Process

Abstract

Provided herein are methods of characterizing a target polypeptide as it moves relative to a nanopore. Related kits, systems, and devices for performing such methods are also provided.

Description

Technical Field

[0001] The present disclosure relates to methods for characterizing a target polypeptide by forming a construct comprising a first and second strand (e.g., a polynucleotide strand) comprising the target polypeptide, moving the construct relative to a nanopore under conditions such that the first and second strands of the construct move through the nanopore, and performing measurements specific to the polypeptide during such movement. The present disclosure also relates to kits, systems, and apparatus for performing such methods. Background Art

[0002] The characterization of biomolecules is increasingly important in biomedical and biotechnological applications. For example, nucleic acid sequencing allows for the study of genomes and the proteins they encode, and for example, allows for the correlation between nucleic acid mutations and observable phenomena (such as disease indications). Nucleic acid sequencing can be used in evolutionary biology to study the relationships between organisms. Metagenomics involves identifying organisms present in a sample, such as microorganisms in a microbiome, where nucleic acid sequencing allows for the identification of such organisms. While technologies for characterizing polynucleotides (e.g., sequencing them) are widely developed, technologies for characterizing polypeptides are less advanced, despite their significant biotechnological importance. For example, knowledge of protein sequence can allow for the establishment of structure-activity relationships and influence 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 may have multiple phosphorylation sites, which can be used to activate or inactivate the protein, promote its degradation, or modulate interactions with protein partners. Summary of the Invention

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

[0004] Protein mass spectrometry involves characterizing whole proteins or fragments thereof in ionized form. Known methods for protein mass spectrometry include electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI). While mass spectrometry offers some benefits, the results obtained can be affected by the presence of contaminants, and it can be difficult to manipulate fragile molecules without fragmenting them. Furthermore, mass spectrometry is not a single-molecule technique and only provides a fraction of the information about the sample being interrogated. Mass spectrometry is not well-suited to characterizing differences within a population of polypeptide samples and is also cumbersome when attempting to distinguish between adjacent residues.

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

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

[0007] An attractive approach for single-molecule characterization of biomolecules such as peptides is nanopore sensing. Nanopore sensing is a method for analyte detection and characterization that relies on the observation of individual binding or interaction events between analyte molecules and ion-conducting channels. Nanopore sensors can be created by placing a single, nanometer-sized pore in an electrically insulating membrane and measuring the voltage-driven ionic current across the pore in the presence of analyte molecules. The presence of an analyte inside or near the nanopore alters the ion flow through the pore, causing a change in the ion or current measured across the channel. The identity of the analyte is revealed by its unique current signature, specifically the duration and extent of the current burst and the change in current level during its interaction with the pore. Nanopore sensing has the potential to enable rapid and inexpensive peptide characterization.

[0008] Nanopore sensing and characterization of polypeptides has been proposed in the art. For example, WO 2013 / 123379 discloses the use of NTP-driven protein processing unfolding enzymes to process proteins to be translocated through nanopores. WO 2021 / 111125 discloses a method for conjugating a target polypeptide to a polynucleotide to form a single-chain polypeptide-polynucleotide conjugate, wherein a polynucleotide processing protein is used to move the conjugate through the nanopore. However, there is still a need for alternative and / or improved methods for characterizing polypeptides.

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

[0010] The present disclosure relates to methods of characterizing a target polypeptide.

[0011] In one aspect, the method involves a conjugate chain comprising the target polypeptide. The target polypeptide is conjugated to one or more polynucleotide flanking chains at each end of the polypeptide. The conjugate is contacted with a polynucleotide carrier chain, thereby forming a polynucleotide-polypeptide construct. The construct is contacted with a nanopore. The contact occurs 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 specific to the polypeptide are performed. In this manner, the target polypeptide contained in the conjugate is characterized.

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

[0013] Therefore, a method for characterizing a target polypeptide is provided herein, comprising

[0014] - contacting (i) a polynucleotide-polypeptide conjugate chain comprising a target polypeptide conjugated at each terminus of the target polypeptide to one or more polynucleotide flanking chains with (ii) a polynucleotide carrier chain, thereby forming a polynucleotide-polypeptide construct;

[0015] - contacting the construct with the nanopore under conditions such that both the polynucleotide-polypeptide conjugate chain and the polynucleotide carrier chain co-translocate through the nanopore; and

[0016] - performing one or more measurements specific to the polypeptide as the construct moves relative to the nanopore,

[0017] The target polypeptide is thereby characterized.

[0018] In some embodiments, the one or more polynucleotide flanking strands are each independently complementary to a region of the polynucleotide carrier strand. In some embodiments, the one or more polynucleotide flanking strands are each independently at least partially hybridized to the polynucleotide carrier strand.

[0019] A method for characterizing a target polypeptide is also provided, comprising

[0020] contacting (i) a polynucleotide-polypeptide conjugate chain comprising a target polypeptide linked to a polynucleotide wing chain with (ii) a polynucleotide handling protein capable of controlling movement of the polynucleotide wing chain relative to the nanopore; and

[0021] - contacting the polynucleotide-polypeptide conjugate chain with the nanopore under conditions such that the polynucleotide handling protein controls movement of the polynucleotide-polypeptide conjugate chain relative to the nanopore; and

[0022] - performing one or more measurements specific to the polypeptide as the polynucleotide flanking strands and the target polypeptide co-translocate through the nanopore,

[0023] The target polypeptide is thereby characterized.

[0024] In certain embodiments, prior to the method, the polynucleotide flanking strand is at least partially hybridized to a polynucleotide carrier strand, thereby forming a polynucleotide-polypeptide construct.

[0025] In some embodiments of the above methods, the polynucleotide-polypeptide conjugate chain comprises a plurality of target polypeptides.

[0026] In some embodiments, during the method, the or each polypeptide independently remains in linearized form.

[0027] In some embodiments, the or each target polypeptide is independently from about 5 peptide units to about 1000 peptide units in length.

[0028] In some embodiments, the method comprises 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 is 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.

[0029] In some embodiments, the method comprises contacting the construct with a polynucleotide handling protein capable of controlling movement of the one or more polynucleotide flanking strands and / or the polynucleotide carrier strand, and wherein the polynucleotide handling protein controls movement of the target polypeptide relative to the nanopore.

[0030] In some embodiments, the method comprises contacting both the polynucleotide-polypeptide conjugate chain and the polynucleotide carrier chain of the construct with a polynucleotide handling protein capable of controlling movement of the polynucleotide-polypeptide conjugate chain and / or the polynucleotide carrier chain, and wherein the polynucleotide handling protein controls movement of the construct relative to the nanopore.

[0031] In some embodiments, the method comprises contacting the polynucleotide-polypeptide conjugate chain with a polynucleotide handling protein capable of controlling movement of the polynucleotide-polypeptide conjugate chain, and wherein the polynucleotide handling protein controls movement of the target polypeptide relative to the nanopore.

[0032] In some embodiments, the method comprises contacting the polynucleotide carrier strand with a polynucleotide handling protein capable of controlling movement of the polynucleotide carrier strand, and wherein the polynucleotide handling protein controls movement of the target polypeptide relative to the nanopore.

[0033] In some embodiments:

[0034] 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, the polynucleotide-polypeptide conjugate chain and / or 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 from the cis side of the nanopore to the trans side of the nanopore; or

[0035] ii) 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 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 from the trans side of the nanopore to the cis side of the nanopore.

[0036] 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 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 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.

[0037] In some embodiments:

[0038] 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, the polynucleotide-polypeptide conjugate chain and / or 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 from the trans side of the nanopore to the cis side of the nanopore; or

[0039] ii) 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 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 from the cis side of the nanopore to the trans side of the nanopore.

[0040] 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.

[0041] In some embodiments, the polynucleotide handling protein binds to the polynucleotide carrier strand in the region of the polynucleotide carrier strand spanned by the non-hybridized regions of the polynucleotide flanking strands prior to contacting the construct with the nanopore.

[0042] In some embodiments, the polynucleotide handling protein is capable of remaining bound to the polynucleotide-polypeptide conjugate chain when the portion of the polynucleotide-polypeptide conjugate chain that contacts the active site of the polynucleotide handling protein comprises the target polypeptide. In some embodiments, the polynucleotide handling protein is modified to prevent the polynucleotide handling protein from disengaging from the construct, the polynucleotide-polypeptide conjugate chain, and / or the polynucleotide carrier chain conjugate when the polynucleotide handling protein contacts the target polypeptide. In some embodiments, the polynucleotide handling protein is modified to completely or partially close an opening present in at least one conformational state of the unmodified protein through which the polynucleotide chain can unbind. In some embodiments, the polynucleotide handling protein is or comprises a helicase, a translocase, or a helicase-nuclease complex.

[0043] In some embodiments, the construct comprises a arrest moiety and the polynucleotide handling protein is positioned such that the arrest moiety is between the polynucleotide handling protein and the target polypeptide prior to translocation of the target polypeptide through the nanopore.

[0044] In some embodiments, one or more adapters and / or one or more tethers and / or one or more anchors are connected 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 comprises a blocking moiety connected via an optional linker, wherein the blocking moiety is unable to translocate through the nanopore.

[0045] In some embodiments, the method comprises:

[0046] i) performing a method as described herein such that the target polypeptide translocates the nanopore in a first direction relative to the nanopore;

[0047] ii) allowing the construct, the polynucleotide-polypeptide conjugate chain and / or the polynucleotide carrier chain to move 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;

[0048] iii) optionally allowing the construct, the polynucleotide-polypeptide conjugate chain and / or the polynucleotide carrier chain to move in the first direction such that the target polypeptide re-translocates the nanopore in the first direction;

[0049] iv) optionally repeating steps (ii) and (iii) to oscillate the polypeptide through the nanopore.

[0050] In some embodiments, the one or more measurements are specific to one or more properties 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.

[0051] In some embodiments, the nanopore is a protein nanopore, preferably a β-barrel protein nanopore.

[0052] This article also provides a system, the system comprising

[0053] - a construct comprising (i) a polynucleotide-polypeptide conjugate chain comprising a target polypeptide conjugated at each terminus of the target polypeptide to one or more polynucleotide flanking chains, and (ii) a polynucleotide carrier chain;

[0054] - a nanopore capable of co-translocating the polynucleotide-polypeptide conjugate strand and the polynucleotide flanking strand of the construct; and

[0055] - Polynucleotide processing proteins.

[0056] The present invention also provides a kit, which comprises:

[0057] - nanopores;

[0058] - a first polynucleotide comprising a reactive functional group for conjugation to a first terminus of a target polypeptide;

[0059] - a second polynucleotide comprising a reactive functional group for conjugation to the second end of the target polypeptide; and

[0060] - Polynucleotide processing proteins.

[0061] In some embodiments of the systems and kits provided herein, the nanopore, the construct and / or the polynucleotide handling protein are as defined herein.

[0062] Also provided are methods of characterizing target polynucleotide sequences. Unless the context suggests otherwise, the methods described herein relating to polypeptide characterization can be similarly applied to the characterization of target polynucleotide sequences, as described in more detail herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1A schematic diagram illustrates a non-limiting example of an embodiment of the disclosed method in which a polynucleotide handling protein that moves on ssDNA at the cis side of a nanopore controls the movement of a construct comprising a polynucleotide-polypeptide conjugate chain hybridized to a polynucleotide carrier chain as described herein from the cis side of the nanopore to the trans side of the nanopore, thereby allowing characterization of the polypeptide as it moves relative to the nanopore. As depicted, the polynucleotide handling protein is initially loaded on the ssDNA opposite an optional bubble region (F). Both ends of the polynucleotide can be captured in the nanopore, for example, from the cis side of the membrane by applying, for example, a positive voltage to the trans side of the membrane (i) to the polynucleotide handling enzyme (ii), which arrests the polynucleotide handling protein (if the optional arresting chemistry is used) (iii) allowing it to translocate on the ssDNA. The movement of the polynucleotide handling protein along the polynucleotide segment of the carrier chain feeds the construct into the pore; as the polynucleotide handling protein moves along the polynucleotide (e.g., in steps driven by 1 nucleotide fuel), it feeds the construct into the nanopore, and the peptide segment passes through the nanopore, allowing it to be characterized. The two ssDNA strands reanneal behind the enzyme, which, as depicted, translocates in a migration bubble corresponding to the polymer chain spanning the polynucleotide handling protein.

[0064] A, peptide conjugated in a dsDNA context; B, optional motor protein arrest chemistry (e.g., BNA, LNA, or RNA); C, optional motor protein arrest chemistry (e.g., Spacer 18 or similar); D, polynucleotide handling enzyme; E, nanopore inserted in the membrane; F, optional bubbling ssDNA, ssRNA, or spacer chemistry opposite enzyme.

[0065] Figure 2A schematic diagram illustrates a non-limiting example of an embodiment of the disclosed method in which a polynucleotide handling protein that moves on ssDNA at the cis side of a nanopore controls the movement of a construct comprising a polynucleotide-polypeptide conjugate chain hybridized to a polynucleotide carrier chain as described herein from the trans side of the nanopore to the cis side of the nanopore, thereby allowing characterization of the polypeptide as it moves relative to the nanopore. As depicted, the polynucleotide handling protein is initially loaded on the ssDNA opposite an optional bubble region (F). Both ends of the polynucleotide are captured in the nanopore, for example, from the cis side of the membrane, by applying, for example, a positive voltage to the trans side of the membrane (i) to the polynucleotide handling enzyme (ii), which removes an optional enzyme-stabilizing blocking moiety (if used), and (iii) allows its translocation on the ssDNA. The movement of the polynucleotide handling protein along the polynucleotide segment of the carrier strand pulls the construct out of the pore (e.g., in the direction from the trans side to the cis side of the pore); as the polynucleotide handling protein moves along the polynucleotide (e.g., in steps driven by 1 nucleotide fuel), it pulls the construct out of the nanopore, and the peptide segment passes through the nanopore, allowing it to be characterized. The two ssDNA strands reanneal behind the enzyme, which translocates in the migration bubble as depicted, thereby controlling the exit of the two strands of the conjugate from the nanopore. All steps can be performed on the trans side of the membrane under positive bias, except for (ii), which can be performed at zero potential or a low negative (trans) bias.

[0066] A, optionally bubbled ssDNA, ssRNA, or spacer chemistry opposite enzyme; B, polynucleotide processing enzyme; C, optional enzyme arrest blocking moiety (e.g., LNA, BNA, or RNA); D, optional enzyme arresting chemistry (e.g., Spacer 18 or similar); E, peptide conjugated in the context of dsDNA; F, nanopore inserted in the membrane.

[0067] Figure 3A schematic diagram illustrates a non-limiting example of an embodiment of the disclosed method, wherein a polynucleotide handling protein at the cis side of a nanopore controls the movement of a construct comprising a polynucleotide-polypeptide conjugate chain hybridized to a polynucleotide carrier chain as described herein from the trans side of the nanopore to the cis side of the nanopore, thereby allowing characterization of the polypeptide as it moves relative to the nanopore. As depicted, the polynucleotide handling protein is initially loaded and optionally arrested on ssDNA opposite an optional bubble (D). An optional leader sequence (A) may be present on the construct to facilitate passage of the construct through the nanopore. For example, both ends of the polynucleotide are captured in the nanopore (e.g., via the optional leader sequence), e.g., from the cis side of the membrane by applying, for example, a positive voltage to the trans side of the membrane (i) until the polynucleotide handling enzyme (ii), after which the enzyme is pushed back to an optional blocking moiety (F), (iii) and then allowed to translocate across the ssDNA (iv). The movement of the polynucleotide handling protein pulls the construct out of the pore (e.g., in the direction from the trans side of the pore to the cis side); as the polynucleotide handling protein moves along the polynucleotide (e.g., in steps driven by 1 nucleotide fuel), it pulls the construct out of the nanopore, and the peptide segment passes through the nanopore, allowing it to be characterized. The two ssDNA chains reanneal behind the enzyme, which translocates in the migration bubble as depicted, thereby controlling the two chains of the conjugate to exit the nanopore. The enzyme can be pushed back to an earlier position at any point by forces acting on the DNA, which resets the cycle. The polynucleotide handling protein can then again control the movement of the construct relative to the nanopore, allowing the target polypeptide to repeatedly "flossed" through the nanopore.

[0068] A, optional leader sequence segment; B, optional C3 segment for stalling the enzyme; C, polynucleotide processing enzyme; D, optionally bubbled ssDNA, ssRNA, or spacer chemically opposed enzyme; E, peptide conjugated in the context of dsDNA; F, optional post-blocker moiety to prevent enzyme dissociation; G, nanopore inserted in the membrane.

[0069] Figure 4 Schematic diagram illustrating a non-limiting example of an embodiment of the disclosed method, wherein a polynucleotide handling protein at the cis side of a nanopore controls the movement of a construct comprising a polynucleotide-polypeptide conjugate chain hybridized to a polynucleotide carrier chain as described herein from the cis side of the nanopore to the trans side of the nanopore, thereby allowing characterization of the polypeptide as it moves relative to the nanopore. The polynucleotide handling protein engages both chains of the construct (e.g., the polynucleotide-polypeptide conjugate chain and the carrier chain) and can actively control the movement of either or both chains. Movement of the construct and characterization of the target polypeptide are as described for Figure 1 discussed.

[0070] Figure 5 Schematic diagram illustrating a non-limiting example of an embodiment of the disclosed method, wherein a polynucleotide handling protein at the cis side of a nanopore controls the movement of a construct comprising a polynucleotide-polypeptide conjugate chain hybridized to a polynucleotide carrier chain as described herein from the trans side of the nanopore to the cis side of the nanopore, thereby allowing characterization of the polypeptide as it moves relative to the nanopore. The polynucleotide handling protein engages both chains of the construct (e.g., the polynucleotide-polypeptide conjugate chain and the carrier chain) and can actively control the movement of either or both chains. Movement of the construct and characterization of the target polypeptide are as described for Figure 2 discussed.

[0071] Figure 6 Schematic diagrams illustrating additional non-limiting examples of embodiments of the disclosed methods. A: The target polypeptide is contained within the polynucleotide-polypeptide conjugate chain by being linked to the polynucleotide flanking chains. The polynucleotide-polypeptide conjugate chain is in contact with the polynucleotide handling protein, thereby allowing the polypeptide to be characterized as it moves relative to the nanopore. As depicted, the movement scheme is as follows: Figure 1 However, those skilled in the art will recognize that the exact movement scheme is not limited, and the movement scheme (as described herein and particularly in Figures 1 to 5 The movement schemes described in each of FIG are also compatible with this embodiment and are specifically disclosed hereby. B: The polynucleotide-polypeptide conjugate chain of FIG. A can optionally be contacted with a polynucleotide carrier chain to form a construct as depicted. The construct is contacted with a polynucleotide handling protein, thereby allowing the polypeptide to be characterized as it moves relative to the nanopore. As depicted, the movement scheme is as described in FIG. Figure 4 However, those skilled in the art will recognize that the exact movement scheme is not limited, and the movement scheme (as described herein and particularly in Figures 1 to 5 The movement schemes described in each of FIG are also compatible with this embodiment and are specifically disclosed hereby. C: The polynucleotide-polypeptide conjugate chain of FIG. A can optionally be contacted with a polynucleotide carrier chain to form a construct as depicted; wherein the carrier chain is optionally excluded from the nanopore during translocation of the construct through the nanopore. As depicted, the movement scheme is as Figure 1 However, those skilled in the art will recognize that the exact movement scheme is not limited, and the movement scheme (as described herein and particularly in Figures 1 to 5 The movement schemes described in each of ) are also compatible with this embodiment and are specifically disclosed hereby.

[0072] Figure 7A schematic diagram illustrates another non-limiting example of a method for the repeated controlled movement of a polynucleotide-polypeptide conjugate from trans to cis, wherein movement is controlled by a polynucleotide-handling enzyme that moves on ssDNA. The enzyme is initially loaded and stalled on the ssDNA overhang. The method is performed under conditions where multiple enzymes can be loaded onto the overhang. The two ends of the polynucleotide are trapped in the nanopore (i) until the polynucleotide-handling enzyme (ii), after which the enzyme controls the movement of the conjugate out of the nanopore until the peptide (iii). The enzyme can then dissociate from the polynucleotide, at which point the position of the conjugate in the nanopore drops to that of a second enzyme loaded at an earlier position on the DNA, resetting the cycle.

[0073] A, polynucleotide handling enzyme loaded on ssDNA overhang; B, peptide conjugated in the context of dsDNA; C, nanopore inserted in the membrane; D, second polynucleotide handling enzyme bound to the ssDNA overhang while the first translocates.

[0074] Figure 8 Schematic diagram showing an additional non-limiting example of a method for the controlled movement of repeats of a polynucleotide-polypeptide conjugate from trans to cis, wherein movement control is performed by a polynucleotide handling enzyme that moves on ssDNA. Figure 7 Same, except the enzyme is loaded on a strand of continuous ssDNA, except for a spacer moiety (A) that restricts the movement of the enzyme and enables the enzyme to detach from the DNA.

[0075] Figure 9 Schematic diagrams illustrate additional non-limiting examples of schemes for characterizing polypeptides 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 outside the nanopore. The polypeptide is attached to two internal sites on the ssDNA oligonucleotide, and either (i) the ssDNA and peptide or (ii) the dsDNA and peptide co-translocate through the nanopore. B: A polynucleotide handling enzyme controls the polynucleotide-polypeptide conjugate entering the nanopore. The polypeptide is attached to a single site on the ssDNA oligonucleotide, and either (i) the ssDNA and peptide or (ii) the dsDNA and peptide co-translocate through the nanopore. C: A polynucleotide handling enzyme controls the polynucleotide-polypeptide conjugate entering the nanopore. The polypeptide is attached to two internal sites on the ssDNA oligonucleotide, and either (i) the ssDNA and peptide or (ii) the dsDNA and peptide co-translocate through the nanopore. In case (ii) in each of the schemes shown, the polynucleotide handling enzyme can translocate on either DNA strand.

[0076] Figure 10Assembly method for polynucleotide-polypeptide constructs. In the first click reaction (step (i)), a hairpin DNA (DNA1) bearing a 3' TCO group reacts with a peptide bearing an N-terminal azide and a C-terminal methyltetrazine group. A second click reaction (step (ii)) is then performed on DNA2; DNA2 carries a 5' BCN group and a 3' biotin group. To generate the final construct (A), monovalent traptavidin is added (step (iii)). The distance x, defined as the distance in base pairs between the monovalent traptavidin and the peptide (excluding any intermediate linker chemistry), was varied in the experiments described in Example 1. In this example, the "carrier" strand (as opposed to the peptide) is 6 nucleotides long. As shown in Figure (A), the hairpin ends are captured in the nanopore.

[0077] Figure 11 . shows an example current-time trace for capture of a polynucleotide-polypeptide conjugate as described in Example 1. The peptide sequence used throughout the figure is N-GGSG XX SGSG-C: In trace (A), XX = DD; in (B), XX = RR; and in (C), XX = YY. Each trace shows the initial stage of open pore current (i), followed by a decrease to a lower level due to the capture of the conjugate (ii). The normalized current (I / I0; Figure 12 ) is scored as the median current of level (ii) divided by the median current of level (i).

[0078] Figure 12 Three peptides (sequence N-GGSG XX Histograms of normalized currents for SGSG-C, where XX = DD in (A), XX = RR in (B), and XX = YY in (C). Figure 11 Define the normalized current.

[0079] Figure 13 .Peptide carrying sequence N-GGSG XX Plot of normalized current versus distance for a series of polynucleotide-polypeptide conjugates with SGSG-C and the central two residues mutated to DD, RR, or YY. Figure 11 ) is plotted as a function of the distance (in base pairs) between monovalent traptavidin and the peptide. The dsDNA level, shown as a dashed line, was determined from a double-stranded DNA control.

[0080] Figure 14An example current-time trace of capture of a polynucleotide-polypeptide conjugate is shown, where the conjugate is moved out of the nanopore by a helicase, as described in Example 2. The peptide sequence used throughout the figure is N-GGSG XX SGSG-C: In trace (A), XX = DD; in (B), XX = RR; and in (C), XX = YY. Each trace shows an initial phase of open-pore current (i), followed by a decrease to a lower level marked by (ii) due to conjugate capture. Each example shows a repeating pattern indicating repeated movement of the conjugate through the nanopore. At (iii), the helicase controlling the movement dissociates from the conjugate, and without the binding of a second helicase, the current trace returns to the open-pore level (i).

[0081] Figure 15 Example current-time traces for capture of a polynucleotide-polypeptide conjugate are shown, where the conjugate is bound by a helicase, but in the absence of ATP, as described in Example 2. The peptide sequence used is N-GGSGDDSGSG-C. The trace shows an initial period of open pore current (i), followed by a decrease to a lower level marked by (ii) due to capture of the conjugate. Figure 14 In contrast, no repetitive motion is seen, demonstrating that the helicase's movement is ATP-dependent. At (iii), the helicase controlling the movement dissociates from the conjugate, and no second helicase binds, so the current trace returns to the open pore level (i).

[0082] Figure 16 .Polynucleotide-polypeptide constructs used in Example 3: a. leader sequence; b. tether; c. hairpin; d. DNA 1 oligonucleotide; e. DNA 2 oligonucleotide; f. peptide; g. DNA 3 oligonucleotide; x. ssDNA overhang for enzyme loading; y. helicase, where the arrow indicates the direction of helicase movement; circle = click chemistry group; vertical line = attachment site.

[0083] Figure 17 . shows the current-time trace of the capture of a polynucleotide-polypeptide conjugate, where the conjugate is moved out of the nanopore by a helicase, as described in Example 3. The peptide sequence tested is RSDSGQQARY ( Figure 17 A, 2D), GGSGSSSGSG ( Figure 17 B, 17E) and EAIYAAPFAKKK ( Figure 17C, 17F). Each trace, A, B, and C, shows an initial phase of open-pore current (i), followed by a drop to a lower level (ii) due to conjugate capture. Each trace, A, B, and C, further shows a repeating pattern indicating repeated movement of the conjugate through the nanopore. At (iii), the helicase controlling the movement dissociates from the conjugate, and no further helicase binds, so the current trace returns to the open-pore current level. Examples of a single read for each peptide are shown in D, E, and F. The single read exhibits a single-stranded DNA phase (iv), followed by a current drop (v) caused by the peptide. The length of this block (iv) depends on the location of enzyme binding along the single-stranded overhang as the conjugate enters the nanopore. The 100 pA current level is shown as a dashed line to highlight the differences in the magnitude of the peptide drop between the different sequences tested.

[0084] Figure 18 Current-time traces from Example 3 showing individual readouts of polynucleotide-polypeptide conjugates containing peptides with different charges. Movement of the conjugate out of the nanopore is controlled by a 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. The individual readouts exhibit a single-stranded DNA phase (iv), followed by a current drop (v) caused by the peptide. The length of the block (iv) depends on the location of enzyme binding along the single-stranded overhang as the conjugate enters the nanopore. The 0 pA current level is shown as a dashed line to highlight the differences in peptide block amplitudes between the different sequences tested.

[0085] Figure 19 Polynucleotide-polypeptide construct used in Example 4: a. leader sequence; b. tether; c. hairpin adaptor; d. DNA 1 oligonucleotide; e. DNA 2 oligonucleotide; f. peptide; g. DNA 3 oligonucleotide; x. stall; y. helicase, where the arrow indicates the direction of helicase movement; z. bubble; circle = click chemistry group; vertical line = attachment site;

[0086] Figure 20Current-time traces from Example 4, showing the capture of a polynucleotide-polypeptide conjugate, where movement of the conjugate into the nanopore is controlled by the Dda helicase, as described in Example 4. The peptide sequence is HDSGDEVHHQK, a fragment of the amyloid-beta protein. Three example traces are shown, demonstrating signal reproducibility. Each trace shows an initial phase of open-pore current (i), followed by a drop to a lower level due to conjugate capture and helicase arrest (ii). Once the enzyme is electrophoretically un-arrested, the signal shifts to the dsDNA level (iii), followed by a drop in signal due to the peptide (iv). Once the helicase moves the peptide through the constriction, the signal returns to the dsDNA level (iii).

[0087] Figure 21 Polynucleotide-polypeptide constructs used in Example 5. a. DNA 1 oligonucleotide; b. DNA 2 oligonucleotide; c. Peptide; d. DNA 3 oligonucleotide; e. DNA 4 oligonucleotide; f. Sequencing adaptor; x. Arrest; y. Helicase, where the arrow indicates the direction of helicase movement; Circle = click chemistry group, located within the DNA 2 and DNA 4 oligonucleotides; Vertical line = ligation site;

[0088] Figure 22 A1: Current-time trace of enzymatically controlled migration of a polypeptide-polynucleotide conjugate, including DNA-peptide co-translocation, as described in Example 5. A2: Zoom in on the highlighted segment in the A1 trace. B1: Current-time trace for a control experiment in which the peptide alone translocated through the nanopore. B2: Zoom in on the highlighted segment in the B1 trace.

[0089] Each trace shows an initial phase of open-pore current (i), followed by a dip to a certain segment of the adaptor sequence after analyte capture (ii), followed by a current spike caused by the peptide (iii). The peptide is flanked by oligonucleotides containing 40 dT bases, which produce flat segments in the signal on either side of the peptide (iv).

[0090] Figure 23 Polynucleotide-polypeptide constructs used in Example 6. a. DNA 1 oligonucleotide; b. DNA 2 oligonucleotide, containing an internal click chemistry group and showing the DNA 'flap' along with the peptide; c. Peptide; d. DNA 3 oligonucleotide; e. DNA 4 oligonucleotide; f. Sequencing adapter; x. Arrest; y. Helicase, with arrows indicating the direction of helicase movement; circle = click chemistry group; vertical line = ligation site;

[0091] Figure 24A1: Current-time traces of enzymatically controlled migration of a polypeptide-polynucleotide conjugate, including co-translocation of the DNA flap and peptide, as described in Example 6. A2: Zoom-in of the highlighted segment in the A1 trace. Each trace shows an initial phase of open-pore current (i), followed by a dip to a certain segment of the adaptor sequence after analyte capture (ii), followed by a current spike caused by the peptide (iii). The peptide is flanked by oligonucleotides containing 40 dT bases, which produce flat segments in the signal on either side of the peptide (iv). DETAILED DESCRIPTION

[0092] The present invention will be described with respect to specific embodiments and with reference to certain drawings, but the invention is not limited thereto but only by the claims. Any reference signs in the claims should not be construed as limiting the scope. Of course, it should be understood that not all aspects or advantages need be achieved according to any particular embodiment of the present invention. Thus, for example, those skilled in the art will recognize that the present invention can be embodied or performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages taught or suggested herein.

[0093] The organization and method of operation of the present invention, as well as its features and advantages, are best understood by reference to the following detailed description when read in conjunction with the accompanying drawings. Aspects and advantages of the present invention will become apparent from and will be elucidated with reference to the embodiments described hereinafter. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Similarly, it should be understood that in the description of exemplary embodiments of the present invention, various features of the present invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding understanding of one or more of the various inventive aspects. However, this method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than expressly recited in each claim. Rather, as reflected in the following claims, inventive aspects lie in less than all features of a single aforementioned disclosed embodiment.

[0094] It should be understood that "embodiments" of the present disclosure may be specifically combined together unless the context dictates otherwise. All specific combinations of disclosed embodiments (unless the context dictates otherwise) are further disclosed embodiments of the claimed invention.

[0095] In addition, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes two or more polynucleotides, reference to a "motor protein" includes two or more such proteins, reference to a "helicase" includes two or more helicases, reference to a "monomer" refers to two or more monomers, reference to a "pore" includes two or more pores, etc.

[0096] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0097] definition

[0098] Where an indefinite or definite article is used when referring to a singular noun (for example, "a" or "an", "the"), this includes the plural form of the noun unless specifically stated otherwise. When the term "comprising" is used in this specification and claims, it does not exclude other elements or steps. In addition, the terms first, second, third, etc. in the specification and claims are used to distinguish similar elements and are not necessarily used to describe a sequential or chronological order. It should be understood that the terms used in this way are interchangeable where appropriate, and that the embodiments of the invention described herein are capable of operating in other sequences than those described or illustrated herein. The following terms or definitions are provided only to assist in understanding the present invention. Unless specifically defined herein, all terms used herein have the same meaning as understood by those skilled in the art in the field of the invention. For definitions and terminology in the art, practitioners are particularly referred to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Suppl. 114), John Wiley & Sons, New York (2016). The definitions provided herein should not be construed to have a scope less than that understood by one of ordinary skill in the art.

[0099] As used herein, the term "about" when referring to a measurable value such as an amount, duration, etc., is meant to encompass deviations of ± 20% or ± 10%, more preferably ± 5%, even more preferably ± 1% and still more preferably ± 0.1% from the specified value, as such deviations are suitable for performing the disclosed methods.

[0100] As used herein, "nucleotide sequence," "DNA sequence," or "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Thus, the term includes double-stranded and single-stranded DNA, as well as RNA. As used herein, the term "nucleic acid" is a single-stranded or double-stranded covalently linked sequence of nucleotides in which the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond. A polynucleotide can be composed of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids can be synthesized in vitro or isolated from natural sources. Nucleic acids can further include modified DNA or RNA, such as DNA or RNA that has been methylated, or RNA that has been subjected to post-translational modifications, such as 5' capping with 7-methylguanosine, 3' processing such as cleavage and polyadenylation, and splicing. Nucleic acids can also include synthetic nucleic acids (XNA), such as hexitol nucleic acids (HNA), cyclohexene nucleic acids (CeNA), threose nucleic acids (TNA), glycerol nucleic acids (GNA), locked nucleic acids (LNA), and peptide nucleic acids (PNA). The size of nucleic acids (also referred to herein as "polynucleotides") is typically expressed as the number of base pairs (bp) for double-stranded polynucleotides or as the number of nucleotides (nt) in the case of single-stranded polynucleotides. One thousand bp or nt equals a kilobase (kb). Polynucleotides less than about 40 nucleotides in length are often referred to as "oligonucleotides" and may include primers used in manipulations of DNA, such as by the polymerase chain reaction (PCR).

[0101] In the context of the present disclosure, the term "amino acid" is used in its broadest sense and is intended to include organic compounds containing amine (NH2) and carboxyl (COOH) functional groups and side chains (e.g., R groups) specific to each amino acid. In some embodiments, amino acids refer to naturally occurring L α-amino acids or residues. The commonly used single-letter and three-letter abbreviations 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 D-amino acids, retro-inverso amino acids, and chemically modified amino acids (e.g., amino acid analogs), naturally occurring amino acids not typically incorporated into proteins (e.g., norleucine), and chemically synthesized compounds with properties known in the art to be unique to amino acids (e.g., β-amino acids). For example, analogs or mimetics of phenylalanine or proline that allow conformational restriction of peptide compounds identical to naturally occurring Phe or Pro are included within the definition of an amino acid. Such analogs and mimetics are referred to herein as "functional equivalents" of the corresponding amino acids. Additional examples of amino acids are listed 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.

[0102] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to polymers of amino acid residues, as well as variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids (e.g., chemical analogs of corresponding naturally occurring amino acids), as well as naturally occurring amino acid polymers. Polypeptides may also undergo maturation or post-translational modification processes, which may include, but are not limited to, glycosylation, proteolytic cleavage, lipidation, signal peptide cleavage, propeptide cleavage, phosphorylation, and the like. Peptides can be prepared using recombinant techniques (e.g., by expressing recombinant or synthetic polynucleotides). Recombinantly produced peptides are typically substantially free of culture medium, e.g., culture medium accounts for less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0103] The term "protein" is used to describe a folded polypeptide having a secondary or tertiary structure. A protein may consist of a single polypeptide or may comprise multiple polypeptides assembled to form a multimer. Multimers may be homo-oligomers or hetero-oligomers. A protein may be a naturally occurring or wild-type protein or a modified or non-naturally occurring protein. For example, a protein may differ from a wild-type protein by the addition, substitution, or deletion of one or more amino acids.

[0104] " Variants " of proteins encompass peptides, oligopeptides, polypeptides, proteins and enzymes that have amino acid substitutions, deletions and / or insertions relative to the unmodified or wild-type protein in question and have biological and functional activities similar to the unmodified protein from which they are derived. As used herein, the term "amino acid identity" refers to the degree to which a sequence is identical on an amino acid to amino acid basis over a comparison window. Therefore, "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the same 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) appear in the two sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to produce percentage of sequence identity.

[0105] For all aspects and embodiments of the invention, a "variant" has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% complete sequence identity with the amino acid sequence of the corresponding wild-type protein. Sequence identity can also be to fragments or portions of a full-length polynucleotide or polypeptide. Thus, a sequence may have only 50% overall sequence identity with a full-length reference sequence, but the sequence of a particular region, domain or subunit may share 80%, 90% or up to 99% sequence identity with the reference sequence.

[0106] The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is the most commonly observed gene in a population and is therefore arbitrarily designated as the "normal" or "wild-type" form of a gene. In contrast, the terms "modified," "mutant," or "variant" refer to a gene or gene product that exhibits sequence modifications (e.g., substitutions, truncations, or insertions), post-translational modifications, and / or functional properties (e.g., altered characteristics) compared to the wild-type gene or gene product. It should be noted that naturally occurring mutants can be isolated; these mutants are identified by having altered properties compared to the 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 replaced 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 non-naturally occurring amino acids are also well known in the art. For example, non-naturally occurring amino acids can be introduced by including synthetic aminoacyl-tRNAs in the IVTT system used to express mutant monomers. Alternatively, the non-naturally occurring amino acids can be introduced by expressing mutant monomers in Escherichia coli (E. coli) that are auxotrophic for specific amino acids in the presence of synthetic (i.e., non-naturally occurring) analogs of those amino acids. If the mutant monomers are produced using partial peptide synthesis, they can also be produced by naked ligation. 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 acid can have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acid it replaces. Alternatively, conservative substitutions can introduce another aromatic or aliphatic amino acid in place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected based on the properties of the 20 major amino acids, as defined in Table 1 below. In cases where amino acids have similar polarity, this can also be determined by reference to the amino acid side chain hydrophilicity scale in Table 2.

[0107] Table 1 - Chemical properties of amino acids

[0108]

[0109] Table 2 - Hydrophilicity Scale

[0110]

[0111] Mutants or modified proteins, monomers or peptides can also be chemically modified in any manner and at any site. Preferably, the mutant or modified monomer or peptide is chemically modified by linking the molecule to one or more cysteines (cysteine ​​linkage), linking the molecule to one or more lysines, linking the molecule to one or more non-natural amino acids, enzymatically modifying the epitope, or modifying the termini. Methods suitable for performing such modifications are well known in the art. Mutants of modified proteins, monomers or peptides can be chemically modified by the linkage of any molecule. For example, mutants of modified proteins, monomers or peptides can be chemically modified by the linkage of a dye or fluorophore.

[0112] The disclosed method

[0113] The present disclosure relates to methods for characterizing polypeptides. The polypeptide is characterized in that it co-translocates through a nanopore along with one or more polynucleotide chains (such as one or more polynucleotide flanking chains and / or carrier chains, as described in more detail herein). The polypeptide and polynucleotide chains together can be referred to as a construct.

[0114] The method exploits the ability of many nanopores to accommodate multiple polymer chains simultaneously.Co-translocation of a polypeptide chain and one or more polynucleotide chains has advantages as discussed in more detail herein.

[0115] In contrast to methods that seek to use polypeptide handling enzymes to control the movement of polypeptides relative to nanopores, certain embodiments of the present disclosure relate to methods that involve using polynucleotide handling enzymes to control the movement of polypeptides relative to nanopores. Other embodiments of the present disclosure do not require the use of enzymes to control the movement of constructs.

[0116] Therefore, in one aspect, the present invention provides a method for characterizing a target polypeptide, the method comprising

[0117] - contacting (i) a polynucleotide-polypeptide conjugate chain comprising a target polypeptide conjugated at each terminus of the target polypeptide to one or more polynucleotide flanking chains with (ii) a polynucleotide carrier chain, thereby forming a polynucleotide-polypeptide construct;

[0118] - contacting the construct with the nanopore under conditions such that both the polynucleotide-polypeptide conjugate chain and the polynucleotide carrier chain co-translocate through the nanopore; and

[0119] - performing one or more measurements specific to the polypeptide as the construct moves relative to the nanopore,

[0120] The target polypeptide is thereby characterized.

[0121] In a related aspect, a method of characterizing a target polypeptide is provided, the method comprising

[0122] - contacting (i) a polynucleotide-polypeptide conjugate chain comprising a target polypeptide conjugated at each terminus of the target polypeptide to one or more polynucleotide flanking chains with (ii) a polynucleotide carrier chain, thereby forming a polynucleotide-polypeptide construct;

[0123] - controlling movement of the construct through the nanopore such that both the polynucleotide-polypeptide conjugate chain and the polynucleotide carrier chain co-translocate through the nanopore; and

[0124] - performing one or more measurements specific to the polypeptide as the construct moves relative to the nanopore,

[0125] The target polypeptide is thereby characterized.

[0126] In another related aspect of the present disclosure, a method for characterizing a target polypeptide is provided, the method comprising

[0127] contacting (i) a polynucleotide-polypeptide conjugate chain comprising a target polypeptide linked to a polynucleotide wing chain with (ii) a polynucleotide handling protein capable of controlling movement of the polynucleotide wing chain relative to the nanopore; and

[0128] - contacting the polynucleotide-polypeptide conjugate chain with the nanopore under conditions such that the polynucleotide handling protein controls movement of the polynucleotide-polypeptide conjugate chain relative to the nanopore; and

[0129] - performing one or more measurements specific to the polypeptide as the polynucleotide flanking strands and the target polypeptide co-translocate through the nanopore,

[0130] The target polypeptide is thereby characterized.

[0131] In a related aspect, a method of characterizing a target polypeptide is provided, the method comprising

[0132] - contacting (i) a polynucleotide-polypeptide conjugate chain comprising a target polypeptide linked to a polynucleotide flanking strand with (ii) a polynucleotide handling protein;

[0133] - controlling the movement of the polynucleotide flanking strands relative to the nanopore using the polynucleotide handling protein; and

[0134] - contacting the polynucleotide-polypeptide conjugate chain with the nanopore under conditions such that the polynucleotide handling protein controls movement of the polynucleotide-polypeptide conjugate chain relative to the nanopore; and

[0135] - performing one or more measurements specific to the polypeptide as the polynucleotide flanking strands and the target polypeptide co-translocate through the nanopore,

[0136] The target polypeptide is thereby characterized.

[0137] It will be apparent to those skilled in the art that a polynucleotide handling protein is not required for the methods provided herein, although in some preferred embodiments, a polynucleotide handling protein is used to control the movement of constructs, polynucleotide-polypeptide conjugate chains and / or carrier chains as described herein. Thus, in some embodiments, for example, in some embodiments that do not necessarily involve a polynucleotide handling protein, provided herein is a method for characterizing a target polypeptide comprising

[0138] - contacting (i) a polynucleotide-polypeptide conjugate chain comprising a target polypeptide linked to a polynucleotide flanking chain with (ii) a nanopore;

[0139] - controlling the movement of the polynucleotide-polypeptide conjugate chain relative to the nanopore; and

[0140] - performing one or more measurements specific to the polypeptide as the polynucleotide flanking strands and the target polypeptide co-translocate through the nanopore,

[0141] The target polypeptide is thereby characterized.

[0142] The above methods may be referred to herein as disclosed methods. For the avoidance of doubt, for the sake of brevity, embodiments of the present disclosure are described herein in conjunction with the disclosed methods. Unless the context otherwise requires, such embodiments are expressly disclosed in conjunction with and as such preferred features of each of the above disclosed methods.

[0143] 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 that obtained by nuclease digestion of the protein or 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.

[0144] Any suitable polynucleotide can be used to form a polynucleotide-polypeptide conjugate chain for use in the methods disclosed herein. The polynucleotide chain connected to the polypeptide chain in the polynucleotide-polypeptide conjugate chain can be referred to as a side chain. Side chains are described in more detail herein.

[0145] In some embodiments, the length of the or each polynucleotide flanking strand is at least as long as the portion of the target polypeptide to be characterized. In some embodiments, the length of the or each polynucleotide flanking strand is greater than the portion of the target polypeptide to be characterized. In some embodiments, the length of the or each polynucleotide flanking strand is shorter than the portion of the target polypeptide to be characterized. Polynucleotides suitable for use in the disclosed methods are disclosed in more detail herein.

[0146] In some embodiments, the or each polynucleotide-polypeptide conjugate chain is complexed with a polynucleotide carrier chain. In some embodiments, the polynucleotide flanking strands of the polynucleotide-polypeptide conjugate chain are each independently complementary to a region of the polynucleotide carrier chain. In some embodiments, the polynucleotide flanking strands of the polynucleotide-polypeptide conjugate chain are each independently complementary to a region of the polynucleotide carrier chain. In some embodiments, the polynucleotide flanking strands of the polynucleotide-polypeptide conjugate chain are each independently 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% complementary to a corresponding region of the polynucleotide carrier chain.

[0147] In some embodiments, the one or more polynucleotide flanking strands are each independently hybridized at least partially with the polynucleotide carrier strand. Each flanking strand can independently hybridize 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%, at least 99.5% with the complementary strand over the length of the flanking strand. For example, in some embodiments, in some embodiments, the one or more polynucleotide flanking strands are each independently hybridized with the polynucleotide carrier strand over a length of at least 5, such as at least 10, such as at least 20, such as at least 30, such as at least 40, such as at least 50, such as at least 60, such as at least 80, such as at least 100, such as at least 150, such as at least 200, such as at least 500 or more nucleotides. The complementary strands described herein can associate and therefore hybridize together by, for example, hydrogen bonds (e.g., base pairing). Those skilled in the art will appreciate that the strength of hybridization between a polynucleotide carrier strand and the polynucleotide flanking strands of a polynucleotide-polypeptide conjugate strand can be controlled by controlling, among other things, the length and degree of complementarity of the hybridization region. Shorter flanking strands can be used to reduce the hybridization force between the flanking strands of a polynucleotide-polypeptide conjugate strand and the carrier strand. Longer strands can be used to increase the hybridization force between the flanking strands of a polynucleotide-polypeptide conjugate strand and the carrier strand. Increasing the degree of complementarity can be used to increase the hybridization force between the flanking strands of a polynucleotide-polypeptide conjugate strand and the carrier strand.

[0148] In some embodiments, a polynucleotide-polypeptide conjugate chain or a carrier chain may protrude beyond the other chain of the construct. For example, in some embodiments, a polynucleotide-polypeptide conjugate chain protrudes beyond the carrier chain at one end. For example, the 3' end of the polynucleotide-polypeptide conjugate chain may protrude beyond the 5' end of the carrier chain. In other embodiments, the carrier chain may protrude beyond the polynucleotide-polypeptide conjugate chain at one end of the polynucleotide-polypeptide conjugate chain. For example, the 3' end of the carrier chain may protrude beyond the 5' end of the polynucleotide-polypeptide conjugate chain. Of course, an overhang may alternatively be present at the other end of the construct, for example, the 5' end of the polynucleotide-polypeptide conjugate chain may protrude beyond the 3' end of the carrier chain; or the 5' end of the carrier chain may protrude beyond the 3' end of the polynucleotide-polypeptide conjugate chain. In some embodiments, an overhang may be present at each end of the construct. In some embodiments, the overhang provides a loading site for the polynucleotide processing protein.

[0149] In the disclosed methods, any suitable means can be used to conjugate the target polypeptide to the polynucleotide. Some exemplary means are described in more detail herein.

[0150] In some embodiments, as discussed in more detail herein, the conjugate formed in the disclosed methods is contacted with a polynucleotide handling protein. The polynucleotide handling protein is generally capable of controlling the movement of the polynucleotide flanking strands and / or the polynucleotide carrier strand (if present) relative to the nanopore. Exemplary polynucleotide handling proteins are described in more detail herein.

[0151] When present, the polynucleotide handling protein controls the movement of the polynucleotide chain relative to the nanopore. As discussed in more detail herein, the polynucleotide handling protein can control the movement of the polynucleotide flanking chains relative to the nanopore. The polynucleotide handling protein can control the movement of the polynucleotide carrier chain relative to the nanopore. The polynucleotide handling protein can control the movement of both the polynucleotide flanking chains and the polynucleotide carrier chain relative to the nanopore. Thus, the polynucleotide handling protein controls the movement of the construct relative to the nanopore. Any suitable nanopore can be used in the disclosed methods. Nanopores suitable for use in the disclosed methods are described in more detail herein.

[0152] The disclosed methods comprise performing one or more measurements specific to the polypeptide as the or each polypeptide portion of a polynucleotide-polypeptide conjugate chain moves relative to a nanopore. The one or more measurements can be any suitable measurement. Typically, the one or more measurements are electrical measurements, such as current measurements, and / or one or more optical measurements. Apparatus for recording suitable measurements and the information that such measurements can provide are described in more detail herein.

[0153] Certain disclosed methods may also be used to characterize target polynucleotides, and unless the context suggests otherwise, features of the disclosed methods may generally apply to such methods. Certain methods of characterizing target polynucleotides are described in more detail herein.

[0154] Characterization of target peptides

[0155] Described herein are methods for characterizing a target polypeptide, the methods comprising: forming a construct comprising a first strand comprising the target polypeptide and a second strand comprising a polynucleotide, moving the construct relative to the nanopore under conditions such that the first and second strands of the construct co-translocate through the nanopore, and performing one or more measurements specific to the polypeptide as the construct moves relative to the nanopore. The methods described herein may comprise the step of controlling the movement of the construct through the nanopore.

[0156] As disclosed herein, polynucleotides can be used to control the movement of a target polypeptide relative to a nanopore. Because 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 polynucleotide flanking chains. The polynucleotide-polypeptide conjugate chain can hybridize to a polynucleotide carrier chain.

[0157] Thus, the methods provided herein involve (i) a target polypeptide and (ii) a polynucleotide chain Co-translocation In some embodiments, the polynucleotide strands that co-translocate through the nanopore are polynucleotide flanking strands as described herein. In some embodiments, the polynucleotide strands that co-translocate through the nanopore are polynucleotide carrier strands as described herein.

[0158] As used herein, the term "co-translocation" refers to translocation together and simultaneously. Thus, with respect to the methods described herein, co-translocation through a nanopore refers to translocation through a nanopore together and simultaneously.

[0159] In the methods described herein, a target polypeptide and a polynucleotide chain are co-translocated through a nanopore. Thus, the target polypeptide and the polynucleotide chain are translocated together and simultaneously through the nanopore. This co-translocation of the two chains necessarily requires that the two chains pass through the nanopore side by side in a double-stranded configuration. Thus, by way of further example, in some embodiments of the methods described herein, the target polypeptide and polynucleotide chain that are co-translocated through the nanopore can do so in the form of a double-stranded polypeptide-polynucleotide chimera.

[0160] The foregoing can be contrasted with prior art methods such as those described in WO 2021 / 111125 and WO 2021 / 133168. In such prior art methods, a polynucleotide handling enzyme is used to move a single-chain polypeptide-polynucleotide conjugate through a nanopore, such that the polypeptide and polynucleotide portions of the conjugate move (i.e., translocate) through the nanopore one by one in a linear manner, without the double-chain polypeptide-polynucleotide moving through the pore. This linear translocation of polypeptides and polynucleotides one by one contrasts with the "parallel" movement of target polypeptide and polynucleotide chains provided by the methods of the present invention disclosed herein.

[0161] The simultaneous co-translocation of the target polypeptide and polynucleotide chains means that the analyte (e.g., construct) moving through the pore at the measurement point must be double-stranded. One strand comprises the polynucleotide. The other strand comprises the target polypeptide. Therefore, as used herein, the term "double-stranded" encompasses polynucleotide strands running parallel to the polypeptide strand. This offers advantages over methods known in the art for characterizing polypeptides.

[0162] For example, the target polypeptide is typically substantially uncharged or has a low net charge and / or charge density, and / or is irregularly charged. In other words, the charge distribution in the target polypeptide is typically irregularly distributed along the length of the target polypeptide. As described in Table 1 above, some amino acids contained in the target polypeptide are polar, and some amino acids are non-polar. Some amino acids are positively or negatively charged under physiological conditions, others are uncharged under physiological conditions but may be charged under conditions where a method such as the method disclosed herein is performed, while still others are uncharged under all relevant conditions. The distribution of amino acids in the target polypeptide is a function of the exact analyte being characterized in the disclosed methods and therefore may not be known in advance by the user.

[0163] In known peptide analysis methods that rely on the electrophoretic movement of peptides through nanopores, this irregular charge along the target peptide can present difficulties because the electrophoretic forces acting on the peptide will vary as the peptide chain moves through the nanopore. As a result, the rate of migration of the peptide through the nanopore can be unpredictable, which hinders accurate characterization. For example, it may be difficult to distinguish two identical amino acids that move rapidly through the pore from a single amino acid that moves more slowly. The low average charge density of the target peptide is a factor that can be included. polynucleotides Methods for detecting or characterizing proteins and their analogs (e.g., PNA; peptide nucleic acids) are generally not suitable for target peptides Therefore, methods that do not rely on the different charges of the polypeptides to determine the movement of polypeptides through the pore are needed.

[0164] Another problem that arises with known methods for peptide analysis is that target polypeptides often possess irregular 3D structures. For example, proteins are known to fold into 3D structures that may be relevant to their biological function. The presence of 3D structures (e.g., secondary or tertiary structures) in target polypeptides can hinder their characterization using nanopores in known methods that rely on translocation of a single strand of the target polypeptide through a pore. This is because different parts of the folded target polypeptide will require varying degrees of force to unfold in order to translocate in this manner. As a result, the movement of the polypeptide through the pore may be irregular, with some parts moving through the pore faster than others, potentially hindering accurate characterization. Furthermore, using nanopores in this manner may not allow for the unfolding of the protein. Therefore, methods that involve decorating a double-stranded polynucleotide with a folded protein alone and translocating the construct through a nanopore (e.g., solid-state nanopores) generally fail to provide detailed information about the protein, such as its sequence.

[0165] A further problem that can arise in some known methods for characterizing polypeptides using nanopores is that motor proteins that can be used to control the movement of such polypeptides can sometimes be inefficient at precisely controlling the movement of long polypeptide chains, even though they can efficiently translocate along such chains. For example, when used to control the movement of very long polypeptide chains (e.g., in the form of polynucleotide-polypeptide conjugates), the motor protein can slip along the polypeptide portion of the chain as it moves through the nanopore. Slippage is problematic because it can lead to inaccurate characterization of the polypeptide.

[0166] The present method solves some or all of these problems. As described above, the simultaneous co-translocation of the target polypeptide and the polynucleotide chain means that the analyte moving through the hole at the measurement point must be double-stranded. In some embodiments, this will bring some or all of the following advantages.

[0167] First, the polynucleotide chain that co-translocates the nanopore with the target polypeptide will generally have a regular charge density and will therefore exert a regular electrophoretic force on the construct as it moves relative to the nanopore. The forces exerted by the polynucleotide moving through the nanopore are well understood and can be accurately modeled. The co-translocated polynucleotide can therefore drive predictable and consistent movement of the target polypeptide through the pore, thereby facilitating the characterization of the polypeptide.

[0168] Second, a polynucleotide chain that co-translocates with the target polypeptide nanopore can be selected or configured to effectively linearize the target polypeptide. For example, the target polypeptide can be connected to a polynucleotide flanking chain at each end of the analyte, and the polynucleotide flanking chain can be hybridized with the polynucleotide carrier chain. The hybridization of the flanking chain prevents the folding of the polypeptide. The flanking chain can be designed to stretch the target polypeptide to a desired degree according to the parameters of the method as operated by the user. Because the folding of the target polypeptide can be reduced or eliminated, the target polypeptide moves through the nanopore more regularly.

[0169] Third, the polynucleotide handling protein can be contacted with the polynucleotide-polypeptide conjugate chain and / or the polynucleotide carrier chain and used to control the construct with respect to the nanopore. In embodiments where the polynucleotide handling protein is used to control the movement of the polynucleotide chain in order to control the movement of the construct, the length of the target polypeptide is not particularly limited because the polynucleotide handling protein progressively processes the polynucleotide chain, which is co-translocated with the polypeptide chain, even as the polypeptide chain moves through the nanopore.

[0170] Thus, the methods provided herein allow for some or all of the following: improved capabilities for detecting target polypeptides; improved accuracy of polypeptide characterization, improved reproducibility of polypeptide data, improved (increased) polypeptide read lengths, and reduced slippage.

[0171] You can refer to Figure 1Understand the method of the present invention, Figure 1 A non-limiting example of the disclosed method is presented.

[0172] The construct can comprise a polynucleotide-polypeptide conjugate chain comprising a target polypeptide flanked by one or more polynucleotide flanking chains. In some embodiments, the construct can be contacted with a polynucleotide handling protein such that the construct passes through the nanopore. Optionally, additional polymers such as leader sequences (described herein) can be used to facilitate passage of the polypeptide through the nanopore. Such use is within the scope of the disclosed methods, however, it is not required. Optionally, spacers and / or stoppers can be used to restrict the movement of the polynucleotide handling protein prior to the user operating the method ( Figure 1 ). This is described herein and is within the scope of the disclosed methods, but is not required.

[0173] In some embodiments, the polynucleotide processing protein can process the polynucleotide carrier strand. When the polynucleotide processing protein processes the polynucleotide carrier strand, the construct passes through the nanopore because the carrier strand hybridizes to the polynucleotide-polypeptide conjugate strand (e.g., hybridizes to a flanking strand portion of the polynucleotide-polypeptide conjugate strand). As a result, the polypeptide passes through the nanopore. As the polypeptide passes through the nanopore, the polypeptide is characterized.

[0174] In some embodiments, the polynucleotide processing protein can process the polynucleotide-polypeptide conjugate chain (e.g., by processing the polynucleotide flanking chains). When the polynucleotide processing protein processes the polynucleotide flanking chains, the construct passes through the nanopore because the flanking chains hybridize to the polynucleotide carrier chain. Thus, the polypeptide passes through the nanopore. As the polypeptide passes through the nanopore, the polypeptide is characterized.

[0175] You can also refer to Figure 6 Understand the method of the present invention, Figure 6 Another non-limiting example of the disclosed method is presented.

[0176] A polynucleotide-polypeptide conjugate chain comprising a target polypeptide flanked by one or more polynucleotide wing chains can pass through a nanopore.

[0177] In some embodiments, the polynucleotide-polypeptide conjugate chain moves relative to the hole. In some embodiments, the target polypeptide is spanned by the polynucleotide flanking chains. Therefore, when the target polypeptide translocates through the nanopore, the target polypeptide is spanned by the polynucleotide flanking chains, and thus the polynucleotide flanking chains and the target polypeptide co-translocate through the hole. In some embodiments, the target polypeptide is spanned by the polynucleotide flanking chains and is connected to the polynucleotide flanking chains at one end of the target polypeptide. In some embodiments, the target polypeptide is spanned by the polynucleotide flanking chains and is connected to the polynucleotide flanking chains at each end of the target polypeptide. In some embodiments, each end of the target polypeptide is conjugated to a polynucleotide flanking chain, and one or two polynucleotide flanking chains further span the target polypeptide. In some embodiments, one end of the target polypeptide is conjugated to a first polynucleotide flanking chain; the other end of the target polypeptide is conjugated to a second polynucleotide flanking chain; and one or both of the first polynucleotide flanking chain and the second polynucleotide flanking chain span the target polypeptide. In some embodiments, the portion of the flanking chain that spans the target polypeptide is a polynucleotide of the same type as the remainder of the flanking chain. In some embodiments, the portion of the flanking chain that spans the target polypeptide is a polynucleotide of a different type than the remainder of the flanking chain.

[0178] In some embodiments, the polynucleotide-polypeptide conjugate strand is hybridized to a polynucleotide carrier strand as described herein. In some embodiments, the resulting construct is translocated through a nanopore.

[0179] In some embodiments, movement of the construct through the nanopore results in co-translocation of three strands: the target polypeptide, a polynucleotide flanking strand spanning the target polypeptide, and a polynucleotide carrier strand. Figure 6 B. However, in some embodiments, the polynucleotide carrier strand is removed (e.g., dehybridized and thereby unzipped), for example, by the nanopore. In such embodiments, when the target polypeptide is translocated through the nanopore, the target polypeptide is spanned by the polynucleotide flanking strands, and thus the polynucleotide flanking strands and target polypeptide are co-translocated through the pore, and the polynucleotide carrier strand is excluded from the nanopore. This is shown in FIG. Figure 6 Shown in C.

[0180] In some embodiments, the polynucleotide-polypeptide conjugate chain and / or the polynucleotide carrier chain are contacted with the polynucleotide handling protein to allow the construct to pass through the nanopore. However, this is not required. Optionally, additional polymers, such as leader sequences (described herein), can be used to facilitate passage of the polypeptide through the nanopore. Such use is within the scope of the disclosed methods, however, this is not required. Optionally, spacers and / or stoppers can be used to restrict the movement of the polynucleotide handling protein (if present) prior to the user performing the method. This is described herein and is within the scope of the disclosed methods, but is not required.

[0181] exist Figure 1and 6 In the examples shown in FIG, from the "point of view" of the polynucleotide handling protein, the polynucleotide handling protein "moves" the construct into the pore. For example, as shown, the polynucleotide handling protein can be located 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 arrangement can also be used.

[0182] In other words, in some embodiments, the polynucleotide handling protein can be 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. 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 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. 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 the polypeptide from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of the polypeptide through the nanopore.

[0183] In other embodiments, 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 trans side of the nanopore to the cis side of 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 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. Thus, 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 polypeptide from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of the polypeptide through the nanopore.

[0184] As explained in more detail herein, the construct may comprise a leader sequence. The leader sequence may be present on the polynucleotide-polypeptide conjugate strand. The leader sequence may be present on the carrier strand (if present).

[0185] The leader sequence can be present on the same strand that is processed by the polynucleotide handling protein (if present). For example, in some embodiments, the polynucleotide-polypeptide conjugate strand comprises the leader sequence, and the polynucleotide handling protein controls the movement of the polynucleotide-polypeptide conjugate strand relative to the nanopore. In other embodiments, the carrier strand comprises the leader sequence, and the polynucleotide handling protein controls the movement of the carrier strand relative to the nanopore.

[0186] The leader sequence can be present on one strand of the construct, and the polynucleotide handling protein can control the movement of the other strand of the construct. For example, in some embodiments, the polynucleotide-polypeptide conjugate strand comprises the leader sequence, and the polynucleotide handling protein controls the movement of the carrier strand relative to the nanopore. In other embodiments, the carrier strand comprises the leader sequence, and the polynucleotide handling protein controls the movement of the polynucleotide-polypeptide conjugate strand relative to the nanopore.

[0187] When a carrier strand is present, a leader sequence can be attached to both the polynucleotide-polypeptide conjugate strand and the carrier strand. For example, a Y-adapter as described herein can be used to connect the polynucleotide-polypeptide conjugate strand and the carrier strand. The stem of the Y-adapter can provide a leader sequence, such as a single-stranded or double-stranded polynucleotide portion.

[0188] When present, the leader sequence can comprise nucleotide units, spacer units and / or other monomeric units that can be linked together to form the leader sequence. For example, the leader sequence can comprise one or more nucleotide units and / or one or more spacer units. Suitable spacer units are described in more detail herein, and the spacer units include, for example, one or more C3, iSp9 and / or iSp18 spacers as described herein.

[0189] Any suitable leader sequence can be used, as explained herein. Optionally, the leader sequence can be a polynucleotide. The leader sequence can be the same as or different from the polynucleotide in the conjugate. As explained above, the leader sequence can facilitate passage of the conjugate through the nanopore.

[0190] In other words, in some embodiments, the polynucleotide-polypeptide conjugate chain comprises a polynucleotide of the form L-{PN}-P m One or more structures, wherein:

[0191] - L is a leader sequence, wherein L is optionally a moiety of N;

[0192] -P is a polypeptide;

[0193] -N comprises a polynucleotide; and

[0194] -m is 0 or 1;

[0195] And the method may comprise passing the leader sequence (L) through the nanopore, thereby contacting the polypeptide (P) with the nanopore.

[0196] A polynucleotide-polypeptide conjugate chain can hybridize to a carrier chain. The N portion of a polynucleotide-polypeptide conjugate chain can hybridize to one or more polynucleotide carrier chains. In some embodiments, each N portion of a polynucleotide-polypeptide conjugate chain hybridizes to a polynucleotide carrier chain.

[0197] In some such embodiments, the polynucleotide handling protein is located on the cis side of the nanopore, and the method comprises allowing the polynucleotide handling protein to control the movement of the polynucleotide portion (N) from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of the polypeptide (P) through the nanopore. In other embodiments, the polynucleotide handling protein is located on the trans side of the nanopore, and the method comprises allowing the polynucleotide handling protein to control the movement of the polynucleotide portion (N) from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of the polypeptide (P) through the nanopore.

[0198] As explained in more detail herein, in some embodiments, the construct comprises a plurality of polynucleotides and polypeptides. In such embodiments, the polynucleotide handling protein sequentially controls the movement of the polynucleotide-polypeptide conjugate chains and / or carrier chains relative to the nanopore, thereby sequentially moving each polypeptide relative to the nanopore. In this manner, each polypeptide within the conjugate can be sequentially characterized in the disclosed methods.

[0199] In some embodiments, a polynucleotide-polypeptide conjugate chain comprises multiple target polypeptides. In some embodiments, a polynucleotide-polypeptide conjugate chain comprises multiple target polypeptides. For example, a polynucleotide-polypeptide conjugate chain may comprise one or more moieties of the form ...NPNPN..., where each N (which may be the same or different) is a polypeptide, and where each N (which may be the same or different) comprises a polynucleotide. Some or all N moieties of the polynucleotide-polypeptide conjugate chain may hybridize to one or more polynucleotide carrier chains. In some embodiments, each N moiety of the polynucleotide-polypeptide conjugate chain hybridizes to a polynucleotide carrier chain.

[0200] For example, a polynucleotide-polypeptide conjugate chain may comprise a chain of the form L-P1-N-{PN} n -P m One or more structures, wherein:

[0201] -n is a positive integer;

[0202] - L is a leader sequence, wherein L is optionally a moiety of N;

[0203] - each P, which may be the same or different, is a polypeptide;

[0204] - each N, which may be the same or different, comprises a polynucleotide; and

[0205] -m is 0 or 1;

[0206] And the method may comprise passing the leader sequence (L) through the nanopore, thereby contacting the polypeptide (P1) with the nanopore.

[0207] Typically, in such embodiments, n is from 1 to about 1000, for example, from 2 to about 100, such as from about 3 to about 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0208] In some such embodiments, the polynucleotide handling protein is located on the cis side of the nanopore, and the method comprises allowing the polynucleotide handling protein to control the movement of each polynucleotide (N) sequentially 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 comprises allowing the polynucleotide handling protein to control the movement of each polynucleotide (N) sequentially 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.

[0209] Those skilled in the art will appreciate that when the conjugate comprises more than one polypeptide, it may be advantageous if the polynucleotide handling protein (as described in more detail herein) can remain associated with the conjugate and not dissociate upon contact with the polypeptide. Figure 4 and 5 As shown in , this allows the polynucleotide handling protein to bypass the polypeptide portions of the conjugate upon contacting them in order to move onto the continuous portion of the polynucleotide, thereby controlling the movement of the conjugate relative to the nanopore.

[0210] Figure 2 Another non-limiting embodiment of the disclosed method is schematically illustrated in FIG. In this example, from the "perspective" of the polynucleotide handling protein, the polynucleotide handling protein moves the conjugate "out of" the pore. For example, as shown, the polynucleotide handling protein is located 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 arrangement can also be used.

[0211] In other words, in some embodiments, the polynucleotide handling protein can be 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. 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 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. 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 the polypeptide from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of the polypeptide through the nanopore.

[0212] In other embodiments, 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. 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 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. Thus, 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 polypeptide from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of the polypeptide through the nanopore.

[0213] Using similar notation as above, in some embodiments, a polynucleotide-polypeptide conjugate chain can comprise one or more moieties of the form ...NPNPN..., where each N (which can be the same or different) is a polypeptide, and where each N (which can be the same or different) comprises a polynucleotide. Some or all of the N moieties of the polynucleotide-polypeptide conjugate chain can hybridize to one or more polynucleotide carrier chains. In some embodiments, each N moiety of the polynucleotide-polypeptide conjugate chain hybridizes to a polynucleotide carrier chain.

[0214] In some embodiments, the polynucleotide-polypeptide conjugate chain comprises a polynucleotide of the form L-{PN}-P m or L-P1-N-{PN} n -P m One or more structures, wherein:

[0215] -n is a positive integer;

[0216] - L is a leader sequence, wherein L is optionally a moiety of N;

[0217] - each P, which may be the same or different, is a polypeptide;

[0218] - each N, which may be the same or different, comprises a polynucleotide;

[0219] -m is 0 or 1;

[0220] And the method may comprise passing the leader sequence (L) through the nanopore, thereby contacting the polypeptide (P) with the nanopore.

[0221] A polynucleotide-polypeptide conjugate chain can hybridize to a carrier chain. The N portion of a polynucleotide-polypeptide conjugate chain can hybridize to one or more polynucleotide carrier chains. In some embodiments, each N portion of a polynucleotide-polypeptide conjugate chain hybridizes to a polynucleotide carrier chain.

[0222] In some such embodiments, the polynucleotide handling protein is located on the cis side of the nanopore, and the method comprises allowing the polynucleotide handling protein to control movement of the polynucleotide portion (N) from the trans side of the nanopore to the cis side of the nanopore, thereby controlling movement of the polypeptide portion (P) through the nanopore. In other such embodiments, the polynucleotide handling protein is located on the trans side of the nanopore, and the method comprises allowing the polynucleotide handling protein to control movement of the polynucleotide portion (N) from the cis side of the nanopore to the trans side of the nanopore, thereby controlling movement of the polypeptide portion (P) through the nanopore.

[0223] In some embodiments, the methods provided herein comprise allowing a construct, a polynucleotide-polypeptide conjugate chain, and / or a polynucleotide carrier chain to move backward and forward relative to a nanopore. The process can be repeated multiple times. In this manner, the polypeptide can oscillate through the pore (i.e., the polypeptide can "shuttle" through the nanopore). This "shuttle" allows the polypeptide portion of the conjugate to be repeatedly characterized by the nanopore. In some embodiments, this allows for increased accuracy of the characterization information. This "shuttle" is also referred to herein as rereading. Figure 3 An example of the rereading method is shown in FIG.

[0224] In some embodiments, the method comprises:

[0225] i) performing a method as described herein such that the target polypeptide translocates the nanopore in a first direction relative to the nanopore;

[0226] ii) allowing the construct, the polynucleotide-polypeptide conjugate chain and / or the polynucleotide carrier chain to move 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;

[0227] iii) optionally allowing the construct, the polynucleotide-polypeptide conjugate chain and / or the polynucleotide carrier chain to move in the first direction such that the target polypeptide re-translocates the nanopore in the first direction;

[0228] iv) optionally repeating steps (ii) and (iii) to oscillate the polypeptide through the nanopore.

[0229] In some embodiments, particularly those involving read-through as described above, the conjugate can comprise a blocking moiety connected to the construct (e.g., to the polynucleotide-polypeptide conjugate strand and / or the carrier strand) via an optional linker.

[0230] The blocking portion is typically too large to pass through the nanopore, and thus when movement of the construct relative to the nanopore brings the blocking portion into contact with the nanopore, the conjugate is prevented from further movement through the nanopore. In embodiments of the disclosed methods, in which the conjugate is moved relative to the nanopore under an applied force (e.g., a voltage potential or a chemical potential), the conjugate can then move "backward" through the pore in a direction opposite to the movement controlled by the polynucleotide handling protein. The backward movement of the conjugate through the pore allows the polypeptide portion of the conjugate to be recharacterized again according to the disclosed methods. This is Figure 3 , where the circle at the top of the chain (here depicted by way of non-limiting example at the top of a vector chain) represents a blocking moiety.

[0231] Any suitable blocking moiety can be used in such embodiments. For example, the conjugate can be modified with biotin, and the blocking moiety can be, for example, streptavidin, avidin, or neutravidin. The blocking moiety can be a large chemical group, such as a dendrimer. The blocking moiety can be a nanoparticle or a bead. Other suitable blocking moieties will be apparent to those skilled in the art.

[0232] Thus, in some embodiments of the disclosed methods, the construct, the polynucleotide-polypeptide conjugate chain, and / or the polynucleotide carrier chain comprises a blocking moiety attached via an optional linker, wherein the blocking moiety is not capable of translocating through the nanopore.

[0233] In some embodiments, the disclosed method further comprises

[0234] i) contacting the polynucleotide-polypeptide conjugate chain or a construct comprising the polynucleotide-polypeptide conjugate chain with the nanopore such that the blocking moiety and the polynucleotide handling protein are located on the same side of the nanopore;

[0235] ii) contacting the polynucleotide region of the construct with the polynucleotide handling protein;

[0236] iii) allowing the polynucleotide handling protein to control the movement of the polynucleotide relative to the nanopore, e.g., in a direction opposite to a force (e.g., a voltage potential) applied across the nanopore; thereby controlling the movement of the polypeptide through the nanopore;

[0237] iv) allowing the construct to move relative to, for example, in the direction of a force (e.g., a voltage potential) applied across the nanopore; i.e., in a direction opposite to the direction of movement controlled by the polynucleotide handling protein;

[0238] v) when the blocking moiety contacts the nanopore, thereby preventing further movement of the conjugate through the nanopore, thereby allowing the polynucleotide handling protein to control movement of the construct through the nanopore; and

[0239] vi) optionally repeating steps (iv) to (iv) to oscillate the polypeptide through the nanopore.

[0240] peptides

[0241] Any suitable polypeptide can be characterized in the disclosed methods.

[0242] As explained 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.

[0243] In some embodiments, the or each target polypeptide is an unmodified protein or portion thereof, or a naturally occurring polypeptide or portion thereof.

[0244] In some embodiments, the target polypeptide or each target polypeptide is secreted by the cell. Alternatively, the target polypeptide or each target polypeptide may be produced intracellularly, such that the target polypeptide must be extracted from the cell to be characterized by the disclosed method. The polypeptide or each polypeptide may comprise a cell expression product of a plasmid, such as a plasmid for cloning proteins according to the methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainview, NY (2012); and Ausubel et al., Molecular Biology: A Laboratory Manual (Suppl. 114), John Wiley & Sons, New York (2016).

[0245] The or each polypeptide may be obtained or extracted from any organism or microorganism. The 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. The or each polypeptide may be obtained from plants (e.g., cereals, legumes, fruits or vegetables).

[0246] The or each target polypeptide may be provided as an impure mixture of one or more polypeptides and one or more impurities. Impurities may comprise truncated forms of the target polypeptide, which are different from the "target polypeptide" used for characterization in the disclosed methods. For example, the or each target polypeptide may be a full-length protein and the impurities may comprise fractions of the protein. Impurities may also comprise proteins other than the target protein, which may, for example, be co-purified from cell culture or obtained from a sample.

[0247] A polypeptide can comprise any combination of any amino acids, amino acid analogs, and modified amino acids (ie, amino acid derivatives). Amino acids (and derivatives, analogs, etc.) in a polypeptide can be distinguished by their physical size and charge.

[0248] The amino acid / derivative / analog may be naturally occurring or artificial. In some embodiments, the polypeptide does not comprise or consist of peptide nucleic acid (PNA).

[0249] In some embodiments, the polypeptide can comprise any naturally occurring amino acid. Twenty amino acids are encoded by the universal genetic code. These amino acids are alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamic acid / glutamic acid (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 pyrrolysine.

[0250] In some embodiments, the or each polypeptide is unmodified, e.g., chemically modified, as described in more detail herein. In some embodiments, the or each polypeptide is unmodified (e.g., chemically modified) for detection. In some embodiments, the or each polypeptide does not comprise modified (e.g., chemically modified) amino acids. In some embodiments, the polypeptide comprises unmodified amino acids. In some embodiments, the polypeptide is an unmodified polypeptide. Thus, the polypeptide may, for example, be a naturally occurring polypeptide or a synthetic polypeptide synthesized using unmodified (e.g., canonical) amino acids (e.g., as listed in Table 1 above).

[0251] In some embodiments, the or each polypeptide is modified. In some embodiments, the or each polypeptide is modified to be detected using the disclosed methods. In some embodiments, the disclosed methods are used to characterize modifications in a target polypeptide.

[0252] In some embodiments, one or more of the amino acids / derivatives / analogs in the or each polypeptide is modified. In some embodiments, one or more of the amino acids / derivatives / analogs in the or each polypeptide is post-translationally modified. Thus, the methods disclosed herein can be used to detect the presence, absence, position, or number of post-translational modifications in a polypeptide. The disclosed methods can be used to characterize the extent to which a polypeptide has been post-translationally modified.

[0253] Any one or more post-translational modifications may be present in the or each polypeptide. Typical post-translational modifications include modification with hydrophobic groups, modification with cofactors, addition of chemical groups, glycosylation (non-enzymatic attachment of sugars), biotinylation, and PEGylation. Post-translational modifications may also be non-natural, such that the post-translational modifications are chemical modifications made in the laboratory for biotechnological or biomedical purposes. This can allow monitoring of the levels of laboratory-made peptides, polypeptides, or proteins compared to their natural counterparts.

[0254] Examples of post-translational modifications with hydrophobic groups include myristoylation, attachment of myristate, C 14 Saturated acid; palmitoylation, palmitate linkage, C 16 saturated acids; prenylation or prenylation, attachment of an isoprenoid group; farnesylation, attachment of a farnesol group; geranylgeranylation, attachment of a geranylgeraniol group; and glypiation, and glycosylphosphatidylinositol (GPI) anchor formation via an amide bond.

[0255] Examples of post-translational modifications with cofactors include fatty acylation, attachment of a lipoate (C8) functional group; flavinylation, attachment of a flavin moiety such as flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD); attachment of heme C, for example via a thioether bond to cysteine; phosphopantetheinylation, attachment of a 4'-phosphopantetheinyl group; and retinyl Schiff base formation.

[0256] Examples of post-translational modifications by the addition of chemical groups include acylation, such as O-acylation (ester), N-acylation (amide), or S-acylation (thioester); acetylation, such as the attachment of an acetyl group to the N-terminus or to lysine; formylation; alkylation, the addition of an alkyl group, such as a methyl or ethyl group; methylation, such as the addition of a methyl group to lysine or arginine; amidation; butyrylation; gamma carboxylation; glycosylation, the enzymatic attachment of a sugar group to, for example, arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, or tryptophan; polysialylation, the attachment of polysialic acid; hydroxylation; iodination; bromination; citrullination. ylation; nucleotide addition, attachment of any nucleotide, such as any of those discussed above, ADP ribosylation; oxidation; phosphorylation, attachment of a phosphate group to, for example, serine, threonine, or tyrosine (O-linked) or histidine (N-linked); adenylation, attachment of an adenylate moiety to, for example, tyrosine (O-linked) or histidine or lysine (N-linked); propionylation; pyroglutamate formation; S-glutathionylation; sumoylation; S-nitrosylation; succinylation, attachment of a succinyl group, for example, to lysine; selenoylation, incorporation of selenium; and ubiquitination, addition of a ubiquitin subunit (N-linked).

[0257] It is within the scope of the method that this paper provides to mark described polypeptide or each polypeptide with molecular marker.Molecular marker can be the modification to polypeptide, and it promotes the detection of polypeptide in the method that this paper provides.For example, mark can be the modification to polypeptide, and it changes the signal that obtains when characterizing conjugate.For example, mark may disturb the ion flux that passes nanopore.In this way, mark can improve the sensitivity of method.

[0258] In some embodiments, the or each polypeptide contains one or more cross-linked segments, eg, C—C bridges. In some embodiments, the polypeptide is not cross-linked prior to characterization using the disclosed methods.

[0259] In some embodiments, the or each polypeptide comprises a sulfide-containing amino acid and thus 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) prior to characterization using the disclosed methods.

[0260] In some embodiments, the or each polypeptide is a full-length protein or naturally occurring polypeptide. In some embodiments, the protein or naturally occurring polypeptide is fragmented prior to conjugation to the polynucleotide. In some embodiments, the protein or polypeptide is chemically or enzymatically fragmented. In some embodiments, polypeptides or polypeptide fragments can be conjugated to form a longer target polypeptide.

[0261] The polypeptide or each polypeptide can be a polypeptide of any suitable length. In certain embodiments, the length of the polypeptide or each polypeptide is independently about 5 to about 5000 peptide units. In certain embodiments, the length of the polypeptide is 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, such as 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 certain embodiments, the length of the polypeptide or each polypeptide is independently about 10 to about 5000 peptide units. In certain embodiments, the length of the polypeptide or each polypeptide is 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, such as 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 or each polypeptide is independently from about 25 to about 1000 peptide units in length, such as from about 50 to about 500 peptide units, for example from about 100 to about 250 peptide units in length.

[0262] Any number of polypeptides can be characterized in the disclosed methods. For example, the methods can comprise characterizing 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, or more polypeptides. If two or more polypeptides are used, they can be different polypeptides or two or more instances of the same polypeptide.

[0263] It will therefore be apparent that the measurements made in the disclosed methods are typically specific to one or more properties of a polypeptide selected from the group consisting of: (i) the length of the polypeptide; (ii) the identity of the polypeptide; (iii) the sequence of the polypeptide; (iv) the secondary structure of the polypeptide; and (v) whether the polypeptide is modified. In typical embodiments, the measurement is specific to the sequence of the polypeptide, or whether the polypeptide is modified, for example, by one or more post-translational modifications. In some embodiments, the measurement is specific to the sequence of the polypeptide.

[0264] In some embodiments, the polypeptide is in a relaxed form. In some embodiments, the polypeptide is maintained in a linearized form. Maintaining the polypeptide in a linearized form can facilitate characterization of the polypeptide on a residue-by-residue basis by preventing "bunching up" of the polypeptide within the nanopore.

[0265] Any suitable means may be used to maintain the polypeptide in linear form.

[0266] In some embodiments, the polypeptide remains in a linearized form because it is linked to a polynucleotide flanking strand at each end of the polypeptide, and the flanking strands hybridize to complementary or substantially complementary portions of one or more polynucleotide carrier strands. The sequences of the flanking strands and / or carrier strands can be selected or designed so that the distance between the regions of the carrier strand to which the flanking strands hybridize is similar to the length of the polypeptide in its linearized form. The length of the polypeptide in its linearized form can be determined by the number of amino acids in the polypeptide (if known), for example, a peptide unit in a polypeptide is generally considered to have a length of approximately 0.35 nm (3.5 Å). Therefore, it is within the ability of one skilled in the art to design flanking strands and carrier strands for any desired target polypeptide, for example, based on the mass of the polypeptide, which can be readily determined by methods known to one skilled in the art, such as denaturing (e.g., SDS-) or native PAGE, or by mass spectrometry; or based on the sequence of the polypeptide (if known).

[0267] Other methods of maintaining a target polypeptide in linearized form are known.

[0268] For example, if the polypeptide is charged, the polypeptide can be maintained in a linearized form by applying a voltage.

[0269] If the polypeptide is uncharged or only weakly charged, the charge can be changed or controlled by adjusting the pH. For example, the polypeptide can be kept in a linearized form by increasing the relative negative charge of the polypeptide using a high pH. Increasing the negative charge of the polypeptide allows it to remain in a linearized form, for example, under a positive voltage. Alternatively, the polypeptide can be kept in a linearized form by increasing the relative positive charge of the polypeptide using a low pH. Increasing the positive charge of the polypeptide allows it to remain in a linearized form, for example, under a negative voltage. In the disclosed method, a polynucleotide processing protein is used to control the movement of the polynucleotide relative to the nanopore. Since polynucleotides are generally negatively charged, it is generally most suitable to increase the linearization of the polypeptide by increasing the pH, thereby making the polypeptide more negatively charged, which is the same as the polynucleotide. In this way, the polynucleotide-polypeptide conjugate chain retains an overall negative charge and can therefore move easily, for example under an applied voltage.

[0270] The polypeptide can be maintained in a linearized form by using appropriate denaturing conditions. Suitable denaturing conditions include, for example, the presence of an appropriate concentration of a denaturing agent, such as guanidine hydrochloride and / or urea. The concentration of such denaturing agents used in the disclosed methods depends on the target polypeptide to be characterized in the method and can be readily selected by one skilled in the art.

[0271] The polypeptide can be maintained in linear form by using a suitable detergent. Suitable detergents for use in the disclosed methods include SDS (sodium dodecyl sulfate).

[0272] The polypeptide can be maintained in a linearized form by performing the disclosed method at an elevated temperature. Increasing the temperature overcomes intrachain bonds and allows the polypeptide to adopt a linearized form.

[0273] The polypeptide can be maintained 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 providing an appropriate charge in the nanopore channel. The charge in the protein nanopore channel can be altered, for example, by mutagenesis. Changing the charge of a nanopore is well within the capabilities of those skilled in the art. When a voltage potential is applied across the nanopore, altering the charge of the nanopore generates a strong electroosmotic force due to the unbalanced flow of cations and anions through the nanopore.

[0274] Conjugate chain formation

[0275] In forming a polynucleotide-polypeptide conjugate chain, the or each target polypeptide may be conjugated to the or each polynucleotide flanking chain at any suitable position. For example, the or each polypeptide may be conjugated to the polynucleotide flanking chain at the N-terminus or C-terminus of the polypeptide. The polypeptide may be conjugated to the polynucleotide via side chain groups of residues (e.g., amino acid residues) in the polypeptide.

[0276] In some embodiments, the target polypeptide has naturally occurring reactive functional groups that can be used to promote conjugation to polynucleotide side chains. For example, cysteine ​​residues can be used to form disulfide bonds with polynucleotide side chains or modified groups thereon.

[0277] In some embodiments, the target polypeptide is modified to facilitate conjugation to the polynucleotide side chains. For example, in some embodiments, the polypeptide is modified by attaching a moiety comprising a reactive functional group for attachment to the polynucleotide side chains. 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) comprising one or more reactive functional groups for reaction with corresponding reactive functional groups on the polynucleotide side chains. 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 can be used for attachment to the polynucleotide portion of the polynucleotide-polypeptide conjugate chain, for example, via maleimide chemistry (e.g., by reacting a cysteine ​​with an azido-maleimide compound (e.g., azido-[Pol]-maleimide, where [Pol] is typically a short-chain polymer such as PEG, e.g., PEG2, PEG3, or PEG4); followed by coupling to an appropriately functionalized polynucleotide, such as a polynucleotide bearing a BCN group, for reaction with an azide). For the avoidance of doubt, where a polypeptide comprises suitable naturally occurring residues at the N-terminus and / or C-terminus (e.g., a naturally occurring cysteine ​​residue at the N-terminus and / or C-terminus), then such residues may be used for linkage to a polynucleotide.

[0278] In some embodiments, residues in the target polypeptide are modified to facilitate attachment of the target polypeptide to the polynucleotide side chains. In some embodiments, residues (e.g., amino acid residues) in the polypeptide are chemically modified for attachment to the polynucleotide side chains. In some embodiments, residues (e.g., amino acid residues) in the polypeptide are enzymatically modified for attachment to the polynucleotide side chains.

[0279] The conjugation chemistry between the polynucleotide side chain and the polypeptide portion of the 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, imidoesters that can react with amines, isocyanates that can react with hydroxyls, 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 thymines, pyridyl disulfides that can react with sulfhydryl groups, vinyl sulfones that can react with sulfhydrylamines and hydroxyls, vinyl sulfonamides, etc.

[0280] Other suitable chemistries for conjugating polypeptides to polynucleotides include 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:

[0281] (a) Copper(I)-catalyzed azide-alkyne cycloaddition (Azide-Alkyne Huisgen Cycloaddition);

[0282] (b) Strain-promoted azide-alkyne cycloadditions; including the [3+2] cycloaddition of alkenes and azides; the reverse-demand Diels-Alder reaction of alkenes and tetrazines; and the photoclick reaction of alkenes and tetrazoles;

[0283] (c) copper-free variants of 1,3-dipolar cycloaddition reactions in which azides react with alkynes under strain, for example in cyclooctane rings, as in bicyclo[6.1.0]nonyne (BCN);

[0284] (d) reaction of an oxygen nucleophile on one linker with an epoxide or aziridine reactive moiety on the other linker; and

[0285] (e) Staudinger ligation, in which the alkyne moiety can be replaced by an aryl phosphine, leading to a specific reaction with an azide to form an amide bond.

[0286] Any reactive group can be used to form a conjugate. Some suitable reactive groups include [1,4-bis[3-(2-pyridyldithio)propionylamino]butane; 1,11-maleimidotriethylene glycol; bis(N-hydroxysuccinimide 3,3'-dithiodipropionate); ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester); 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt; bis[2-(4-azidosalicylamido)ethyl] disulfide; N-hydroxysuccinimide 3-(2-pyridyldithio)propionate; N-hydroxysuccinimide 4-maleimidobutyrate; N-hydroxysuccinimide iodoacetate; N-hydroxysuccinimide S-acetylthioglycolate; azide-PEG-maleimide; and alkyne-PEG-maleimide. The reactive group may be any of the reactive groups disclosed in WO 2010 / 086602, in particular in Table 3 of said application.

[0287] In some embodiments, prior to the conjugation step, the reactive functional group is contained in the polynucleotide and the target functional group is contained in the polypeptide. In other embodiments, prior to the conjugation step, the reactive functional group is contained in the polypeptide and the target functional group is contained in the polynucleotide. In some embodiments, the reactive functional group is directly linked to the polypeptide. In some embodiments, the reactive functional group is linked to the polypeptide via a spacer. Any suitable spacer can be used. Suitable spacers include, for example, alkyl diamines, such as ethylenediamine.

[0288] As explained above, in some embodiments, the conjugate comprises multiple polypeptide segments and / or multiple polynucleotide segments. For example, the conjugate may comprise a structure of the form ...-PNPNPN..., where P is a polypeptide and N is a polynucleotide. In such embodiments, the multiple polynucleotides and polypeptides may be conjugated together using the same or different chemistries.

[0289] As explained herein, the construct may comprise a leader sequence. The leader sequence may be present on the polynucleotide-polypeptide conjugate strand (e.g., on a flanking strand) or on the carrier strand (if present). Any suitable leader sequence may be used, as explained herein. In some embodiments, the leader sequence is or comprises a polynucleotide. In embodiments where the leader sequence is a polynucleotide, the leader sequence may be of the same type as the polynucleotide used in the conjugate, or the leader sequence may be of a different type. For example, the polynucleotide in the conjugate may be DNA, and the leader sequence may be RNA, or vice versa.

[0290] In some embodiments, the leader sequence can be about 10 to 150 nucleotides in length (e.g., DNA and / or RNA nucleotides), such as 20 to 120, for example 30 to 100, for example 40 to 80, such as 50 to 70 nucleotides in length, or about 10 to about 60 nucleotides in length, for example about 20 to about 50, such as about 20 to about 40, for example about 30 nucleotides in length.

[0291] In some embodiments, the leader sequence is a charged polymer, such as a negatively charged polymer. In some embodiments, the leader sequence comprises a polymer, such as PEG or a polysaccharide. In such embodiments, the leader sequence can be 10 to 150 monomer units (e.g., ethylene glycol or sugar units) in length, such as 20 to 120, such as 30 to 100, such as 40 to 80, such as 50 to 70 monomer units (e.g., ethylene glycol or sugar units) in length.

[0292] polynucleotides

[0293] As explained in more detail herein, the methods provided herein comprise conjugating one or more polypeptides to one or more polynucleotide flanking chains. The resulting polynucleotide-polypeptide conjugate chains can be hybridized or otherwise linked to polynucleotide carrier chains.

[0294] In the disclosed methods, any suitable polynucleotide can be used as a flanking chain and / or carrier chain. It is within the capabilities of those skilled in the art to select appropriate polynucleotides for use as flanking chains and carrier chains based on the target polypeptide to be characterized in the disclosed methods. For example, in some embodiments, the flanking chain or each flanking chain is at least as long as the target polypeptide. In some embodiments, the flanking chain or each flanking chain is shorter than the target polypeptide. In some embodiments, the flanking chain spans the target polypeptide.

[0295] In some embodiments, the or each polynucleotide used is secreted by the cell. Alternatively, the polynucleotide may be produced intracellularly, such that the polynucleotide must be extracted from the cell for use in the disclosed methods.

[0296] Polynucleotides can be provided as an impure mixture of one or more polynucleotides and one or more impurities. Impurities may comprise truncated polynucleotides that are different from the polynucleotides used in forming the conjugate. For example, the polynucleotides used as flanking strands or carrier strands may be genomic DNA, and impurities may comprise fractions of genomic DNA, plasmids, etc. The desired polynucleotide may be a coding region of genomic DNA, and undesirable polynucleotides may comprise non-coding regions of DNA.

[0297] Examples of polynucleotides include DNA and RNA. The bases in DNA and RNA can be distinguished by their physical size.

[0298] Polynucleotides or nucleic acids suitable for use as flanking or carrier strands may comprise any combination of 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 comprise pyrimidine dimers. Such dimers are commonly associated with damage caused by ultraviolet light and are a major cause of cutaneous melanoma.

[0299] One or more nucleotides in a polynucleotide can be modified, for example, with a marker or label, suitable examples of which are known to those skilled in the art. Polynucleotides suitable for use as flanking or carrier strands can include one or more spacers. Adapters, such as sequencing adapters, can be included in the polynucleotide. Adapters, labels, and spacers are described in more detail herein.

[0300] Examples of modified bases are disclosed herein and can be incorporated into polynucleotides by means known in the art, such as by polymerase incorporation of modified nucleotide triphosphates during chain replication (e.g., in PCR) or by polymerase filling methods. In some embodiments, one or more bases can be chemically modified using reagents known in the art.

[0301] Nucleotides typically contain a nucleobase, a sugar, and at least one phosphate group. The nucleobase and sugar form a nucleoside. Nucleobases are typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, and more specifically, adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar is preferably deoxyribose. Polynucleotides preferably contain 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 monophosphates, diphosphates, or triphosphates. Nucleotides may contain more than three phosphates, such as four or five phosphates. The phosphates may be attached to the 5' or 3' side of the nucleotide. The nucleotides in a polynucleotide may be linked to each other in any manner. Nucleotides are typically linked via their sugar and phosphate groups, as in nucleic acids. Nucleotides may be linked via their nucleobases, as in pyrimidine dimers.

[0302] The polynucleotide may be double-stranded or single-stranded. In the disclosed methods, the flanking strands are typically single-stranded. The carrier strand is typically single-stranded. Thus, the construct formed by ligation (e.g., hybridization) between the flanking strands and the carrier strand is double-stranded.

[0303] Single-stranded polynucleotides suitable for use as flanking or carrier strands are typically single-stranded DNA. Single-stranded RNA can also be used. In some embodiments, polynucleotides suitable for use as flanking or carrier strands are single-stranded DNA-RNA hybrids. DNA-RNA hybrids can be prepared by linking single-stranded DNA to RNA, or vice versa. The polynucleotide is most typically single-stranded deoxyribonucleic acid (DNA) or single-stranded ribonucleic acid (RNA), typically DNA.

[0304] In some embodiments, the construct comprising the polynucleotide-polypeptide conjugate chain and the vector chain construct comprise double-stranded DNA. In some embodiments, they comprise double-stranded RNA. In some embodiments, they comprise double-stranded DNA-RNA hybrids. Double-stranded DNA-RNA hybrids can be prepared from single-stranded RNA by reverse transcription of the cDNA complement.

[0305] The polynucleotides suitable for use as flanking or carrier strands can be of any length. For example, the polynucleotides can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides or nucleotide pairs in length. The polynucleotides can be 1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotides or nucleotide pairs, or 100,000 or more nucleotides or nucleotide pairs in length.

[0306] More typically, polynucleotides suitable for use as flanking or carrier strands have a length of from about 1 to about 10,000 nucleotides or nucleotide pairs, such as from about 1 to about 1000 nucleotides or nucleotide pairs (e.g., from about 10 to about 1000 nucleotides or nucleotide pairs), for example from about 5 to about 500 nucleotides or nucleotide pairs, such as from about 10 to about 100 nucleotides or nucleotide pairs, for example from about 20 to about 80 nucleotides or nucleotide pairs, such as from about 30 to about 50 nucleotides or nucleotide pairs.

[0307] Typically, a polynucleotide suitable for use as a carrier strand is longer than a polynucleotide suitable for use as a flanking strand. Thus, typically multiple flanking strands and polypeptide segments can be associated with a single polynucleotide carrier strand.

[0308] For example, in some embodiments:

[0309] - the or each target polypeptide has a length of from about 5 to about 1000 peptide units, for example from about 5 to about 500 peptide units, for example from about 5 to about 250 peptide units, such as from about 5 to about 100 peptide units, for example from about 5 to about 50 peptide units;

[0310] - each flanking strand has a length of about 5 to about 1000 nucleotides, e.g., about 5 to about 500 nucleotides, for example about 5 to about 250 nucleotides, such as about 5 to about 100 nucleotides, for example about 5 to about 50 nucleotides; and

[0311] Each carrier strand has a length of about 50 to about 50,000 nucleotides, such as about 100 to about 10,000 nucleotides, for example about 5000 nucleotides. Much longer carrier strands, such as about 1000 nucleotides to about 10 millibases, such as about 10,000 to about 1,000,000 nucleotides, for example about 100,000 nucleotides, can also be used.

[0312] In some embodiments, before the construct is contacted with the nanopore, the polynucleotide handling protein binds to the polynucleotide carrier strand in the region of the polynucleotide carrier strand spanned by the non-hybridized region of the polynucleotide flanking strand. The flanking strands can thus 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 flanking strands and the carrier strand. This is particularly important in Figure 1 and 2 The invention is shown in schematic form by way of non-limiting example.

[0313] The bubble region spanning the polynucleotide processing protein (when present) can have any suitable length. The length is generally a function of the size of the polynucleotide processing protein used. The length of the bubble region spanning the polynucleotide processing protein (when present) can be, for example, about 2 to about 50 nucleotides, such as about 5 to about 20 nucleotides, for example, about 19 nucleotides. In certain embodiments, the length of the bubble region is, for example, about 2 to about 100 nucleotides, such as about 10 to about 50 nucleotides, such as about 20 to about 50 nucleotides, such as about 30 to about 40, for example, about 35 nucleotides.

[0314] In some embodiments, prior to the methods disclosed herein, the length of the portion of the corresponding strand around which the bubble is formed (e.g., when the bubble is part of a flanking strand, the corresponding strand can be the carrier strand) can be about 5 to about 20, such as about 8 to about 12, for example, about 10 nucleotide units. Thus, the bubble region is typically longer than the portion of the corresponding strand around which the bubble region is formed prior to the methods disclosed herein. For example, in some embodiments, the bubble region is about 10 to about 50 nucleotides in length, such as about 30 to about 40 nucleotides in length, and the length of the portion of the corresponding strand around which the bubble is formed is about 5 to about 20, such as about 8 to about 12, for example, about 10 nucleotide units in length.

[0315] The bubble region that spans the polynucleotide handling protein (prior to initiating the methods disclosed herein) can comprise or consist of any suitable type of polynucleotide. In some embodiments, the portion of the polynucleotide flanking strand that spans the polynucleotide handling protein is the same type of polynucleotide as the remainder of the flanking strand. In some embodiments, the portion of the polynucleotide flanking strand that spans the polynucleotide handling protein is a different type of polynucleotide than the remainder of the flanking strand. In some embodiments, the flanking strand comprises a DNA polynucleotide and the bubble region (prior to initiating the disclosed methods) comprises or consists of RNA. In some embodiments, the flanking strand comprises RNA and the bubble region (prior to initiating the disclosed methods) comprises or consists of DNA. In some embodiments, using a different type of polynucleotide to form the initial bubble (as opposed to the remainder of the flanking strand) can be used to preferentially position the polynucleotide handling protein prior to initiating the methods provided herein. In some embodiments, the construct comprises a bubble region on one strand (e.g., a flanking strand or a carrier strand) and the polynucleotide handling protein is bound to the other strand of the construct (e.g., the carrier strand or the flanking strand). In some embodiments, the polynucleotide handling protein is arrested at an arresting moiety, such as a spacer, as described in more detail herein. In some embodiments, the stagnation portion is opposite to the bubble region. For example, in some embodiments, the carrier strand comprises a spacer portion that is used to stall the polynucleotide processing protein prior to commencing the methods provided herein, and the polynucleotide processing protein is bound to the carrier strand and flanked by the bubble region on the polynucleotide-polypeptide conjugate strand. In some embodiments, the polynucleotide-polypeptide conjugate strand comprises a spacer portion that is used to stall the polynucleotide processing protein prior to commencing the methods provided herein, and the polynucleotide processing protein is bound to the polynucleotide-polypeptide conjugate strand and flanked by the bubble region on the carrier strand.

[0316] When the polynucleotide processing protein processes the construct, the polynucleotide processing protein moves along the chain to which it is attached. Therefore, the movement of the polynucleotide processing protein along the chain causes the bubble region to migrate along the chain with the polynucleotide processing protein. Figure 1 and 2 It is shown schematically in FIG.

[0317] Any number of polynucleotides (e.g., any number of flanking chains) can be used in the disclosed methods. For example, the methods can comprise the use of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, or more polynucleotides (e.g., polynucleotide flanking chains). If two or more polynucleotides are used, they can be different polynucleotides or two instances of the same polynucleotide. The polynucleotides can be naturally occurring or artificial. Typically, the polynucleotide-polypeptide conjugate chain comprises one flanking chain separating each target polypeptide; thus, the number of polynucleotide flanking chains is typically the same or substantially the same as the number of polypeptides.

[0318] 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 abasic (i.e., lack a nucleobase). Nucleotides can also lack a nucleobase and a sugar (i.e., be a C3 spacer).

[0319] The polynucleotides may comprise products of PCR reactions, genomic DNA, products of endonuclease digestion, and / or DNA libraries. The polynucleotides may be obtained or extracted from any organism or microorganism. The 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. The polynucleotides may be obtained from plants (e.g., cereals, beans, fruits, or vegetables). The polynucleotides may comprise genomic DNA. The genomic DNA may be fragmented. The DNA may be fragmented by any suitable method. For example, methods for fragmenting DNA are known in the art. Such methods may utilize transposases, such as MuA transposase. Typically, genomic DNA is not fragmented.

[0320] It is within the scope of the method that this paper provides to mark polynucleotide with molecular marker.Molecular marker can be the modification to polynucleotide, and it promotes the detection of polynucleotide or conjugate in the method that this paper provides.For example, labeling can be the modification to polynucleotide, and it changes the signal that obtains when characterizing conjugate.For example, labeling may interfere with the ion flux that passes nanopore.In this way, labeling can improve the sensitivity of method.

[0321] adapter

[0322] In some embodiments of the methods provided herein, a polynucleotide (e.g., a flanking strand or a carrier strand) can have a polynucleotide adaptor attached thereto. An adaptor typically comprises a polynucleotide chain that can be attached to the end of a polynucleotide.

[0323] In some embodiments, the adaptor is attached to the flanking strand prior to forming a conjugate with the polypeptide. In some embodiments, the adaptor is attached to the conjugate of the polynucleotide and the polypeptide.

[0324] Thus, in some embodiments, the methods comprise linking an adaptor (e.g., an adaptor as described herein) to a polynucleotide and forming a polynucleotide-polypeptide conjugate chain by conjugating the polynucleotide / adapter construct to a target polypeptide. In some embodiments, the polynucleotide-polypeptide conjugate chain is formed by linking an adaptor (e.g., an adaptor as described herein) to a polynucleotide and forming a conjugate by linking the adaptor to a target polypeptide.

[0325] In some embodiments, adaptors can be selected or modified to provide specific sites for conjugation to a polynucleotide.

[0326] An adapter can be attached to only one end of a polynucleotide or conjugate. A polynucleotide adapter can be added to both ends of a polynucleotide or conjugate. Alternatively, different adapters can be added to both ends of a polynucleotide or conjugate.

[0327] Adaptors can be added to both strands of a double-stranded polynucleotide (e.g., a construct as described herein). Adaptors can be added to single-stranded polynucleotides (e.g., a polynucleotide-polypeptide conjugate strand as described herein). Methods for adding adaptors to polynucleotides are known in the art. Adaptors can be attached to polynucleotides, for example, by ligation, by click chemistry, by tagmentation, by topoisomerization, or by any other suitable method.

[0328] In one embodiment, the adapter or each adapter is synthetic or human. 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. The polynucleotide adapter or each polynucleotide adapter may comprise DNA, RNA, modified DNA (such as abasic 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 a polynucleotide of the same type as the polynucleotide chain to which it is attached. The adapter may comprise a polynucleotide of a different type than the polynucleotide chain to which it is attached. In some embodiments, the polynucleotide chain used in the disclosed methods 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, for example, double-stranded or single-stranded DNA.

[0329] In some embodiments, the adapter can be a bridging moiety. A bridging moiety can be used to connect two strands of a double-stranded polynucleotide. For example, in some embodiments, a bridging moiety is used to connect the template strand of a double-stranded polynucleotide to the complement strand of the double-stranded polynucleotide. For example, a bridging adapter can be used to connect a flanking strand to a carrier strand.

[0330] The bridging moiety typically covalently connects the two strands of a double-stranded polynucleotide. The bridging moiety can be any substance capable of connecting the two strands of a double-stranded polynucleotide, provided that the bridging moiety does not interfere with the movement of the polynucleotide relative to the nanopore. Suitable bridging moieties include, but are not limited to, polymer linkers, chemical linkers, polynucleotides, or polypeptides. Preferably, the bridging moiety comprises DNA, RNA, modified DNA (e.g., abasic DNA), RNA, PNA, LNA, or PEG. More preferably, the bridging moiety is DNA or RNA.

[0331] In some embodiments, the bridging portion is a hairpin adapter. A hairpin adapter is an adapter comprising a single polynucleotide chain, wherein the ends of the polynucleotide chain can hybridize with each other or are hybridized with each other, and wherein the middle section of the polynucleotide forms a loop. Suitable hairpin adapters can be designed using methods known in the art. In some embodiments, the length of the hairpin loop is generally 4 to 100 nucleotides, for example, 4 to 50, such as 4 to 20, for example 4 to 8 nucleotides in length. In some embodiments, the bridging portion (e.g., a hairpin adapter) is connected to one end of a double-stranded polynucleotide. The bridging portion (e.g., a hairpin adapter) is not typically connected to both ends of a double-stranded polynucleotide.

[0332] In some embodiments, the adapter is a linear adapter. A linear adapter can bind to either or both ends of a single-stranded polynucleotide. When the polynucleotide is a double-stranded polynucleotide, a linear adapter can bind to either or both ends of either or both strands of the double-stranded polynucleotide. A linear adapter can be attached to either or both ends of the polynucleotide-polypeptide conjugate strand and the carrier strand (if present).

[0333] The linear adapter may comprise a leader sequence as described herein. The linear adapter may comprise a portion for hybridizing to a tag as described herein (e.g., a pore tag). The linear adapter may be 10 to 150 nucleotides in length, such as 20 to 120, for example 30 to 100, for example 40 to 80, such as 50 to 70 nucleotides in length. The linear adapter may be single-stranded. The linear adapter may be double-stranded.

[0334] In some embodiments, the adapter can be a Y adapter. A Y adapter is typically a polynucleotide adapter. A Y adapter is typically double-stranded and comprises (a) a region at one end where the two strands hybridize together and (b) a region at the other end where the two strands are non-complementary. The non-complementary portions of the strands typically form overhangs. The presence of the non-complementary region in the Y adapter gives the adapter its Y shape because, unlike the double-stranded portion, the two strands typically do not hybridize to each other. The two single-stranded portions of the Y adapter can be the same length or different lengths. For example, one single-stranded portion of the Y adapter can be 10 to 150 nucleotides in length, such as 20 to 120, e.g., 30 to 100, e.g., 40 to 80, e.g., 50 to 70 nucleotides in length, and the other single-stranded portion of the Y adapter can independently be 10 to 150 nucleotides in length, such as 20 to 120, e.g., 30 to 100, e.g., 40 to 80, e.g., 50 to 70 nucleotides in length. The double-stranded "stem" portion of the Y-adapter can be, for example, 10 to 150 nucleotides in length, such as 20 to 120, for example 30 to 100, for example 40 to 80, such as 50 to 70 nucleotides in length. The Y-adapter can be attached to either or both ends of the constructs described herein.

[0335] The adapter can be connected to the polynucleotide (e.g., carrier strand or flanking strand) by any suitable means known in the art. The adapter can be synthesized separately and chemically or enzymatically connected to the polynucleotide. Alternatively, the adapter can be produced during the processing of the polynucleotide. In certain embodiments, the adapter is connected to the target polynucleotide at or near one end of the target polynucleotide. In certain embodiments, the adapter is connected to the polynucleotide within 50 nucleotides, such as within 20 nucleotides, or within 10 nucleotides of the polynucleotide end. In certain embodiments, the adapter is connected to the polynucleotide at the end of the polynucleotide. When the adapter is connected to the polynucleotide, the adapter may comprise nucleotides of the same type as the polynucleotide or may comprise nucleotides different from the polynucleotide.

[0336] Adaptors particularly suitable for use with the disclosed methods can comprise a linear homopolymer region (e.g., about 5 to about 20 nucleotides, such as about 10 to about 30 nucleotides, e.g., thymine or cytidine) and / or a hybridization site for hybridization to one or more tethers or anchors (as described in more detail herein). Such adaptors can also comprise reactive functional groups for binding to target polypeptides. Click chemistry groups are particularly suitable in this regard. For example, exemplary groups for inclusion in adaptors include those that can be micronized in copper-free click chemistry, such as those based on BCN (bicyclo[6.1.0]nonyne) and its derivatives, dibenzocyclooctyne (DBCO) groups, and the like. 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. DBCO groups are highly reactive toward azide groups. Other particularly suitable chemical groups include 2-pyridinecarboxaldehyde (2-PCA) groups and their derivatives. For example, 6-(azidomethyl)-2-pyridinecarboxaldehyde can react with the N-terminal amino group of a peptide.

[0337] spacer

[0338] In some embodiments of the methods provided herein, the polynucleotides, the conjugates formed by reacting the polynucleotides with polypeptides, leader sequences, or adapters as described herein, can include spacers. For example, one or more spacers can be present in a polynucleotide adapter. One or more spacers can be present in an adapter connected to a flanking strand or a carrier strand.

[0339] For example, the polynucleotide adaptor can comprise 1 to about 20 spacers, such as about 1 to about 10, such as 1 to about 5 spacers, such as 1, 2, 3, 4 or 5 spacers. The spacer can comprise any suitable number of spacer units. The spacer can provide an energy barrier that hinders the movement of the polynucleotide processing protein. For example, the spacer can stagnate the polynucleotide processing protein by reducing the traction force of the polynucleotide processing protein on the polynucleotide. This can be achieved, for example, by using a baseless spacer, i.e., a spacer in which a base is removed from one or more nucleotides in the polynucleotide adaptor. The spacer can physically block the movement of the polynucleotide processing protein, for example, by introducing a bulky chemical group to physically hinder the movement of the polynucleotide processing protein.

[0340] In some embodiments, one or more spacers are included in a polynucleotide or conjugate or adapter used in the methods claimed herein in order to provide a unique signal when the polynucleotide or conjugate or adapter passes through or spans the nanopore, i.e., when the polynucleotide or conjugate or adapter moves relative to the nanopore.

[0341] In some embodiments, the spacer may comprise a linear molecule, such as a polymer. Typically, such a spacer has a structure different from that of the polynucleotide used in the conjugate. For example, if the polynucleotide is DNA, the spacer or each spacer typically does not comprise DNA. Specifically, if the polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), the spacer or each spacer preferably comprises a peptide nucleic acid (PNA), a glycerol nucleic acid (GNA), a threose nucleic acid (TNA), a locked nucleic acid (LNA), or a synthetic polymer having nucleotide side chains. In some embodiments, the spacer can comprise one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more trans-thymidines (trans-dT), one or more trans-dideoxythymidines (ddT), one or more dideoxycytidines (ddC), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2'-O-methyl RNA bases, one or more isodeoxycytidines (iso-dC), one or more isodeoxyguanosines (iso-dG), one or more C3 (OC3H6OPO3) groups, one or more photodegradable (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 sulfhydryl linkages. The spacer can 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®. The spacer can contain any number of the above groups as spacer units.

[0342] In some embodiments, the spacer may comprise one or more chemical groups that arrest the polynucleotide processing protein. In some embodiments, suitable chemical groups are one or more side chemical groups. The one or more chemical groups may be attached to one or more nucleobases in the polynucleotide, construct, or adaptor. The one or more chemical groups may be attached to the backbone of the polynucleotide adaptor. Any number of suitable chemical groups may be present, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more. Suitable groups include, but are not limited to, fluorophores, streptavidin and / or biotin, cholesterol, methylene blue, dinitrophenol (DNP), digoxigenin and / or anti-digoxigenin, and dibenzylcyclooctyne 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).

[0343] In some embodiments, the spacer may comprise one or more abasic nucleotides (i.e., nucleotides lacking a nucleobase), such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more abasic nucleotides. In an abasic nucleotide, the nucleobase may be replaced by -H(idSp) or -OH. An abasic spacer can be inserted into a target polynucleotide by removing a nucleobase from one or more adjacent nucleotides. For example, a polynucleotide can be modified to include 3-methyladenine, 7-methylguanine, 1,N6-vinylidene adenine inosine, or hypoxanthine, and the nucleobase can be removed from these nucleotides using human alkyladenine DNA glycosylase (hAAG). Alternatively, a polynucleotide can be modified to include uracil, and the nucleobase can be removed using uracil-DNA glycosylase (UDG). In one embodiment, the one or more spacers do not contain any abasic nucleotides.

[0344] Methods for using spacers to stall polynucleotide processing proteins, such as helicases, on polynucleotide adaptors are described in WO 2014 / 135838, which is hereby incorporated by reference in its entirety.

[0345] In some embodiments, the construct comprises a stagnation moiety, and the polynucleotide processing protein is positioned such that the stagnation moiety is located between the polynucleotide processing protein and the target polypeptide prior to translocation of the target polypeptide through the nanopore. In some embodiments, this can be advantageous because it prevents the polynucleotide processing protein from processing the portion of the construct corresponding to the target polypeptide prior to initiation of measurement.

[0346] In some embodiments, the construct comprises a stalling moiety at the junction between the target polypeptide and one or more polynucleotide flanking chains. In some embodiments, the one or more flanking chains are conjugated to the target polypeptide chain via click chemistry, as described in greater detail herein. In some embodiments, the polynucleotide processing protein stalls at the click chemistry junction.

[0347] anchor

[0348] In some embodiments, the polynucleotide, its conjugate to the polypeptide, or the adaptor linked thereto can comprise, for example, a membrane anchor or a transmembrane pore anchor linked to the adaptor. In one embodiment, the anchor facilitates characterization of the conjugate according to the methods disclosed herein. For example, the membrane anchor or transmembrane pore anchor can facilitate localization of the conjugate around a nanopore in a membrane.

[0349] The anchor can 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, such as cholesterol, palmitate or tocopherol. The anchor can comprise a thiol, biotin or a surfactant.

[0350] In one embodiment, the anchor can be biotin (for binding to streptavidin), amylose (for binding to maltose binding protein or fusion protein), Ni-NTA (for binding to polyhistidine or polyhistidine-tagged proteins), or a peptide (such as an antigen).

[0351] In one embodiment, the anchor comprises 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 can be linear, branched, or cyclic. For example, the linker can be a cyclic polynucleotide. The adapter can hybridize to a complementary sequence on a cyclic polynucleotide linker. The one or more anchors or one or more linkers can contain components that can be cleaved or decomposed, such as restriction sites or photolabile groups. The linker can be functionalized with a maleimide group to attach to cysteine ​​residues in proteins. Suitable linkers are described in WO 2010 / 086602.

[0352] In one embodiment, the anchor is cholesterol or a fatty acyl chain. For example, any fatty acyl chain with a length of 6 to 30 carbon atoms, such as hexadecanoic acid, can be used. Examples of suitable anchors and methods for attaching anchors to adaptors are disclosed in WO 2012 / 164270 and WO 2015 / 150786.

[0353] Controlling the movement of the conjugate relative to the nanopore

[0354] As explained above, the methods provided herein comprise controlling the movement of a polynucleotide-polypeptide conjugate strand, optionally bound (eg, hybridized) to a carrier strand, as the conjugate or construct moves relative to a nanopore.

[0355] 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 (electrical potential). In some embodiments, the physical force is provided by an electrical potential (e.g., voltage) or a temperature gradient, etc.

[0356] In some embodiments, movement of the construct comprises mechanically manipulating the construct, polynucleotide-polypeptide conjugate chain, and / or polynucleotide carrier chain, thereby moving the construct, polynucleotide-polypeptide conjugate chain, and / or polynucleotide carrier chain relative to the nanopore. In some embodiments, movement of the construct by mechanical manipulation does not comprise use of a polynucleotide handling protein.

[0357] 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 are moved relative to the nanopore as described in WO 2020 / 128517, the entire contents of which are hereby incorporated by reference, particularly for its discussion of the movement of polynucleotides relative to the nanoreactor.

[0358] In some embodiments, when a potential is applied across the nanopore, the conjugate moves relative to the nanopore. The polynucleotide is negatively charged, and therefore applying a voltage potential across the nanopore will cause the polynucleotide to move relative to the nanopore under the influence of the applied voltage potential. For example, if a positive voltage potential is applied to the trans side of the nanopore relative to the cis side of the nanopore, this will induce the negatively charged analyte to move from the cis side of the nanopore to the trans side of the nanopore. Similarly, if a positive voltage potential is applied to the trans side of the nanopore relative to the cis side of the nanopore, this will hinder the negatively charged analyte from moving from the trans side of the nanopore to the cis side of the nanopore. If a negative voltage potential is applied to the trans side of the nanopore relative to the cis side of the nanopore, the opposite will occur. The apparatus and method for applying an appropriate voltage are described in more detail herein.

[0359] In some embodiments, the chemical force is provided by a concentration (eg, pH) gradient.

[0360] In some embodiments, movement of the conjugate or construct relative to the nanopore is controlled using methods as described in WO 2020 / 016573, the entire contents of which are incorporated herein by reference.

[0361] In some embodiments, a pause group can be non-covalently attached to the construct prior to performing the method. A pause group (e.g., a short oligonucleotide capable of hybridizing to the construct) can transiently pause the movement of the construct relative to the nanopore. This can be useful for allowing the polypeptide portion of the polynucleotide-polypeptide conjugate chain to transiently remain in the pore for an increased period of time to facilitate its characterization.

[0362] In some embodiments, the polynucleotide handling protein controls the movement of the construct, polynucleotide-polypeptide conjugate chain, and / or carrier chain in the same direction as the physical or chemical force (electrical potential). For example, in some embodiments, a positive voltage potential is applied to the trans side of the nanopore relative to the cis side of the nanopore, and the polynucleotide handling protein controls the movement of the construct from the cis side of the nanopore 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 of the nanopore, and the polynucleotide handling protein controls the movement of the construct from the trans side of the nanopore to the cis side of the nanopore.

[0363] In some embodiments, the polynucleotide handling protein controls the movement of a construct, a polynucleotide-polypeptide conjugate chain, and / or a carrier chain in a direction opposite to a physical or chemical force (electrical potential). For example, in some embodiments, a positive voltage potential is applied to the trans side of the nanopore relative to the cis side of the nanopore, and the polynucleotide handling protein controls the movement of a construct from the trans side of the nanopore 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 of the nanopore, and the polynucleotide handling protein controls the movement of a construct from the cis side of the nanopore to the trans side of the nanopore.

[0364] In some embodiments, in the absence of an applied potential, movement of the construct, polynucleotide-polypeptide conjugate strands, and / or carrier strands is driven by a polynucleotide handling protein.

[0365] In the disclosed methods, the polynucleotide handling protein is typically capable of controlling the movement of the construct, polynucleotide-polypeptide conjugate chain and / or carrier chain relative to the nanopore. In other words, the polynucleotide handling protein is capable of controlling the movement of the construct.

[0366] In some embodiments, the disclosed methods comprise contacting the construct with a polynucleotide handling protein capable of controlling movement of the one or more polynucleotide flanking strands and / or the polynucleotide carrier strand, wherein the polynucleotide handling protein controls movement of the conjugate relative to the nanopore. In some embodiments, the disclosed methods comprise contacting the construct with a polynucleotide handling protein, and using the polynucleotide handling protein to control movement of the one or more polynucleotide flanking strands and / or the polynucleotide carrier strand, thereby controlling movement of the construct relative to the nanopore.

[0367] In some embodiments, the disclosed methods comprise 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 relative to the nanopore. In some embodiments, the disclosed methods comprise contacting both the polynucleotide-polypeptide conjugate chain and the polynucleotide carrier chain of the construct with a polynucleotide handling protein, and controlling the movement of the polynucleotide-polypeptide conjugate chain and / or the polynucleotide carrier chain using the polynucleotide handling protein, thereby controlling the movement of the construct relative to the nanopore.

[0368] In some embodiments, the disclosed methods comprise contacting the polynucleotide-polypeptide conjugate chain with a polynucleotide handling protein capable of controlling movement of the polynucleotide-polypeptide conjugate chain, wherein the polynucleotide handling protein controls movement of the construct relative to the nanopore. In some embodiments, the disclosed methods comprise contacting the polynucleotide-polypeptide conjugate chain with a polynucleotide handling protein, and controlling movement of the polynucleotide-polypeptide conjugate chain using the polynucleotide handling protein, thereby controlling movement of the construct relative to the nanopore.

[0369] In some embodiments, the disclosed methods comprise contacting the polynucleotide carrier strand with a polynucleotide handling protein capable of controlling movement of the polynucleotide carrier strand, wherein the polynucleotide handling protein controls movement of the construct relative to the nanopore. In some embodiments, the disclosed methods comprise contacting the polynucleotide carrier strand with a polynucleotide handling protein, and controlling movement of the polynucleotide carrier strand using the polynucleotide handling protein, thereby controlling movement of the construct relative to the nanopore.

[0370] When the construct moves relative to the nanopore, the target polypeptide of the construct moves relative to the nanopore and can be characterized thereby.

[0371] Suitable polynucleotide processing proteins are also referred to as motor proteins or polynucleotide handling enzymes. Suitable polynucleotide processing proteins are known in the art, and some exemplary polynucleotide processing proteins are described in more detail below.

[0372] In one embodiment, the motor protein is or is derived from a polynucleotide-handling enzyme. A polynucleotide-handling enzyme is a polypeptide that interacts with a polynucleotide and modifies at least one property of the polynucleotide. Enzymes can modify polynucleotides by cleaving them to form individual nucleotides or shorter nucleotide chains (e.g., dinucleotides or trinucleotides). Enzymes can modify polynucleotides by directing or moving them to a specific location.

[0373] In some embodiments, the polynucleotide handling protein can be present on the construct prior to contacting the construct with the nanopore. For example, the polynucleotide handling protein can be present on a polynucleotide in the conjugate (e.g., a flanking strand or a carrier strand). In some embodiments, the polynucleotide handling protein is present on an adaptor that comprises a portion of the conjugate, or can be otherwise present on a portion of the conjugate.

[0374] In some embodiments, when the portion of the polynucleotide-polypeptide conjugate chain that contacts the active site of the polynucleotide handling protein comprises a polypeptide, the polynucleotide handling protein is able to remain associated with the polynucleotide-polypeptide conjugate chain. In other words, in some embodiments, when the polynucleotide handling protein contacts the polypeptide portion of the polynucleotide-polypeptide conjugate chain, the polynucleotide handling protein does not dissociate from the polynucleotide-polypeptide conjugate chain. In some embodiments, the polynucleotide handling protein is free to move relative to the polypeptide portion until one or more subsequent polynucleotide portions of the polynucleotide-polypeptide conjugate chain are contacted.

[0375] In some embodiments, when a polynucleotide handling protein contacts a portion of a conjugate comprising a polypeptide, the polynucleotide handling protein is modified to prevent the polynucleotide handling protein from detaching from the construct (e.g., from a polynucleotide-polypeptide conjugate chain or a carrier chain) (except by removing the end of the construct or chain). Such modified polynucleotide handling proteins are particularly suitable for use in the disclosed methods.

[0376] The polynucleotide processing protein can be adjusted in any suitable manner.For example, the polynucleotide processing protein can be loaded onto a construct, a polynucleotide-polypeptide conjugate chain and / or a polynucleotide carrier chain, and then modified to prevent the polynucleotide processing protein from disengaging. Alternatively, the polynucleotide processing protein can be modified to prevent the polynucleotide processing protein from disengaging before being loaded onto a construct, a polynucleotide-polypeptide conjugate chain and / or a polynucleotide carrier chain. Modification of the polynucleotide processing protein to prevent it from disengaging from a construct, a polynucleotide-polypeptide conjugate chain and / or a polynucleotide carrier chain can be performed using methods known in the art (such as the method discussed in WO 2014 / 013260, which is hereby incorporated by reference in its entirety) and with particular reference to describing the modification of polynucleotide processing proteins (polynucleotide binding proteins) such as helicases to prevent the polynucleotide processing proteins from disengaging from the polynucleotide chain.

[0377] For example, a polynucleotide handling protein can have a polynucleotide unbinding opening; for example, a cavity, cleft, or gap through which a polynucleotide chain can pass when the polynucleotide handling protein is disengaged from the chain. In some embodiments, the polynucleotide unbinding opening for a given motor protein (polynucleotide handling protein) can be determined by reference to its structure (e.g., 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 location of the polynucleotide unbinding opening in a given polynucleotide handling protein can be inferred or confirmed by molecular modeling using standard methods known in the art. In some embodiments, the polynucleotide unbinding opening can be generated transiently by the movement of one or more moieties (e.g., one or more domains of a polynucleotide handling protein).

[0378] Polynucleotide handling proteins (motor proteins) can be modified by closing the polynucleotide unbinding opening. Thus, closing the polynucleotide unbinding opening can prevent the polynucleotide handling protein from dissociating from the polypeptide portion of the conjugate and from the polynucleotide or adaptor. For example, a motor protein can be modified by covalently closing the polynucleotide unbinding opening. In some embodiments, the motor protein used for addressing in this manner is a helicase as described herein. Thus, in some embodiments of the disclosed methods, the polynucleotide handling protein is modified to fully or partially close an opening present in at least one conformational state of the unmodified protein, through which the polynucleotide chain can unbind.

[0379] The polynucleotide handling protein can be chosen or selected based on the polynucleotide of the polynucleotide-polypeptide conjugate chain and / or carrier chain to be used in the methods disclosed herein. Alternatively, the polynucleotide of the polynucleotide-polypeptide conjugate chain and / or carrier chain can be chosen or selected based on the polynucleotide handling protein used to control the movement of the conjugate. For example, when the polynucleotide is DNA, a DNA motor protein can generally be used. When the polynucleotide is RNA, an RNA motor protein can be used. When the polynucleotide is a hybrid of DNA and RNA, a motor protein that can process both DNA and RNA can be used.

[0380] In one embodiment, the motor protein is derived from a member of any 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.

[0381] In some embodiments of the claimed methods, the motor protein is a helicase, a polymerase, an exonuclease, a topoisomerase, or a variant thereof.

[0382] In one embodiment, the motor protein is an exonuclease. Suitable enzymes include, but are not limited to, exonuclease I from Escherichia coli (SEQ ID NO: 1), exonuclease III from Escherichia coli (SEQ ID NO: 2), RecJ from Thermus thermophilus (SEQ ID NO: 3), bacteriophage lambda exonuclease (SEQ ID NO: 4), TatD exonuclease, and variants thereof. Three subunits comprising the sequence shown in SEQ ID NO: 3 or variants thereof interact to form a trimeric exonuclease.

[0383] In one embodiment, the motor protein is a polymerase. The polymerase can be PyroPhage® 3173 DNA polymerase (commercially available from Lucigen®), SD polymerase (commercially available from Bioron®), Klenow from NEB, or a variant thereof. In one embodiment, the enzyme is Phi29 DNA polymerase (SEQ ID NO: 5) or a variant thereof. Modified versions of Phi29 polymerase that can be used in the disclosed methods are disclosed in U.S. Patent No. 5,576,204.

[0384] In embodiments provided herein comprising methods for controlling the movement of a construct, a polynucleotide-polypeptide conjugate chain, and / or a polynucleotide vector chain by synthesizing a chain complementary to a polynucleotide chain, the polynucleotide handling protein is typically a polymerase, e.g., as described herein.

[0385] In one embodiment, the polynucleotide processing protein is a topoisomerase. In one embodiment, the topoisomerase is a member of any of the partial classification (EC) groups: 5.99.1.2 and 5.99.1.3. The topoisomerase can be a reverse transcriptase, which is an enzyme capable of catalyzing the formation of cDNA from an RNA template. Such topoisomerases are commercially available from, for example, New England Biolabs® and Invitrogen®.

[0386] In one embodiment, the polynucleotide handling protein is a translocase. Examples include translocases in the FtsK and SpoIII families.

[0387] In one embodiment, the polynucleotide processing protein is a helicase. Any suitable helicase can be used according to the method provided herein. For example, the motor protein used according to the present disclosure or each motor protein can be independently selected from Hel308 helicase, RecD helicase, Tal helicase, TrwC helicase, XPD helicase and Dda helicase or its variant. Monomeric helicases can comprise several domains linked together. For example, Tal helicase and Tal subgroup helicase can contain two RecD helicase domains, a relaxase domain and a C-terminal domain. These domains usually form monomeric helicases that can work and can not form oligomers. The specific examples of suitable helicases include Hel308, NS3, Dda, UvrD, Rep, PcrA, Pif1 and Tal. These helicases usually act on single-stranded DNA. Examples of helicases that can move along both strands of double-stranded DNA include FtsK and hexameric enzyme complexes, or multi-subunit complexes such as RecBCD, and are particularly suitable for use in some embodiments disclosed herein. NS3 helicase is particularly suitable for use in the disclosed methods because it can process both DNA and RNA, and thus can be used in embodiments of the disclosed methods in which the target double-stranded nucleic acid is a DNA-RNA hybrid.

[0388] The Hel308 helicase is described in publications such as WO 2013 / 057495, the entire contents of which are incorporated by reference. The RecD helicase is described in publications such as WO 2013 / 098562, the entire contents of which are incorporated by reference. The XPD helicase is described in publications such as WO 2013 / 098561, the entire contents of which are incorporated by reference. The Dda helicase is described in publications such as WO 2015 / 055981 and WO 2016 / 055777, the entire contents of each of which are incorporated by reference.

[0389] In one embodiment, the helicase comprises the sequence set forth in SEQ ID NO: 6 (Trwc Cba) or a variant thereof, the sequence set forth in SEQ ID NO: 7 (Hel308 Mbu) or a variant thereof, or the sequence set forth in SEQ ID NO: 8 (Dda) or a variant thereof. The variant may differ from the native sequence in any of the manners discussed herein. An exemplary variant of SEQ ID NO: 8 comprises E94C / A360C. Another exemplary variant of SEQ ID NO: 8 comprises E94C / A360C followed by (ΔM1)G1G2 (i.e., deletion of M1 and subsequent addition of G1 and G2).

[0390] In some embodiments, a motor protein (e.g., a helicase) can control the movement of a construct, a polynucleotide-polypeptide conjugate chain, and / or a polynucleotide carrier chain in at least two active modes of operation (when the motor protein is provided with all necessary components to facilitate movement, e.g., fuel and cofactors such as ATP and Mg as discussed herein). 2+ ) and an inactive mode of operation when the motor protein is not provided with the necessary components to promote movement.

[0391] When provided with all necessary components to facilitate movement (i.e., in active mode), a motor protein (e.g., a helicase) moves along the polynucleotide in the 5' to 3' or 3' to 5' direction (depending on the motor protein). The motor protein can be used to move a construct, polynucleotide-polypeptide conjugate chain, and / or polynucleotide carrier chain away from (e.g., out of) the pore (e.g., against an applied force) or toward (e.g., into) the pore (e.g., by an applied force). For example, when the end of the construct, polynucleotide-polypeptide conjugate chain, and / or polynucleotide carrier chain toward which the motor protein is moving is captured by the pore, the motor protein acts against the direction of the force and pulls the passing chain out of the pore (e.g., into the cis chamber). However, when the end toward which the motor protein is moving is captured in the pore, the motor protein acts in the direction of the force and pushes the passing chain into the pore (e.g., into the trans chamber).

[0392] When a motor protein (e.g., a helicase) is not provided with the necessary components to facilitate movement (i.e., in an inactive mode), the motor protein can bind to the construct, polynucleotide-polypeptide conjugate chain, and / or polynucleotide carrier chain and act as a brake to slow the movement of the construct, polynucleotide-polypeptide conjugate chain, and / or polynucleotide carrier chain as it moves relative to the nanopore, for example by being pulled into the pore with force. In the inactive mode, it does not matter which end of the construct, polynucleotide-polypeptide conjugate chain, and / or polynucleotide carrier chain is captured; the force applied determines movement relative to the pore, and the polynucleotide binding protein acts as a brake. When in the inactive mode, the control of movement of the polynucleotide binding protein can be described in a variety of ways, including ratcheting, sliding, and braking.

[0393] Motor proteins typically require fuel to process polynucleotides. The fuel is typically free nucleotides or free nucleotide analogs. Free nucleotides can be, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (DAMP), deoxyadenosine diphosphate (UDP), deoxyadenosine diphosphate (DAP ... The free nucleotide is typically selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, or dCMP. The free nucleotide is typically adenosine triphosphate (ATP).

[0394] A cofactor for a motor protein is a factor that allows 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 Co 2+ The cofactor is most preferably Mg 2+ .

[0395] Nanopore

[0396] As explained above, the methods disclosed herein comprise the use of a polynucleotide handling protein to control the movement of the conjugate relative to the nanopore.

[0397] In the disclosed methods, any suitable nanopore may be used.In one embodiment, the nanopore is a transmembrane pore.

[0398] A transmembrane pore is a structure that penetrates the membrane to some extent. The transmembrane pore allows hydrated ions to flow across or within the membrane, driven by an applied potential. A transmembrane pore typically passes through the entire membrane, allowing hydrated ions to flow from one side of the membrane to the other. However, a transmembrane pore does not necessarily pass through the membrane. The transmembrane pore may be closed at one end. For example, the pore may be a well, gap, channel, groove, or slit in the membrane, along which hydrated ions can flow or flow.

[0399] Any suitable transmembrane pore can be used in the methods provided herein. The pore can be biological or artificial. Suitable pores include, but are not limited to, protein pores, polynucleotide pores, and solid-state pores.

[0400] In one embodiment, the solid-state pore may comprise a nanochannel. In some embodiments, the solid-state pore is a pore disclosed in WO 2003 / 003446, WO 2009 / 020682, or WO 2016 / 187519, each of which is incorporated by reference in its entirety.

[0401] In some embodiments, the pore can be a DNA origami pore (Langecker et al., Science, 2012; 338:932-936). Suitable DNA origami pores are disclosed in WO 2013 / 083983, WO 2018 / 011603, and WO 2020 / 025974, each of which is incorporated by reference in its entirety.

[0402] In one embodiment, the nanopore is a scaffold polypeptide nanopore. In some embodiments, the pore is a scaffold polypeptide nanopore disclosed in WO 2020 / 025909 or WO 2020 / 074399, each of which is incorporated by reference in its entirety.

[0403] In one embodiment, the nanopore is a transmembrane protein pore. A transmembrane protein pore is a polypeptide or collection of polypeptides that allows hydrated ions (such as polynucleotides) to flow from one side of a membrane to the other side of the membrane. In the methods provided herein, a transmembrane protein pore can form a pore that allows hydrated ions driven by an applied potential to flow from one side of the membrane to the other side. The transmembrane protein pore preferably allows polynucleotides to flow from one side of a membrane (such as a triblock copolymer membrane) to the other side. The transmembrane protein pore allows the polynucleotide to move through the pore.

[0404] In one embodiment, the nanopore is a transmembrane protein pore that is a monomer or oligomer. The pore is 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 pore is preferably a hexamer, heptamer, octamer, or nonamer. The pore can be a homo-oligomer or a hetero-oligomer.

[0405] In one embodiment, the transmembrane protein pore comprises a barrel or channel through which ions can flow. The subunits of the pore typically surround a central axis and contribute strands to a transmembrane β-barrel or channel or a transmembrane α-helical bundle or channel.

[0406] Typically, the barrel or channel of a transmembrane protein pore comprises amino acids that promote 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. Transmembrane protein pores typically comprise 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 promote interaction between the pore and the nucleotide, polynucleotide, or nucleic acid.

[0407] In one embodiment, the nanopore is a transmembrane protein pore derived from a β-barrel pore or an α-helical bundle pore. β-barrel pores comprise a barrel or channel formed by β-strands. Suitable β-barrel pores include, but are not limited to, β-toxins such as α-hemolysin, anthrax toxin, and leukocidin, as well as bacterial outer membrane proteins / porins such as Mycobacterium smegmatis porins (Msp), such as MspA, MspB, MspC, or MspD, CsgG, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, and Neisseria autotransporter (NalP), as well as other pores such as lysins. α-helical bundle pores comprise a barrel or channel formed by α-helices. Suitable α-helical bundle pores include, but are not limited to, inner membrane proteins and α-outer membrane proteins such as WZA and ClyA toxins.

[0408] In one embodiment, the nanopore is a transmembrane pore derived from or based on Msp, α-hemolysin (α-HL), lysenin, CsgG, ClyA, Sp1 or the hemolytic protein fragaceatoxin C (FraC).

[0409] In one embodiment, the nanopore is a transmembrane protein pore derived from CsgG, for example, from CsgG from Escherichia coli strain K-12 substrain MC4100. Such pores are oligomeric and typically comprise 7, 8, 9, or 10 monomers derived from CsgG. The pore may be a homo-oligomeric pore derived from CsgG comprising identical monomers. Alternatively, the pore may be a hetero-oligomeric pore derived from CsgG comprising at least one monomer that is different from the other monomers. Examples of suitable pores derived from CsgG are disclosed in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318, and WO 2019 / 002893, each of which is hereby incorporated by reference in its entirety.

[0410] In one embodiment, the nanopore is a transmembrane pore derived from lysenin. Examples of suitable pores derived from lysenin are disclosed in WO 2013 / 153359, which is hereby incorporated by reference in its entirety.

[0411] In one embodiment, the nanopore is a transmembrane pore derived from or based on α-hemolysin (α-HL). The wild-type α-hemolysin pore is formed from seven identical monomers or subunits (i.e., it is heptameric). The α-hemolysin pore can be α-hemolysin-NN or a variant thereof. The variant preferably comprises N residues at positions E111 and K147.

[0412] In one embodiment, the nanopore is a transmembrane protein pore derived from Msp, such as from MspA. Examples of suitable pores derived from MspA are disclosed in WO 2012 / 107778.

[0413] In one embodiment, the nanopore is a transmembrane pore derived from or based on ClyA. Examples of suitable pores derived from ClyA are disclosed in Soskine et al., Nano Letters 2012 12 (9), 4895-4900; WO 2014 / 153625; and WO 2017 / 098322, each of which is hereby incorporated by reference.

[0414] In one embodiment, the nanopore is a transmembrane pore derived from Phi 29. Examples of suitable pores derived from Phi 29 are disclosed in Wendell et al., Nature Nanotech 4, 765–772 (2009), WO 2010 / 062697, WO 2019 / 157365, and WO 2019 / 157424, each of which is hereby incorporated by reference.

[0415] In some embodiments, the nanopore is selected from the group consisting of M-loop protein, perforin-2, PlyAB (pleurotolysin), SpoIIIAG, VirB7, type II secretion system protein D, GspD, InvG, PilQ, penetratin, and portal proteins, including T4, T7, P23-45, G20c, and Phi29 nanopores.

[0416] In one embodiment, the nanopore is a transmembrane pore derived from or based on the bacteria Rhodococcus, such as Rhodococcus corynebacteroides or Rhodococcus ruber, such as PorARr, PorBRr, or PorARc. Examples of such pores are described in Piselli et al., EurBiophys J 51, 309–323 (2022).

[0417] As explained above, in some embodiments, the nanopore comprises a constriction. A constriction is generally a narrowing in the channel running through the nanopore that can determine or control the signal obtained when the conjugate moves relative to the nanopore. As used herein, both protein and solid-state nanopores can comprise a "constriction."

[0418] In some embodiments, the nanopore is designed, modified, or selected to have a constriction sized according to the diameter of the construct. In some embodiments, the diameter of the constriction of the pore is at least 1 nm, for example, at least 1.5 nm, such as at least 2 nm, for example, at least 2.5 nm, for example, at least 3 nm. In some embodiments, the diameter of the constriction of the pore is about 1.5 nm to about 2.5 nm. In some embodiments, the pore has a constriction capable of translocating double-stranded DNA. The diameter of the double-stranded DNA is about 2 nm. The DNA-peptide chimera can be narrower or wider than the duplex DNA, depending on the amino acids and strand interactions.

[0419] In some embodiments, the nanopore is modified to extend the distance between the polynucleotide processing protein and the constriction region of the nanopore. Methods for doing so are disclosed in WO 2021 / 111125.

[0420] Label

[0421] In some embodiments of the methods provided herein, tags on the nanopore can be used, for example, to facilitate nanopore capture constructs.

[0422] The interaction between the tag on the nanopore and a binding site on the construct, polynucleotide-polypeptide conjugate chain, and / or polynucleotide carrier chain (e.g., a binding site present in the polynucleotide portion of the conjugate or in an adaptor attached to the conjugate, where the binding site can be provided by an anchor or leader sequence of the adaptor or by a capture sequence within the duplex stem of the adaptor) can be reversible. For example, a polynucleotide can be bound to a tag on the nanopore, for example, via its adaptor, and released at certain points, for example, during characterization of the polynucleotide by the nanopore and / or during processing by the motor protein. Strong non-covalent binding (e.g., biotin / avidin) is still reversible and can be used in some embodiments of the methods described herein. For example, a pair of pore tags and polynucleotide adaptors can be designed to provide sufficient interaction between the complement of the double-stranded polynucleotide (or the portion of the adaptor to which the complement is attached) and the nanopore so that the complement remains close to the nanopore (does not dissociate from the nanopore and diffuse), but can be released from the nanopore during processing.

[0423] The pore tag and polynucleotide adaptor can be configured such that the binding strength or affinity of a binding site on the polynucleotide (e.g., a binding site provided by an anchor or leader sequence of the adaptor or by a capture sequence within the duplex stem of the adaptor) to the tag on the nanopore is sufficient to maintain the connection between the nanopore and the polynucleotide until an applied force is placed thereon to release the bound polynucleotide from the nanopore.

[0424] In some embodiments, the tag or tether is uncharged to ensure that the tag or tether is not pulled into the nanopore under the influence of a potential difference, if any.

[0425] One or more molecules that attract or bind to the construct, polynucleotide-polypeptide conjugate chain and / or polynucleotide carrier chain can be connected to the nanopore. Any molecule that hybridizes to the conjugate, adapter and / or polynucleotide can be used. The molecule connected to the pore can be selected from PNA tags, PEG linkers, short oligonucleotides, positively charged amino acids and aptamers. Pores that allow such molecules to be connected to them are known in the art. For example, pores to which short oligonucleotides are connected are disclosed in Howarka et al. (2001) Nature Biotech. 19:636-639 and WO 2010 / 086620, and pores containing PEG connected to the lumen of the pore are disclosed in Howarka et al. (2000) J. Am. Chem. Soc. 122(11):2411-2416.

[0426] Short oligonucleotides comprising a sequence complementary to a sequence in the conjugate (e.g., in a leader sequence or another single-stranded sequence in an adaptor) attached to a nanopore can be used to enhance capture of constructs, polynucleotide-polypeptide conjugate chains, and / or polynucleotide carrier chains in the methods described herein.

[0427] In some embodiments, the tag or tether may comprise or may be an oligonucleotide (e.g., DNA, RNA, LNA, BNA, PNA, or morpholino). The oligonucleotide may be about 10-30 nucleotides or about 10-20 nucleotides in length. In some embodiments, the oligonucleotide may have at least one end (e.g., the 3' or 5' end) modified for conjugation to other modifications or solid substrate surfaces (including, for example, beads). The end modifier may add 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 can be combined with spacers of different lengths (e.g., C3, C9, C12, spacers 9 and 18) to increase the physical distance between the functional group and the end of the oligonucleotide sequence.

[0428] Examples of modifications on the 3' and / or 5' termini of the oligonucleotide include, but are not limited to, 3' affinity tags and functional groups for chemical attachment (including, for example, 3'-biotin, 3'-primary amine, 3'-disulfide amide, 3'-pyridyldisulfide, and any combination thereof); 5' terminus modifications (including, for example, 5'-primary amine and / or 5'-dabcyl), modifications for click chemistry (including, for example, 3'-azide, 3'-alkyne, 5'-azide, 5'-alkyne), and any combination thereof.

[0429] In some embodiments, the tag or tether may further comprise a polymer linker, for example, to facilitate coupling to the nanopore. Exemplary polymer linkers include, but are not limited to, polyethylene glycol (PEG). The molecular weight of the polymer linker may be between about 500 Da and about 10 kDa (inclusive) or between about 1 kDa and about 5 kDa (inclusive). The polymer linker (e.g., PEG) may be functionalized with various functional groups, including, for example, but not limited to, maleimide, NHS ester, dibenzocyclooctyne (DBCO), azide, biotin, amine, alkyne, aldehyde, and any combination thereof.

[0430] Other examples of tags or tethers include, but are not limited to, a His tag, biotin or streptavidin, an antibody that binds to the analyte, an aptamer that binds to the analyte, an analyte binding domain, such as a DNA binding domain (including, for example, a peptide zipper, such as a leucine zipper, a single-stranded DNA binding protein (SSB)), and any combination thereof.

[0431] Any method known in the art can be used to connect the label or tether to the outer surface of the nanopore, for example, on the cis side of the membrane. For example, one or more labels or tethers can be connected to the nanopore by 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 biotins or streptavidins, one or more antibody-based labels, one or more enzyme modifications of epitopes (including, for example, acetyltransferases), and any combination thereof. Methods suitable for performing such modifications are well known in the art. Suitable non-natural amino acids include, but are not limited to, 4-azido-L-phenylalanine (Faz), and Liu C. C. and Schultz PG, Annu. Rev. Biochem., 2010, 79, 413-444. Figure 1 Any of the amino acids numbered 1-71 in .

[0432] In some embodiments where one or more tags or tethers are attached to the nanopore via cysteine ​​bonds, one or more cysteines can be introduced into one or more monomers that form the nanopore by substitution. In some embodiments, the nanopore can be chemically modified by attaching: (i) maleimides, including dibromomaleimides, such as 4-phenylazomaleimide, 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, Amine, N-methoxycarbonylmaleimide, N-tert-butylmaleimide, N-(2-aminoethyl)maleimide, 3-maleimido-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-dimethylphenyl)maleimide, N-(2,4-difluorophenyl)maleimide, N-(3-chloro-p-methylphenyl)-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-2,5-dihydro-1H-pyrrole-2,5-dione, N-(4-phenoxyphenyl)maleimide, N-(4-nitrophenyl)maleimide;(ii) Iodoacetamides, such as 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-iodoacetamide, N-(1,3-benzothiazol-2-yl)-2-iodoacetamide, N-(2,6-difluoro- (iii) bromoacetamides such as 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-fluoro ...4-chlorophenyl)acetamide, 2-bromo-N-(2-chlorophenyl)acetamide, 2-bromo-N-(2-chlorophenyl)acetamide, 2-bromo-N-(4-chlorophenyl)acetamide, 2-bromo-N-(2-chlorophenyl)acetamide, 2-bromo-N-(2-chlorophenyl)acetamide, 2-bromo- (iv) disulfides, such as aldehyde thiol-2, aldehyde thiol-4, isopropyl disulfide ethers, 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 (v) thiols, such as 4-phenylthiazole-2-thiol, Purpald, and 5,6,7,8-tetrahydro-quinazoline-2-thiol.

[0433] In some embodiments, the tag or tether can be connected to the nanopore directly or through one or more linkers. The tag or tether can be connected to the nanopore using a hybrid linker described in WO 2010 / 086602. Alternatively, a peptide linker can be used. A peptide linker is an amino acid sequence. The length, flexibility and hydrophilicity of the peptide linker are generally designed so that it does not interfere with the function of the monomer and the pore. Preferred flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16 serine and / or glycine amino acids. More preferred flexible linkers include (SG)1, (SG)2, (SG)3, (SG)4, (SG)5 and (SG)8, wherein S is serine and G is glycine. Preferred rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24 proline amino acids. More preferred rigid linkers include (P) 12 , where P is proline.

[0434] membrane

[0435] Typically, in the disclosed methods, the nanopores are typically present in the membrane.Any suitable membrane can be used in the system.

[0436] The membrane is preferably an amphiphilic layer. An amphiphilic layer is formed from amphiphilic molecules (such as phospholipids) that have both hydrophilic and lipophilic properties. Amphiphilic molecules can be synthetic or naturally occurring. Non-naturally occurring amphiphiles and amphiphiles that form monolayers are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). A block copolymer is a polymer material in which two or more monomer subunits are polymerized together to form a single polymer chain. Block copolymers generally have properties contributed by each monomer subunit. However, block copolymers can have unique properties that polymers formed from individual subunits do not possess. Block copolymers can be designed so that one of the monomer subunits is hydrophobic (i.e., lipophilic) in an aqueous medium, while the other subunits are hydrophilic. In this case, the block copolymer can possess amphiphilic properties and form a structure that mimics a biological membrane. Block copolymers can be diblock (composed of two monomer subunits), but can also be constructed from more than two monomer subunits to form more complex arrangements that behave as amphiphiles. The copolymers can be triblock, tetrablock, or pentablock copolymers. The membrane is preferably a triblock copolymer membrane.

[0437] Archaeal bipolar tetraether lipids are naturally occurring lipids that are constructed to form lipid monolayers. These lipids are generally found in extreme microorganisms, thermophiles, halophiles, and acidophiles that survive in harsh biological environments. Their stability is believed to be derived from the fusogenic properties of the final bilayer. It is simple to construct block copolymer materials that mimic these biological entities by producing triblock polymers with the general motif hydrophilicity-hydrophobicity-hydrophilicity. This material can form monomeric membranes that behave similarly to lipid bilayers and encompass a series of stage performances from vesicles to lamellar membranes. The membranes formed by these triblock copolymers maintain several advantages over biological lipid membranes. Because the triblock copolymers are synthetic, accurate construction can be carefully controlled to provide the correct chain length and properties required to form a membrane and interact with pores and other proteins.

[0438] Block copolymers can also be constructed from subunits that are not classified as lipid submaterials; for example, hydrophobic polymers can be made from siloxanes or other non-hydrocarbon-based monomers. The hydrophilic subsegments of the block copolymers can also possess low protein binding properties, which allows for the creation of membranes that are highly resistant when exposed to raw biological samples. The head group unit can also be derived from non-classical lipid head groups.

[0439] Compared to biological lipid membranes, triblock copolymer membranes also have increased mechanical and environmental stability, such as much higher operating temperature or pH ranges.The synthetic nature of block copolymers provides a platform for tailoring polymer-based membranes for a wide range of applications.

[0440] In some embodiments, the membrane is one of the membranes disclosed in International Application Nos. WO 2014 / 064443 or WO 2014 / 064444.

[0441] The amphiphilic molecules can be chemically modified or functionalized to facilitate coupling of the polynucleotide. The amphiphilic layer can be a single layer or a double layer. The amphiphilic layer is typically planar. The amphiphilic layer can be curved. The amphiphilic layer can be supported.

[0442] Amphiphilic membranes are usually naturally mobile, essentially at a rate of about 10 -8 cm s -1 The lipid diffusion rate is 2.5 Å, which acts as a two-dimensional liquid. This means that the pore and the coupled polynucleotide can generally move within the amphiphilic membrane.

[0443] The membrane can be a lipid bilayer. The lipid bilayer is a model for the cell membrane and is used as an excellent platform for a range of experimental studies. For example, the lipid bilayer can be used to study membrane proteins in vitro by single-channel recording. Alternatively, the lipid bilayer can be used as a biosensor to detect the presence of a range of 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 WO 2008 / 102121, WO 2009 / 077734, and WO 2006 / 100484.

[0444] Methods for forming lipid bilayers are known in the art. Lipid bilayers are typically 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 an aqueous solution / air interface on either side of an aperture perpendicular to the interface. Lipids are typically added to the surface of an aqueous electrolyte solution by first dissolving the lipids in an organic solvent and then allowing the solvent to evaporate on the surface of the aqueous solution on either side of the aperture. Once the organic solvent evaporates, the solution / air interface on either side of the aperture physically moves back and forth through the aperture until a bilayer is formed. A planar lipid bilayer can be formed across an aperture in the membrane or across an opening in a groove.

[0445] The method of Montal and Mueller is popular because it is a cost-effective and relatively simple method for forming high-quality lipid bilayers suitable for protein pore insertion. Other common methods for bilayer formation include tip dipping of liposomal bilayers, bilayer painting, and patch clamping.

[0446] Tip-immersion bilayer formation requires contacting an open pore surface (e.g., a pipette tip) with the surface of a test solution carrying a lipid monolayer. Similarly, a lipid monolayer is first generated at the solution / air interface by allowing a drop of lipid dissolved in an organic solvent to evaporate on the solution surface. The bilayer is then formed via the Langmuir-Schaefer method, requiring robotic automation to move the pore relative to the solution surface.

[0447] For a painted bilayer, a drop of lipid dissolved in an organic solvent is applied directly to an open pore, which is immersed in an aqueous test solution. A paintbrush or equivalent is used to thinly paint the lipid solution onto the pore. This thinning of the solvent allows the lipid bilayer to form. However, completely removing the solvent from the bilayer is difficult, and as a result, bilayers formed using this method are less stable and more susceptible to noise during electrochemical measurements.

[0448] Patch clamping is commonly used in the study of biological cell membranes. The cell membrane is clamped to the end of a pipette by pumping, and the membrane patch becomes connected in the perforation. The method is suitable for producing a lipid bilayer by clamping and then bursting liposomes to leave the lipid bilayer sealed in the perforation of the pipette. The method requires stable, large and unilamellar liposomes and the manufacture of small perforations in materials with glass surfaces.

[0449] Liposomes can be formed by sonication, extrusion, or the Mozafari method (Colas et al. (2007) Micron 38:841–847).

[0450] In certain embodiments, the lipid bilayer is formed as described in International Application No. WO 2009 / 077734. Advantageously, in this method, the lipid bilayer is formed by dried lipids. In certain embodiments, the lipid bilayer is formed across an opening as described in WO 2009 / 077734.

[0451] The lipid bilayer is formed by two relative lipid layers. The two lipid layers are arranged so that their hydrophobic tail groups face each other to form a hydrophobic interior. The hydrophilic head groups of the lipids face outwards towards the aqueous environment on each side of the bilayer. The bilayer can be present in many lipid phases, including but not limited to liquid disordered phase (fluid lamellar), liquid ordered phase, solid ordered phase (lamellar gel phase, interdigitated gel phase) and planar bilayer crystals (lamellar subgel phase, lamellar crystalline phase).

[0452] Any lipid composition that forms a lipid bilayer can be used. The lipid composition is selected so as to form a lipid bilayer with desired properties (such as surface charge, ability to support membrane proteins, bulk density or mechanical properties). The lipid composition can comprise one or more different lipids. For example, the lipid composition can contain up to 100 lipids. The lipid composition preferably contains 1 to 10 lipids. The lipid composition can comprise naturally occurring lipids and / or artificial lipids.

[0453] Lipids typically comprise a head group, an interfacial moiety, and two hydrophobic tail groups that can be the same or different. Suitable head groups include, but are not limited to, neutral head groups such as diacylglycerides (DG) and ceramides (CM); zwitterionic head groups such as phosphatidylcholines (PC), phosphatidylethanolamines (PE), and sphingomyelins (SM); negatively charged head groups such as phosphatidylglycerols (PG); phosphatidylserines (PS), phosphatidylinositols (PI), phosphatidic acid (PA), and cardiolipin (CA); and positively charged head groups such as trimethylammoniumpropane (TAP). Suitable interfacial moieties include, but are not limited to, naturally occurring interfacial moieties such as glycerol-based or ceramide-based moieties. Suitable hydrophobic tail groups include, but are not limited to, saturated hydrocarbon chains such as lauric acid (n-dodecanoic acid), myristic acid (n-tetradecanoic 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 phytanyl. The chain length and the position and number of double bonds in the unsaturated hydrocarbon chain can vary. The chain length and the position and number of branches (e.g., methyl groups) in the branched hydrocarbon chain can vary. The hydrophobic tail group can be attached to the interfacial moiety as an ether or ester. The lipid can be a mycolic acid.

[0454] The lipids can also be chemically modified. The head group or tail group of the lipid can be chemically modified. Suitable lipids whose head groups have been chemically modified include, but are not limited to, PEG-modified lipids such as 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]; functionalized PEG lipids such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)2000]; and lipids modified for conjugation such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl). Suitable lipids whose tail groups have been chemically modified include, but are not limited to, polymerizable lipids such as 1,2-bis(10,12-tricosadiynyl)-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-linked lipids such as 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine. The lipids may be chemically modified or functionalized to facilitate coupling of polynucleotides.

[0455] The amphiphilic layer, such as a lipid composition, typically comprises 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 palmityl, myristyl alcohol, and oleyl alcohol; sterols such as cholesterol, ergosterol, lanosterol, sitosterol, and stigmasterol; lysophospholipids such as 1-acyl-2-hydroxy-sn-glycero-3-phosphocholine; and ceramides.

[0456] In another embodiment, the membrane comprises 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), as well as glass. The solid-state layer can be formed from graphene. Suitable graphene layers are disclosed in WO 2009 / 035647. If the membrane comprises a solid-state layer, the pores are typically present in the amphiphilic membrane or layer contained within the solid-state layer, such as within holes, wells, gaps, channels, grooves, or slits within the solid-state layer. Suitable solid-state / amphiphilic hybrid systems can be prepared by one skilled in the art. Suitable systems are disclosed in WO 2009 / 020682 and WO 2012 / 005857. Any of the amphiphilic membranes or layers discussed above can be used.

[0457] The methods disclosed herein are typically performed using: (i) an artificial amphiphilic layer comprising a pore, (ii) an isolated naturally occurring lipid bilayer comprising a pore, or (iii) a cell into which a pore is inserted. The methods are typically performed using an artificial amphiphilic layer, such as an artificial triblock copolymer layer. The layer may contain other transmembrane and / or intramembrane proteins, as well as other molecules in addition to the pore. Suitable equipment and conditions are discussed below. The disclosed methods are typically performed in vitro.

[0458] condition

[0459] The disclosed characterization methods can be performed using any apparatus suitable for studying membrane / pore systems in which pores are inserted into the membrane. The characterization methods can be performed using any apparatus suitable for transmembrane pore sensing. For example, the apparatus can include a chamber containing an aqueous solution and a barrier dividing the chamber into two sections. The barrier typically has an opening in which a membrane containing a transmembrane pore is formed. Transmembrane pores are described herein.

[0460] The characterisation method may be performed using the apparatus described in WO 2008 / 102120, WO 2010 / 122293 or WO 00 / 28312.

[0461] Characterization methods may comprise optical measurements, for example as described in WO 2016 / 009180 and WO 2021 / 198695.

[0462] The characterization method may involve measuring the ionic current flowing through the pore, typically by measuring the current. Alternatively, the ionic current through the pore may be measured optically, as disclosed in Heron et al., Journal of the American Chemical Society, Vol. 9, No. 5, 2009. Thus, the apparatus may also include circuitry capable of applying a potential across the membrane and the pore and measuring the electrical signal. 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.

[0463] The characterization method can be performed on silicon-based well arrays, where each array contains 128, 256, 512, 1024, 2000, 3000, 4000, 6000, 10000, 12000, 15000 or more wells.

[0464] Characterization method can relate to measuring the electric current flowing through hole.Described method is usually performed when transmembrane and hole apply voltage.The voltage used is usually +2 V to -2 V, usually -400 mV to +400 mV.The voltage used is preferably in the scope with lower limit and upper limit, and described lower limit is selected from -400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, -20 mV and 0 mV, and described upper limit is independently selected from +10 mV, + 20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV.The voltage used is more preferably in the scope of 100 mV to 240 mV, and most preferably in the scope of 120 mV to 220 mV.By using the applied potential increased, the degree of differentiation between different nucleotides can be increased by hole.

[0465] Characterization methods are typically performed in the presence of any charge carrier, such as a metal salt, for example, an alkali metal salt, or a halide salt, for example, a chloride salt, such as an alkali metal chloride salt. The charge carrier can include an ionic liquid or an organic salt, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, or 1-ethyl-3-methylimidazolium chloride. In the exemplary apparatus discussed above, the salt is present in an aqueous solution within the chamber. Potassium chloride (KCl), sodium chloride (NaCl), or cesium chloride (CsCl) is typically used. KCl is preferred. The salt can be an alkaline earth metal salt, such as calcium chloride (CaCl2). The salt concentration can be at saturation. The salt concentration can be 3 M or less, and is typically 0.1 M to 2.5 M, 0.3 M to 1.9 M, 0.5 M to 1.8 M, 0.7 M to 1.7 M, 0.9 M to 1.6 M, or 1 M to 1.4 M. The salt concentration is preferably 150 mM to 1 M. The characterization method can be performed using a salt concentration of at least 0.3 M, such as 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. High salt concentrations provide a high signal-to-noise ratio and allow identification of currents indicative of binding / absence against the background of normal current fluctuations.

[0466] The characterization method is typically performed in the presence of a buffer. In the exemplary apparatus discussed above, the buffer is present in the aqueous solution in 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 a pH of 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7, or 7.0 to 8.8, or 7.5 to 8.5. The pH used may be approximately 7.5.

[0467] The characterization method can be performed at a temperature 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. The characterization method is typically performed at room temperature. Optionally, the characterization method is performed at a temperature that supports enzyme function, such as approximately 37°C.

[0468] system

[0469] A system is also provided, comprising

[0470] - a construct comprising (i) a polynucleotide-polypeptide conjugate chain comprising a target polypeptide conjugated at each terminus of the target polypeptide to one or more polynucleotide flanking chains, and (ii) a polynucleotide carrier chain;

[0471] - a nanopore capable of co-translocating the polynucleotide-polypeptide conjugate strand and the polynucleotide flanking strand of the construct; and

[0472] - Polynucleotide processing proteins.

[0473] In some embodiments, the construct, nanopore, and / or polynucleotide handling protein are as described in more detail herein.

[0474] Also provided is a kit comprising:

[0475] - nanopores;

[0476] - a first polynucleotide comprising a reactive functional group for conjugation to a first terminus of a target polypeptide;

[0477] - a second polynucleotide comprising a reactive functional group for conjugation to the second end of the target polypeptide; and

[0478] - Polynucleotide processing proteins.

[0479] In some embodiments, the nanopore and polynucleotide handling protein are each as described in more detail herein.In some embodiments, the first polynucleotide and the second polynucleotide are each a flanking strand as described herein.

[0480] Each of the systems and kits can be independently configured for use with an algorithm as provided herein, which is suitable for running on a computer system. The algorithm can be adapted to detect information specific to a polypeptide (e.g., information specific to the polypeptide sequence and / or whether the polypeptide is modified) and selectively process the signal obtained when a construct comprising a polypeptide conjugated to polynucleotide flanking strands and hybridized to a polynucleotide carrier strand moves relative to a nanopore (i.e., when the strands co-translocate the pore).

[0481] In some embodiments, the system comprises a computing device configured to detect information specific to the polypeptide (e.g., information specific to the polypeptide sequence and / or whether the polypeptide is modified) and selectively process signals obtained when a conjugate comprising the polypeptide conjugated to a polynucleotide flanking strand and a polynucleotide carrier strand co-translocates through the nanopore. In some embodiments, the system comprises a receiving device for receiving data from detection of the polypeptide, a processing device for processing signals obtained when the conjugate moves relative to the nanopore, and an output device for outputting the characterizing information thereby obtained.

[0482] Characterization of target polynucleotide sequences

[0483] The method discussed above can also be used to characterize a polynucleotide chain, which can be, for example, a polynucleotide chain that does not comprise a polypeptide sequence. It will be understood by those skilled in the art that many of the advantages described for the characterization and mobile methods related to polynucleotide-polypeptide constructs disclosed above are equally applicable to methods in which the construct being assessed does not comprise a polypeptide. For example, in some embodiments, the target polypeptide portion of the construct as described herein can be exchanged for a target polynucleotide sequence, and the method disclosed above can be used accordingly to characterize the target polynucleotide sequence. In such embodiments, unless the context indicates otherwise, the features described above can generally be applied. Therefore, for example, a polynucleotide chain and its formation, a construct and its formation, a polynucleotide processing protein, a nanopore and general settings and mobile schemes can all be as described above. This is further described below.

[0484] Therefore, a method of characterizing a target polynucleotide sequence is provided, the method comprising

[0485] - contacting (i) a target polynucleotide strand comprising said target polynucleotide sequence with (ii) a polynucleotide carrier strand, thereby forming a double-stranded polynucleotide construct;

[0486] - contacting the construct with the nanopore under conditions such that both the target polynucleotide strand and the polynucleotide carrier strand co-translocate through the nanopore; and

[0487] - performing one or more measurements specific to the target polynucleotide as the construct moves relative to the nanopore,

[0488] The target polynucleotide is thereby characterized.

[0489] In some embodiments, the target polynucleotide sequence is conjugated to one or more polynucleotide flanking strands at each end of the target polynucleotide sequence. In some embodiments, the one or more polynucleotide flanking strands are each independently complementary to a region of the polynucleotide carrier strand. In some embodiments, the one or more polynucleotide flanking strands are each independently at least partially hybridized to the polynucleotide carrier strand.

[0490] In some embodiments, the target polynucleotide sequence is complementary to a region of the polynucleotide carrier strand. Thus, in some embodiments, in the construct formed by the target polynucleotide strand and the polynucleotide carrier strand, the target polynucleotide strand and the polynucleotide carrier strand hybridize together in the region of the target polynucleotide sequence.

[0491] In some embodiments, the target polynucleotide sequence is non-complementary to the polynucleotide carrier strand. Thus, in some embodiments, in the construct formed by the target polynucleotide strand and the polynucleotide carrier strand, the target polynucleotide strand and the polynucleotide carrier strand do not hybridize together in the region of the target polynucleotide. In some embodiments, although the target polynucleotide strand and the polynucleotide carrier strand hybridize together in the region of the polynucleotide flanking strand, they do not hybridize together in the region of the target polynucleotide sequence.

[0492] Therefore, a method for characterizing a target polynucleotide sequence is also provided, the method comprising

[0493] - contacting (i) a target polynucleotide chain comprising a target polynucleotide sequence linked to the polynucleotide flanking chain with (ii) a polynucleotide handling protein capable of controlling movement of the polynucleotide flanking chain relative to the nanopore; and

[0494] - contacting the target polynucleotide chain with the nanopore under conditions such that the polynucleotide handling protein controls movement of the target polynucleotide chain relative to the nanopore; and

[0495] - performing one or more measurements specific to the target polynucleotide sequence as the polynucleotide flanking strands and the target polynucleotide analyte co-translocate through the nanopore,

[0496] The target polypeptide is thereby characterized.

[0497] In some embodiments, prior to such methods, the polynucleotide flanking strand is at least partially hybridized to the polynucleotide carrier strand.

[0498] In some embodiments of the above methods, the target polynucleotide chain comprises a plurality of target polynucleotide sequences. For example, in some embodiments, the length of the target polynucleotide sequence or each target polynucleotide sequence can independently be from about 5 to about 1000 nucleotide units. The polynucleotide chain used in the above methods can be any of the polynucleotide chains described in more detail herein.

[0499] In certain embodiments, the target polynucleotide sequence comprises nucleotides of different types from the polynucleotide carrier chain and / or polynucleotide flanking chains. For example, the target polynucleotide sequence can comprise RNA nucleotides or consist of them, and the polynucleotide carrier chain and / or flanking chains can comprise DNA nucleotides or consist of them. In certain embodiments, the target polynucleotide sequence can comprise DNA nucleotides or consist of them, and the polynucleotide carrier chain and / or flanking chains can comprise RNA nucleotides or consist of them. In certain embodiments, the target polynucleotide sequence comprises nucleotides of the same type as the polynucleotide carrier chain and / or polynucleotide flanking chains. For example, the target polynucleotide sequence can comprise DNA nucleotides or consist of them, and the polynucleotide carrier chain and / or flanking chains can comprise DNA nucleotides or consist of them.

[0500] The target polynucleotide chain, polynucleotide carrier chain and / or construct can be mechanically manipulated in the manner discussed above in the context of the polynucleotide-polypeptide conjugate chain. Thus, the above method can comprise mechanically manipulating the construct, target polynucleotide chain and / or polynucleotide carrier chain to thereby move the construct, target polynucleotide chain and / or polynucleotide carrier chain relative to the nanopore. The movement can be in a direction opposite to the potential applied across the nanopore (e.g., the voltage potential applied across the nanopore).

[0501] The above methods can comprise contacting the construct or its polynucleotide strands with a polynucleotide handling protein capable of controlling the movement of one or more strands of the construct (e.g., the one or more polynucleotide flanking strands and / or the target polynucleotide strand), thereby controlling the movement of the construct relative to the nanopore. As discussed in more detail herein, in some embodiments, the polynucleotide handling protein controls the movement of both strands of the construct. In some embodiments, the polynucleotide handling protein controls the movement of only one of the strands of the construct (e.g., the target polynucleotide strand or the polynucleotide carrier strand).

[0502] The polynucleotide handling protein can be located on the cis side or the trans side of the nanopore and can control the movement of the construct in the direction from the cis side of the nanopore to the trans side of the nanopore or from the trans side of the nanopore to the cis side, as described in more detail herein in the context of methods involving the movement and characterization of polynucleotide-polypeptide chains and constructs comprising the same.

[0503] The polynucleotide processing protein can be any of the polynucleotide processing proteins described in more detail herein. In some embodiments, prior to the above method, the polynucleotide processing protein is bound to the polynucleotide carrier chain in the region of the polynucleotide carrier chain spanned by the non-hybridized region of the polynucleotide flanking chain. In some embodiments, the polynucleotide processing protein is capable of remaining bound to the construct as described in more detail herein. For example, the polynucleotide processing protein can be modified to prevent the polynucleotide processing protein from disengaging from the construct, the target polynucleotide chain, and / or the polynucleotide carrier chain. The polynucleotide processing protein can be modified in any manner described herein, such as by being modified to completely or partially close an opening that exists in at least one conformational state of the unmodified protein, through which the polynucleotide chain can unbind. As discussed in more detail herein, in some embodiments, the polynucleotide processing protein is or comprises a helicase, a translocase, or a helicase-nuclease complex.

[0504] As discussed in more detail herein in the context of methods involving the movement and characterization of polynucleotide-polypeptide chains and constructs comprising the same, in some embodiments of the methods discussed above, the construct comprises a stagnation moiety that can be positioned prior to the method such that the stagnation moiety is between the polynucleotide processing protein and the target polynucleotide sequence. Adapters, tethers, anchors, and / or blocking moieties can further be included in the constructs as discussed in more detail above.

[0505] As discussed in more detail herein in the context of methods involving the movement and characterization of polynucleotide-polypeptide chains and constructs comprising the same, in some embodiments of the methods discussed above, the construct or one or more of its constituent chains can be "shuttled" through the nanopore by performing the method such that (i) the chain translocates the nanopore in a first direction relative to the nanopore; (ii) the chain is allowed to move in a direction opposite to the direction of movement relative to the nanopore in step (i); (iii) the chain is optionally allowed to move in the first direction; and steps (ii) and (iii) are optionally repeated to oscillate the chain through the nanopore.

[0506] Properties that can be measured in the above methods can include, for example, (i) the length of the target polynucleotide sequence, (ii) the identity 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 sequence is modified.

[0507] The nanopore can be any of the nanopores discussed herein, such as a protein nanopore, for example a beta-barrel protein nanopore.

[0508] It should be understood that although specific embodiments, specific configurations, and materials and / or molecules have been discussed herein for the methods according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of the present invention. The foregoing embodiments and the following examples are provided for illustration only and should not be construed as limiting the present application. The present application is limited only by the claims.

[0509] Examples

[0510] Example 1

[0511] This example demonstrates the discrimination between three different peptides, each contained in a construct comprising a peptide analyte and a polynucleotide chain as described above.

[0512] The distinction was demonstrated using a modified transmembrane protein nanopore derived from Rhodococcus bacteria. The inner diameter of the nanopore is wide enough to accommodate double-stranded DNA (dsDNA). The pore is formed from a monomer with the following amino acid sequence:

[0513] MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPGLNLSVGNGVAATVTNVLPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSSAWSHPQFEK.

[0514] (SEQ ID NO: 9)

[0515] The peptide was conjugated between two dsDNA oligonucleotides, where a single-stranded DNA (ssDNA) strand served as the "carrier" strand and a terminal biotin was used to prevent the conjugate from completely translocating through the nanopore. To simulate the stepwise movement of this conjugate through the nanopore, a series of conjugates were prepared in which the distance in dsDNA base pairs between the terminal biotin and the peptide was varied (distance "x," as Figure 10 ).

[0516] Construct assembly

[0517] All constructs were formed from two DNA oligonucleotides (DNA1 and DNA2) and a peptide, e.g. Figure 10 As shown in , DNA1 carries a hairpin and a 3' terminal TCO group, the peptide carries an N-terminal azide and a C-terminal methyl tetrazine group, and DNA2 carries a 5' BCN group and a 3' biotin group. XX The SGSG-C peptide, in which the middle two amino acid residues (XX) are varied: DD, RR, or YY, is modified with an N-terminal azide and a C-terminal methyltetrazine connected via an ethylenediamine linker.

[0518] The SPRI bead mix was prepared by exchanging SpeedBead magnetic carboxylate-modified particles (Merck, catalog number GE65152105050250) into SPRI wash 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).

[0519] The construct was assembled in a two-stage process in which click reactions were performed sequentially. DNA1 oligonucleotide (1 µM) was first reacted with 200 µM peptide in 25 mM HEPES-NaOH (pH 7.5), 500 mM NaCl, and 0.01% (v / v) Tween-20. The reaction was covered with foil and incubated at 37°C with shaking at 800 rpm for 2 hours. The reaction was purified by adding a 3.7-fold excess of SPRI bead mix; incubating the mixture at room temperature on a Hula mixer with shaking for 5 minutes; pelleting the beads and washing them twice with SPRI wash buffer before elution in 25 mM HEPES-NaOH (pH 7.5), 50 mM NaCl. The sample was heated to 95°C for 3 minutes and then rapidly cooled on ice for at least 10 minutes before further processing.

[0520] A second click reaction was then assembled consisting of 1 µM peptide-DNA conjugate (the product of the first click reaction) and 4 µM BCN-modified DNA strand (DNA2) with a 3.7-fold excess of SPRI wash buffer. The reaction was incubated at 37°C with shaking at 1500 rpm for 2 hours. The reaction was purified as in the first click reaction to produce a polynucleotide-polypeptide conjugate. The assembly was analyzed by denaturing and native PAGE. As a control, a conjugate was prepared by annealing two oligonucleotides, one of which contained a 3'-terminal biotin group. The sequences of all oligonucleotides used in this example are listed in Table 3.

[0521] Data collection

[0522] Electrical data were collected using a MinION flow cell (Oxford Nanopore Technologies plc) containing the modified Rhodococcus pore as described above.

[0523] Samples were prepared by incubating equimolar amounts of monovalent traptavidin with the polynucleotide-polypeptide conjugate in running buffer (25 mM HEPES-KOH (pH 8.0), 500 mM KCl). The flow cell was first flushed with at least 1 mL of running buffer. Running buffer alone or with each sample was sequentially added to the flow cell via the SpotON port, with the running buffer flushed between each sample addition. Data were acquired at a sampling rate of 5 kHz and an applied potential of 180 mV, with a 120 mV reverse potential applied at one-minute intervals to dislodge each conjugate from the nanopore. Data were analyzed by determining the normalized blockade level caused by the sample relative to the open pore level.

[0524] Results and Conclusions

[0525] Figure 11 Shown are raw data collected from a series of samples where the distance between monovalent traptavidin and the peptide was 11 bp, and the peptide sequence (read N to C) was GGSGDDSGSG (SEQ ID NO: 10), GGSGRRSGSG (SEQ ID NO: 11), or GGSGYYSGSG (SEQ ID NO: 12). Each example shows an open pore current of approximately 275 pA, followed by a unique blockade level for the peptide when the analyte was captured in the nanopore. This data demonstrates that the nanopore can distinguish between the three peptides tested.

[0526] Figure 12Shown are histograms of normalized blocking levels for a range of analytes where the distance between monovalent traptavidins was 11 bp and the peptide sequence was GGSGDDSGSG (SEQ ID NO: 10), GGSGRRSGSG (SEQ ID NO: 11), or GGSGYYSGSG (SEQ ID NO: 12).

[0527] Figure 13 Aggregated data for distances from 6 bp to 36 bp in 5 bp increments are shown, expressed as a plot of normalized current (I / I0) versus the distance between monovalent traptavidin and the peptide. The data demonstrate that 11 bp is the most sensitive position for peptide discrimination using this nanopore. At peptides 21 bp or more away from monovalent traptavidin, blockade levels are indistinguishable from dsDNA alone. The data demonstrate that stepwise movement of polynucleotide-polypeptide conjugates through the nanopore can generate unique current-distance traces dependent on the peptide being analyzed.

[0528]

[0529]

[0530] Table 3: Oligonucleotides used in Example 1. 3 represents amino C3 labeled with TCO. 2 represents 5' amino C3 labeled with BCN. 1 represents 3' biotin modification. "+" before the base represents LNA.

[0531] Example 2

[0532] This example demonstrates the repetitive movement of a polynucleotide-polypeptide conjugate through a nanopore with an inner diameter wide enough to accommodate double-stranded DNA (dsDNA). The peptide was conjugated between two dsDNA oligonucleotides, with single-stranded DNA (ssDNA) serving as the "carrier" strand.

[0533] Construct assembly

[0534] The oligonucleotide sequences used are summarized in Table 4. Three different constructs were generated based on the polypeptide sequences used. XX The SGSG-C peptide, in which the middle two amino acid residues (XX) were varied: DD, RR, or YY, was modified with an N-terminal azide and a C-terminal methyltetrazine connected via an ethylenediamine linker. The construct used in this example was assembled according to the method described in Example 1. The construct differed from the construct used in Example 1 in that it carried a 3' poly(dA) tail instead of a terminal biotin, which facilitated loading of the helicase from solution.

[0535] Data collection

[0536] Electrical data were collected using a MinION flow cell (Oxford Nanopore Technologies) containing a modified Rhodococcus pore, as used in Example 1. Samples were prepared by incubating 0.2 µM of polynucleotide-polypeptide conjugate with 1 µM wild-type Hel308 helicase in either running 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 running buffer lacked ATP and served as a control for ATP-dependent enzymatic mobilization. The flow cell was first flushed with at least 1 mL of running buffer or sequencing buffer. Sample (75 µL) was then introduced into the flow cell via the SpotON port. Electrical data were acquired at a sampling rate of 5 kHz and an applied potential of 180 mV, with a reverse potential of 120 mV applied at one minute intervals to dislodge each conjugate from the nanopore.

[0537] Results and discussion

[0538] Figure 14 An example trace of enzymatically driven movement of a polypeptide-polynucleotide conjugate using a helicase is shown. The experiment was performed under conditions where there was an excess of helicase relative to the conjugate. The presence of repeated traces indicates that the conjugate is trapped by the hairpin end. The bound helicase prevents the conjugate from completely translocating, and the helicase then moves 3'-5' along the ssDNA to control the movement of the dsDNA out of the nanopore. When 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 quickly falls back to the dissociated second helicase bound to the 3'-distal end of the enzyme, so that the enzyme repeatedly shuttles the conjugate through the nanopore. The movement is ATP-dependent, as shown in the control lacking ATP ( Figure 15 ), the control showed only blockade and no repetitive pattern. The data show that helicases can be used to repeatedly move polypeptide-polynucleotide conjugates through nanopores.

[0539] Table 4: Oligonucleotides used in Example 2. 3 represents amino C3 labeled with TCO. 2 represents 5' amino C3 labeled with BCN. "+" before a base represents LNA.

[0540]

[0541] Example 3

[0542] This example demonstrates the repeated movement of a polynucleotide-polypeptide conjugate through a nanopore. The conjugate moves in the 'out' direction of the nanopore by the Hel308 helicase. The peptide was conjugated between two dsDNA oligonucleotides.

[0543] The conjugate contains a single-stranded overhang onto which a helicase is loaded from solution. The bound helicase prevents the conjugate from completely translocating through the nanopore; the helicase moves along the single strand of DNA in the direction 3' to 5' and controls the movement of the dsDNA and peptide in the direction 'outside' of the nanopore. When the helicase approaches the junction between the DNA and the peptide in the construct, it dissociates from the single strand of DNA, and the position of the conjugate in the nanopore quickly falls back to a second helicase loaded from solution onto the construct at the 3'-distal end of the first helicase. The cycle is then repeated, with the second helicase again controlling the movement of the double-stranded portion of the construct in the direction 'outside' of the nanopore. This mechanism can then be repeated with one or more additional helicases, allowing the conjugate to repeatedly move through the nanopore.

[0544] Construct assembly

[0545] The oligonucleotide sequences used are summarized in Table 5. Multiple constructs were generated by combining different peptide sequences. Peptides were synthesized with terminal modifications: an N-terminal azide and a C-terminal methyltetrazine attached via an ethylenediamine linker. The constructs contained a 3' poly(dA) tail (which facilitated loading of the helicase from solution) and a hairpin (containing the tethering oligonucleotide); both were attached to the ends of the constructs. Figure 16 A schematic of the assembled construct is shown in Figure 2 . The construct used in this example was assembled according to the general method described in Example 1. A DNA oligonucleotide containing a terminal BCN click group and a negatively charged C3 spacer (a; SEQ ID NO: 29) was first annealed to a tether sequence (b; SEQ ID NO: 30). This tethering increased the rate of analyte capture on the flow cell. The annealed oligonucleotide was then clicked to a hairpin DNA (c; SEQ ID NO: 31) carrying an internal azide click group. The hairpin sequence contains four base sticky ends to which the DNA 1 (d; SEQ ID NO: 32) and DNA 2 (e; SEQ ID NO: 33) oligonucleotides were attached. The DNA 2 oligonucleotide has a terminal 3' TCO group, which reacts with the tetrazine at the C-terminus of the peptide (f). The peptide carries an azide group at its N-terminus, which is then clicked to DNA 3 (g; SEQ ID NO: 34) via a terminal 5' BCN group.

[0546]

[0547] Table 5: Oligonucleotides used in Example 3; Figure 16 The corresponding marks ag are shown in .

[0548] Data collection

[0549] Electrical data were collected using a MinION flow cell (Oxford Nanopore Technologies) containing a modified Rhodococcus pore, as used in Example 1. Samples were prepared by incubating 0.2 µM of polynucleotide-polypeptide conjugate with 1 µM wild-type Hel308 helicase in either running 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 running buffer lacked ATP and served as a control for ATP-dependent enzymatic mobilization. The flow cell was first flushed with at least 1 mL of running buffer or sequencing buffer. Samples (75 µL) were then introduced into the flow cell via the SpotON port. Electrical data were acquired at a sampling rate of 3 kHz and an applied potential of 160 mV.

[0550] Results and discussion

[0551] Figure 17 Figures AF show example current-time traces of enzymatically controlled movement of a polypeptide-polynucleotide conjugate using the 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). Both repeated and single read traces are shown for each peptide. The experiment was performed under conditions in which there was an excess of helicase relative to the conjugate. The presence of repeated traces demonstrates that the conjugate was captured and read multiple times via the hairpin end. These traces demonstrate that peptides of different sequences produce unique signals.

[0552] Figure 18 AC shows example current-time traces for peptides with varying charges ranging from eight negatively charged residues to eight positively charged residues. The following peptides were tested:

[0553] A. SRRRRRRRRS (charge: +8) (SEQ ID NO: 38)

[0554] B. SEEEEEEEES (charge: –8) (SEQ ID NO: 39)

[0555] C. HDSGYEVHHQK* (charge: –2). (SEQ ID NO: 40)

[0556] *: Indicates that the peptide is linked via the C-terminal lysine R group rather than the C-terminal carboxyl group.

[0557] Figure 18 The current-time traces shown in AC demonstrate that peptides with different charges can be moved through the nanopore. Most notably, a peptide with a large positive charge (exemplified by peptide A with a charge of +8) can move through the pore against electrophoretic forces.

[0558] Example 4

[0559] This example demonstrates a 'single-pass' movement of a polynucleotide-polypeptide conjugate through a nanopore. The conjugate is moved in the direction 'entering' the pore by the Dda helicase. The peptide is conjugated between two dsDNA oligonucleotides. The conjugate comprises a hairpin adaptor loaded with the Dda helicase, wherein the helicase is topologically closed around the single-stranded polynucleotide before docking. The presence of the bound helicase initially prevents the conjugate from completely translocating through the nanopore. Once the analyte enters the pore, the helicase is arrested by electrophoretic forces and moves along the single-stranded DNA in the direction 5' to 3' to control the movement of the dsDNA and peptide in the direction 'entering' the nanopore. The helicase does not directly encounter the peptide located on the complementary DNA "carrier" strand and moves through the pore together with the strand on which the helicase translocates. When the helicase reaches the end of the single-stranded polynucleotide on which it is moving, it falls off the construct, thereby releasing the remainder of the conjugate through the pore and terminating the reading.

[0560] Construct assembly

[0561] The oligonucleotide sequences used are summarized in Table 6. The constructs comprised an enzyme-loading hairpin adaptor with a tethering site. Figure 19A schematic of the assembled construct is shown in Figure 2 . The construct used in this example was assembled according to the general methods described in Example 1. A DNA oligonucleotide containing a terminal BCN click group and a negatively charged C3 spacer (a; SEQ ID NO: 29) was first annealed to a tether sequence (b; SEQ ID NO: 30). This tethering increased the rate of analyte capture on the flow cell. The annealed oligonucleotide was then clicked to a hairpin DNA carrying an internal azide click group (c; SEQ ID NO: 41). The hairpin contains the Dda helicase loaded onto a single-stranded portion and held in place by disulfide bridges that close the enzyme around the strand topology. The loading site is opposite a 35-nucleotide 2'-O-methyl RNA bubble to provide loading space for the enzyme. The use of RNA bases prevents the helicase from binding to the bubble instead of the intended loading site. The hairpin adaptor contains four base sticky ends to which the DNA 1 (d; SEQ ID NO: 42) and DNA 2 (e; SEQ ID NO: 43) oligonucleotides are attached. The DNA 2 oligonucleotide has a terminal 3' TCO group, which reacts with the tetrazine at the C-terminus of the peptide (f; SEQ ID NO: 44). The peptide carries an azide group at its N-terminus, which is then attached to DNA 3 (g; SEQ ID NO: 45) via a terminal 5' BCN group.

[0562]

[0563] Table 6: Oligonucleotides used in Example 4; Figure 19 The corresponding marks ag are shown in .

[0564] *: Indicates that the peptide is linked via the C-terminal lysine R group rather than the C-terminal carboxyl group.

[0565] Data collection

[0566] Electrical data were collected using a MinION flow cell (Oxford Nanopore Technologies) containing a modified Rhodococcus pore, as used in Example 1. Samples were prepared by mixing 0.2 µM of a 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). The flow cell was first rinsed with at least 1 mL of sequencing buffer. The sample (75 µL) was then introduced into the flow cell via the SpotON port. Electrical data were acquired at a sampling rate of 1 kHz and an applied potential of 180 mV. The recording temperature was 21°C.

[0567] Results and discussion

[0568] Figure 20 Shown are example traces of enzymatically controlled migration of a polypeptide-polynucleotide conjugate using Dda helicase in single-pass mode. The presence of reproducible signal deviation from the dsDNA level (three examples shown) demonstrates that the peptide (HDSGDEVHHQK; SEQ ID NO: 46) on the "carrier" strand co-translocates through the pore. Placing the peptide on the "carrier" strand can impose fewer restrictions on the length of the peptide that can be analyzed, as the enzyme does not have to diffuse on the peptide to move the conjugate through the pore.

[0569] Example 5

[0570] This example demonstrates a 'single pass' movement of a polynucleotide-polypeptide conjugate through a nanopore. Movement of the conjugate in the 'entry' direction of the nanopore is controlled by the Dda helicase. The peptide was conjugated between two double-stranded (dsDNA) oligonucleotides containing internal rather than terminal click groups, resulting in a single-stranded DNA stretch next to the peptide (see Figure 6 A sequencing adapter loaded with helicase is ligated to the ends of the construct. The helicase is topologically enclosed within the single-stranded DNA portion surrounding the oligonucleotide before stalling. The presence of the bound helicase initially prevents the conjugate from fully translocating through the nanopore. Once the conjugate enters the nanopore, the helicase is unstalled by electrophoretic forces and then moves along the single-stranded DNA in the 5' to 3' direction. As the conjugate moves through the nanopore, the annealed complementary strands are 'unzipped' (separated), allowing only the DNA strand with the attached peptide to move through the constriction. When the helicase encounters the covalently linked DNA-peptide segment, it diffuses over the two strands and reattaches to the DNA on the other side of the DNA-peptide segment. It then continues to control the movement of the DNA-peptide, followed by the single-stranded DNA, through the nanopore in the 5' to 3' direction. When the enzyme reaches the end of the bound DNA oligonucleotide, it detaches, releasing the remainder of the conjugate through the nanopore and terminating the read. With this system, the nanopore only needs to have a diameter wide enough to accommodate the co-translocating DNA-peptide (the cross-section of which can be narrower than that of the double-stranded DNA).

[0571] Construct assembly

[0572] The oligonucleotide sequences used are summarized in Table 7. The constructs contained an adaptor with an enzyme loading site attached to the ends of the construct, and the peptide spanning DNA consisted of segments from two oligonucleotides that could optionally be ligated together. Figure 21A schematic of the assembled construct is shown in Figure 2 . The construct used in this example was assembled according to the general method described in Example 1. Two DNA oligonucleotides were annealed (a, b; SEQ ID NOs: 47, 48), one of which contained a 3' TCO click group (b). The annealed oligonucleotides were then reacted with a peptide bearing a C-terminal tetrazine click group (c; SEQ ID NO: 49). The peptide contained an azide group at its N-terminus, which was then clicked to another set of annealed oligonucleotides (d, e; SEQ ID NOs: 50-51), one of which contained a 5' BCN click group (e). The polynucleotide-polypeptide conjugate was ligated to a standard sequencing adapter (f), which is loaded with a topologically closed helicase (using a disulfide closure) and contains a negatively charged leader sequence and a tether sequence. The tethering increases the rate of analyte capture on the flow cell.

[0573]

[0574] Table 7: Oligonucleotides used in Example 5; Figure 21 Also shown are the oligonucleotides used in an alternative construct, in which DNA oligonucleotide (b) and DNA oligonucleotide (e) are replaced by a single oligonucleotide spanning the peptide (alternative strand: b + e; SEQ ID NO: 52).

[0575] *: Indicates that the peptide is linked via the C-terminal lysine R group rather than the C-terminal carboxyl group.

[0576] Data collection

[0577] Electrical data were collected using a MinION flow cell (Oxford Nanopore Technologies) containing a modified Rhodococcus pore. Samples were prepared by mixing 0.2 µM of a polynucleotide-peptide 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). The flow cell was first rinsed with at least 1 mL of sequencing buffer. The sample (75 µL) was then introduced into the flow cell via the SpotON port. Electrical data were acquired at a sampling rate of 1 kHz and an applied potential of 200 mV. The recording temperature was 21°C.

[0578] Results and discussion

[0579] Figure 22A shows an example current-time trace of Dda helicase-controlled movement of a polypeptide-polynucleotide conjugate, including DNA-peptide cotranslocation. The peptide (HDSGDEVHHQK) is covalently linked to the DNA at its C- and N-termini, and thus both the DNA and peptide cotranslocate together through the nanopore to produce the observed signal; see Figure 22 A1, 22A2.

[0580] Control experiments were performed in which a construct in which a peptide bridging the gap between two flanking DNA oligonucleotides (as described in WO2021 / 111125) was used to translocate a peptide of the same sequence (HDSGDEVHHQK) alone, i.e., not co-translocated with the DNA strand. Figure 22 The current-time traces for the peptide segments obtained in this control experiment are shown in B1(iii) and 22B2(iii). Figure 22 The current-time traces shown in A1(iii) and 22A2(iii) are different, confirming that Figure 22 Co-translocation of DNA and peptide is shown in A1 and 22A2, and it is demonstrated that the presence of negative charges in the co-translocated DNA can modulate the peptide signal.

[0581] Example 6

[0582] This example demonstrates the 'single-pass' movement of a polynucleotide-polypeptide conjugate through a nanopore. The movement of the conjugate in the 'entry' direction of the nanopore is controlled by the Dda helicase. A peptide was conjugated between two double-stranded (dsDNA) oligonucleotides, one of which contained an internal rather than terminal click group, creating a 'flap' of single-stranded DNA next to the peptide. Standard sequencing adapters loaded with the helicase were ligated to the ends of the construct. The helicase was topologically enclosed within the ssDNA portion surrounding the oligonucleotides before stalling. The presence of the bound helicase initially prevented the conjugate from fully translocating through the nanopore. Once the conjugate entered the nanopore, the helicase was unstalled by electrophoretic forces and then moved 5'-3' along the ssDNA. As the conjugate moved through the nanopore, the annealed complementary strands were 'unzipped' (separated), allowing only the DNA strand with the attached peptide to move through the constriction. When the helicase encountered the covalently linked DNA-peptide segment, it diffused across both the peptide and the DNA 'flap', rejoining the DNA on the other side of the DNA-peptide segment. It then proceeds to control the movement of the DNA-peptide, followed by the movement of the single-stranded DNA in the 5' to 3' direction through the nanopore. When the enzyme reaches the end of the bound DNA oligonucleotide, the enzyme falls off, thereby releasing the remainder of the conjugate through the nanopore and terminating the reading. As in Example 5 above, with this system, the nanopore only needs to have a diameter wide enough to accommodate the co-translocated DNA-peptide.

[0583] Construct assembly

[0584] The oligonucleotide sequences used are summarized in Table 8. The construct contained an adaptor with a tethering site for loading the enzyme linked to one end of the construct, and a DNA 'flap' spanning the peptide consisted of 6 bases linked to the C-terminus of the peptide. Figure 23 A schematic of the assembled construct is shown in Figure 2 . The construct used in this example was assembled according to the general method described in Example 1. Two DNA oligonucleotides were annealed (a, b; SEQ ID NOs: 53, 54), one of which contained an internal 3' TCO click group (b). The annealed oligonucleotides were then reacted with a peptide bearing a C-terminal tetrazine click group (c; SEQ ID NO: 49). The peptide contained an azide group at its N-terminus, which was then clicked to another set of annealed oligonucleotides (d, e; SEQ ID NOs: 55, 56), one of which contained a terminal 5' BCN click group (e). The polynucleotide-polypeptide conjugate was then ligated with a standard sequencing adapter (f), which is loaded with a topologically closed helicase (using a disulfide closure) and contains a negatively charged leader sequence and a tether sequence. The tethering increases the rate of analyte capture on the flow cell.

[0585]

[0586] Table 8: Oligonucleotides used in Example 6; Figure 23 The corresponding marks af are shown in .

[0587] *: Indicates that the peptide is linked via the C-terminal lysine R group rather than the C-terminal carboxyl group.

[0588] Data collection

[0589] Electrical data were collected using a MinION flow cell (Oxford Nanopore Technologies) containing a modified Rhodococcus pore. Samples were prepared by mixing 0.2 µM of a polynucleotide-peptide 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). The flow cell was first rinsed with at least 1 mL of sequencing buffer. The sample (75 µL) was then introduced into the flow cell via the SpotON port. Electrical data were acquired at a sampling rate of 1 kHz and an applied potential of 200 mV. The recording temperature was 21°C.

[0590] Results and discussion

[0591] Figure 24Current-time traces of Dda helicase-controlled movement of a polypeptide-polynucleotide conjugate, including co-translocation of the DNA flap and the peptide, are shown. The peptide (HDSGDEVHHQK) is covalently linked 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 co-translocate through the nanopore to produce the observed signal. The peptide signal generated by this protocol is distinct from the peptide signal generated by a control experiment of peptide translocation alone (using a corresponding construct containing the same peptide sequence, HDSGDEVHHQK, but lacking the DNA flap), as described in Example 5 above and Figure 22 As shown in B.

[0592] Description of Sequence Listing

[0593] SEQ ID NO: 1 shows the amino acid sequence of (hexahistidine-tagged) exonuclease I (EcoExo I) from Escherichia coli.

[0594] SEQ ID NO: 2 shows the amino acid sequence of the exonuclease III enzyme from Escherichia coli.

[0595] SEQ ID NO: 3 shows the amino acid sequence of the RecJ enzyme from Thermus thermophilus (TthRecJ-cd).

[0596] SEQ ID NO: 4 shows the amino acid sequence of bacteriophage lambda exonuclease, which represents one of three identical subunits that assemble into a trimer (http: / / www.neb.com / nebecomm / products / productM0262.asp).

[0597] SEQ ID NO: 5 shows the amino acid sequence of Phi29 DNA polymerase from Bacillus subtilis.

[0598] SEQ ID NO: 6 shows the amino acid sequence of the Trwc Cba (Citromicrobium bathyomarinum) helicase.

[0599] SEQ ID NO: 7 shows the amino acid sequence of the Hel308 Mbu (Methanococcoides burtonii) helicase.

[0600] SEQ ID NO: 8 shows the amino acid sequence of Dda helicase 1993 from enterobacteriaceae phage T4.

[0601] SEQ ID NO: 9 shows the amino acid sequence of a monomer of the transmembrane protein nanopore derived from Rhodococcus as described in Example 1.

[0602] SEQ ID NOs: 10-12 show the polypeptide sequences used in Example 1 (read N to C).

[0603] SEQ ID NOs: 13-26 show the polynucleotide sequences used in Example 1. 3 represents amino C3 labeled with TCO. 2 represents 5' amino C3 labeled with BCN. 1 represents 3' biotin modification. A "+" before a base represents LNA.

[0604] SEQ ID NOs: 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. A “+” before a base represents LNA.

[0605] SEQ ID NOs: 29-56 show the polynucleotide and polypeptide sequences used in Examples 3 to 6.

[0606] Sequence Listing

[0607] SEQ ID NO: 1 - Exonuclease I from Escherichia coli

[0608] MMNDGKQQSTFLFHDYETFGTHPALDRPAQFAAIRTDSEFNVIGEPEVFYCKPADDYLPQPGAVLITGITPQEARAKGENEAAFAARIHSLFTVPKTCILGYNNVRFDDEVTRNIFYRNFYDPYAWSWQHDNSRWDLLDVMRACYALRPEGINWPENDDGLPSFRLEHLTKANGIEHSNAHDAMADVYATIAMAKLVKTRQPRLFDYLFTHRNKHKLMALIDVPQMKPLVHVSGMFGAWRGNTSWVAPLAWHPENRNAVIMVDLAGDISPLLELDSDTLRERLYTAKTDLGDNAAVPVKLVHINKCPVLAQANTLRPEDADRLGINRQHCLDNLKILRENPQVREKVVAIFAEAEPFTPSDNVDAQLYNGFFSDADRAAMKIVLETEPRNLPALDITFVDKRIEKLLFNYRARNFPGTLDYAEQQRWLEHRRQVFTPEFLQGYADELQMLVQQYADDKEKVALLKALWQYAEEIVSGSGHHHHHH

[0609] SEQ ID NO: 2 - Exonuclease III enzyme from Escherichia coli

[0610] MKFVSFNINGLRARPHQLEAIVEKHQPDVIGLQETKVHDDMFPLEEVAKLGYNVFYHGQKGHYGVALLTKETPIAVRRGFPGDDEEAQRRIIMAEIPSLLGNVTVINGYFPQGESRDHPIKFPAKAQFYQNLQNYLETELKRDNPVLIMGDMNISPTDLDIGIGEENRKRWLRTGKCSFLPEEREWMDRLMSWGLVDTFRHANPQTADRFSWFDYRSKGFDDNRGLRIDLLLASQPLAECCVETGIDYEIRSMEKPSDHAPVWATFRR

[0611] SEQ ID NO: 3 - RecJ enzyme from Thermus thermophilus

[0612] MFRRKEDLDPPLALLPLKGLREAAALLEEALRQGKRIRVHGDYDADGLTGTAILVRGLAALGADVHPFIPHRLEEGYGVLMERVPEHLEASDLFLTVDCGITNHAELRELLENGVEVIVTDHHTPGKTPPPGLVVHPALTPDLKEKPTGAGVAFLLLWALHERLGLPPPLEYADLAAVGTIADVAPLWGWNRALVKEGLARIPASSWVGLRLLAEAVGYTGKAVEVAFRIAPRINAASRLGEAEKALRLLLTDDAAEAQALVGELHRLNARRQTLEEAMLRKLLPQADPEAKAIVLLDPEGHPGVMGIVASRILEATLRPVFLVAQGKGTVRSLAPISAVEALRSAEDLLLRYGGHKEAAGFAMDEALFPAFKARVEAYAARFPDPVREVALLDLLPEPGLLPQVFRELALLEPYGEGNPEPLFL

[0613] SEQ ID NO: 4 - Lambda phage exonuclease

[0614] MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPRSGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESPIIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMWVTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFGEQWR

[0615] SEQ ID NO: 5 - Phi29 DNA polymerase

[0616] MKHMPRKMYSCAFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKSGGSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK

[0617] SEQ ID NO: 6 - Trwc Cba Helicase

[0618] MLSVANVRSPSAAASYFASDNYYASADADRSGQWIGDGAKRLGLEGKVEARAFDALLRGELPDGSSVGNPGQAHRPGTDLTFSVPKSWSLLALVGKDERIIAAYREAVVEALHWAEKNAAETRVVEKGMVVTQATGNLAIGLFQHDTNRNQEPNLHFHAVIANVTQGKDGWRTLKNDRLWQLNTTLNSIAMARFRVAVEKLGYEPGPVLKHGNFEARGISREQVMAFSTRRKEVLEARRGPGGLDAGRIAALDTRASKEGIEDRATLSKQWSEAAQSIGLDLKPLVDRARTKALGQGMEATRIGSLVERGRAWLSRFAAHVRGDPADPLVPPSVLKQDRQTIAAAQAVASAVRHLSQREAAFERTALYKAALDFGLPTTIADVEKRTRALVRSGDLIAGKGEHKGWLASRDAVVTEQRILSEVAAGKGDSSPAITPQKAAASVQAAALTGQGFRLNEGQLAAARLILISKDRTIAVQGIAGKS SVLKPVAEVLRDEGHPVIGLAIQNTLVQMLERDTGIGSQTLARFLGGWNKLLDDPGNVALRAEAQASLKDHVLVLDEASMVSNEDKEKLVRLANLAGVHRLVLIGDRKQLGAVDAGKPFALLQRAGIARAEMATNLRARDPVVREAQAAAQAGDVRKARLHLKSHTVEARGDGAQVAAETWLALDKETRARTSIYASGRAIRSAVNAAVQQGLLASREIGPAKMKLEVLDRVNTTREELRHLPAYRAGRVLEVSRKQQALGLFIGEYRVIGQDRKGKLVEVEDKRGKRFRFDPARIRAGKGDDNLTLLEPRKLEIHEGDRIRWTRNDHRRGLFNADQARVVEIANGKVTFETSKGDLVELKKDDPMLKRIDLAYALNVHMAQGLTSDRGIAVMDSRERNLSNQKTFLVTVTRLRDHLTLVVDSADKLGAAVARNKGEKASAIEVTGSVKPTATKGSGVDQPKSVEANKAEKELTRSKSKTLDFGI

[0619] SEQ ID NO: 7 - Hel308 Mbu Helicase

[0620] MMIRELDIPRDIIGFYEDSGIKELYPPQAEAIEMGLLEKKNLLAAIPTASGKTLLAELAMIKAIREGGKALYIVPLRALASEKFERFKELAPFGIKVGISTGDLDSRADWLGVNDIIVATSEKTDSLLRNGTSWMDEITTVVVDEIHLLDSKNRGPTLEVTITKLMRLNPDVQVVALSATVGNAREMADWLGAALVLSEWRPTDLHEGVLFGDAINFPGSQKKIDRLEKDDAVNLVLDTIKAEGQCLVFESSRRNCAGFAKTASSKVAKILDNDIMIKLAGIAEEVESTGETDTAIVLANCIRKGVAFHHAGLNSNHRKLVENGFRQNLIKVISSTPTLAAGLNLPARRVIIRSYRRFDSNFGMQPIPVLEYKQMAGRAGRPHLDPYGESVLLAKTYDEFAQLMENYVEADAEDIWSKLGTENALRTHVLSTIVNGFASTRQELFDFFGATFFAYQQDKWMLEEVINDCLEFLIDKAMVSETEDIEDASKLFLRGTRLGSLVSMLYIDPLSGSKIVDGFKDIGKSTGGNMGSLEDDKGDDITVTDMTLLHLVCSTPDMRQLYLRNTDYTIVNEYIVAHSDEFHEIPDKLKETDYEWFMGEVKTAMLLEEWVTEVSAEDITRHFNVGEGDIHALADTSEWLMHAAAKLAELLGVEYSSHAYSLEKRIRYGSGLDLMELVGIRGVGRVRARKLYNAGFVSVAKLKGADISVLSKLVGPKVAYNILSGIGVRVNDKHFNSAPISSNTLDTLLDKNQKTFNDFQ

[0621] SEQ ID NO: 8 - Dda Helicase

[0622] MTFDDLTEGQKNAFNIVMKAIKEKKHHVTINGPAGTGKTTLTKFIIEALISTGETGIILAAPTHAAKKILSKLSGKEASTIHSILKINPVTYEENVLFEQKEVPDLAKCRVLICDEVSMYDRKLFKILLSTIPPWCTIIGIGDNKQIRPVDPGENTAYISPFFTHKDFYQCELTEVKRSNAPIIDVATDVRNGKWIYDKVVDGHGVRGFTGDTALRDFMVNYFSIVKSLDDLFENRVMAFTNKSVDKLNSIIRKKIFETDKDFIVGEIIVMQEPLFKTYKIDGKPVSEIIFNNGQLVRIIEAEYTSTFVKARGVPGEYLIRHWDLTVETYGDDEYYREKIKIISSDEELYKFNLFLGKTAETYKNWNKGGKAPWSDFWDAKSQFSKVKALPASTFHKAQGMSVDRAFIYTPCIHYADVELAQQLLYVGVTRGRYDVFYV

[0623] SEQ ID NO: 9

[0624] MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPGLNLSVGNGVAATVTNVLPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0625] SEQ ID NO: 10

[0626] GGSGDDSGSG

[0627] SEQ ID NO: 11

[0628] GGSGRRSGSG

[0629] SEQ ID NO: 12

[0630] GGSGYYSGSG

[0631] SEQ ID NO: 13

[0632] +G+T+C+C+A+CTTTTTTGCTGCGGCTTAGCTGTGATAGCTTTTGCTATCACAGCTAAGCCGCAGC3

[0633] SEQ ID NO: 14

[0634] GTCCATTATTCTTTTTTGCTGCGGCTTAGCTGTGATAGCTTTTGCTATCACAGCTAAGCCGCAGC3

[0635] SEQ ID NO: 15

[0636] GTATTGTCCATTATTCTTTTTTGCTGCGGCTTAGCTGTGATAGCTTTTGCTATCACAGCTAAGCCGCAGC3

[0637] SEQ ID NO: 16

[0638] GTATTGTATTGTCCATTATTCTTTTTTGCTGCGGCTTAGCTGTGATAGCTTTTGCTATCACAGCTAAGCCGCAGC3

[0639] SEQ ID NO: 17

[0640] GTATTGTATTGTATTGTCCATTATTCTTTTTTGCTGCGGCTTAGCTGTGATAGCTTTTGCTATCACAGCTAAGCCGCAGC3

[0641] SEQ ID NO: 18

[0642] GTATTGTATTGTATTGTATTGTCCATTATTCTTTTTTGCTGCGGCTTAGCTGTGATAGCTTTTGCTATCACAGCTAAGCCGCAGC3

[0643] SEQ ID NO: 19

[0644] GTATTGTATTGTATTGTATTGTATTGTCCATTATTCTTTTTTGCTGCGGCTTAGCTGTGATAGCTTTTGCTATCACAGCTAAGCCGCAGC3

[0645] SEQ ID NO: 20

[0646] 2GTGGAC1

[0647] SEQ ID NO: 21

[0648] 2GAATATGGAC1

[0649] SEQ ID NO: 22

[0650] 2QUESTGQUEST1

[0651] SEQ ID NO: 23

[0652] 2COMPETITIONGCOMPATRATIONAC1

[0653] SEQ ID NO: 24

[0654] 2RATEGQRATCHRAP1

[0655] SEQ ID NO: 25

[0656] 2COMMUNICATIONSGQCHAPTERCHAPTERCHAPTER1

[0657] SEQ ID NO: 26

[0658] 2COUNTSGCHAPTERCHAPTERCHAPTERCHAPTER1

[0659] SEQ ID NO: 27

[0660] +G+T+C+C+ATTATTCTTTTTGCTGCGGCTTAGCTGTGATAGCTTTTGCTATCACAGCTAAGCCGCAGC3

[0661] SEQ ID NO: 28

[0662] 2GAATATGGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAHAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA.

[0663] SEQ ID NO: 29

[0664] / 5SpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / / iSpC3 / GTTATTCAAGACTTCTTTAATACACTTT / BCN /

[0665] SEQ ID NO: 30

[0666] GTCCATTATTCTTTTTTGCTGCGGCTTAGCTGTGATAGCAGGA

[0667] SEQ ID NO: 31

[0668] / 5Phos / CGCAATACGTAACTGAACGAAGTACT / iBCN / TGTACTTCGTTCAGTTACGTATTGCGTCCT

[0669] SEQ ID NO: 32

[0670] / 5Phos / GTGTATTAAAGAAGTCTTGAATAACTTTGAGGCGAGCGGTCAA

[0671] SEQ ID NO: 33

[0672] / 5Phos / GCTATCACAGCTAAGCCGCAGC / TCO /

[0673] SEQ ID NO: 34

[0674] / BCN / GAATAATGGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0675] SEQ ID NO: 35

[0676] RSDSGQQARY

[0677] SEQ ID NO: 36

[0678] GGSGSSSGSG

[0679] SEQ ID NO: 37

[0680] EAIYAAPFAKKK

[0681] SEQ ID NO: 38

[0682] SRRRRRRRRS

[0683] SEQ ID NO: 39

[0684] SEEEEEEEES

[0685] SEQ ID NO: 40

[0686] HDSGYEVHHQK

[0687] SEQ ID NO: 41

[0688] / 5Phos / GCAATACGTAACTGAACGAAGT / iBNA-T / / iBNA-G / / iBNA-T / / iBNA-G / / iBNA-G / mUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUmUGGTTAAACACCCAAGCAGACGCCT7TGGCGTCTGCTTGGGTGTTTAACCTTTTTTTTTT8CCACAACTTCGTTCAGTTACGTATTGCTCCT

[0689] SEQ ID NO: 42

[0690] CTCAAGTGCCTGGTATATTACATCCACAGTGAAGACCTGGACACTGGACGTCCATTATTC / TCO /

[0691] SEQ ID NO: 43

[0692] / 5Phos / GCTATCACAGGCAATAAGAATAACGTCATAATGCGTAACTGACTAAGCCGCAGCTTTTTTGAATAATGGACGTCCAGTGTCCAGGTCTTCACTGTGGATGTAATATACCAGGCACTTGAG

[0693] SEQ ID NO: 44

[0694] Peptide with N-terminal azide and C-terminal tetrazine [HDSGDEVHHQK]

[0695] SEQ ID NO: 45

[0696] / BCN / GCTGCGGCTTAGTCAGTTACGCATTATGACGTTATTCTTATTGCCTGTGATAGCAGGA

[0697] SEQ ID NO: 46

[0698] HDSGDEVHHQK

[0699] SEQ ID NO: 47

[0700] AAAAAAAAAAAAAAAAAAAAAAAAAGGTTAAACACCCAAGCAGCAAT

[0701] SEQ ID NO: 48

[0702] GCTTGGGTGTTTAACCTTTTTTTTTTTTTTTTTTTTTTTTT / iC3-TCO / TTT

[0703] SEQ ID NO: 49

[0704] Azide-HDSGDEVHHQK-Tetrazine

[0705] SEQ ID NO: 50

[0706] CCCTGGACACTGGACAAAAAAAAAAAAAAAAAAAAAAAAA

[0707] SEQ ID NO: 51

[0708] / 5Phos / TTT / iC3-BCN / TTTTTTTTTTTTTTTTTTTTTTTTTGTCCAGTGTCCAGGG

[0709] SEQ ID NO: 52

[0710] CCCTGGACACTGGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGTTAAACACCCAAGCAGCAAT

[0711] SEQ ID NO: 53

[0712] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGTTAAACACCCAAGCAGCAAT

[0713] SEQ ID NO: 54

[0714] GCTTGGGTGTTTAACCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT / iC3-TCO / TTTTTT

[0715] SEQ ID NO: 55

[0716] CCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0717] SEQ ID NO: 56

[0718] / BCN / TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGG

Claims

1. A method for characterizing a target polypeptide, the method comprising - contacting (i) a polynucleotide-polypeptide conjugate strand with (ii) a polynucleotide carrier strand, thereby forming a polynucleotide-polypeptide construct, the polynucleotide-polypeptide conjugate strand comprising the target polypeptide conjugated to one or more polynucleotide flanking strands at each end of the target polypeptide; - contacting the construct with the nanopore under conditions such that both the polynucleotide-polypeptide conjugate strand and the polynucleotide carrier strand co-translocate through the nanopore; and - performing one or more measurements characteristic of the polypeptide as the construct moves relative to the nanopore, thereby characterizing the target polypeptide.

2. The method according to claim 1, wherein each of the one or more polynucleotide flanking strands is independently complementary to a region of the polynucleotide carrier strand.

3. The method according to claim 1 or claim 2, wherein each of the one or more polynucleotide flanking strands is independently at least partially hybridized to the polynucleotide carrier strand.

4. A method for characterizing a target polypeptide, the method comprising - contacting (i) a polynucleotide-polypeptide conjugate strand with (ii) a polynucleotide-processing protein, the polynucleotide-polypeptide conjugate strand comprising a target polypeptide linked to a polynucleotide flanking strand, the polynucleotide-processing protein being capable of controlling the movement of the polynucleotide flanking strand relative to the nanopore; and - contacting the polynucleotide-polypeptide conjugate strand with the nanopore under conditions such that the polynucleotide-processing protein controls the movement of the polynucleotide-polypeptide conjugate strand relative to the nanopore; and - performing 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.

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

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

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

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

9. The method according to any one of the preceding claims, which comprises mechanically manipulating the construct, the polynucleotide-polypeptide conjugate strand, and / or the polynucleotide carrier strand, thereby moving the construct, the polynucleotide-polypeptide conjugate strand, and / or the polynucleotide carrier strand relative to the nanopore.

10. The method according to claim 9, wherein the construct, the polynucleotide-polypeptide conjugate strand, and / or the polynucleotide carrier strand are moved by mechanical manipulation in a direction opposite 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 processing protein capable of controlling the movement of the one or more polynucleotide flanking strands and / or the polynucleotide carrier strand, and wherein the polynucleotide processing protein controls the movement of the construct relative to the nanopore.

13. The method according to claim 12, comprising contacting both the polynucleotide - polypeptide conjugate strand and the polynucleotide carrier strand of the construct with a polynucleotide processing protein capable of controlling the movement of the polynucleotide - polypeptide conjugate strand and / or the polynucleotide carrier strand, and wherein the polynucleotide processing protein controls the movement of the construct relative to the nanopore.

14. The method according to any one of claims 1 to 11, comprising contacting the polynucleotide - polypeptide conjugate strand with a polynucleotide processing protein capable of controlling the movement of the polynucleotide - polypeptide conjugate strand, and wherein the polynucleotide processing protein controls the movement of the construct relative to the nanopore.

15. The method according to any one of claims 1 to 3 or 5 to 11, comprising contacting the polynucleotide carrier strand with a polynucleotide processing protein capable of controlling the movement of the polynucleotide carrier strand, and wherein the polynucleotide processing protein controls the movement of the construct relative to the nanopore.

16. The method according to any one of claims 12 to 15, wherein: i) the polynucleotide processing protein is located on the cis side of the nanopore, and the polynucleotide processing protein controls the movement of the construct, the polynucleotide - polypeptide conjugate strand and / or 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 from the cis side of the nanopore to the trans side of the nanopore; or ii) the polynucleotide processing protein is located on the trans side of the nanopore, and the polynucleotide processing protein controls the movement of the construct, the polynucleotide - polypeptide conjugate strand and / or 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 from the trans side of the nanopore to the cis side of the nanopore.

17. The method according to claim 6, wherein the polynucleotide processing protein is located on the cis side of the nanopore, and the polynucleotide processing 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.

18. The method according to claim 16, wherein the polynucleotide processing protein is located on the trans side of the nanopore, and the polynucleotide processing 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.

19. The method according to any one of claims 12 to 15, wherein: i) the polynucleotide processing protein is located on the cis side of the nanopore, and the polynucleotide processing protein controls the movement of the construct, the polynucleotide-polypeptide conjugate strand, and / or 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 from the trans side of the nanopore to the cis side of the nanopore; or ii) the polynucleotide processing protein is located on the trans side of the nanopore, and the polynucleotide processing protein controls the movement of the construct, the polynucleotide-polypeptide conjugate strand, and / or 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 from the cis side of the nanopore to the trans side of the nanopore.

20. The method according to claim 19, wherein the polynucleotide processing protein is located on the cis side of the nanopore, and the polynucleotide processing 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.

21. The method according to claim 19, wherein the polynucleotide processing protein is located on the trans side of the nanopore, and the polynucleotide processing 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.

22. The method according to any one of claims 12 to 21, wherein before the construct contacts the nanopore, the polynucleotide processing protein binds to the polynucleotide carrier strand in the region spanned by the non-hybridized region of the polynucleotide flanking strand.

23. The method according to any one of claims 12 to 21, wherein when the portion of the polynucleotide-polypeptide conjugate strand that contacts the active site of the polynucleotide processing protein contains the target polypeptide, the polynucleotide processing protein is capable of remaining bound to the polynucleotide-polypeptide conjugate strand.

24. The method according to any one of claims 12 to 23, wherein the polynucleotide processing protein is modified to prevent the polynucleotide processing protein from dissociating from the construct, the polynucleotide-polypeptide conjugate strand, and / or the polynucleotide carrier strand when the polynucleotide processing protein contacts the target polypeptide.

25. The method according to any one of claims 12 to 24, wherein the polynucleotide-processing protein is modified to completely or partially close an opening that exists in at least one conformational state of the unmodified protein, through which the polynucleotide strand can dissociatively bind.

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

27. The method according to any one of the preceding claims, wherein the construct comprises a stalling portion, and before the target polypeptide translocates through the nanopore, the polynucleotide-processing protein is positioned such that the stalling portion is located between the polynucleotide-processing protein and the target polypeptide.

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

29. The method according to any one of the preceding claims, wherein the construct, the polynucleotide-polypeptide conjugate chain, and / or the polynucleotide carrier chain comprise a blocking portion linked by an optional linker, wherein the blocking portion is not capable of translocating through the nanopore.

30. The method according to any one of the preceding claims, wherein the method comprises: i) performing the method according to any one of the preceding claims such that the target polypeptide translocates the nanopore in a first direction relative to the nanopore; ii) allowing the construct, the polynucleotide-polypeptide conjugate chain, and / or the polynucleotide carrier chain to move 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 allowing the construct, the polynucleotide-polypeptide conjugate chain, and / or the polynucleotide carrier chain to move 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.

31. The method according to any one of the preceding claims, wherein the one or more measurements are specific to one or more properties 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.

32. The method according to any one of the preceding claims, wherein the nanopore is a protein nanopore, preferably a β-barrel protein nanopore.

33. A system comprising - a construct 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 nanopore capable of co-translocating the polynucleotide-polypeptide conjugate chain of the construct and the polynucleotide flanking chain; and - A polynucleotide processing protein.

34. A kit comprising: - A nanopore; - A first polynucleotide comprising a reactive functional group for conjugating to the first end of a target polypeptide; - A second polynucleotide comprising a reactive functional group for conjugating to the second end of the target polypeptide; and - A polynucleotide processing protein.

35. The system according to claim 33 or the kit according to claim 34, wherein the nanopore, the construct and / or the polynucleotide processing protein are as defined in any one of claims 2 to 32.

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