about 100 nucleotides, for example, about 10 to about 50 nucleotides, for example, about 20 to about 50 nucleotides, for example, about 30 to about 40 nucleotides, or for example, about 35 nucleotides.

[0197] In some embodiments, the length of the corresponding chain segment in which the bubble can form (for example, if the bubble is a segment of an adjacent chain, the corresponding chain may be a carrier chain) may be about 5 to about 20, e.g., about 8 to about 12, or about 10 nucleotide units prior to the methods disclosed herein. Thus, the bubble region is typically longer than the surrounding segment of the corresponding chain formed prior to the methods disclosed herein. For example, in some embodiments, the bubble region may have a length of about 10 to about 50 nucleotides, e.g., about 30 to about 40 nucleotides, while the segment of the corresponding chain in which the bubble forms may have a length of about 5 to about 20, e.g., about 8 to about 12, or e.g., about 10 nucleotide units.

[0198] The bubble region extending over the polynucleotide handling protein (before initiating the method disclosed herein) may or may consist of any preferred type of polynucleotide. In some embodiments, the portion of the polynucleotide flanking chain extending over the polynucleotide handling protein is the same type of polynucleotide as the rest of the flanking chain. In some embodiments, the portion of the polynucleotide flanking chain extending over the polynucleotide handling protein is the same type of polynucleotide but different from the rest of the flanking chain. In some embodiments, the flanking chain contains a DNA polynucleotide, and the bubble region (before initiating the method disclosed herein) contains or consists of RNA. In some embodiments, the flanking chain contains RNA, and the bubble region (before initiating the method disclosed herein) contains or consists of DNA. In some embodiments, the use of different types of polynucleotides to form the initial bubble (as opposed to the rest of the flanking chain) may help to preferentially position the polynucleotide handling protein before initiating the method provided herein. In some embodiments, the construct includes a bubble region on one chain (e.g., on the flanking chain or carrier chain), and the polynucleotide handling protein is bound to the other chain of the construct (e.g., the carrier chain or flanking chain). In some embodiments, the polynucleotide handling protein is stalled at a stall portion, such as a spacer, which is described in more detail herein. In some embodiments, the stall portion is on the opposite side of the bubble region. For example, in some embodiments, the carrier chain includes a spacer portion for stalling the polynucleotide handling protein before the start of the method provided herein, and the polynucleotide handling protein is bound to the carrier chain and adjacent to the bubble region on the polynucleotide-polypeptide conjugate chain.In some embodiments, the polynucleotide-polypeptide conjugate chain includes a spacer portion for stalling a polynucleotide handling protein before the commencement of the method provided herein, the polynucleotide handling protein being bound to the polynucleotide-polypeptide conjugate chain and adjacent to a bubble region on the carrier chain.

[0199] As polynucleotide handling proteins process constructs, they migrate along the strand(s) to which they are attached. Therefore, the migration of polynucleotide handling proteins along the strand results in a bubble region that migrates along the strand with the polynucleotide handling protein. This is schematically illustrated in Figures 1 and 2.

[0200] Any number of polynucleotides (e.g., any number of adjacent chains) can be used in the disclosed manner. For example, the method may involve using 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, or more polynucleotides (e.g., polynucleotide adjacent chains). If two or more polynucleotides are used, they may be different polynucleotides or two examples of the same polynucleotide. Polynucleotides may be naturally occurring or artificial. Typically, a polynucleotide-polypeptide conjugate chain contains one adjacent chain separating each target polypeptide. Therefore, in many cases, the number of polynucleotide adjacent chains is the same as, or substantially the same as, the number of polypeptides.

[0201] Nucleotides can have any identity and include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate (dCMP), and deoxymethylcytidine monophosphate. Nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP, and dUMP. Nucleotides can be debased (i.e., lacking a nucleic acid base). Nucleotides can also lack both a nucleic acid base and a sugar (i.e., they are C3 spacers).

[0202] Polynucleotides may include products of PCR reactions, genomic DNA, products of endonuclease digestion, and / or DNA libraries. Polynucleotides may be obtained from or extracted from any organism or microorganism. Polynucleotides may be obtained from humans or animals, for example, from urine, lymph, saliva, mucus, semen, or amniotic fluid, or from whole blood, plasma, or serum. Polynucleotides may be obtained from plants, for example, cereals, legumes, fruits, or vegetables. Polynucleotides may include genomic DNA. Genomic DNA may be fragmented. DNA may be fragmented by any suitable method. For example, methods for fragmenting DNA are known in the art, and such methods may use transposases such as MuA transposase. Genomic DNA is often not fragmented.

[0203] Labeling of polynucleotides with molecular labels is within the scope of the methods provided herein. Molecular labeling may be modifications to polynucleotides that facilitate the detection of polynucleotides or conjugates in the methods provided herein. For example, labeling may be modifications to polynucleotides that alter the signal obtained when a conjugate is characterized. For example, labeling may interfere with the flow of ions through nanopores. In this way, labeling may improve the sensitivity of the method.

[0204] adapter In some embodiments of the methods provided herein, a polynucleotide (e.g., an adjacent chain or a carrier chain) may have a polynucleotide adapter attached thereto. The adapter typically includes a polynucleotide chain that can be attached to the ends of the polynucleotide.

[0205] In some embodiments, the adapter is attached to an adjacent chain before a conjugate with the polypeptide is formed. In some embodiments, the adapter is attached to a conjugate of polynucleotides and polypeptides.

[0206] Therefore, in some embodiments, the method includes forming a polynucleotide-polypeptide conjugate chain by attaching an adapter (e.g., the adapter described herein) to a polynucleotide and conjugating the polynucleotide / adapter construct to a target polypeptide. In some embodiments, the polynucleotide-polypeptide conjugate chain is formed by attaching an adapter (e.g., the adapter described herein) to a polynucleotide and forming a conjugate by attaching the adapter to a target polypeptide.

[0207] In some embodiments, the adapter may be selected or modified to provide a specific site for conjugation to polynucleotides.

[0208] Adapters can be attached to only one end of a polynucleotide or conjugate. Polynucleotide adapters can be attached to both ends of a polynucleotide or conjugate. Alternatively, different adapters can be attached to two ends of a polynucleotide or conjugate.

[0209] Adapters can be attached to both strands of a double-stranded polynucleotide (e.g., the constructs described herein). Adapters can also be attached to a single-stranded polynucleotide (e.g., the polynucleotide-polypeptide conjugate chains described herein). Methods for attaching adapters to polynucleotides are known in the art. Adapters can be attached to polynucleotides, for example, by ligation, click chemistry, tagmentation, topoisomerase conversion, or any other suitable method.

[0210] In one embodiment, the adapter or each adapter is synthetic or artificial. Typically, the adapter or each adapter comprises a polymer as described herein. In some embodiments, the adapter or each adapter comprises a spacer as described herein. In some embodiments, the adapter or each adapter comprises a polynucleotide. A polynucleotide adapter or each polynucleotide adapter may comprise DNA, RNA, modified DNA (e.g., debased DNA), RNA, PNA, LNA, BNA, and / or PEG. Typically, the adapter or each adapter comprises single-stranded and / or double-stranded DNA or RNA. The adapter may comprise the same type of polynucleotide as the polynucleotide chain to which it is attached. The adapter may comprise a different type of polynucleotide than the polynucleotide chain to which it is attached. In some embodiments, the polynucleotide chain used in the disclosed method is a single-stranded DNA chain, and the adapter comprises DNA or RNA, typically single-stranded DNA. In some embodiments, the polynucleotide is a double-stranded DNA chain, and the adapter comprises DNA or RNA, e.g., double-stranded or single-stranded DNA.

[0211] In some embodiments, the adapter may be a crosslinking portion. A crosslinking portion can be used to connect two strands of a double-stranded polynucleotide. For example, in some embodiments, the crosslinking portion is used to connect a template strand of a double-stranded polynucleotide to a complementary strand of the same double-stranded polynucleotide. For example, a crosslinking adapter can be used to connect an adjacent strand to a carrier strand.

[0212] The crosslinking portion typically covalently bonds two strands of a double-stranded polynucleotide. The crosslinking portion can be any material capable of linking two strands of a double-stranded polynucleotide, provided that the crosslinking portion does not interfere with the movement of the polynucleotide into the nanopore. Suitable crosslinking portions include, but are not limited to, polymer linkers, chemical linkers, polynucleotides, or polypeptides. Preferably, the crosslinking portion includes DNA, RNA, modified DNA (e.g., debasticated DNA), RNA, PNA, LNA, or PEG. The crosslinking portion is more preferably DNA or RNA.

[0213] In some embodiments, the crosslinking portion is a hairpin adapter. The hairpin adapter is an adapter comprising a single polynucleotide chain, where both ends of the polynucleotide chain can or are hybridized to each other, and the central portion of the polynucleotide forms a loop. A suitable hairpin loop adapter may be designed using methods known in the art. In some embodiments, the hairpin loop is typically 4 to 100 nucleotides long, e.g., 4 to 50, e.g., 4 to 20, e.g., 4 to 8 nucleotides long. In some embodiments, the crosslinking portion (e.g., hairpin adapter) is attached to one end of the double-stranded polynucleotide. Typically, the crosslinking portion (e.g., hairpin adapter) is not attached to either end of the double-stranded polynucleotide.

[0214] In some embodiments, the adapter is a linear adapter. The linear adapter can be bound to either or both ends of a single-stranded polynucleotide. If the polynucleotide is a double-stranded polynucleotide, the linear adapter can be bound to either or both ends of either or both strands of the double-stranded polynucleotide. If present, the linear adapter can be attached to either or both ends of the polynucleotide-polypeptide conjugate chain and the carrier chain.

[0215] The linear adapter may include a leader sequence as described herein. The linear adapter may include a portion for hybridization with a tag (such as a pore tag) as described herein. The linear adapter may be 10 to 150 nucleotides long, e.g., 20 to 120, e.g., 30 to 100, e.g., 40 to 80, e.g., 50 to 70 nucleotides long. The linear adapter may be single-stranded. The linear adapter may be double-stranded.

[0216] In some embodiments, the adapter may be a Y-adapter. A Y-adapter is typically a polynucleotide adapter. A Y-adapter is typically double-stranded and includes (a) a region at one end in which the two strands hybridize together, and (b) a region at the other end in which the two strands are not complementary. The non-complementary portion of the strands typically forms an overhang. Because the two strands do not typically hybridize with each other, unlike the double-stranded portion, the presence of the non-complementary region in the Y-adapter gives the adapter a Y shape. The two single-stranded portions of the Y-adapter may be of the same length or of different lengths. For example, one single-stranded portion of the Y-adapter may be 10 to 150 nucleotides long, e.g., 20 to 120, e.g., 30 to 100, e.g., 40 to 80, e.g., 50 to 70 nucleotides long, and the other single-stranded portion of the Y-adapter may independently be 10 to 150 nucleotides long, e.g., 20 to 120, e.g., 30 to 100, e.g., 40 to 80, e.g., 50 to 70 nucleotides long. The double-stranded "stem" portion of the Y-adapter may be 10 to 150 nucleotides long, e.g., 20 to 120, e.g., 30 to 100, e.g., 40 to 80, e.g., 50 to 70 nucleotides long. The Y-adapter may be attached to either or both ends of the constructs described herein.

[0217] The adapter may be ligated to a polynucleotide (e.g., to a carrier chain or adjacent chain) by any suitable means known in the art. The adapter may be synthesized separately and chemically attached to a target polynucleotide, or enzymatically ligated to it. Alternatively, the adapter may be generated during the processing of the polynucleotide. In some embodiments, the adapter is ligated to the target polynucleotide at or near one end of the polynucleotide. In some embodiments, the adapter is ligated to the polynucleotide within 50, e.g., 20, e.g., 10 nucleotides from the end of the polynucleotide. In some embodiments, the adapter is ligated to the polynucleotide at the end of the polynucleotide. When the adapter is ligated to a polynucleotide, the adapter may contain nucleotides of the same type as the polynucleotide, or nucleotides different from those of the polynucleotide.

[0218] Adapters particularly suitable for use in the disclosed method may include a linear homopolymer region (e.g., about 5 to about 20 nucleotides, e.g., about 10 to about 30 nucleotides, e.g., thymine or cytidine) and / or a hybridization site (as described in more detail herein) for hybridizing to one or more tethers or anchors. Such adapters may also include reactive functional groups for binding to a target polypeptide. Click chemistry groups are particularly preferred in this regard. For example, exemplary groups for inclusion in adapters include groups that can particulate in copper-free click chemistry, e.g., groups based on BCN (bicyclo[6.1.0]nonine) and its derivatives, dibenzocyclooctin (DBCO) groups, and the like. The reactions of such groups are well known in the art. For example, BCN groups typically react with groups such as azides, tetrazines, and nitrones, which can be incorporated into polypeptides, for example. DBCO groups exhibit high reactivity with azide groups. Other particularly suitable chemical groups include 2-pyridinecarboxyaldehyde (2-PCA) groups and their derivatives. For example, 6-(azidomethyl)-2-pyridinecarboxyaldehyde can react with the N-terminal amino group of a peptide.

[0219] Spacer In some embodiments of the methods provided herein, the conjugate, leader, or adapter described herein, formed by reaction with a polynucleotide or polypeptide, may include spacers. For example, one or more spacers may be present in the polynucleotide adapter. One or more spacers may be present in the adapter attached to an adjacent chain or carrier chain.

[0220] For example, a polynucleotide adapter may include 1 to about 20 spacers, e.g., about 1 to about 10, e.g., 1 to about 5 spacers, e.g., 1, 2, 3, 4, or 5 spacers. The spacers may include any suitable number of spacer units. The spacers may provide an energy barrier that hinders the movement of polynucleotide handling proteins. For example, spacers may stall polynucleotide handling proteins by reducing the traction force on the polynucleotides. This can be achieved, for example, by using debased spacers, i.e., spacers from which a base has been removed from one or more nucleotides in the polynucleotide adapter. Spacers may physically block the movement of polynucleotide handling proteins, for example, by introducing a large chemical group that physically hinders the movement of polynucleotide handling proteins.

[0221] In some embodiments, one or more spacers are included in a polynucleotide or conjugate, or in an adapter, as used herein, to provide a specific signal when they pass through or traverse a nanopore, i.e., when they move relative to a nanopore.

[0222] In some embodiments, the spacers may include linear molecules such as polymers. Typically, such spacers have a different structure from the polynucleotides used in the conjugate. For example, when the polynucleotide is DNA, the spacers or each spacer typically do not contain DNA. In particular, when the polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), the spacers or each spacer preferably include peptide nucleic acids (PNA), glycerol nucleic acids (GNA), threose nucleic acids (TNA), locked nucleic acids (LNA), or synthetic polymers having nucleotide side chains. In some embodiments, the spacer is one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromodeoxyuridines, one or more inverted thymidines (inverted dT), one or more inverted dideoxythymidines (ddT), one or more dideoxycytidines (ddC), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2'-O-methylRNA bases, one or more iso - May contain deoxycytidine (Iso-dC), one or more iso-deoxyguanosine (Iso-dG), one or more C3 (OC3H6OPO3) groups, one or more photocleavable (PC)[OC3H6-C(O)NHCH2-C6H3NO2-CH(CH3)OPO3] groups, one or more hexanediol groups, one or more spacer 9 (iSp9)[(OCH2CH2)3OPO3] groups, or one or more spacer 18 (iSp18)[(OCH2CH2)6OPO3] groups, or one or more thiol bonds. Spacers may contain any combination of these groups. Many of these groups are commercially available from IDT® (Integrated DNA Technologies®). For example, C3, iSp9, and iSp18 spacers are all available from IDT®. Spacers may contain any number of the above groups as spacer units.

[0223] In some embodiments, the spacer may comprise one or more chemical groups that stall the polynucleotide handling protein. In some embodiments, preferred chemical groups are one or more pendant chemical groups. One or more chemical groups may attach to one or more nucleic acid bases in the polynucleotide, construct, or adapter. One or more chemical groups may attach to the backbone of the polynucleotide adapter. Any number of suitable chemical groups may be present, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. Preferred groups include, but are not limited to, fluorophores, streptavidin and / or biotin, cholesterol, methylene blue, dinitrophenol (DNP), digoxigenin and / or anti-digoxigenin, and dibenzylcyclooctin groups. In some embodiments, the spacer may comprise a polymer. In some embodiments, the spacer may comprise a polymer that is a polypeptide or polyethylene glycol (PEG).

[0224] In some embodiments, the spacer may contain one or more debasalized nucleotides (i.e., nucleotides lacking a nucleic acid base), for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more debasalized nucleotides. The nucleic acid base may be replaced by -H (idSp) or -OH in the debasalized nucleotide. The debasalized spacer may be inserted into the target polynucleotide by removing a nucleic acid base from one or more adjacent nucleotides. For example, the polynucleotide may be modified to contain 3-methyladenine, 7-methylguanine, 1,N6-ethenoadenine inosine, or hypoxanthine, and the nucleic acid base may be removed from these nucleotides using human alkyladenine DNA glycosylase (hAAG). Alternatively, the polynucleotide may be modified to contain uracil, and the nucleic acid base may be removed by uracil DNA glycosylase (UDG). In one embodiment, the one or more spacers do not contain any debasalized nucleotides.

[0225] A method for stalling polynucleotide handling proteins, such as helicases, on a polynucleotide adapter using a spacer is described in WO2014 / 135838, which is incorporated in its entirety herein by reference.

[0226] In some embodiments, the construct includes a stalled portion, and the polynucleotide handling protein is positioned so that the stalled portion is located between the polynucleotide handling protein and the target polypeptide before the target polypeptide is rearranged through the nanopores. In some embodiments, this may be advantageous because it prevents the polynucleotide handling protein from processing the portion of the construct corresponding to the target polypeptide before the start of the measurement.

[0227] In some embodiments, the construct includes a stalled portion at the junction between the target polypeptide and one or more polynucleotide flanking chains. In some embodiments, one or more flanking chains are conjugated to the target polypeptide chain via click chemistry, as described in more detail herein. In some embodiments, the polynucleotide handling protein stalls at the click chemistry junction.

[0228] anchor In some embodiments, the polynucleotide, its conjugate with a polypeptide, or an adapter attached thereto may include, for example, a membrane anchor or transmembrane pore anchor attached to the adapter. In one embodiment, the anchor assists in the characterization of the conjugate by the methods disclosed herein. For example, the membrane anchor or transmembrane pore anchor may facilitate the localization of the conjugate around nanopores in the membrane.

[0229] The anchor may be a polypeptide anchor and / or a hydrophobic anchor that can be inserted into the membrane. In one embodiment, the hydrophobic anchor is a lipid, fatty acid, sterol, carbon nanotube, polypeptide, protein, or amino acid, for example, cholesterol, palmitate, or tocopherol. The anchor may also include thiols, biotin, or surfactants.

[0230] In one embodiment, the anchor may be biotin (for binding to streptavidin), amylose (for binding to maltose-binding proteins or fusion proteins), Ni-NTA (for binding to polyhistidine or polyhistidine-tagged proteins), or a peptide (such as an antigen).

[0231] In one embodiment, the anchor may comprise one linker, or two, three, four or more linkers. Preferred linkers include, but are not limited to, polymers such as polynucleotides, polyethylene glycol (PEG), polysaccharides, and polypeptides. These linkers may be linear, branched, or cyclic. For example, the linker may be a cyclic polynucleotide. The adapter may hybridize to a complementary sequence on the cyclic polynucleotide linker. One or more anchors or one or more linkers may comprise components that can be cleaved or degraded, such as limiting sites or photodissociable groups. The linker is functionalized with maleimide groups to attach to cysteine ​​residues of the protein. Preferred linkers are described in WO2010 / 086602.

[0232] In one embodiment, the anchor is cholesterol or a fatty acyl chain. For example, any fatty acyl chain having a carbon atom length of 6 to 30, such as hexadecanoic acid, can be used. Examples of suitable anchors and methods for attaching the anchor to the adapter are disclosed in WO2012 / 164270 and WO2015 / 150786.

[0233] Control of conjugate migration to nanopores As described above, the methods provided herein include controlling the movement of a polynucleotide-polypeptide conjugate chain optionally attached (e.g., hybridized) to a carrier chain as the conjugate or construct moves relative to the nanopore.

[0234] The movement of the conjugate relative to the nanopore can be driven by any suitable means. In some embodiments, the movement of the conjugate is driven by a physical or chemical force (potential). In some embodiments, the physical force is provided by an electrical (e.g., voltage) potential or a temperature gradient, etc.

[0235] In some embodiments, the movement of the construct includes mechanically manipulating the construct, the polynucleotide-polypeptide conjugate chain, and / or the polynucleotide carrier chain, thereby moving the construct, the polynucleotide-polypeptide conjugate chain, and / or the polynucleotide carrier chain relative to the nanopore. In some embodiments, the movement of the construct by mechanical manipulation does not include using a polynucleotide handling protein.

[0236] In some embodiments, the construct, the polynucleotide-polypeptide conjugate chain, and / or the polynucleotide carrier chain are moved by mechanical manipulation in a direction opposite to the potential applied across the nanopore. In some embodiments, the potential is a voltage potential applied across the nanopore. In some embodiments, the construct, the polynucleotide-polypeptide conjugate chain, and / or the polynucleotide carrier chain move relative to the nanopore as described in WO2020 / 128517, the entire contents of which are incorporated herein by reference, particularly with respect to the discussion in that document of the movement of polynucleotides relative to nanoreactors.

[0237] In some embodiments, when a potential is applied across the nanopore, the conjugate moves toward the nanopore. Since polynucleotides are negatively charged, when a potential is applied across the nanopore, the polynucleotide moves toward the nanopore under the influence of the applied potential. For example, when a positive potential is applied to the trans side of the nanopore relative to the cis side, the negatively charged analyte is induced to move from the cis side to the trans side of the nanopore. Similarly, when a positive potential is applied to the trans side of the nanopore relative to the cis side, this prevents the negatively charged analyte from moving from the trans side to the cis side of the nanopore. The opposite occurs when a negative potential is applied to the trans side of the nanopore relative to the cis side. Apparatus and methods for applying appropriate voltages are described in more detail herein.

[0238] In some embodiments, the chemical force is provided by a concentration (e.g., pH) gradient.

[0239] In some embodiments, the movement of conjugates or constructs into nanopores is controlled using methods described in WO2020 / 016573, the entire content of which is incorporated herein by reference.

[0240] In some embodiments, the termination group may be non-covalently bonded to the construct before the method is carried out. A termination group, such as a short oligonucleotide that can hybridize to the construct, can temporarily halt the movement of the construct into the nanopore. This can be usefully used to temporarily retain the polypeptide portion of a polynucleotide-polypeptide conjugate chain within the pore, increasing the time available for its characterization.

[0241] In some embodiments, polynucleotide handling proteins control the movement of constructs, polynucleotide-polypeptide conjugate chains, and / or carrier chains in the same direction as a physical or chemical force (electric potential). For example, in some embodiments, a positive voltage potential is applied to the trans side of the nanopore relative to the cis side, and the polynucleotide handling protein controls the movement of constructs from the cis side to the trans side of the nanopore. In some embodiments, a positive voltage potential is applied to the cis side of the nanopore relative to the trans side, and the polynucleotide handling protein controls the movement of constructs from the trans side to the cis side of the nanopore.

[0242] In some embodiments, polynucleotide handling proteins control the movement of constructs, polynucleotide-polypeptide conjugate chains, and / or carrier chains in the opposite direction to a physical or chemical force (potential). For example, in some embodiments, a positive voltage potential is applied to the trans side of the nanopore relative to the cis side, and the polynucleotide handling protein controls the movement of constructs from the trans side to the cis side of the nanopore. In some embodiments, a positive voltage potential is applied to the cis side of the nanopore relative to the trans side, and the polynucleotide handling protein controls the movement of constructs from the cis side to the trans side of the nanopore.

[0243] In some embodiments, the movement of the construct, polynucleotide-polypeptide conjugate chain and / or carrier chain is driven by a polynucleotide handling protein in the absence of an applied potential.

[0244] In the disclosed method, polynucleotide handling proteins can typically control the movement of constructs, polynucleotide-polypeptide conjugate chains, and / or carrier chains into nanopores. In other words, polynucleotide handling proteins can control the movement of constructs.

[0245] In some embodiments, the disclosed method comprises contacting a construct with a polynucleotide handling protein capable of controlling the movement of one or more polynucleotide adjacent chains and / or polynucleotide carrier chains, the polynucleotide handling protein controlling the movement of the construct into nanopores.

[0246] In some embodiments, the disclosed method comprises contacting both the polynucleotide-polypeptide conjugate chain and the polynucleotide carrier chain of a construct with a polynucleotide handling protein that can control the movement of the polynucleotide-polypeptide conjugate chain and / or the polynucleotide carrier chain, the polynucleotide handling protein controlling the movement of the construct into nanopores.

[0247] In some embodiments, the disclosed method comprises contacting a polynucleotide-polypeptide conjugate chain with a polynucleotide handling protein that can control the movement of the polynucleotide-polypeptide conjugate chain, the polynucleotide handling protein controlling the movement of the construct into the nanopores.

[0248] In some embodiments, the disclosed method comprises contacting a polynucleotide carrier chain with a polynucleotide handling protein that can control the movement of the polynucleotide carrier chain, the polynucleotide handling protein controlling the movement of a construct into nanopores.

[0249] As the construct moves into the nanopores, the target polypeptide of the construct moves into the nanopores, thereby allowing for characterization.

[0250] Suitable polynucleotide handling proteins are also known as motor proteins or polynucleotide handling enzymes. Several suitable polynucleotide handling proteins are known in the art, and some exemplary polynucleotide handling proteins are described in more detail below.

[0251] In one embodiment, the motor protein is or derived from a polynucleotide handling enzyme. A polynucleotide handling enzyme is a polypeptide that interacts with a polynucleotide and can modify at least one of its properties. The enzyme may modify the polynucleotide by cleaving it to form individual nucleotides or shorter chains of nucleotides, such as di- or trinucleotides. The enzyme may also modify the polynucleotide by orienting it or moving it to a specific position.

[0252] In some embodiments, the polynucleotide handling protein may be present on the construct before contact with the nanopores. For example, the polynucleotide handling protein may be present on the polynucleotides in the conjugate (e.g., adjacent strands or carrier strands). In some embodiments, the polynucleotide handling protein may be present on an adapter containing part of the conjugate, or on a portion of the conjugate.

[0253] In some embodiments, if a portion of the polynucleotide-polypeptide conjugate chain that comes into contact with the active site of the polynucleotide handling protein contains a polypeptide, the polynucleotide handling protein can maintain its binding to the polynucleotide-polypeptide conjugate chain. In other words, in some embodiments, the polynucleotide handling protein does not dissociate from the polynucleotide-polypeptide conjugate chain when it comes into contact with the polypeptide portion of the polynucleotide-polypeptide conjugate chain. In some embodiments, the polynucleotide handling protein moves freely relative to the polypeptide portion until it comes into contact with one or more subsequent polynucleotide portions of the polynucleotide-polypeptide conjugate chain.

[0254] In some embodiments, polynucleotide handling proteins are modified to prevent them from unassociating with a construct (e.g., from the polynucleotide-polypeptide conjugate chain or carrier chain) (other than by passing off) when they come into contact with a portion of a polypeptide-containing conjugate. Such modified polynucleotide handling proteins are particularly suitable for use in the disclosed methods.

[0255] Polynucleotide handling proteins can be adapted in any preferred manner. For example, a polynucleotide handling protein may be modified to prevent it from being loaded onto a construct, a polynucleotide-polypeptide conjugate chain, and / or a polynucleotide carrier chain. Alternatively, the protein may be modified to prevent it from being unassociated before being loaded onto the construct, the polynucleotide-polypeptide conjugate chain, and / or the polynucleotide carrier chain. Modification of a polynucleotide handling protein to prevent it from unassociating from a construct, the polynucleotide-polypeptide conjugate chain, and / or the polynucleotide carrier chain can be achieved using methods known in the art, for example, the methods discussed in WO2014 / 013260, which is incorporated entirely herein by reference, and with particular reference to the section describing the modification of polynucleotide handling proteins (polynucleotide-binding proteins), such as helicases, to prevent the polynucleotide handling protein from unassociating from a polynucleotide chain.

[0256] For example, a polynucleotide-handling protein may have a polynucleotide-unbinding aperture through which a polynucleotide strand can pass when the polynucleotide-handling protein dissociates from the polynucleotide strand, such as a cavity, groove, or void. In some embodiments, the polynucleotide-unbinding aperture of a given motor protein (polynucleotide-handling protein) can be determined by reference to its structure, such as by reference to its X-ray crystal structure. The X-ray crystal structure can be obtained in the presence and / or absence of a polynucleotide substrate. In some embodiments, the position of the polynucleotide-unbinding aperture in a given polynucleotide-handling protein can be estimated or confirmed by molecular modeling using standard packages known in the art. In some embodiments, the polynucleotide-unbinding aperture can be transiently generated by the movement of one or more portions of the polynucleotide-handling protein, such as one or more domains.

[0257] A polynucleotide-handling protein (motor protein) can be modified by closing the polynucleotide-unbinding aperture. Thus, closing the polynucleotide-unbinding aperture can prevent the polynucleotide-handling protein from dissociating from the polypeptide portion of the conjugate and can also prevent it from dissociating from the polynucleotide or adapter. For example, a motor protein can be modified by covalently closing the polynucleotide-non-binding aperture. In some embodiments, a preferred motor protein for dealing with this in this way is a helicase as described herein. Thus, in some embodiments of the disclosed methods, a polynucleotide-handling protein is modified to completely or partially close an aperture that is present in at least one conformational state of the unmodified protein through which the polynucleotide strand can break its bond.

[0258] The polynucleotide handling protein is selected or may be selected according to the polynucleotide used in the polynucleotide-polypeptide conjugate chain and / or carrier chain used in the methods disclosed herein. Alternatively, the polynucleotide of the polynucleotide-polypeptide conjugate chain and / or carrier chain is selected or may be selected according to the polynucleotide handling protein used to control the movement of the conjugate. For example, if the polynucleotide is DNA, a DNA motor protein can typically be used. If the polynucleotide is RNA, an RNA motor protein can be used. If the polynucleotide is a DNA-RNA hybrid, a motor protein capable of processing both DNA and RNA can be used.

[0259] In one embodiment, the motor protein is derived from one of the members of the enzyme classification (EC) groups 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 3.1.31.

[0260] In some embodiments of the claimed method, the motor protein is a helicase, polymerase, exonuclease, topoisomerase, or a variant thereof.

[0261] In one embodiment, the motor protein is an exonuclease. Preferred enzymes include, but are not limited to, exonuclease I (SEQ ID NO: 1) derived from E. coli, exonuclease III enzyme (SEQ ID NO: 2) derived from E. coli, RecJ (SEQ ID NO: 3) and bacteriophage lambda exonuclease (SEQ ID NO: 4) derived from T. thermophilus, TatD exonuclease, and their variants. Three subunits, including the sequence shown in SEQ ID NO: 3 or its variants, interact to form a trimer exonuclease.

[0262] In one embodiment, the motor protein is a polymerase. The polymerase may be PyroPhage® 3173 DNA polymerase (commercially available from Lucigen® Corporation), SD polymerase (commercially available from Bioron®), Klenow from NEB, or variants thereof. In one embodiment, the enzyme is Phi29 DNA polymerase (SEQ ID NO: 5) or a variant thereof. A modified version of Phi29 polymerase that can be used in the disclosed method is disclosed in U.S. Patent No. 5,576,204.

[0263] In embodiments of the methods provided herein, which include controlling the movement of constructs, polynucleotide-polypeptide conjugate chains and / or polynucleotide carrier chains by synthesizing chains complementary to polynucleotides, the polynucleotide handling protein is typically a polymerase, such as the polymerase described herein.

[0264] In one embodiment, the polynucleotide handling protein is a topoisomerase. In one embodiment, the topoisomerase is a member of either subgroup (EC) 5.99.1.2 or 5.99.1.3. The topoisomerase may be a reverse transcriptase, which is an enzyme capable of catalyzing the formation of cDNA from an RNA template. These are commercially available, for example, from New England Biolabs® and Invitrogen®.

[0265] In one embodiment, the polynucleotide handling protein is a translocase. Examples include translocases in the FtsK and SpoIII families.

[0266] In one embodiment, the polynucleotide handling protein is a helicase. Any suitable helicase can be used according to the methods provided herein. For example, the motor protein or each motor protein used according to this disclosure may be independently selected from Hel308 helicase, RecD helicase, TraI helicase, TrwC helicase, XPD helicase, and Dda helicase, or variants thereof. Monomer helicases may comprise several domains attached together. For example, TraI helicase and TraI subgroup helicases may comprise two RecD helicase domains, a relaxase domain, and a C-terminal domain. These domains typically form a monomer helicase that can function without forming an oligomer. Specific examples of suitable helicases include Hel308, NS3, Dda, UvrD, Rep, PcrA, Pif1, and TraI. These helicases typically act on single-stranded DNA. Examples of helicases that can move along both strands of double-stranded DNA include the FtsK and hexamer enzyme complex, or multi-subunit complexes such as RecBCD, which are particularly suitable for some embodiments disclosed herein. NS3 helicases are particularly suitable for use in the disclosed methods because they can process both DNA and RNA and can therefore be used in embodiments of the disclosed methods where the target double-stranded nucleic acid is a DNA-RNA hybrid.

[0267] The Hel308 helicase is described in its entirety in publications such as WO2013 / 057495, which is incorporated by reference. The RecD helicase is described in its entirety in publications such as WO2013 / 098562, which is incorporated by reference. The XPD helicase is described in its entirety in publications such as WO2013 / 098561, which is incorporated by reference. The Dda helicase is described in its entirety in publications such as WO2015 / 055981 and WO2016 / 055777, which are incorporated by reference.

[0268] In one embodiment, the helicase includes the sequence shown in SEQ ID NO: 6 (Trwc Cba) 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. The variant may differ from the natural sequence in any of the ways discussed herein. An exemplary variant of SEQ ID NO: 8 includes E94C / A360C. Further exemplary variants of SEQ ID NO: 8 include E94C / A360C followed by (ΔM1)G1G2 (i.e., deletion of M1 followed by addition of G1 and G2).

[0269] In some embodiments, a motor protein (e.g., helicase) has at least two active operating modes (all the components necessary for the motor protein to facilitate movement, e.g., ATP and Mg as discussed herein). 2+ The movement of constructs, polynucleotide-polypeptide conjugate chains, and / or polynucleotide carrier chains can be controlled in a single inactive mode (when fuels and cofactors are present) and in a single inactive mode (when the motor protein does not have the components necessary to facilitate movement).

[0270] When all the necessary components for facilitating movement are present (i.e., in active mode), a motor protein (egericase) moves along the polynucleotide in a 5' to 3' or 3' to 5' direction (depending on the motor protein). A motor protein can be used to move a construct, polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain away from a pore (e.g., against an applied force) or toward a pore (e.g., toward a cis chamber). For example, if the ends of a construct, polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain toward which a motor protein is moving are trapped by a pore, the motor protein will act against the direction of the force and pull the threaded chain out of the pore (e.g., into a cis chamber). However, if the detached end of the motor protein is trapped within the pore, the motor protein acts according to the direction of the force, pushing the threaded chain into the pore (for example, into the trans chamber).

[0271] When a motor protein (e.g., a helicase) lacks the necessary components to facilitate movement (i.e., in inactive mode), the motor protein can act as a brake, delaying the movement of the construct, polynucleotide-polypeptide conjugate chain, and / or polynucleotide carrier chain when it moves toward a nanopore, for example by being drawn into the pore by force. In inactive mode, it doesn't matter which end of the construct, polynucleotide-polypeptide conjugate chain, and / or polynucleotide carrier chain is captured; it is the applied force that determines movement toward the pore, and the polynucleotide-binding protein acts as a brake. In inactive mode, the control of movement by the polynucleotide-binding protein can be described in several ways, including ratcheting, sliding, and braking.

[0272] Motor proteins typically require fuel to handle the processing of polynucleotides. This fuel is typically free nucleotides or free nucleotide analogs. Free nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), and deoxyadenosine monophosphate. The free nucleotide may be one or more of the following: adenosine triphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), and deoxycytidine triphosphate (dCTP). The free nucleotide is usually selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, or dCMP. The free nucleotide is typically adenosine triphosphate (ATP).

[0273] A motor protein cofactor is a factor that enables the motor protein to function. The cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg 2+ Mn 2+ Ca 2+ , or Cole 2+ The cofactor is most preferably Mg 2+ That is the case.

[0274] nanopores As described above, the methods disclosed herein include using polynucleotide handling proteins to control the movement of conjugates into nanopores.

[0275] The disclosed method can use any suitable nanopores. In one embodiment, the nanopores are transmembrane pores.

[0276] Transmembrane pores are structures that extend to some extent across a membrane. They allow hydrated ions, driven by an applied potential, to flow across or within the membrane. Transmembrane pores typically traverse the entire membrane, allowing hydrated ions to flow from one side of the membrane to the other. However, transmembrane pores do not necessarily have to traverse the membrane; they may be closed at one end. For example, pores can be wells, gaps, channels, trenches, or slits within a membrane, through which hydrated ions can flow or into which they flow.

[0277] Any suitable transmembrane pore can be used in the manner provided herein. The pore may be biological or artificial. Suitable pores include, but are not limited to, protein pores, polynucleotide pores, and solid-state pores.

[0278] In one embodiment, the solid-state pore may include nanochannels. In some embodiments, the solid-state pore is a pore disclosed in WO2003 / 003446, WO2009 / 020682, or WO2016 / 187519, each of which is incorporated by reference.

[0279] In one embodiment, the pores may be DNA origami pores (Langecker et al., Science, 2012;338:932-936). Preferred DNA origami pores are disclosed in WO2013 / 083983, WO2018 / 011603, and WO2020 / 025974, each of which is incorporated by reference.

[0280] In one embodiment, the nanopores are scaffolded polypeptide nanopores. In some embodiments, the pores are scaffolded polypeptide nanopores disclosed in WO2020 / 025909 or WO2020 / 074399, each of which is incorporated by reference as a whole.

[0281] In one embodiment, the nanopores are transmembrane protein pores. Transmembrane protein pores are polypeptides or aggregates of polypeptides that allow hydrated ions, such as polynucleotides, to flow from one side of a membrane to the other. In the methods provided herein, transmembrane protein pores can form pores that allow hydrated ions, driven by an applied potential, to flow from one side of a membrane to the other. The transmembrane protein pores preferably allow polynucleotides to flow from one side of a membrane to the other, such as a triblock copolymer membrane. The transmembrane protein pores allow polynucleotides to move through the pores.

[0282] In one embodiment, the nanopores are transmembrane protein pores that are monomers or oligomers. The pores are preferably composed of several repeating subunits, such as 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 14, at least 15, or at least 16 subunits. The pores are preferably hexamers, heptamers, octamers, or nanomeric pores. The pores may be homooligomers or heterooligomers.

[0283] In one embodiment, a transmembrane protein pore includes a barrel or channel through which ions can flow. The pore subunit typically surrounds a central axis and contributes chains to a transmembrane β-barrel or channel or a transmembrane α-helix bundle or channel.

[0284] Typically, the barrel or channel of a transmembrane protein pore contains amino acids that facilitate interaction with an analyte, such as a target polypeptide (as described herein). These amino acids are preferably located near the constriction of the barrel or channel. The transmembrane protein pore typically contains one or more positively charged amino acids, such as arginine, lysine, or histidine, or aromatic amino acids, such as tyrosine or tryptophan. These amino acids typically facilitate interaction between the pore and nucleotides, polynucleotides, or nucleic acids.

[0285] In one embodiment, the nanopores are transmembrane protein pores derived from β-barrel pores or α-helix bundle pores. β-barrel pores include barrels or channels formed from β-chains. Suitable β-barrel pores include, but are not limited to, β-toxins such as α-hemolytic toxins, anthrax toxins, and leucocidines, as well as bacterial outer membrane proteins / porins such as Mycobacterium smegmatis porins (Msp), e.g., MspA, MspB, MspC, or MspD, CsgG, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A and Neisseria autotransporter lipoprotein (NalP), and other pores such as lysenin. α-helix bundle pores include barrels or channels formed from α-helices. Suitable α-helix bundle pores include, but are not limited to, inner membrane proteins and α-outer membrane proteins, e.g., WZA and ClyA toxins.

[0286] In one embodiment, the nanopores are transmembrane pores derived from or based on Msp, α-hemolysin (α-HL), lysenin, CsgG, ClyA, Sp1, or the hemolytic protein fragaceatoxin C (FraC).

[0287] In one embodiment, the nanopores are transmembrane protein pores derived from CsgG, for example, E. coli strain K-12 sub-strain MC4100. Such pores are oligomers and typically contain 7, 8, 9, or 10 monomers derived from CsgG. The pores may be homooligomeric pores derived from CsgG containing the same monomers. Alternatively, the pores may be heterooligomeric pores derived from CsgG containing at least one monomer distinct from the others. Examples of suitable pores derived from CsgG are disclosed in WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2017 / 149318, and WO2019 / 002893, each of which is incorporated herein in whole by reference.

[0288] In one embodiment, the nanopores are transmembrane pores derived from lysenine. Examples of suitable lysenine-derived pores are disclosed in WO2013 / 153359, which is incorporated in whole by reference herein.

[0289] In one embodiment, the nanopores are transmembrane pores derived from or based on α-hemolysin (α-HL). Wild-type α-hemolysin pores are formed from seven identical monomers or subunits (i.e., they are heptamers). The α-hemolysin pores may be α-hemolysin-NN or a variant thereof. The variants preferably contain N residues at positions E111 and K147.

[0290] In one embodiment, the nanopores are transmembrane protein pores derived from Msp, for example, MspA. Examples of suitable pores derived from MspA are disclosed in WO2012 / 107778.

[0291] In one embodiment, the nanopores are transmembrane pores derived from or based on ClyA. Examples of suitable ClyA-derived pores are disclosed in Soskine et al., Nano Letters 2012 12(9), 4895-4900, WO 2014 / 153625, and WO 2017 / 098322, respectively, which are incorporated herein by reference.

[0292] In one embodiment, the nanopores are transmembrane pores derived from Phi29. Examples of suitable pores derived from Phi29 are disclosed in Wendell et al., Nature Nanotech 4, 765-772 (2009), WO 2010 / 062697, WO 2019 / 157365 and WO 2019 / 157424, each incorporated herein by reference.

[0293] In some embodiments, the nanopores are selected from portal proteins including M-ring protein, perforin-2, PlyAB (pleurotolysin), SpoIIIAG, VirB7, type II secretory protein D, GspD, InvG, PilQ, pentraxin, and T4, T7, P23_45, G20c, and Phi29 nanopores.

[0294] In one embodiment, the nanopores are transmembrane pores derived from or based on Rhodococcus species bacteria, such as Rhodococcus corynebacteroides or Rhodococcus ruber, such as PorARr, PorBRr, or PorARc. Examples of such pores are described in Piselli et al., Eur Biophys J 51, 309-323 (2022).

[0295] As described above, in some embodiments, nanopores include constriction. Constriction is typically a narrowing of the channel through the nanopore, which can determine or control the signal obtained when a conjugate moves through the nanopore. As used herein, both protein and solid-state nanopores may include "constriction".

[0296] In some embodiments, nanopores are designed, modified, or selected to have constrictions sized according to the diameter of the construct. In some embodiments, the pores have constrictions with a diameter of at least 1 nm, e.g., at least 1.5 nm, e.g., at least 2 nm, e.g., at least 2.5 nm, e.g., at least 3 nm. In some embodiments, the pores have constrictions with a diameter of about 1.5 to about 2.5 nm. In some embodiments, the pores have constrictions that can transpose double-stranded DNA. Double-stranded DNA has a diameter of about 2 nm. The DNA peptide chimera may be narrower or wider than the double-stranded DNA, depending on the interactions of amino acids and strands.

[0297] In some embodiments, the nanopores are modified to extend the distance between the polynucleotide handling protein and the constricted region of the nanopore. Methods for doing so are disclosed in WO2021 / 111125.

[0298] tag In some embodiments of the methods provided herein, for example, tags on nanopores can be used to facilitate the capture of a structure by the nanopores.

[0299] The interaction between the tag on the nanopore and the construct, the binding site on the polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain (e.g., the binding site present on the polynucleotide portion of the conjugate or on the adapter attached to the conjugate, where the binding site can be provided by the anchor or leader sequence of the adapter or by a capture sequence in the double-stranded stem of the adapter) may be reversible. For example, a polynucleotide can bind to the tag on the nanopore, for example, via its adapter, and be released at some point, for example, during the characterization of the polynucleotide by the nanopore and / or during processing by the motor protein. Strong non-covalent bonds (e.g., biotin / avidin) are still reversible and may be useful in some embodiments of the methods described herein. For example, a pair of pore tag and polynucleotide adapter may be designed to provide sufficient interaction between the double-stranded polynucleotide complement (or the portion of the adapter attached to its complement) and the nanopore so that the complement is retained near the nanopore (without separating and diffusing from the nanopore) but can be released from the nanopore when processed.

[0300] The pore tag and polynucleotide adapter may be configured such that the binding strength or affinity of the binding site on the polynucleotide to the tag on the nanopore (e.g., the binding site provided by the adapter's anchor or leader sequence, or by a capture sequence in the adapter's double-stranded stem) is sufficient to maintain the coupling between the nanopore and the polynucleotide until an applied force is applied and the bound polynucleotide is released from the nanopore.

[0301] In some embodiments, the tag or tether is uncharged. This ensures that, if a potential difference exists, the tag or tether will not be drawn into the nanopore under its influence.

[0302] One or more molecules that attract or bind to constructs, polynucleotide-polypeptide conjugate chains and / or polynucleotide carrier chains may be ligated to the nanopores. Any molecule that hybridizes to the conjugate, adapter and / or polynucleotide may be used. The molecules attached to the pores may be selected from PNA tags, PEG linkers, short oligonucleotides, positively charged amino acids and aptamers. Pores having such molecules ligated to them are known in the art. For example, pores with attached short oligonucleotides are disclosed in Howarka et al (2001) Nature Biotech. 19: 636-639 and WO2010 / 086620, and pores containing PEG attached to the lumen of the pore are disclosed in Howarka et al (2000) J.Am.Chem.Soc. 122(11):2411-2416.

[0303] The capture of the construct, polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain may be enhanced in the method herein by using short oligonucleotides attached to nanopores, which contain a sequence complementary to the sequence in the conjugate (e.g., in the leader sequence in the adapter or in another single-stranded sequence).

[0304] In some embodiments, the tag or tether may include or be composed of an oligonucleotide (e.g., DNA, RNA, LNA, BNA, PNA, or morpholino). The oligonucleotide may have a length of about 10 to 30 nucleotides or about 10 to 20 nucleotides. In some embodiments, the oligonucleotide may have at least one modified end (e.g., 3'- or 5'-end) for conjugation to other modification sites or to the surface of a solid substrate, such as beads. The end modifier may be a reactive functional group that can be used for conjugation. Examples of functional groups that can be added include, but are not limited to, amino, carboxyl, thiol, maleimide, aminooxy, and any combination thereof. The functional group may be combined with spacers of different lengths (e.g., C3, C9, C12, spacers 9 and 18) to add physical distance from the ends of the oligonucleotide sequence to the functional group.

[0305] Examples of 3' and / or 5'-terminal modifications of oligonucleotides include, but are not limited to, 3' affinity tags and functional groups for chemical bonding (e.g., 3'-biotin, 3'-primary amine, 3'-disulfideamide, 3'-pyridyldithio, and any combination thereof); 5'-terminal modifications (e.g., 5'-primary ammine, and / or 5'-dabucil); modifications for click chemistry (e.g., 3'-azide, 3'-alkyne, 5'-azide, 5'-alkyne), and any combination thereof.

[0306] In some embodiments, the tag or tether may further include a polymer linker, for example, to facilitate coupling to nanopores. Exemplary polymer linkers include, but are not limited to, polyethylene glycol (PEG). The polymer linker may have a molecular weight of about 500 Da to about 10 kDa (inclusive of both ends) or about 1 kDa to about 5 kDa (inclusive of both ends). The polymer linker (e.g., PEG) can be functionalized with different functional groups, including, but not limited to, maleimide, NHS esters, dibenzocyclooctin (DBCO), azides, biotin, amines, alkynes, aldehydes, and any combination thereof.

[0307] Other examples of tags or tethers include, but are not limited to, His tags, biotin or streptavidin, antibodies that bind to the analyte, aptamers that bind to the analyte, analyte-binding domains such as DNA-binding domains (including peptide zippers like leucine zippers, single-stranded DNA-binding proteins (SSBs)), and any combination thereof.

[0308] Tags or tethers may be attached to the outer surface of nanopores, for example, on the cis side of the membrane, using any method known in the art. For example, one or more tags or tethers can be attached to nanopores via one or more cysteines (cysteine ​​bonds), one or more primary amines such as lysine, one or more non-natural amino acids, one or more histidines (His tags), one or more biotin or streptavidins, one or more antibody-based tags, one or more enzymatic modifications of epitopes (e.g., including acetyltransferases), and any combination thereof. Preferred methods for carrying out such modifications are well known in the art. Preferred non-natural amino acids include, but are not limited to, 4-azido-L-phenylalanine (Faz) and any one of the amino acids numbered 1 to 71 in Figure 1 of Liu CC and Schultz PG, Annu. Rev. Biochem., 2010, 79, 413-444.

[0309] In some embodiments, where one or more tags or tethers are attached to the nanopores via cysteine ​​bonds, one or more cysteines can be introduced by substitution into one or more monomers forming the nanopores. In some embodiments, the nanopores can be chemically modified by the attachment of: (i) 4-phenylazomareinanyl, 1.N-(2-hydroxyethyl)maleimide, N-cyclohexylmaleimide, 1.3-maleimidopropionic acid, 1.1-4-aminophenyl-1H-pyrrole,2,5,dione, 1.1-4-hydroxyphenyl-1H-pyrrole,2,5,dione, N-ethylmaleimide, N-methoxycarbonylmaleimide N-tert-butylmaleimide, N-(2-aminoethyl)maleimide, 3-maleimide-proxyl, N-(4-chlorophenyl)maleimide, 1-[4-(dimethylamino)-3,5-dinitrophenyl]-1H-pyrrole-2,5-dione, N-[4-(2-benzimidazolyl)phenyl]maleimide, N-[4-(2-benzoxazolyl)phenyl]maleimide, N-(1-naphthyl)-maleimide, N-(2,4 -Xylyl)maleimide, N-(2,4-difluorophenyl)maleimide, N-(3-chloro-para-tolyl)-maleimide, 1-(2-amino-ethyl)-pyrrole-2,5-dione hydrochloride, 1-cyclopentyl-3-methyl-2,5-dihydro-1H-pyrrole-2,5-dione, 1-(3-aminopropyl)-2,5-dihydro-1H-pyrrole-2,5-dione hydrochloride, 3-methyl-1-[2-oxo-2 -(piperazin-1-yl)ethyl]-2,5-dihydro-1H-pyrrole-2,5-dione hydrochloride, 1-benzyl-2,5-dihydro-1H-pyrrole-2,5-dione, 3-methyl-1-(3,3,3-trifluoropropyl)-2,5-dihydro-1H-pyrrole-2,5-dione, 1-[4-(methylamino)cyclohexyl]-2,5-dihydro-1H-pyrrole-2,5-dione trifluoroacetic acid, SMILES O=C1C=CC(=O)N1CC=2C=CN=CC2, SMILES O=C1C=CC(=O)N1CN2CCNCC2, 1-benzyl-3-methyl-2,5-dihydro-1H-pyrrole-2,5-dione, 1-(2-fluorophenyl)-3-methyl-2,Maleimides including diabromomaleimides such as 5-dihydro1H-pyrrole-2,5-dione, N-(4-phenoxyphenyl)maleimide, N-(4-nitrophenyl)maleimide, (ii)3-(2-iodoacetamide)-proxyl, N-(cyclopropylmethyl)-2-iodoacetamide, 2-iodo-N-(2-phenylethyl)acetamide, 2-iodo-N-(2,2,2-trifluoroethyl)acetamide, N-(4-acetylphenyl)-2-iodoacetamide, N-(4-(aminosulfonyl)phenyl)-2- iodoacetamides such as iodoacetamide, N-(1,3-benzothiazole-2-yl)-2-iodoacetamide, N-(2,6-(diethylphenyl)-2-iodoacetamide, N-(2-benzoyl-4-chlorophenyl)-2-iodoacetamide, (iii)N-(4-(acetylamino)phenyl)-2-bromoacetamide, N-(2-acetylphenyl)-2-bromoacetamide, 2-bromo-n-(2-cyanophenyl)acetamide, 2-bromo-N-(3-(trifluoromethyl)phenyl)acetamide, N -(2-benzoylphenyl)-2-bromoacetamide, 2-bromo-N-(4-fluorophenyl)-3-methylbutanamide, N-benzyl-2-bromo-N-phenylpropionamide, N-(2-bromo-butyryl)-4-chlorobenzenesulfonamide, 2-bromo-N-methyl-N-phenylacetamide, 2-bromo-N-phenethyl-acetamide, 2-adamantan-1-yl-2-bromo-N-cyclohexyl-acetamide, 2-bromo-N-(2-methylphenyl)butanamide, monobromoacetanilide, etc. Bromoacetamide, (iv) Aldrithiol-2, Aldrithiol-4, Isopropyl disulfide, 1-(Isobutyldisulfanyl)-2-methylpropane, Dibenzyl disulfide, 4-Aminophenyl disulfide, 3-(2-Pyridyldithio)propionic acid, 3-(2-Pyridyldithio)propionic acid hydrazide, 3-(2-Pyridyldithio)propionic acid N-succinimidyl ester, am6amPDP1-βCD and other disulfides, as well as (v) 4-Phenylthiazole-2-thiol, Purpald, 5,6,7,Thiols such as 8-tetrahydroquinazoline-2-thiol.

[0310] In some embodiments, the tag or tether may be attached directly to the nanopore or via one or more linkers. The tag or tether may be attached to the nanopore using a hybridization linker as described in WO2010 / 086602. Alternatively, a peptide linker may be used. The peptide linker is an amino acid sequence. The length, flexibility, and hydrophilicity of the peptide linker are typically designed so as not to interfere with the function of the monomer and the pore. A preferred flexible peptide linker is a stretch of 2 to 20, e.g., 4, 6, 8, 10, or 16 serine and / or glycine amino acids. A more preferred flexible linker comprises (SG)1, (SG)2, (SG)3, (SG)4, (SG)5, and (SG)8, where S is serine and G is glycine. A preferred rigid linker is a stretch of 2 to 30, e.g., 4, 6, 8, 16, or 24 proline amino acids. A more preferred rigid linker is (P) 12 It contains, and P is proline.

[0311] film Typically, in the disclosed method, nanopores are typically present in a membrane. Any suitable membrane can be used in the system.

[0312] The membrane is preferably an amphiphilic layer. The amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, that have both hydrophilic and lipophilic properties. The amphiphilic molecules may be synthetic or naturally occurring. Amphiphilic substances that do not exist naturally and amphiphilic substances that form monolayers are known in the art, and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). A block copolymer is a polymer material in which two or more monomer subunits are polymerized together to produce a single polymer chain. Block copolymers typically have properties contributed by each monomer subunit. However, block copolymers may have unique properties that polymers formed from individual subunits do not have. A block copolymer may be designed such that one of the monomer subunits is hydrophobic (i.e., lipophilic) and the other subunit(s) are hydrophilic in an aqueous medium. In this case, the block copolymer may have amphiphilic properties and may form a structure that mimics a biological membrane. The block copolymer may be a diblock (consisting of two monomer subunits), but may be composed of more than two monomer subunits to form a more complex arrangement that functions as an amphiphilic organism. The copolymer may be a triblock, tetrablock, or pentablock copolymer. The membrane is preferably a triblock copolymer membrane.

[0313] Archaeal bipolar tetraether lipids are naturally occurring lipids that are constructed to form monolayer membranes. These lipids are commonly found in extremophilic, thermophilic, halophilic, and acidophilic bacteria that survive in harsh biological environments. Their stability is thought to stem from the fused properties of the final bilayer. It is straightforward to construct block copolymers that mimic these biological entities by creating triblock polymers with a common hydrophilic-hydrophobic-hydrophilic motif. This material forms monomer membranes that behave similarly to lipid bilayers and can encompass a wide range of phase behaviors, from vesicles to layered membranes. Membranes formed from these triblock copolymers offer several advantages over biological lipid membranes. Because triblock copolymers are synthetic, their precise structure can be carefully controlled to provide the correct chain length and properties necessary for membrane formation and interaction with pores and other proteins.

[0314] Block copolymers may also be constructed from subunits not classified as lipid submaterials; for example, hydrophobic polymers may be made from siloxanes or other non-hydrocarbon monomers. The hydrophilic subsections of block copolymers may also have low protein-binding properties, which allows for the creation of films that are highly resistant when exposed to raw biological samples. These head group units may also originate from non-classical lipid head groups.

[0315] Triblock copolymer membranes also possess increased mechanical and environmental stability compared to biolipid membranes, such as a much higher operating temperature or pH range. The synthetic properties of block copolymers provide a basis for customizing polymer-based membranes for a wide range of applications.

[0316] In some embodiments, the film is one of the films disclosed in International Application No. 2014 / 064443 or 2014 / 064444.

[0317] The amphiphilic molecules may be chemically modified or functionalized to facilitate the coupling of polynucleotides. The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic layer is typically planar. The amphiphilic layer may be curved. The amphiphilic layer may be supported.

[0318] Amphiphilic membranes are typically about 10 -8 cm·s -1 It is naturally mobile, essentially acting as a two-dimensional fluid with a lipid diffusion rate. This means that pores and coupled polynucleotides can move typically within amphiphilic membranes.

[0319] The membrane may be a lipid bilayer. Lipid bilayers are a model of cell membranes and serve as an excellent base for a wide range of experimental studies. For example, lipid bilayers can be used for in vitro investigations of membrane proteins by single-channel recording. Alternatively, lipid bilayers can be used as biosensors for detecting the presence of various substances. The lipid bilayer can be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, planar lipid bilayers, supported bilayers, or liposomes. The lipid bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers are disclosed in WO2008 / 102121, WO2009 / 077734, and WO2006 / 100484.

[0320] Methods for forming lipid bilayers are known in the art. Lipid bilayers are generally formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972; 69: 3561-3566), in which a lipid monolayer is supported on the aqueous solution / air interface, passing through one side of the opening perpendicular to the interface. The lipid is usually added to the surface of the aqueous electrolyte by first dissolving it in an organic solvent and then evaporating a small amount of solvent over the aqueous solution interface on both sides of the opening. As the organic solvent evaporates, the solution / air interfaces on both sides of the opening physically move up and down across the opening until the bilayer is formed. Planar lipid bilayers can be formed in the membrane across the opening or in the recess across the opening.

[0321] The Montal & Mueller method is popular because it is a cost-effective, relatively simple method for forming high-quality lipid bilayers suitable for protein pore insertion. Other common methods for bilayer formation include tip-dipping, painting bilayers, and liposome bilayer patch clamping.

[0322] Tip-immersion bilayer formation involves bringing an opening (e.g., a pipette tip) into contact with the surface of a test solution supporting a lipid monolayer. In this case, too, the lipid monolayer is formed at the solution / air interface by evaporating a small amount of lipid initially dissolved in an organic solvent at the solution surface. The bilayer is then formed by the Langmuir-Shafer method, requiring mechanical automation to move the opening relative to the solution surface.

[0323] In the case of bilayer coating, a small amount of lipid dissolved in an organic solvent is applied directly to the opening immersed in the test aqueous solution. The lipid solution is spread thinly across the opening using a paint brush or equivalent. Diluting the solvent leads to the formation of a lipid bilayer. However, it is difficult to completely remove the solvent from the bilayer, and as a result, the bilayer formed by this method is less stable and prone to generating noise during electrochemical measurements.

[0324] Patch clamping is commonly used in the study of biological cell membranes. The cell membrane is retained at the end of the pipette by aspiration, allowing a patch of membrane to adhere across the opening. This method has been adapted to produce lipid bilayers by retaining and then rupturing liposomes, sealing the lipid bilayer across the pipette opening. This method requires the creation of stable, large monolayer liposomes and small openings in materials with glass surfaces.

[0325] Liposomes can be formed by sonication, extrusion, or the Mozafari method (Colas et al. (2007) Micron 38:841-847).

[0326] In some embodiments, the lipid bilayer is formed as described in International Application No. 2009 / 077734. Advantageously, in this method, the lipid bilayer is formed from dry lipids. In some embodiments, the lipid bilayer is formed across the opening as described in WO2009 / 077734.

[0327] A lipid bilayer is formed from two opposing layers of lipids. These two lipid layers are arranged such that their hydrophobic tail groups face each other, forming a hydrophobic interior. The hydrophilic head groups of the lipids face outward toward the aqueous environment on both sides of the bilayer. Bilayers may exist in several lipid phases, including, but not limited to, liquid disordered phases (fluid layered), liquid ordered phases, solid ordered phases (layered gel phases, comb-shaped gel phases), and planar bilayer crystals (layered subgel phases, lamellar crystal phases).

[0328] Any lipid composition may be used to form a lipid bilayer. The lipid composition is selected so as to form a lipid bilayer having the desired properties, such as surface charge, ability to support membrane proteins, packing density, or mechanical properties. The lipid composition may contain one or more different lipids. For example, the lipid composition may contain up to 100 lipids. The lipid composition preferably contains 1 to 10 lipids. The lipid composition may contain naturally occurring lipids and / or synthetic lipids.

[0329] Lipids typically comprise a head group, an interfacial moiety, and two hydrophobic tail groups, which may be the same or different. Suitable head groups include, but are not limited to, neutral head groups, e.g., diacylglycerides (DG) and ceramides (CM); zwitterionic head groups, e.g., phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM); negatively charged head groups, e.g., phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), and cardiolipin (CA); and positively charged head groups, e.g., trimethylammonium-propane (TAP). Suitable interfacial moieties include, but are not limited to, naturally occurring interfacial moieties, e.g., glycerol-based moieties or ceramide-based moieties. Suitable hydrophobic tail groups include, but are not limited to, saturated hydrocarbon chains such as lauric acid (n-dodecanolic acid), myristic acid (n-tetradecononic acid), palmitic acid (n-hexadecanoic acid), stearic acid (n-octadecanoic acid), and arachidic acid (n-eicosanoic acid), unsaturated hydrocarbon chains such as oleic acid (cis-9-octadecanoic acid), and branched hydrocarbon chains such as phytanoyl. The length of the chain and the position and number of double bonds in the unsaturated hydrocarbon chains may vary. The length of the chain and the position and number of branches, such as methyl groups in the branched hydrocarbon chains, may also vary. The hydrophobic tail groups may be linked to the interface as ethers or esters. The lipids may be mycolic acids.

[0330] Lipids can also be chemically modified. The head or tail groups of lipids can be chemically modified. Suitable lipids with chemically modified head groups include, but are not limited to, PEG-modified lipids, e.g., 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], functionalized PEG lipids, e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)2000], and lipids modified for conjugation, e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl). Suitable lipids with chemically modified tail groups include, but are not limited to, polymerizable lipids such as 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine, fluorinated lipids such as 1-palmitoyl-2-(16-fluoropalmitoyl)-sn-glycero-3-phosphocholine, deuterated lipids such as 1,2-dipalmitoyl-D62-sn-glycero-3-phosphocholine, and ether-binding lipids such as 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine. Lipids may be chemically modified or functionalized to facilitate polynucleotide coupling.

[0331] The amphiphilic layer, for example, a lipid composition, typically contains one or more additives that will affect the properties of the layer. Suitable additives include, but are not limited to, fatty acids, such as palmitic acid, myristic acid, and oleic acid; fatty alcohols, such as palmitic alcohol, myristic alcohol, and oleic alcohol; sterols, such as cholesterol, ergosterol, lanosterol, sitosterol, and stigmasterol; lysophospholipids, such as 1-acyl-2-hydroxy-sn-glycero-3-phosphocholine; and ceramides.

[0332] In another embodiment, the film includes a solid state layer. The solid state layer can be formed from both organic and inorganic materials, including, but not limited to, microelectronic materials, insulating materials such as Si3N4, Al2O3, and SiO, organic and inorganic polymers such as polyamides, plastics such as Teflon®, or elastomers such as two-component addition-cured silicone rubber, and glass. The solid state layer may be formed from graphene. A suitable graphene layer is disclosed in WO2009 / 035647. When the film includes a solid state layer, pores are typically present in the amphiphilic film or layer contained within the solid state layer, for example, in holes, wells, gaps, channels, trenches, or slits within the solid state layer. Those skilled in the art can prepare suitable solid-state / amphiphilic hybrid systems. Suitable systems are disclosed in WO2009 / 020682 and WO2012 / 005857. Any of the amphiphilic membranes or layers discussed above may be used.

[0333] The methods disclosed herein are typically carried out using (i) an artificial amphiphilic layer containing pores, (ii) an isolated, naturally occurring lipid bilayer containing pores, or (iii) a cell into which pores have been inserted. The methods are typically carried out using an artificial amphiphilic layer, such as an artificial triblock copolymer layer. In addition to pores, the layer may contain other transmembrane proteins and / or intramembrane proteins, as well as other molecules. Preferred apparatus and conditions are discussed below. The disclosed methods are typically carried out in vitro.

[0334] conditions The feature evaluation method disclosed may be performed using any apparatus suitable for investigating membrane / pore systems in which pores are inserted into a membrane. The feature evaluation method may be performed using any apparatus suitable for transmembrane pore sensing. For example, the apparatus may include a chamber containing an aqueous solution and a barrier separating the chamber into two sections. The barrier may have openings on which a membrane containing transmembrane pores is formed. Transmembrane pores are described herein.

[0335] The feature evaluation method may be carried out using the apparatus described in WO2008 / 102120, WO2010 / 122293, or WO00 / 28312.

[0336] The feature evaluation method may include, for example, optical measurements as described in WO2016 / 009180 and WO2021 / 198695.

[0337] The characterization method may typically involve measuring the ion current through the pores by measuring an electric current. Alternatively, the flow of ions through the pores may be measured optically, for example, as disclosed in Heron et al: J.Am.Chem.Soc.9 Vol.131, No.5, 2009. Thus, the apparatus may also include an electrical circuit capable of applying a potential and measuring electrical signals across the membrane and pores. The characterization method may be performed using a patch clamp or a voltage clamp. The characterization method preferably involves the use of a voltage clamp. The feature evaluation method can be performed on a silicon-based well array, where each array has 128, 256, 512, 1024, 2000, 3000, 4000, 6000, 10000, 12000, 15000, or more wells.

[0338] The feature evaluation method may include measuring the current flowing through the pores. This method is typically performed using a voltage applied across the membrane and pores. The voltage used is typically +2V to -2V, and typically -400mV to +400mV. The voltage used is preferably in a range having a lower limit selected from -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV, and 0mV, and an upper limit independently selected from +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300mV, and +400mV. The voltage used is more preferably in the range of 100mV to 240mV, and most preferably in the range of 120mV to 220mV. By using increased applied potentials, it is possible to increase the differentiation between different nucleotides in each pore.

[0339] The characterization method is typically performed in the presence of any charge carrier, such as metal salts, e.g., alkali metal salts, halide salts, chloride salts, e.g., alkali metal chloride salts. Examples of charge carriers include ionic liquids or organic salts, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, or 1-ethyl-3-methylimidazolium chloride. In the exemplary apparatus considered above, the salt is present in an aqueous solution within the chamber. Potassium chloride (KCl), sodium chloride (NaCl), or cesium chloride (CsCl) are typically used. KCl is preferred. The salt may be an alkaline earth metal salt such as calcium chloride (CaCl2). The salt concentration may be saturated. The salt concentration may be 3 M or less, and is typically 0.1–2.5 M, 0.3–1.9 M, 0.5–1.8 M, 0.7–1.7 M, 0.9–1.6 M, or 1 M–1.4 M. The salt concentration is preferably 150 mM to 1 M. Characterization methods may be performed using salt concentrations of at least 0.3 M, e.g., at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M, or at least 3.0 M. Higher salt concentrations provide a high signal-to-noise ratio, enabling the identification of currents showing coupling / uncoupling to the background of normal current fluctuations.

[0340] The characterization method is typically performed in the presence of a buffer. In the exemplary apparatus discussed above, 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-HCl buffer. The method is typically performed at pH values ​​of 4.0–12.0, 4.5–10.0, 5.0–9.0, 5.5–8.8, 6.0–8.7, or 7.0–8.8, or 7.5–8.5. The pH used may be approximately 7.5.

[0341] Characterization methods may be performed at temperatures of 0°C to 100°C, 15°C to 95°C, 16°C to 90°C, 17°C to 85°C, 18°C ​​to 80°C, 19°C to 70°C, or 20°C to 60°C. Characterization methods are typically performed at room temperature. Optionally, characterization methods may be performed at temperatures supporting enzyme function, e.g., around 37°C.

[0342] system Furthermore, what is provided is a system, -(i) a polynucleotide-polypeptide conjugate chain comprising the target polypeptide conjugated to one or more polynucleotide flanking chains at each end of the target polypeptide, and (ii) a polynucleotide carrier chain, -Nanopores that can co-transpose the polynucleotide-polypeptide conjugate chain and adjacent polynucleotide chain of the construct, - A system that includes polynucleotide handling proteins.

[0343] In some embodiments, the constructs, nanopores, and / or polynucleotide handling proteins are as described in detail herein.

[0344] Furthermore, what is provided is a kit, - Nanopores, - A first polynucleotide containing a reactive functional group for conjugation to the first terminus of a target polypeptide, - A second polynucleotide containing a reactive functional group for conjugation to the second terminus of the target polypeptide, - This kit includes polynucleotide handling proteins.

[0345] In some embodiments, the nanopore and polynucleotide handling proteins are as described in detail herein. In some embodiments, the first and second polynucleotides are adjacent chains as described herein.

[0346] The systems and kits may be configured to be used independently with algorithms provided herein, which are adapted to run on a computer system. The algorithms can be adapted to detect information characteristic of polypeptides (e.g., characteristic of polypeptide sequence and / or whether the polypeptide is modified) and to selectively process the signals obtained when a construct containing polypeptides conjugated to polynucleotide flanking chains and hybridized to polynucleotide carrier chains moves into nanopores (i.e., when the chains co-transpose through the pores).

[0347] In some embodiments, the system includes computational means configured to detect information characteristic of polypeptides (e.g., characteristic of polypeptide sequence and / or whether the polypeptide is modified) and to selectively process signals obtained when a conjugate, comprising a polypeptide conjugated to a polynucleotide adjacent chain and a polynucleotide carrier chain, co-transposes through a nanopore. In some embodiments, the system includes receiving means for receiving data from polypeptide detection, processing means for processing signals obtained when the conjugate moves through the nanopore, and output means for outputting the resulting feature evaluation information.

[0348] Characterize target polynucleotide sequences. The methods discussed above can also be applied to the characterization of polynucleotide chains, which may, for example, not contain polypeptide sequences. Those skilled in the art will understand that many of the advantages set forth in the methods disclosed above for characterizing and transporting polynucleotide-polypeptide constructs are equally applicable to methods in which the construct being evaluated does not contain polypeptides. For example, in some embodiments, the target polypeptide portion of the construct described herein may be replaced with a target polynucleotide sequence, and therefore the methods disclosed above may be applied to characterize a target polynucleotide sequence. In such embodiments, the features described above may be generally applicable unless otherwise indicated by context. Thus, for example, polynucleotide chains and their formation, constructs and their formation, polynucleotide handling proteins, nanopores, and general setup and transport schemes may all be as described above. This is further shown below.

[0349] Therefore, what is provided is a method for characterizing a target polynucleotide sequence, -(i) a target polynucleotide chain containing a target polynucleotide sequence is brought into contact with (ii) a polynucleotide carrier chain, thereby forming a double-stranded polynucleotide construct. - The construct is brought into contact with nanopores under conditions such that both the target polynucleotide chain and the polynucleotide carrier chain co-transform through the nanopores. - This includes obtaining one or more measurements characteristic of the target polynucleotide as the construct moves into the nanopores, This method is used to characterize target polynucleotides.

[0350] In some embodiments, the target polynucleotide sequence is conjugated at each end of the target polynucleotide sequence to one or more polynucleotide facies. In some embodiments, each of these one or more polynucleotide facies is independently complementary to a region of the polynucleotide carrier chain. In some embodiments, each of these one or more polynucleotide facies is independently at least partially hybridized to the polynucleotide carrier chain.

[0351] In some embodiments, the target polynucleotide sequence is complementary to the region of the polynucleotide carrier chain. Therefore, in some embodiments, in the construct formed by the target polynucleotide chain and the polynucleotide carrier chain, the target polynucleotide chain and the polynucleotide carrier chain are hybridized together in the region of the target polynucleotide sequence.

[0352] In some embodiments, the target polynucleotide sequence is non-complementary to the polynucleotide carrier chain. Therefore, in some embodiments, in the construct formed by the target polynucleotide chain and the polynucleotide carrier chain, the target polynucleotide chain and the polynucleotide carrier chain are not hybridized together in the region of the target polynucleotide sequence. In some embodiments, the target polynucleotide chain and the polynucleotide carrier chain are hybridized together in the region of the polynucleotide flanking chain, but not in the region of the target polynucleotide sequence.

[0353] Furthermore, what is provided is a method for characterizing a target polynucleotide sequence, -(i) a target polynucleotide chain containing a target polypeptide attached to a polynucleotide flanking chain, and (ii) a polynucleotide handling protein capable of controlling the movement of the polynucleotide flanking chain into a nanopore, - The target polynucleotide chain is brought into contact with a nanopore under conditions such that a polynucleotide handling protein controls the movement of the target polynucleotide chain to the nanopore. - Obtaining one or more measurements characteristic of the target polynucleotide sequence as the polynucleotide adjacent chain and the target polypeptide analyte co-transition through nanopores, This method is used to characterize target polypeptides.

[0354] In some embodiments, prior to such a method, the adjacent polynucleotide strands are at least partially hybridized to the polynucleotide carrier strand.

[0355] In some embodiments of the methods described above, the target polynucleotide chain comprises multiple target nucleotide sequences. For example, in some embodiments, the target nucleotide sequences or each target nucleotide sequence may independently have a length of about 5 to about 1000 nucleotide units. The polynucleotide chain used in the methods described above may be any of the polynucleotide chains described in more detail herein.

[0356] In some embodiments, the target polynucleotide sequence contains different types of nucleotides than the polynucleotide carrier chain and / or the polynucleotide flanking chain. For example, the target polynucleotide sequence may contain or be composed of RNA nucleotides, and the polynucleotide carrier chain and / or flanking chain may contain or be composed of DNA nucleotides. In some embodiments, the target polynucleotide sequence may contain or be composed of DNA nucleotides, and the polynucleotide carrier chain and / or flanking chain may contain or be composed of RNA nucleotides. In some embodiments, the target polynucleotide sequence contains the same types of nucleotides as the polynucleotide carrier chain and / or the polynucleotide flanking chain. For example, the target polynucleotide sequence may contain or be composed of DNA nucleotides, and the polynucleotide carrier chain and / or flanking chain may contain or be composed of DNA nucleotides.

[0357] The target polynucleotide chain, polynucleotide carrier chain, and / or construct can be mechanically manipulated in the context of a polynucleotide-polypeptide conjugate chain in the manner described above. Therefore, the method may include mechanically manipulating the construct, target polynucleotide chain, and / or polynucleotide carrier chain to move the construct, target polynucleotide chain, and / or polynucleotide carrier chain toward a nanopore. The movement may be in the opposite direction to the potential applied across the nanopore, such as a voltage potential applied across the nanopore.

[0358] The above method may involve contacting a construct or its polynucleotide chains with a polynucleotide handling protein capable of controlling the movement of one or more chains of the construct (e.g., one or more adjacent polynucleotide chains and / or target polynucleotide chains), thereby controlling the movement of the construct into nanopores. As will be discussed in detail herein, in some embodiments, the polynucleotide handling protein controls the movement of both chains of the construct. In some embodiments, the polynucleotide handling protein controls the movement of only one of the chains of the construct, e.g., a target polynucleotide chain or a polynucleotide carrier chain.

[0359] The polynucleotide handling proteins may be located on either the cis or trans side of the nanopore and may control the movement of the construct from the cis side to the trans side of the nanopore, or from the trans side to the cis side of the nanopore, as described in more detail herein in the context of methods involving the movement and characterization of polynucleotide-polypeptide chains and constructs containing them.

[0360] The polynucleotide handling protein may be any of the polynucleotide handling proteins described in detail herein. In some embodiments, prior to the method described above, the polynucleotide handling protein is bound to a polynucleotide carrier chain within a region of the polynucleotide carrier chain that extends to the non-hybridization region of the polynucleotide flanking chain. In some embodiments, the polynucleotide handling protein can maintain binding to the construct as described in detail herein. For example, the polynucleotide handling protein may be modified to prevent it from unassociating from the construct, target polynucleotide chain and / or polynucleotide carrier chain. The polynucleotide handling protein may be modified in any manner described herein by being modified to completely or partially close an opening present in at least one conformational state of the unmodified protein that can unassociate a polynucleotide chain. As will be discussed in more detail herein, in some embodiments, the polynucleotide handling protein is or comprises a helicase, translocase, or helicase-nuclease complex.

[0361] In the context of methods involving the movement and characterization of polynucleotide-polypeptide chains and constructs containing them, in some embodiments of the methods discussed above, as will be discussed in more detail herein, the construct includes a stall portion which may be positioned between the polynucleotide handling protein and the target polynucleotide sequence before the method. Adapters, tethers, anchors, and / or blocking portions may further be included in the construct, as will be discussed in more detail above.

[0362] In the context of methods for the movement and characterization of polynucleotide-polypeptide chains and constructs comprising them, as will be discussed in more detail herein, in some embodiments of the methods discussed above, a construct and one or more of its constituent chains may be "floating" through nanopores such that (i) a chain(s) displaces the nanopore in a first direction relative to the nanoparticles, (ii) a chain(s) moves in a direction opposite to the direction of movement relative to the nanoparticles in step (i), and (iii) a chain(s) optionally moves in the first direction, and steps (ii) and (iii) are optionally repeated to cause the chain(s) to vibrate through the nanopores.

[0363] Features that can be measured by the above method may include, for example, (i) the length of the target polynucleotide sequence, (ii) the identification of the target polynucleotide sequence, (iii) the sequence of the target polynucleotide sequence, (iv) the secondary structure of the target polynucleotide sequence, and (v) whether the target polynucleotide is modified.

[0364] The nanopores may be any of the nanopores discussed herein, such as protein nanopores, for example, β-barrel protein nanopores.

[0365] While specific embodiments, configurations, and materials and / or molecules are discussed herein in relation to the methods according to the present invention, it should be understood that various changes or modifications of form and detail may be made without departing from the scope and spirit of the invention. The embodiments described above and the following examples are provided for illustrative purposes only and should not be considered as limiting the use. This application is limited solely by the claims. [Examples]

[0366] Example 1 This example illustrates the distinction between three different peptides contained in peptide analytes and polynucleotide chain-containing constructs, as described herein.

[0367] Distinction was demonstrated using modified transmembrane protein nanopores derived from Rhodococcus. The inner diameter of the nanopores was wide enough to accommodate double-stranded DNA (dsDNA). The pores were formed from monomers having the following amino acid sequence. MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPGLNLSVGNGVAATVTNVLPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK (Sequence ID 9)

[0368] The peptide was conjugated between two dsDNA oligonucleotides using a single-stranded DNA (ssDNA) strand acting as a "carrier" chain and terminal biotin to prevent complete transposition of the conjugate through the nanopore. To simulate the stepwise movement of this conjugate through the nanopore, a series of conjugates were prepared with varying distances between the terminal biotin and the dsDNA base pair (distance "x" as defined in Figure 10).

[0369] Construction All constructs are formed from two DNA oligonucleotides (DNA1 and DNA2) and a peptide, as shown in Figure 10, where DNA1 has a hairpin and a 3'-terminal TCO group, the peptide has an N-terminal azide and a C-terminal methyltetrazine group, and DNA2 has a 5'BCN group and a 3'-biotin group. [ka] A peptide having the following sequence was synthesized. Here, the two central amino acid residues (XX) are changed to DD, RR, or YY, modified with an N-terminal azide, and a C-terminal methyltetrazine is attached via an ethylenediamine linker.

[0370] The SPRI bead mix was prepared by replacing SpeedBead magnetic carboxylate-modified particles (Merck, catalog number GE65152105050250) in SPRI washing buffer (25 mM Tris-Cl (pH 7.5), 2.5 M NaCl, 28% (w / v) PEG-8000) to a final concentration of 0.25% (w / v).

[0371] The construct was assembled in a two-step process involving sequential click reactions. DNA1 oligonucleotide (1 μM) was first reacted with 25 mM HEPES-NaOH (pH 7.5), 500 mM NaCl, and 200 μM peptide in 0.01% (v / v) Tween-20. The reaction mixture was covered with foil and incubated at 37°C for 2 hours, shaking at 800 rpm. The reaction mixture was purified as follows: 3.7-fold excess SPRI bead mix was added, and the mixture was incubated at room temperature for 5 minutes with shaking on a Hula mixer to pelletize the beads. The beads were then washed twice with SPRI washing buffer and eluted with 25 mM HEPES-NaOH (pH 7.5) and 50 mM NaCl. The sample was heated to 95°C for 3 minutes, then rapidly cooled on ice and allowed to stand for at least 10 minutes before further processing.

[0372] Next, a second click reaction was assembled, consisting of a 1 μM peptide-DNA conjugate (product of the first click reaction) and a 4 μM BCN-modified DNA strand (DNA2) in 3.7-fold excess SPRI wash buffer. The reaction mixture was incubated at 37°C for 2 hours with shaking at 1500 rpm. The reaction mixture was purified according to the first click reaction to obtain a polynucleotide-polypeptide conjugate. The assembly was analyzed by denatured and undenatured PAGE. As a control, a conjugate was prepared by annealing two oligonucleotides together, one of which had a 3'-terminal biotin group. All sequences of the oligonucleotides used in this example are listed in Table 3.

[0373] Data acquisition Electrical data was collected using a minion flow cell (Oxford Nanopore Technologies plc) containing the modified Rhodococcus pores described above.

[0374] Samples were prepared by incubating equimolar amounts of monovalent traptabidine with polynucleotide-polypeptide conjugates in electrophoresis buffer (25 mM HEPES-KOH (pH 8.0), 500 mM KCl). At least 1 mL of electrophoresis buffer was first flowed into the flow cell. Electrophoresis buffer alone, or electrophoresis buffer containing each sample, was sequentially added to the flow cell via the SpotON port, with the buffer flowing between each sample addition. Data were acquired at a sample velocity of 5 kHz and an applied potential of 180 mV. A reversal potential of 120 mV was applied at 1-minute intervals to expel each conjugate from the nanopores. The data were analyzed by determining the normalized blockage level caused by the sample relative to the open pore level.

[0375] Results and Conclusions Figure 11 shows raw data collected from a series of samples where the distance between monovalent traptabidine and polypeptide was 11 bp, and the polypeptide sequence (read from N to C) was GGSGDDSGSG (SEQ ID NO: 10), GGSGRRSGSG (SEQ ID NO: 11), or GGSGYYSGSG (SEQ ID NO: 12). Each example shows a peptide-specific blockage level following an open-pore current of approximately 275 pA as the analyte is trapped in the nanopore. This data demonstrates that the nanopore can distinguish between the three peptides tested.

[0376] Figure 12 shows a histogram of normalized blockage levels for a series of analytes where the distance between monovalent traptabidines was 11 bp and the peptide sequence was one of GGSGDDSGSG (SEQ ID NO: 10), GGSGRRSGSG (SEQ ID NO: 11), or GGSGYYSGSG (SEQ ID NO: 12).

[0377] Figure 13 shows aggregated distance data from 6 bp to 36 bp in 5 bp increments, represented as a plot of normalized current (I / I0) against the distance between monovalent traptabidine and the peptide. The data indicates that 11 bp is the most sensitive location for distinguishing peptides using this nanopore. When the distance of the peptide from monovalent traptabidine was greater than 21 bp, the blockage level could not be distinguished from dsDNA alone. The data indicates that the stepwise movement of polynucleotide-polypeptide conjugates through nanopores can generate a unique current-distance trace that depends on the analyzed peptide. [Table 3]

[0378] Example 2 This example demonstrates the repetitive transfer of polynucleotide-polypeptide conjugates through nanopores having an inner diameter wide enough to accommodate double-stranded DNA (dsDNA). The peptide was conjugated between two dsDNA oligonucleotides using single-stranded DNA (ssDNA) as a "carrier" chain.

[0379] Construction Table 4 outlines the sequences of the oligonucleotides used. Three different constructs were generated based on the polypeptide sequences used. [ka] A peptide having the following sequence was synthesized. Here, the two central amino acid residues (XX) are changed to DD, RR, or YY, modified with an N-terminal azide, and linked to a C-terminal methyltetrazine via an ethylenediamine linker. The construct used in this example was assembled according to the method described in Example 1. This construct differs from the one used in Example 1 in that it has a 3' poly(dA) tail instead of terminal biotin to assist in the loading of helicase from solution.

[0380] Data acquisition Electrodynamic data were collected using a minion flow cell (Oxford Nanopore Technologies plc) containing modified Rhodococcus pores, as used in Example 1. Samples were prepared by incubating a 0.2 μM polynucleotide-polypeptide conjugate with 1 μM wild-type Hel308 helicase in either electrophoresis buffer (25 mM HEPES-KOH (pH 8.0), 500 mM KCl) or sequencing buffer (25 mM HEPES-KOH (pH 8.0), 350 mM KCl, 50 mM ATP, 50 mM MgCl2, 2.2 mM EDTA). The electrophoresis buffer lacked ATP and was run as a control for ATP-dependent enzyme motility. At least 1 mL of electrophoresis buffer or sequencing buffer was first run through the flow cell. Then, the sample (75 μL) was introduced into the flow cell via the SpotON port. Electrical data was acquired at a sample velocity of 5 kHz and an applied potential of 180 mV. A reversal potential of 120 mV was applied at 1-minute intervals to expel each conjugate from the nanopore.

[0381] Results and Discussion Figure 14 shows an exemplary trace of the enzyme-driven movement of a polypeptide-polynucleotide conjugate using helicase. The experiment was performed under conditions of excess helicase relative to the conjugate. The presence of repeating traces indicates that the conjugate is captured via its hairpin ends. The bound helicase prevents complete transposition of the conjugate, and then the helicase moves 3'-5' along the ssDNA to control the movement of the dsDNA out of the nanopore. As the helicase approaches the junction between the ssDNA and the peptide, the helicase dissociates from the ssDNA, and the position of the conjugate in the nanopore rapidly descends and returns to a second helicase bound 3' distal to the enzyme, which dissociates, thus the enzyme repeatedly shuttles the conjugate through the nanopore. This movement is ATP-dependent, as shown by the ATP-deficient control which shows only blockage and not a repeating pattern (Figure 15). The data demonstrate that it is possible to repeatedly move polypeptide-polynucleotide conjugates through nanopores using helicase. [Table 4]

[0382] Example 3 This example demonstrates the repetitive movement of a polynucleotide-polypeptide conjugate through a nanopore. The conjugate moves "out" of the nanopore by the Hel308 helicase enzyme. The peptide is conjugated between two dsDNA oligonucleotides.

[0383] The conjugate contains a single-stranded overhang into which a helicase is loaded from solution. The bound helicase prevents the conjugate from completely dislocating through the nanopore, and the helicase moves along the single strand of DNA in the 3' to 5' direction, controlling the movement of the dsDNA and peptide "out" the nanopore. As the helicase approaches the junction in the construct between the DNA and peptide, the helicase dissociates from the single strand of DNA, and the position of the conjugate in the nanopore rapidly descends back to a second helicase loaded from solution onto the construct at a position 3' distal to the first helicase. The cycle then repeats, with the second helicase again controlling the movement of the double-stranded portion of the construct "out" the nanopore. This mechanism can then be repeated with one or more further helicases so that the conjugate repeatedly moves through the nanopore.

[0384] Construction Table 5 outlines the sequences of the oligonucleotides used. Multiple constructs incorporating different peptide sequences were prepared. Peptides were synthesized using the following terminal modifications: an N-terminal azide and a C-terminal methyltetrazine linked via an ethylenediamine linker. This construct includes a 3' poly(dA) tail to assist in helicase loading from solution and a hairpin containing a tethering oligo, both of which are ligated to the ends of the construct. A diagram of the assembled construct is shown in Figure 16. The constructs used in this example were assembled according to the general method described in Example 1. A DNA oligo (a, SEQ ID NO: 29) containing a terminal BCN click group and a negatively charged C3 spacer was first annealed to a tethering sequence (b, SEQ ID NO: 30). The tether increases the analyte capture rate on the flow cell. The annealed oligo was then clicked onto a hairpin DNA (c, SEQ ID NO: 31) having an internal azide click group. The hairpin sequence contains four-base attachment ends to which DNA1(d, SEQ ID NO: 32) and DNA2(e, SEQ ID NO: 33) oligos are ligated. The DNA2 oligo has a terminal 3'TCO group that reacts with tetrazine at the C-terminus of peptide(f). The N-terminus of the peptide has an azide group, which is then clicked to DNA3(g, SEQ ID NO: 34) via a terminal 5'BCN group. [Table 5]

[0385] Data acquisition Electrodynamic data were collected using a minion flow cell (Oxford Nanopore Technologies plc) containing modified Rhodococcus pores, as used in Example 1. Samples were prepared by incubating a 0.2 μM polynucleotide-polypeptide conjugate with 1 μM wild-type Hel308 helicase in either electrophoresis buffer (25 mM HEPES-KOH (pH 8.0), 500 mM KCl) or sequencing buffer (25 mM HEPES-KOH (pH 8.0), 350 mM KCl, 50 mM ATP, 50 mM MgCl2, 2.2 mM EDTA). The electrophoresis buffer lacked ATP and was run as a control for ATP-dependent enzyme motility. At least 1 mL of electrophoresis buffer or sequencing buffer was first run through the flow cell. Then, the sample (75 μL) was introduced into the flow cell via the SpotON port. Electrical data was acquired at a sample velocity of 3 kHz and an applied potential of 160 mV.

[0386] Results and Discussion Figures 17A–F show examples of current-time traces of enzymatically controlled transfer of polypeptide-polynucleotide conjugates using Hel308 helicase. Three different peptide sequences were tested: RSDSGQQARY (SEQ ID NO: 35), GGSGSSSGSG (SEQ ID NO: 36), and EAIYAAPFAKKK (SEQ ID NO: 37) (see legend in the figure). Both reread traces and single-read traces are shown for each peptide. The experiments were performed under conditions of helicase excess with respect to the conjugate. The presence of repetitive traces indicates that the conjugate was captured via the hairpin ends and read multiple times. These traces demonstrate that peptides with different sequences generate their own signals.

[0387] Figures 18A-C show examples of current-time traces of peptides with various charge configurations, ranging from eight negatively charged residues to eight positively charged residues. The following peptides were tested. [Table A]

[0388] The current-time traces shown in Figures 18A-C demonstrate that peptides with various charges can be moved through nanopores. Most notably, peptides with large positive charges (exemplified by peptide A with a charge of +8) can be moved through the pores against electrophoretic forces.

[0389] Example 4 This embodiment demonstrates the "one-pass" migration of a polynucleotide-polypeptide conjugate through a nanopore. The conjugate is moved in the direction of "entering" the pore by Dda helicase. The peptide is conjugated between two dsDNA oligonucleotides. The conjugate contains a hairpin adapter loaded with Dda helicase, which is topologically closed around a single strand of polynucleotide before stalling. The presence of the bound helicase initially prevents the conjugate from completely dislocating through the nanopore. Once the analyte enters the pore, the helicase is released from stall by electrophoretic force and moves along the single strand of DNA in the 5' to 3' direction, controlling the movement of the dsDNA and peptide in the direction of "entering" the nanopore. The helicase is located on the complementary DNA "carrier" strand and does not directly encounter the peptide moving through the pore with the strand that the helicase is dislocating. When the helicase reaches the end of a single strand of the moving polynucleotide, it detaches from the construct, releasing the rest of the conjugate through the pore and ending the reading process.

[0390] Construction Table 6 outlines the sequences of the oligonucleotides used. The construct included a hairpin adapter loaded with an enzyme having a tethering site. A diagram of the assembled construct is shown in Figure 19. The construct used in this example was assembled according to the general method described in Example 1. A DNA oligo (a, SEQ ID NO: 29) containing a terminal BCN click group and a negatively charged C3 spacer was first annealed to a tethering sequence (b, SEQ ID NO: 30). The tether increases the analyte capture rate on the flow cell. The annealed oligo was then clicked onto a hairpin DNA (c, SEQ ID NO: 41) having an internal azido click group. The hairpin contains a Dda helicase that is loaded onto a single-stranded portion and held in place by a disulfide bridge that topologically closes the enzyme around the strand. The loading site is opposed by a 35-nucleotide 2'-O-methyl RNA bubble to provide space for loading the enzyme. The use of the RNA base prevents the helicase from binding to the bubble instead of the intended loading site. The hairpin adapter contains DNA1(d, SEQ ID NO: 42) and DNA2(e, SEQ ID NO: 43) oligos with ligated 4-base attachment ends. The DNA2 oligo has a terminal 3'TCO group that reacts with tetrazine at the C-terminus of peptide(f, SEQ ID NO: 44). The N-terminus of the peptide has an azide group, which is then clicked to DNA3(g, SEQ ID NO: 45) via a terminal 5'BCN group. [Table 6]

[0391] Data acquisition Electroelectric data were collected using a minion flow cell (Oxford Nanopore Technologies plc) containing modified Rhodococcus pores, as used in Example 1. Samples were prepared by mixing a 0.2 μM polynucleotide-polypeptide conjugate loaded with Dda helicase with sequencing buffer (25 mM HEPES-KOH (pH 8.0), 350 mM KCl, 50 mM ATP, 50 mM MgCl2, 2.2 mM EDTA). At least 1 mL of sequencing buffer was first flowed into the flow cell. Then, the sample (75 μL) was introduced into the flow cell via the SpotON port. Electroelectric data were acquired at a sample velocity of 1 kHz and an applied potential of 180 mV. The recording temperature was 21 °C.

[0392] Results and Discussion Figure 20 shows an exemplary trace of the enzymatically controlled movement of a polypeptide-polynucleotide conjugate using Dda helicase in a single-pass mode. The presence of reproducible signal deviations from the dsDNA level (three examples are shown) indicates that the peptide (HDSGDEVHHQK, SEQ ID NO: 46) on the "carrier" chain is co-transposed through the pore. Placing the peptide on the "carrier" reduces the constraints on the length of the peptide that can be analyzed, as the enzyme does not need to diffuse onto the peptide to move the conjugate through the pore.

[0393] Example 5 This embodiment demonstrates the "single-pass" movement of a polynucleotide-polypeptide conjugate through a nanopore. The movement of the conjugate in the direction of "entering" the nanopore is controlled by the Dda helicase enzyme. The peptide is conjugated between two double-stranded (dsDNA) oligonucleotides containing internal click groups rather than terminal click groups, forming a single-strand stretch of DNA alongside the peptide (see Figure 6). A sequencing adapter loaded with helicase is attached to the end of the construct. The helicase is topologically closed around the single-strand DNA portion of the oligonucleotide before stalling. The presence of bound helicase initially prevents the conjugate from completely dislocating through the nanopore. Once the conjugate enters the nanopore, it is released from stall by electrophoretic force, and the helicase then moves along the single-strand of DNA in the 5' to 3' direction. The annealed complementary strand "opens" (separates) as the conjugate moves through the nanopore, resulting in only the DNA strand with the attached peptide moving through the constriction. When the helicase encounters a covalently bound DNA-peptide segment, it diffuses to both strands and re-engages with the DNA on the other side of the DNA-peptide segment. It then continues to control the movement of the DNA peptide, and subsequently controls the movement of the single-stranded DNA in the 5' to 3' direction through the nanopore. The enzyme detaches when it reaches the end of the bound DNA oligonucleotide, releasing the rest of the conjugate through the pore and ending the reading. In this system, the nanopore only needs to have a diameter wide enough to accommodate the co-transposing DNA-peptide (which may have a cross-section narrower than the cross-section of the double-stranded DNA).

[0394] Construction Table 7 outlines the sequences of the oligonucleotides used. The construct included an enzyme-loaded adapter with a ligated tethering site at the end of the construct, and the DNA spreading to the peptide consisted of sections from two oligos that could be optionally ligated together. A diagram of the assembled construct is shown in Figure 21. The construct used in this example was assembled according to the general method described in Example 1. Two DNA oligos were annealed (a, b; SEQ ID NOs. 47, 48), one of which contained a 3'TCO click group (b). The annealed oligo was then reacted with a peptide (c, SEQ ID NO. 49) having a C-terminal tetrazine click group. The N-terminus of the peptide contained an azide group and was then clicked to another set of annealed oligos (d, e; SEQ ID NOs. 50-51), one of which contained a 5'BCN click group (e). A polynucleotide-polypeptide conjugate was ligated to a standard sequencing adapter (f) containing a negatively charged leader sequence and tether sequence, loaded with a topologically closed helicase (using disulfide closure). The tether increases the analyte capture rate on the flow cell. [Table 7]

[0395] Data acquisition Electroelectric data were collected using a minion flow cell (Oxford Nanopore Technologies plc) containing modified Rhodococcus pores. Samples were prepared by mixing a 0.2 μM polynucleotide-polypeptide conjugate loaded with Dda helicase with sequencing buffer (25 mM HEPES-KOH (pH 8.0), 350 mM KCl, 50 mM ATP, 50 mM MgCl2, 2.2 mM EDTA). At least 1 mL of sequencing buffer was first flowed through the flow cell. Then, the sample (75 μL) was introduced into the flow cell via the SpotON port. Electroelectric data were acquired at a sample velocity of 1 kHz and an applied potential of 200 mV. The recording temperature was 21°C.

[0396] Results and Discussion Figure 22A shows an example of current-time tracing of Dda helicase-controlled migration of a polypeptide-polynucleotide conjugate, including DNA-peptide cotransposition. The peptide (HDSGDEVHHQK) is covalently bound to DNA at its C-terminus and N-terminus, so that both the DNA and the peptide cotransposition together through nanopores to produce the observed signal. See Figures 22A1 and 22A2.

[0397] A control experiment was conducted in which the same peptide sequence (HDSGDEVHHQK) was rearranged alone, i.e., without co-rearrangement with the DNA strand, using a construct in which the peptide cross-links the gap between two adjacent DNA oligonucleotides (as described in WO2021 / 111125). As shown in Figures 22B1(iii) and 22B2(iii), the current-time traces obtained for the peptide section in this control experiment differ from those shown in Figures 22A1(iii) and 22A2(iii), which confirms that co-rearrangement of DNA and peptide occurs as shown in Figures 22A1 and 22A2, and that the presence of a negative charge in the co-rearranged DNA modulates the peptide signal.

[0398] Example 6 This embodiment demonstrates the "single-pass" movement of a polynucleotide-polypeptide conjugate through a nanopore. The movement of the conjugate in the direction of "entering" the nanopore is controlled by the Dda helicase enzyme. The peptide is conjugated between two double-stranded (dsDNA) oligonucleotides, one of which contains an internal click group rather than a terminal click group, creating a "flap" of single-stranded DNA alongside the peptide. A standard sequencing adapter loaded with helicase is attached to the end of the construct. The helicase is topologically closed around the single-stranded DNA portion of the oligonucleotide before stalling. The presence of bound helicase initially prevents the conjugate from completely transposing through the nanopore. Once the conjugate enters the nanopore, it is released from stall by electrophoretic force, and the helicase then moves 5'-3' along the single-stranded DNA. The annealed complementary strand "opens" (separates) as the conjugate moves through the nanopore, resulting in only the DNA strand with the attached peptide moving through the constriction. When the helicase encounters the covalently bound DNA-peptide segment, it diffuses to both the peptide and the DNA "flap" and re-engages with the DNA on the other side of the DNA-peptide segment. It then continues to control the movement of the DNA peptide, and subsequently controls the movement of the single-stranded DNA in the 5' to 3' direction through the nanopore. The enzyme detaches when it reaches the end of the bound DNA oligonucleotide, releasing the rest of the conjugate through the pore and ending the reading. As in Example 5 above, in this system, the nanopore only needs to have a diameter wide enough to accommodate the co-transposing DNA peptide.

[0399] Construction Table 8 outlines the sequences of the oligonucleotides used. The construct consisted of an enzyme-loaded adapter with a ligated tethering site at the end of the construct, and a DNA "flap" extending over the peptide, consisting of six bases attached to the C-terminus of the peptide. A diagram of the assembled construct is shown in Figure 23. The construct used in this example was assembled according to the general method described in Example 1. Two DNA oligos were annealed (a, b; SEQ ID NOs. 53, 54), one of which contained an internal 3'TCO click group (b). The annealed oligo was then reacted with a peptide (c, SEQ ID NO: 49) having a C-terminal tetrazine click group. The N-terminus of the peptide contained an azide group and was then clicked to another set of annealed oligos (d, e; SEQ ID NOs. 55, 56), one of which contained a terminal 5'BCN click group (e). Next, the polynucleotide-polypeptide conjugate was loaded into a topologically closed helicase (using disulfide closure) and ligated into a standard sequencing adapter (f) containing a negatively charged leader sequence and a tether sequence. The tether increases the analyte capture rate on the flow cell. [Table 8]

[0400] Data acquisition Electroelectric data were collected using a minion flow cell (Oxford Nanopore Technologies plc) containing modified Rhodococcus pores. Samples were prepared by mixing a 0.2 μM polynucleotide-polypeptide conjugate loaded with Dda helicase with sequencing buffer (25 mM HEPES-KOH (pH 8.0), 350 mM KCl, 50 mM ATP, 50 mM MgCl2, 2.2 mM EDTA). At least 1 mL of sequencing buffer was first flowed through the flow cell. Then, the sample (75 μL) was introduced into the flow cell via the SpotON port. Electroelectric data were acquired at a sample velocity of 1 kHz and an applied potential of 200 mV. The recording temperature was 21°C.

[0401] Results and Discussion Figure 24 shows the current-time trace of Dda helicase-controlled migration of a polypeptide-polynucleotide conjugate, including cotransposition of the DNA flap and peptide. The peptide (HDSGDEVHHQK) covalently binds to the DNA flap at its C-terminus and passes through the nanopore from the C-terminus to the N-terminus. Thus, both the DNA flap and the peptide cotranspose together through the nanopore, producing the observed signal. The peptide signal produced by this scheme differs from that produced by a control experiment of peptide-only transposition (using a corresponding construct containing the same peptide sequence HDSGDEVHHQK but lacking the DNA flap), as described in Example 5 above and shown in Figure 22B.

[0402] Explanation of the sequence list Sequence ID 1 shows the amino acid sequence of E. coli-derived (hexahistidine-tagged) exonuclease I (EcoExo I). Sequence ID 2 shows the amino acid sequence of the exonuclease III enzyme derived from E. coli. Sequence ID 3 shows the amino acid sequence of the RecJ enzyme (TthRecJ-cd) derived from T. thermophilus. Sequence ID 4 shows the amino acid sequence of bacteriophage lambda exonuclease. The sequence is one of three identical subunits that assemble into a trimer. (http: / / www.neb.com / nebecomm / products / productM0262.asp). Sequence ID 5 shows the amino acid sequence of Phi29 DNA polymerase derived from Bacillus subtilis. Sequence ID 6 shows the amino acid sequence of Trwc Cba (Citromicrobium bathyomarinum) helicase. Sequence ID 7 shows the amino acid sequence of Hel308 Mbu (Methanococcoides burtonii) helicase. Sequence ID 8 shows the amino acid sequence of Dda helicase 1993 derived from the intestinal bacterium phage T4. Sequence ID 9 shows the amino acid sequence of a monomer of a transmembrane protein nanopore derived from Rhodococcus, as described in Example 1. Sequence IDs 10-12 show the polypeptide sequences used in Example 1 (read from N to C). Sequence IDs 13-26 show the polynucleotide sequences used in Example 1. 3 represents TCO-labeled amino C3, 2 represents BCN-labeled 5'-amino C3, and 1 represents 3'-biotin modification. A "+" before a base indicates LNA. Sequence IDs 27-28 show the polynucleotide sequences used in Example 2. 3 represents amino C3 labeled with TCO. 2 represents 5' amino C3 labeled with BCN. The "+" before a base indicates LNA. Sequence IDs 29-56 show the polynucleotide and polypeptide sequences used in Examples 3-6.

[0403] Sequence List Exonuclease I derived from Sequence ID No. 1-E. coli MMNDGKQQSTFLFHDYETFGTHPALDRPAQFAAIRTDSEFNVIGEPEVFYCKPADDYLPQPGAVLITGITPQEARAKGENEAAFAARIHSLFTVPKTCILGYNNV RFDDEVTRNIFYRNFYDPYAWSWQHDNSRWDLLDVMRACYALRPEGINWPENDDGLPSFRLEHLTKANGIEHSNAHDAMADVYATIAMAKLVKTRQPRLFDYLFT HRNKHKLMALIDVPQMKPLVHVSGMFGAWRGNTSWVAPLAWHPENRNAVIMVDLAGDISPLLELDSDTLRERLYTAKTDLGDNAAVPVKLVHINKCPVLAQANTLRPEDADRLGINRQHCLDNLKILRENPQVREKVVAIFAEAEPFTPSDNVDAQLYNGFFSDADRAAMKIVLETEPRNLPALDITFVDKRIEKLLFNYRARNFPGTLD YAEQQRWLEHRRQVFTPEFLQGYADELQMLVQQYADDKEKVALLKALWQYAEEIVSGSGHHHHHH Exonuclease III enzyme derived from SEQ ID NO: 2-E. coli MKFVSFNINGLRARPHQLEAIVEKHQPDVIGLQETKVHDDMFPLEEVAKLGYNVFYHGQKGHYGVALLTKETPIAVRRGFPGDDEEAQRRIIMAEIPSLLGNVTVINGYFPQGESRDHPIKFPAKAQFYQNLQN YLETELKRDNPVLIMGDMNISPTDLDIGIGEENRKRWLRTGKCSFLPEEREWMDRLMSWGLVDTFRHANPQTADRFSWFDYRSKGFDDNRGLRIDLLLASQPLAECCVETGIDYEIRSMEKPSDHAPVWATFRR Sequence ID 3 - RecJ enzyme derived from T. thermophilus MFRRKEDLDPPLALLPLKGLREAAALLEEALRQGKRIRVHGDYDADGLTGTAILVRGLAALGADVHPFIPHRLEEGYGVLMERVPEHLEASDLFLTVDCGITNHAELRELLENGVEVIVTDHHTPGKTPPPGLVVHPALTPDLKKEKPTGAGVAFLLLWALHERLGLPPPLEYADLAAVGTIADVAPLWGWNRALVKEGLARIPASSWVGLRL LAEAVGYTGKAVEVAFRIAPRINAASRLGEAEKALRLLLTDDAAEAQALVGELHRLNARRQTLEEAMLRKLLPQADPEAKAIVLLDPEGHPGVMGIVASRILEATLRPVFLVAQGKGTVRSLAPISAVEALRSAEDLLLRYGGHKEAAGFAMDEALFPAFKARVEAYAARFPDPVREVALLLDLLPEPGLLPQVFRELALLEPYGEGNPEPLFL Sequence ID 4-bacteriophagelambdaexonuclease MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPRSGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESPIIYRDESMR TACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMWVTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFGEQWR Sequence ID 5-Phi29 DNA polymerase MKHMPRKMYSCAFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKER PVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLLDKEVRYAYRGGFTWLNDRFKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQI KRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCD TDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKSGGSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK Sequence ID 6-Trwc Cba helicase MLSVANVRSPSAAASYFASDNYYASADADRSGQWIGDGAKRLGLEGKVEARAFDALLRGELPDGSSVGNPGQAHRPGTDLTFSVPKSWSLLALVGKDERIIAAYREAVVEALHWAEKNAAETRVVEKGMVVTQATGNLAIGLFQHDTNRNQEPNLHFHAVIANVTQGKDGWRTLKNDRLWQLNTTLNSIAMARFRVAVEKLGYEPGPVLKHGNFEARGISREQVMAFSTRRKEVLEARRGPGLGLDAGRIAALDTRASKEGIEDRATLSKQWSEAAQSIGLDLKPLVDRARTKALGQGMEATRIGSLVERGRAWLSRFAAHVRGDPADPLVPPSVLKQDRQTIAAAQAVASAVRHLSQREAAFERTALYKAALDFGLPTTIADVEKRTRALVRSGDLIAGKGEHKGWLASRDAVVTEQRILSEVAAGKGDSSPAITPQKAAASVQAAALTGQGFRLNEGQLAAARLILISKDRTIAVQGIA GAGKSSVLKPVAEVLRDEGHPVIGLAIQNTLVQMLERDTGIGSQTLARFLGGWNKLLDDPGNVALRAEAQASLKDHVLVLDEASMVSNEDKEKLVRLANLAGVHRLVLIGDRKQLGAVDAGKPFALLQRAGIARAEMATNLRARDPVVREAQAAAQAGDVRKARLHLKSHTVEARGDGAQVAAETWLALDKETRARTSIYASGRAIRSAVNAAVQQGLLASREIGPAKMKLEVLDRVNTTREELRHLPAYRAGRVLEVSRKQQALGLFIGEYRVIGQDRKGKLVEVEDKRGKRFRFDPARIRAGKGDDNLTLLEPRKLEIHEGDRIRWTRNDHRRGLFNADQARVVEIANGKVTFETSKGDLVELKKDDPMLKRIDLAYALNVHMAQGLTSDRGIAVMDSRERNLSNQKTFLVTVTRLRDHLTLVVDSADKLGAAVARNKGEKASAIEVTGSVKPTATKGSGVDQPKSVEANKAEKELTR SKSKTLDFGI Sequence ID 7-Hel308 Mbu helicase MMIRELDIPRDIIGFYEDSGIKELYPPQAEAIEMGLLEKKNLLAAIPTASGKTLLAELAMIKAIREGGKALYIVPLRALASEKFERFKELAPFGIKVGISTGDLDSRADWLGVNDIIVATSEKTDSLLRNGTSWMDEITTVVVDEIHLLDSKNRGPTLEVTITKLMRLNPDVQVVALSATVGNAREMADW LGAALVLSEWRPTDLHEGVLFGDAINFPGSQKKIDRLEKDDAVNLVLDTIKAEGQCLVFESSRRNCAGFAKTASSKVAKILDNDIMIKLAGIAEEVESTGETDTAIVLANCIRKGVAFHHAGLNSNHRKLVENGFRQNLIKVISSTPTLAAGLNLPARRVIIRSYRRFDSNFGMQPIPVLEYKQMAGRAG RPHLDPYGESVLLAKTYDEFAQLMENYVEADAEDIWSKLGTENALRTHVLSTIVNGFASTRQELFDFFGATFAYQQDKWMLEEVINDCLEFLIDKAMVSETEDIEDASKLFLRGTRLGSLVSMLYIDPLSGSKIVDGFKDIGKSTGGNMGSLEDDKGDDITVTDMTLLHLVCSTPDMRQLYLRNTDYTI VNEYIVAHSDEFHEIPDKLKETDYEWFMGEVKTAMLLEEWVTEVSAEDITRHFNVGEGDIHALADTSEWLMHAAAKLAELLGVEYSSHAYSLEKRIRYGSGLDLMELVGIRGVGRVRARKLYNAGFVSVAKLKGADISVLSKLVGPKVAYNILSGIGVRVNDKHFNSAPISSNTLDTLLDKNQKTFNDFQ Sequence ID 8-Dda helicase MTFDDLTEGQKNAFNIVMKAIKEKKHHVTINGPAGTGKTTLTKFIIEALISTGETGIILAAPTHAAKKILSKLSGKEASTIHSILKINPVTYEENVLFEQKEVPDLAKC RVLICDEVSMYDRKLFKILLSTIPPWCTIIGIGDNKQIRPVDPGENTAYISPFFTHKDFYQCELTEVKRSNAPIIDVATDVRNGKWIYDKVVDGHGVRGFTGDTALRDFM VNYFSIVKSLDDLFENRVMAFTNKSVDKLNSIIRKKIFETDKDFIVGEIIVMQEPLFKTYKIDGKPVSEIIFNNGQLVRIIEAEYTSTFVKARGVPGEYLIRHWDLTVET YGDDEYYREKIKIISSDEELYKFNLFLGKTAETYKNWNKGGKAPWSDFWDAKSQFSKVKALPASTFHKAQGMSVDRAFIYTPCIHYADVELAQQLLYVGVTRGRYDVFYV Sequence ID 9 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPGLNLSVGNGVAATVTNVLPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSSGSAWSHPQFEK Sequence ID 10 GGSGDDSGSG Sequence ID 11 GGSGRRSGSG Sequence ID 12 GGSGYYSGSG Sequence ID 13 +G+T+C+C+A+CTTTTTTGCTGCGGCTTAGCTGTGATAGCTTTTGCTATCACAGCTAAGCCGCAGC3 Sequence ID 14 GTCCATTATTCTTTTTTGCTGCGGCTTAGCTGTGATAGCTTTTGCTATCACAGCTAAGCCGCAGC3 Sequence ID 15 GTATTGTCCATTATTCTTTTTTGCTGCGGCTTAGCTGTGATAGCTTTTGCTATCACAGCTAAGCCGCAGC3 Sequence ID 16 GTATTGTATTGTCCATTATTCTTTTTTGCTGCGGCTTAGCTGTGATAGCTTTTGCTATCACAGCTAAGCCGCAGC3 Sequence ID 17 GTATTGTATTGTATTGTCCATTATTCTTTTTTGCTGCGGCTTAGCTGTGATAGCTTTTGCTATCACAGCTAAGCCGCAGC3 Sequence ID 18 GTATTGTATTGTATTGTATTGTCCATTATTCTTTTTTGCTGCGGCTTAGCTGTGATAGCTTTTGCTATCACAGCTAAGCCGCAGC3 Sequence ID 19 GTATTGTATTGTATTGTATTGTATTGTCCATTATTCTTTTTTGCTGCGGCTTAGCTGTGATAGCTTTTGCTATCACAGCTAAGCCGCAGC3 Sequence ID 20 2GTGGAC1 Sequence ID 21 2GAATAATGGAC1 Sequence ID 22 2GAATAATGGACAATAC1 Sequence ID 23 2GAATAATGGACAATACAATAC1 Sequence ID 24 2GAATAATGGACAATACAATACAATAC1 Sequence ID 25 2GAATAATGGACAATACAATACAATACAATAC1 Sequence ID 26 2GAATAATGGACAATACAATACAATACAATACAATAC1 Sequence ID 27 +G+T+C+C+ATTATTCTTTTTTGCTGCGGCTTAGCTGTGATAGCTTTTGCTATCACAGCTAAGCCGCAGC3 Sequence ID 28 2GAATAATGGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Sequence ID 29 / 5SpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / i SpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / GTTATTCAAGACTTCTTTAATACACTTT / BCN / Sequence ID 30 GTCCATTATTCTTTTTTGCTGCGGCTTAGCTGTGATAGCAGGA Sequence ID 31 / 5Phos / CGCAATACGTAACTGAACGAAGTACT / iBCN / TGTACTTCGTTCAGTTACGTATTGCGTCCT Sequence ID 32 / 5Phos / GTGTATTAAAGAAGTCTTGAATAACTTTGAGGCGAGCGGTCAA Sequence ID 33 / 5Phos / GCTATCACAGCTAAGCCGCAGC / TCO / Sequence ID 34 / BCN / GAATAATGGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Sequence ID 35 RSDSGQQARY Sequence ID 36 GGSGSSSGSG Sequence ID 37 EAIYAAPFAKKK Sequence ID 38 SRRRRRRRRS Sequence ID 39 SEEEEEEEES Sequence ID 40 HDSGYEVHHQK Sequence ID 41 / 5Phos / GCAATACGTAACTGAACGAAGT / iBNA-T / / iBNA-G / / iBNA-T / / iBNA-G / / iBNA-G / mUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmU mUmUmUmUmUmUmUmUmUmUmUmUGGTTAAACACCCAAGCAGACGCCT7TGGCGTCTGCTTGGGTGTTTAACCTTTTTTTTTT8CCACAACTTCGTTCAGTTACGTATTGCTCCT Sequence ID 42 CTCAAGTGCCTGGTATATTACATCCACAGTGAAGACCTGGACACTGGACGTCCATTATTC / TCO / Sequence ID 43 / 5Phos / GCTATCACAGGCAATAAGAATAACGTCATAATGCGTAACTGACTAAGCCGCAGCTTTTTTGAATAATGGACGTCCAGTGTCCAGGTCTTCACTGTGGATGTAATATACCAGGCACTTGAG Sequence ID 44 Peptide having an N-terminal azide and a C-terminal tetrazine [HDSGDEVHHQK] Sequence ID 45 / BCN / GCTGCGGCTTAGTCAGTTACGCATTATGACGTTATTCTTATTGCCTGTGATAGCAGGA Sequence ID 46 HDSGDEVHHQK Sequence ID 47 AAAAAAAAAAAAAAAAAAAAAAAAAGGTTAAACACCCAAGCAGCAAT Sequence ID 48 GCTTGGGTGTTTAACCTTTTTTTTTTTTTTTTTTTTTTTT / iC3-TCO / TTT Sequence ID 49 Azido-HDSGDEVHHQK-Tetrazine Sequence ID 50 CCCTGGACACTGGACAAAAAAAAAAAAAAAAAAAAAAAAA Sequence ID 51 / 5Phos / TTT / iC3-BCN / TTTTTTTTTTTTTTTTTTTTTTTTTGTCCAGTGTCCAGGG Sequence ID 52 CCCTGGACACTGGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGTTAAACACCCAAGCAGCAAT Sequence ID 53 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGTTAAACACCCAAGCAGCAAT Sequence ID 54 GCTTGGGTGTTTAACCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT / iC3-TCO / TTTTTT Sequence ID 55 CCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Sequence ID 56 / BCN / TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGG

Claims

1. A method for characterizing a target polypeptide, - (i) a polynucleotide-polypeptide conjugate chain containing the target polypeptide, which is conjugated to one or more adjacent polynucleotide chains at each end of the target polypeptide, is brought into contact with a polynucleotide carrier chain, thereby forming a polynucleotide-polypeptide construct. - The construct and the nanopores are brought into contact under conditions such that both the polynucleotide-polypeptide conjugate chain and the polynucleotide carrier chain co-transform through the nanopores, - The process includes obtaining one or more characteristic measurements of the polypeptide as the construct moves into the nanopores, A method for characterizing the target polynucleotide thereby.

2. The method according to claim 1, wherein one or more adjacent polynucleotide chains are each independently complementary to the region of the polynucleotide carrier chain.

3. The method according to claim 1 or 2, wherein one or more adjacent polynucleotide chains are independently at least partially hybridized to the polynucleotide carrier chain.

4. A method for characterizing a target polypeptide, - (i) a polynucleotide-polypeptide conjugate chain containing a target polypeptide attached to a polynucleotide adjacent chain, (ii) contact with a polynucleotide handling protein capable of controlling the movement of the polynucleotide adjacent chain to a nanopore, - The polynucleotide-polypeptide conjugate chain is brought into contact with a nanopore under conditions such that the polynucleotide handling protein controls the movement of the polynucleotide-polypeptide conjugate chain to and from the nanopore. - Obtaining one or more characteristic measurements of the polypeptide when the adjacent polynucleotide chain and the target polypeptide co-transform through the nanopore, A method for characterizing the target polynucleotide thereby.

5. The method according to claim 4, wherein, prior to the above method, the adjacent polynucleotide chain is at least partially hybridized to the polynucleotide carrier chain, thereby forming a polynucleotide-polypeptide construct.

6. The method according to any one of the prior claims, wherein the polynucleotide-polypeptide conjugate chain comprises a plurality of target polypeptides.

7. The method according to any one of the prior claims, wherein during the method, the polypeptide or each polypeptide is held independently in a linearized form.

8. The method according to any one of the prior claims, wherein the target polypeptide or each target polypeptide independently has a length of about 5 to about 1000 peptide units.

9. The method according to any one of the prior claims, comprising mechanically manipulating the construct, the polynucleotide-polypeptide conjugate chain and / or the polynucleotide carrier chain to move the construct, the polynucleotide-polypeptide conjugate chain and / or the polynucleotide carrier chain into the nanopores.

10. The method according to claim 9, wherein the construct, polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain are moved by mechanical operation in the opposite direction to the potential applied across the nanopore.

11. The method according to claim 10, wherein the potential is a voltage potential applied across the nanopore.

12. The method according to any one of claims 1 to 3 or 5 to 11, comprising contacting the construct with a polynucleotide handling protein capable of controlling the movement of one or more polynucleotide adjacent chains and / or the polynucleotide carrier chain, wherein the polynucleotide handling protein controls the movement of the construct with respect to the nanopores.

13. The method according to claim 12, comprising contacting both the polynucleotide-polypeptide conjugate chain and the polynucleotide carrier chain of the construct with a polynucleotide handling protein capable of controlling the movement of the polynucleotide-polypeptide conjugate chain and / or the polynucleotide carrier chain, wherein the polynucleotide handling protein controls the movement of the construct into the nanopores.

14. The method according to any one of claims 1 to 11, comprising contacting the polynucleotide-polypeptide conjugate chain with a polynucleotide handling protein capable of controlling the movement of the polynucleotide-polypeptide conjugate chain, wherein the polynucleotide handling protein controls the movement of the structure toward the nanopores.

15. The method according to any one of claims 1 to 3 or 5 to 11, comprising contacting the polynucleotide carrier chain with a polynucleotide handling protein capable of controlling the movement of the polynucleotide carrier chain, wherein the polynucleotide handling protein controls the movement of the structure toward the nanopores.

16. i) The polynucleotide handling protein is located on the cis side of the nanopore, and the polynucleotide handling protein controls the movement of the construct, polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of the target polypeptide from the cis side of the nanopore to the trans side of the nanopore, or ii) The method according to any one of claims 12 to 15, wherein the polynucleotide handling protein is located on the trans side of the nanopore, and the polynucleotide handling protein controls the movement of the construct, the polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of the target polypeptide from the trans side of the nanopore to the cis side of the nanopore.

17. The method according to claim 6, wherein the polynucleotide handling protein is located on the cis side of the nanopore, and the polynucleotide handling protein controls the movement of the polynucleotide carrier chain from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of the target polypeptide through the nanopore.

18. The method according to claim 16, wherein the polynucleotide handling protein is located on the trans side of the nanopore, and the polynucleotide handling protein controls the movement of the polynucleotide carrier chain from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of the target polypeptide through the nanopore.

19. i) The polynucleotide handling protein is located on the cis side of the nanopore, and the polynucleotide handling protein controls the movement of the construct, polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of the target polypeptide from the trans side of the nanopore to the cis side of the nanopore, or ii) The method according to any one of claims 12 to 15, wherein the polynucleotide handling protein is located on the trans side of the nanopore, and the polynucleotide handling protein controls the movement of the construct, polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of the target polypeptide from the cis side of the nanopore to the trans side of the nanopore.

20. The method according to claim 19, wherein the polynucleotide handling protein is located on the cis side of the nanopore, and the polynucleotide handling protein controls the movement of the polynucleotide carrier chain from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of the target polypeptide through the nanopore.

21. The method according to claim 19, wherein the polynucleotide handling protein is located on the trans side of the nanopore, and the polynucleotide handling protein controls the movement of the polynucleotide carrier chain from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of the target polypeptide through the nanopore.

22. The method according to any one of claims 12 to 21, wherein, before the construct is brought into contact with the nanopores, the polynucleotide handling protein is bound to the polynucleotide carrier chain in a region of the polynucleotide carrier chain that extends into the non-hybridization region of the adjacent polynucleotide chain.

23. The method according to any one of claims 12 to 21, wherein if a portion of the polynucleotide-polypeptide conjugate chain that has come into contact with the active site of the polynucleotide handling protein contains the target polypeptide, the polynucleotide handling protein can maintain its binding to the polynucleotide-polypeptide conjugate chain.

24. The method according to any one of claims 12 to 23, wherein when the polynucleotide handling protein comes into contact with the target polypeptide, the polynucleotide handling protein is modified to prevent it from unassociating with the construct, the polynucleotide-polypeptide conjugate chain and / or the polynucleotide carrier chain.

25. The method according to any one of claims 12 to 24, wherein the polynucleotide handling protein is modified to completely or partially close an opening present in at least one conformational state of an unmodified protein capable of detaching polynucleotide chains.

26. The method according to any one of claims 12 to 25, wherein the polynucleotide handling protein is a helicase, a translocase, or a helicase-nuclease complex, or comprises the same.

27. The method according to any one of the prior claims, wherein the construct includes a stalled portion, and the polynucleotide handling protein is positioned such that the stalled portion is located between the polynucleotide handling protein and the target polypeptide before the rearrangement of the target polypeptide through the nanopore.

28. The method according to any one of the prior claims, wherein one or more adapters and / or one or more tethers and / or one or more anchors are attached to the construct, the polynucleotide-polypeptide conjugate chain and / or the polynucleotide carrier chain.

29. The method according to any one of the prior claims, wherein the construct, polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain includes a blocking portion attached via an optional linker, the blocking portion being unable to displace through the nanopores.

30. The method described above is i) The method according to any one of the prior claims is performed such that the target polypeptide displaces the nanopores in a first direction relative to the nanopores, ii) To enable the construct, polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain to move in a direction opposite to the direction of movement to the nanopore in step (i) such that the target polypeptide displaces the nanopore in a second direction opposite to the first direction, iii) To enable the construct, polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain to move in the first direction such that the target polypeptide re-transposes the nanopore in the first direction, iv) optionally repeating steps (ii) and (iii) to cause the polypeptide to reciprocate through the nanopores, the method according to any one of the prior claims.

31. The method according to any one of the prior claims, wherein the one or more measured values ​​are characterized by one or more features of the target polypeptide selected from (i) the length of the target polypeptide, (ii) identification of the target polypeptide, (iii) sequence of the target polypeptide, (iv) secondary structure of the target polypeptide, and (v) whether the target polypeptide is modified.

32. The method according to any one of the prior claims, wherein the nanopores are protein nanopores, preferably β-barrel protein nanopores.

33. It is a system, - A construct comprising (i) a polynucleotide-polypeptide conjugate chain containing the target polypeptide, which is conjugated to one or more adjacent polynucleotide chains at each end of the target polypeptide, and (ii) a polynucleotide carrier chain, - Nanopores that can co-transpose the polynucleotide-polypeptide conjugate chain and the adjacent polynucleotide chain of the construct, - A system including polynucleotide handling proteins.

34. It's a kit, - Nanopores, - A first polynucleotide containing a reactive functional group for conjugation to the first terminus of a target polypeptide, - A second polynucleotide comprising a reactive functional group for conjugation to the second terminus of the target polypeptide, - A kit containing polynucleotide handling proteins.

35. The system according to claim 33 or the kit according to claim 34, wherein the nanopores, constructs and / or polynucleotide handling proteins are as defined in any one of claims 2 to 32.

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