A method for characterizing target peptides using nanopores.

Abstract

The present invention provides a method for characterizing a target polypeptide as it moves toward a nanopore, as well as related kits, systems, and apparatus for carrying out such a method. [Solution] A method for characterizing a target polypeptide comprises: conjugating the target polypeptide to a polynucleotide to form a polynucleotide-polypeptide conjugate; contacting the conjugate with a polynucleotide handling protein capable of controlling the movement of the polynucleotide to a nanopore; and performing one or more measurements characteristic of the polypeptide as the conjugate moves toward the nanopore, thereby characterizing the polypeptide.

Description

[Technical Field]

[0001] This disclosure describes how to form a conjugate between a target polypeptide and a polynucleotide, and how to form a polynucleotide. Using creotide handling proteins to transfer conjugates to nanopores This disclosure relates to a method for characterizing a target polypeptide by control. This relates to kits, systems, and apparatus for performing such methods. [Background technology]

[0002] The characterization of biological molecules is becoming increasingly important in biomedical and bioengineering applications. For example, nucleic acid sequencing allows for the study of genomes and the proteins they encode. This makes it possible, for example, to link nucleic acid mutations with observable phenomena such as disease symptoms. It becomes possible. Nucleic acid sequencing is used in evolutionary biology to study relationships between organisms. Yes, it is possible. Metagenomics involves identifying organisms present in a sample, such as microorganisms in the microbiome. This includes identifying organisms, and nucleic acid sequencing makes such identification possible. Techniques for characterizing (e.g., sequencing) polynucleotides have been widely developed, Despite the significant bioengineering importance of peptide characterization techniques, There hasn't been much progress. For example, by knowing the protein sequence, it is possible to establish structure-activity relationships. This can lead to rational drug development strategies for developing ligands for specific receptors. It is becoming increasingly important. Identifying post-translational modifications is also key to understanding the functional characteristics of many proteins. For example, in eukaryotes, typically 30-50% of protein species are phosphorylated. Some proteins activate or inactivate other proteins, or break them down. Multiple proteins that help promote or modulate interactions with protein partners They may have oxidation sites. Therefore, they are characterized by proteins and other polypeptides. A method for attaching it is urgently needed.

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

[0004] Protein mass spectrometry characterizes the entire protein or its fragments in their ionized form. This includes the following. Known methods of protein mass spectrometry include electrospray ionization (E This includes SI (Single Injection) and matrix-assisted laser desorption / ionization (MALDI). While the analysis has several advantages, the results obtained are affected by the presence of contaminants. It is possible that processing fragile molecules without fragmenting them can be difficult. Furthermore, mass spectrometry is not a single-molecule technique, and it does not provide bulk information about the sample being investigated. Mass spectrometry is unsuitable for characterizing intrapopulation differences in polypeptide samples. This is a cleavage issue, making it difficult to distinguish between adjacent residues.

[0005] Edman degradation is an alternative to mass spectrometry that enables residue-level sequencing of polypeptides. Edman degradation involves sequentially cleaving the N-terminal amino acid and chromatographically or By using electrophoresis to characterize individually cleaved residues, polypeptides can be sequenced. Decision made. However, Edman sequencing is slow and requires the use of expensive reagents. Yes, but it's not a single-molecule technique like mass spectrometry.

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

[0007] One attractive approach for single molecule characterization of biomolecules such as polypeptides is nanopore sensing. Nanopore sensing is an approach to the detection and characterization of analytes that relies on observing individual binding or interaction events between an analyte molecule and an ion-conducting channel A nanopore sensor can be fabricated by placing a single nanometer-sized pore in an insulating membrane and measuring the voltage-driven ionic current through the pore in the presence of the analyte molecule When an analyte is present inside or near the nanopore, the ion flow through the pore is altered resulting in a change in the ionic current measured across the channel The identity of the analyte is revealed through its characteristic current signature, particularly the duration and magnitude of the current block as well as fluctuations in the current level during interaction with the pore. Nanopore sen sing has the potential to enable rapid and inexpensive characterization of polypeptides

[0008] Nanopore sensing and characterization of polypeptides has been proposed in the art. For exam ple, WO2013 / 123379 uses an NTP-driven protein processing unfold ase enzyme to process a protein to cause translocation through a nanopore However, there remains a need for alternative and / or improved methods for characterizing polypeptides . Summary of the Invention

[0009] The present disclosure relates to a method of characterizing a target polypeptide. The method comprises conjugating a target polypept ide to a polynucleotide to form a polypeptide-polynucleotide conjuga te. The method comprises contacting the conjugate with a polynucleotide handling protein. The polynucleotide handling protein is capable of controlling movement of the polynucleotide with respect to a nanopore. One or more measurements characteristic of the polypeptide are taken as the conjugate moves with respect to the nanopore. In this way, the target polypeptide comprised in the conjugate is characterized.

[0010] Accordingly, provided herein is a method of characterizing a target polypeptide, , - conjugating a target polypeptide to a polynucleotide to form a polynucleotide- polypeptide conjugate; - contacting the conjugate with a polynucleotide handling protein capable of controlling movement of the polynucleotide with respect to a nanopore; - taking one or more measurements characteristic of the polypeptide as the conjugate moves with respect to the nanopore; thereby characterizing the polypeptide.

[0011] In some embodiments, the nanopore comprises a constricted region. In some embodiments, the nanopore is modified to increase the distance between the polynucleotide handling protein and the constricted region of the nanopore. In some embodiments, the polynucleotide han The doring protein was separated from the nanopore using a displacer unit, and This increases the distance between the active site of the polynucleotide handling protein and the nanopore. To extend. In some embodiments, the displacer unit has one or more proteins It contains quality. In some embodiments, the polynucleotide handling protein is Extend the distance from the active site of polynucleotide handling proteins to the nanopore. It is modified in such a way.

[0012] In some embodiments, the polynucleotide handling protein is polynucleotide The conjugate portion in contact with the active site of the rheotide handling protein is poly If peptides are present, they can remain bound to the conjugate. In this embodiment, the polynucleotide handling protein is a polynucleotide handling protein. When the binding protein comes into contact with the conjugate portion containing the polypeptide, This is modified to prevent it from detaching from the conjugate. In some embodiments, Therefore, polynucleotide handling proteins are responsible for unbinding polynucleotide chains. An opening present in at least one conformational state of the unmodified protein. It is modified to close completely or partially. In some embodiments, the polynucle Otidylose handling proteins are helicases.

[0013] In some embodiments, the conjugate consists of multiple polypeptide sections and It contains one / or more polynucleotide sections.

[0014] In some embodiments, the polypeptide has a length of 2 to about 50 peptide units. In some embodiments, the polypeptide is held in a linearized form.

[0015] In some embodiments, polynucleotides have a length of about 10 to about 1000 nucleotides. It has one or more adapters and / or one or more The tether and / or one or more anchors above are polynucleotides in the conjugate. It is attached to it.

[0016] In some embodiments of the disclosed method, i) Polynucleotide handling proteins are located on the cis side of the nanopore, Oxide handling proteins control the transition from the cis side of the nanopore to the trans side of the nanopore. Control the movement of the Jugate, or ii) The polynucleotide handling protein is located on the trans side of the nanopore, Nucleotide handling proteins move from the trans side of the nanopore to the cis side of the nanopore. Controls the movement of the conjugate.

[0017] In some embodiments, polynucleotide handling proteins are nanopores Located on the cis side, the polynucleotide handling protein moves from the cis side of the nanopore. Controlling the movement of polynucleotides to the trans side of the nanopore, thereby controlling the passage of polynucleotides through the nanopore. Controls the movement of lipeptides. In some embodiments, polynucleotide handlers The nucleating protein is located on the trans side of the nanopore, and the polynucleotide handling protein The substance controls the movement of polynucleotides from the trans side of the nanopore to the cis side of the nanopore. This controls the movement of polypeptides through the nanopore.

[0018] In some embodiments, the conjugate is L-{PN}-P m One or more forms It includes the structure, -L is the leader, and L is optionally part N. -P is a polypeptide, -N contains a polynucleotide, -m is 0 or 1, The method involves threading the leader (L) through a nanopore, thereby creating polypeptides. This includes bringing (P) into contact with the nanopore, i) The polynucleotide handling protein is located on the cis side of the nanopore, and the method is to The renucleotide handling protein moves from the cis side of the nanopore to the trans side of the nanopore. This enables control over the movement of the polynucleotide moiety (N) to the nanopore, thereby enabling control over the movement of the nanonucleotide moiety (N). This includes controlling the movement of polypeptides (P) through or ii) The polynucleotide handling protein is located on the trans side of the nanopore, and the method This is because the polynucleotide handling protein moves from the trans side of the nanopore to the nanopore. This makes it possible to control the movement of the polynucleotide (N) to the cis side, thereby enabling the This includes controlling the movement of polypeptides (P) through nopores.

[0019] In some embodiments, the conjugate is L-P1-N-{PN} n -P m Shape It includes one or more structures of the state, -n is a positive integer, -L is the leader, and L is optionally part N. -Each P may be the same or different; -Each N, which may be the same or different, contains a polynucleotide, and -m is 0 or 1, The method involves threading the leader (L) through a nanopore, thereby creating polypeptides. This includes bringing (P1) into contact with the nanopore, i) The polynucleotide handling protein is located on the cis side of the nanopore, and the method is to The renucleotide handling protein moves from the cis side of the nanopore to the trans side of the nanopore. This makes it possible to continuously control the movement of each polynucleotide (N) to and thereby This includes continuously controlling the movement of each polypeptide (P) through the nopore, or ii) The polynucleotide handling protein is located on the trans side of the nanopore, and the method This is because the polynucleotide handling protein moves from the trans side of the nanopore to the nanopore. This makes it possible to continuously control the movement of each polynucleotide (N) to the cis side, and This involves continuously controlling the movement of each polypeptide (P) through the nanopore.

[0020] In some embodiments of the disclosed method, i) Polynucleotide handling proteins are located on the cis side of the nanopore, Oxide handling proteins control the transition from the trans side of the nanopore to the cis side of the nanopore. Control the movement of the Jugate, or ii) The polynucleotide handling protein is located on the trans side of the nanopore, Nucleotide handling proteins move from the cis side of the nanopore to the trans side of the nanopore. Controls the movement of the conjugate.

[0021] In some embodiments, polynucleotide handling proteins are nanopores Located on the cis side, the polynucleotide handling protein is on the trans side of the nanopore. This controls the movement of polynucleotides to the cis side of the nanopore, thereby allowing the polynucleotides to pass through the nanopore. Controls the movement of lipeptides. In some embodiments, polynucleotide handlers The nucleating protein is located on the trans side of the nanopore, and the polynucleotide handling protein The substance controls the movement of polynucleotides from the cis side of the nanopore to the trans side of the nanopore. This controls the movement of polypeptides through the nanopore.

[0022] In some embodiments, the conjugate is L-{PN}-P m One or more forms It includes the structure, -L is the leader, and L is optionally part N. -P is a polypeptide, -N contains polynucleotides, -m is 0 or 1, The method involves threading the leader (L) through a nanopore, thereby creating polypeptides. This includes bringing (P) into contact with the nanopore, i) The polynucleotide handling protein is located on the cis side of the nanopore, and the method is to The renucleotide handling protein moves from the trans side of the nanopore to the cis side of the nanopore. This makes it possible to control the movement of polynucleotides (N) to and thereby through nanopores. This includes controlling the movement of polypeptides (P), or i) The polynucleotide handling protein is located on the trans side of the nanopore, and the method The polynucleotide handling protein moves from the cis side of the nanopore through the nanopore. This enables control over the movement of polynucleotides (N) to the nanopore, thereby allowing for the nanopore to be controlled. This includes controlling the movement of polypeptides (P) through a certain path.

[0023] In some embodiments, the conjugate is connected via an optional linker to a polypeptide The method includes the blocking portion attached to the dot, i) The blocking portion is a nanopore relative to the polynucleotide handling protein As shown on the opposite side, the conjugate is brought into contact with the nanopore, ii) Polynucleotide handling proteins for the conjugate polynucleotides To bring it into contact with, iii) Polynucleotide handling proteins against nanopores This enables control over the movement of nanopores, thereby controlling the movement of polypeptides through nanopores. Controlling and iv) The blocking portion comes into contact with the nanopore, thereby allowing the conjugate to pass through the nanopore. If further movement of polynucleotides is prevented, polynucleotide handling proteins will be used. The bond is temporarily broken from the nucleotide, thereby, under the applied force, the polynucleotide The conjugate moves in the opposite direction to the direction of movement controlled by the doring protein in the nanopore. To enable movement through, v) Optionally, repeat steps (ii) to (iv) to pass the polypeptide through the nanopore. This includes causing the cyd to vibrate.

[0024] In some embodiments, one or more measurements are (i) the length of the polypeptide, (ii) (iii) polypeptide identity, (iv) polypeptide sequence, (iv) polypeptide secondary Polypeptides selected based on their structure and (v) whether the polypeptide is modified. It is characterized by one or more features.

[0025] Nanopores including a constricted region are also provided herein, which have a constricted region and a nanopore It increases the distance between the polynucleotide handling protein that is in contact with A. It is modified by "uni".

[0026] Also provided are: -Nanopores including a constricted region, - Conjugates containing polypeptides conjugated to polynucleotides, -Polynucleotide handling proteins, A system including, i) When the polynucleotide handling enzyme is in contact with this nanopore This increases the distance between the constricted region and the active site of the polynucleotide handling protein. Modified to add, and / or ii) This system is connected between the nanopore and the polynucleotide handling protein. They are positioned, thereby connecting the nanopore and the active site of the polynucleotide handling protein. It further includes one or more displacer units that extend the distance between them.

[0027] In some embodiments, nanopores, conjugates and / or polynucleotides Chip handling proteins, and optionally, if present, one or more dispersive The racer unit is as defined herein.

[0028] Also provided are: -Nanopores including a constricted region, -Polynucleotides containing reactive functional groups for conjugation of target polynucleotides Ochido and, - This kit includes polynucleotide handling proteins.

[0029] In some embodiments, (i) the nanopore is used for polynucleotide handling When the enzyme is in contact with the nanopore, the constricted region and the polynucleotide handling protein Modified to increase the distance between the quality and / or (ii) this kit is nano To extend the distance between the pore and the active site of the polynucleotide handling protein The further includes one or more displacer units. In some embodiments, Nopore, polynucleotide and / or polynucleotide handling proteins, Furthermore, if optional, one or more displacer units, if present, are specified herein. It is as defined. [Brief explanation of the drawing]

[0030] [Figure 1]A schematic diagram illustrating a non-limiting example of an embodiment of the disclosed method, in which a polynucleotide handling protein on the cis side of a nanopore controls the movement of a conjugate containing a polypeptide-conjugated polynucleotide (DNA2) from the cis side of the nanopore to the trans side of the nanopore, thus enabling characterization of the polypeptide as it moves toward the nanopore. As shown, an optional leader (DNA1) is attached to the conjugate to facilitate threading of the polypeptide through the nanopore. RED (as discussed herein) is shown as the conceptual distance between the constriction in the nanopore and the active site of the polynucleotide handling protein. (A) The substrate can be captured into the nanopore from the cis side of the membrane, for example, by applying a positive voltage to the trans side of the membrane. The polynucleotide handling protein moves along the polynucleotide section in the direction indicated by the dotted arrow, supplying the substrate to the pore, and proceeding to state (B). As the polynucleotide handling protein moves along the polynucleotide (for example, in a single-nucleotide fuel-driven step), the conjugate is supplied into the nanopore and the peptide section passes through the nanopore. [Figure 2] A schematic diagram illustrating a typical setup embodiment shown in Figure 1. In this non-limiting example, the polynucleotide handling protein first stalls at the spacer (X) of the polynucleotide portion (DNA) of the conjugate. The adapter is attached to the polynucleotide portion of the conjugate, to which a tether is attached, localizing the conjugate to a nanopore region of the membrane for characterization. Steps (A) and (B) are as described in Figure 1. As the polynucleotide handling protein processes the polynucleotide, this protein may replace the adapter. [Figure 3]A schematic diagram illustrating a further embodiment of the general setup shown in Figure 1. In this non-limiting example, the conjugate includes multiple polynucleotide and polypeptide sections that are sequentially moved through a nanopore under the control of a polynucleotide handling protein. As shown, the polynucleotide handling protein is first loaded onto the first polynucleotide portion (DNA1) of the conjugate and moves that portion of the conjugate through the nanopore. The polynucleotide handling protein then moves the first polypeptide section of the conjugate through the nanopore without dissociating from the conjugate. Next, the polynucleotide handling protein comes into contact with the second polynucleotide portion (DNA2) of the conjugate and controls its movement through the nanopore. Further polypeptide and polynucleotide portions (not shown) may similarly be sequentially moved through the nanopore. [Figure 4-1] Schematic diagram showing non-limiting examples of substrates for use in the embodiments described in Figure 3. A: The first polynucleotide portion of the conjugate (DNA1) includes a sequencing Y adapter with a leader (dotted line) to facilitate capture in the nanopore, a tether that allows tethering to the membrane to localize the conjugate to a region in the nanopore, and a polynucleotide handling protein stalled by a spacer (X). As shown, the polynucleotide portion of the conjugate contains double-stranded DNA. B: A variation of the embodiment of the substrate shown in (A). A tether (or additional tether) can be placed on the second polynucleotide portion of the conjugate (DNA2). The notations "top" and "bottom" are purely for ease of understanding. C: Schematic diagram showing a method of the present invention using the substrate shown in 4(A). [Figure 4-2] This is a continuation of Figure 4-1. [Figure 5]A schematic diagram illustrating further non-limiting examples of substrates for use in the disclosed method. The conjugate may comprise multiple polynucleotide and polypeptide sections (n>0), which can be sequentially processed by a polynucleotide handling protein for nanopore characterization, as described herein. [Figure 6] A schematic diagram illustrating a non-limiting example of an embodiment of the disclosed method, in which a polynucleotide handling protein on the cis side of a nanopore controls the movement of a conjugate containing a polynucleotide (DNA2) conjugated to a polypeptide from the trans side of the nanopore to the cis side of the nanopore, thus enabling the characterization of the polypeptide as it moves toward the nanopore. As shown, an optional leader is attached to the conjugate to facilitate the initial threading of the polypeptide through the nanopore. (A) The substrate can be captured into the nanopore from the cis side of the membrane, for example, by applying a positive voltage to the trans side of the membrane. The polynucleotide handling protein moves along the polynucleotide section in the direction indicated by the dotted arrow, thereby moving the substrate out of the pore and proceeding to state (B). As the polynucleotide handling protein moves along the polynucleotide (e.g., in a 1-nucleotide fuel-driven step), the protein evictes the conjugate from the nanopore. Thus, the polypeptide section of the conjugate passes through the nanopore (state C) and is therefore characterized. [Figure 7]A schematic diagram illustrating a non-limiting example of the use of a blocking region (black square) that prevents the movement of the conjugate through the nanopore when it comes into contact with the nanopore. In the non-limiting example shown, the polynucleotide handling protein is a polymerase that can control the movement of the conjugate by elongating the polynucleotide portion of the conjugate. Chain elongation may continue until the blocking region reaches the nanopore. Dissociation of the newly synthesized chain allows the conjugate to return from cis to trans through the nanopore, and the polynucleotide can recycle the trans-to-cis movement of the conjugate through the nanopore. In this way, the conjugate can be "flossed" through the nanopore. Other polynucleotide handling proteins can be used in a similar manner. [Figure 8] Schematic diagrams illustrating non-limiting examples of strategies for increasing the distance between a nanopore (e.g., a constriction within the nanopore) and the active site of a polynucleotide handling protein used to control the movement of the conjugate relative to the nanopore. A: Schematic diagram of an unmodified pore showing an unmodified RED. B: A nanopore may be modified to expand the RED. C: A displacer unit may be used to replace the polynucleotide handling protein from the nanopore, thus expanding the RED. D: Multiple polynucleotide handling proteins may be used to replace the active polynucleotide handling protein that controls the movement of the conjugate from the nanopore relative to the nanopore. These embodiments are described in more detail herein. [Figure 9]For clarity, a representative current-to-time trace of Example 1 is shown using schematic diagrams of the corresponding constructs. States A-D correspond to the states described in Figure 4F. A - Leader strand capture by nanopore, B - Y adapter translocation across nanopore leader head (RED), C - polypeptide translocation across RED, D - polynucleotide tail (DNA2) translocation. The first trace represents a partial translocation event of only the Y adapter (states A and B only), while the second trace shows the translocation of the entire conjugated polynucleotide-polypeptide across the nanopore. Data obtained as described in Example 1 (this data pertains to a polynucleotide-peptide conjugate containing the peptide sequence of SEQ ID NO: 20). [Figure 10] Current-vs-time traces demonstrating high throughput of data acquisition. Within a 3-second period, there are five capture events, four corresponding to complete polynucleotide-polypeptide conjugates (event 3 is a partial translocation of only the Y adapter). Data described in Example 1 (this data pertains to a polynucleotide-peptide conjugate containing the peptide sequence of SEQ ID NO: 20). [Figure 11] Current traces of the translocation of the polynucleotide-peptide conjugate described in Figure 4B and Example 1, corresponding to the peptide sequence GGSGRRSGSG (SEQ ID NO: 21). A: Eleven examples of traces aligned with respect to the states described in Figures 4 and 9. B: Overlay of the same eleven traces. C: Stacked plot of the eleven trace examples showing time axis normalization using a dynamic time stretching algorithm to facilitate optimal alignment of key trace features. [Figure 12] Current traces of the translocation of the polynucleotide-peptide conjugate described in Figure 4B and Example 1, corresponding to the peptide sequence GGSGYYSGSG (SEQ ID NO: 22). A: Twelve examples of traces aligned with respect to the state described in Figures 4 and 9. B: Overlay of the same twelve traces. C: Stacked plot of the twelve trace examples. [Figure 13]Current traces of the translocation of the polynucleotide-peptide conjugate described in Figure 4B and Example 1, corresponding to the peptide sequence GGSGDDSGSG (SEQ ID NO: 20). A: Eleven examples of traces aligned with respect to the states described in Figures 4 and 9. B: Overlay of the same eleven traces. C: Stacked plot of the eleven trace examples. [Figure 14] Schematic structure of the construct obtained using the peptide of SEQ ID NO: 22. Y adapter containing polynucleotide chains of SEQ ID NOs: 11, 12, and 13; and polynucleotide tail containing polynucleotide chains of SEQ ID NOs: 14 and 16 (described in Example 1). [Figure 15] Representative current-versus-time traces of Example 2 compared to Example 1. States A-D correspond to the states described in Figure 4F. A - Leader strand capture by nanopore, B - Y adapter translocation across nanopore leader head (RED), C - polypeptide translocation across RED, D - polynucleotide tail (DNA2) translocation. Traces in the upper panel were collected according to the protocol of Example 1 (using pre-modified peptides during synthesis). Traces in the lower panel show polynucleotide-polypeptide translocations conjugated according to the protocol of Example 2 (unmodified peptide of SEQ ID NO: 23; i.e., using the same sequence as the corresponding trace in Example 1). [Figure 16] Representative current-vs-time traces of translocation of a polynucleotide-peptide conjugate of a 10-amino acid peptide (top panel: SEQ ID NO: 20) compared to a 21-amino acid peptide (bottom panel: SEQ ID NO: 24). States A-D correspond to the states described in Figure 4F. A - Leader chain capture by nanopore, B - Translocation of the Y adapter across the nanopore leader head (RED), C - Translocation of the polypeptide across the RED, D - Translocation of the polynucleotide tail. The results are shown in Example 3. [Modes for carrying out the invention]

[0031] The present invention will be described in relation to specific embodiments and with reference to certain drawings, however The invention is not limited to those, but is limited only by the claims. None of these reference symbols should be interpreted as limiting their scope. Needless to say, Not necessarily all aspects or advantages are achieved according to any particular embodiment of the present invention. Please understand that this is not always possible. Therefore, for example, a person skilled in the art would know this Without necessarily achieving other embodiments or advantages that may be taught or suggested in the specification, The present invention is provided in a manner that achieves or optimizes one or a group of advantages as taught in the details. They will recognize that it can be embodied or performed.

[0032] The present invention relates to both organization and operation methods, along with their features and advantages, and the attached... When read in conjunction with the drawings, the most appropriate mode for carrying out the invention can be found by referring to the following modes for carrying out the invention. This can be easily understood. The aspects and advantages of the present invention will become apparent with reference to the embodiments described below. This will be clarified. Throughout this specification, "one embodiment" or "a certain embodiment" References to the present invention refer to specific features, structures, or properties described in relation to its embodiments. This means that it is included in at least one embodiment of the specification. The appearance of phrases like "in one embodiment" or "in one embodiment" in various places is not necessarily While not all instances of "zushi" refer to the same embodiment, they do in some cases. Similarly, this invention In describing the exemplary embodiments of this disclosure, we have simplified the description and explained one of the various embodiments of the invention. To support the above understanding, various features of the present invention may be illustrated in a single embodiment, figure, or otherwise. Please understand that this may be summarized in the explanations provided. However, the method of this disclosure The claimed invention has more features than those explicitly listed in each patent claim. It should not be interpreted as reflecting an intention to require it. Rather, the following patent request To reflect the scope of the request, the embodiments of the invention include all the features of a single aforementioned disclosed embodiment. It is not a sufficient indicator.

[0033] Unless otherwise indicated by the context, the “embodiments” of this disclosure may be specifically combined together. Please understand that all specific combinations of the disclosed embodiments are (depending on the context) (Unless otherwise implied) these are further disclosed embodiments of the claimed invention.

[0034] In addition, as used herein and in the appended claims, the singular "a", "n" and "the" refer to multiple objects unless the context explicitly indicates otherwise. Therefore, for example, a reference to "polynucleotide" includes two or more polynucleotides. Furthermore, the reference to "motor protein" includes two or more such proteins, and "helicar References to "ze" include two or more helicases, and references to "monomer" include two or more monomers. —This refers to "poa," and a reference to "poa" includes two or more instances of "poa."

[0035] All publications, patents, and patent applications cited above or below in this specification are referenced by the relevant authorities. By reference, the entirety of these elements is incorporated herein.

[0036] definition When referring to a singular noun, use either the indefinite or definite article, for example, "a" or "an" or "th". When "e" is used, unless otherwise specified, it includes the plural form of the noun. Where the term "including" is used in this description and claims, it means that other requirements Elements or steps are not excluded. Furthermore, the first and third in this description and claims. Terms such as 2, 3, etc., are used to distinguish similar elements and do not necessarily reflect the order in which they occurred. or they are not necessarily used to describe chronological order. The terms are interchangeable under appropriate circumstances, and the embodiments of the present invention described herein are Please understand that the components may operate in an order other than that described or illustrated in this specification. The following terms or definitions are provided solely to aid in understanding the present invention. Specifically, as used herein, Unless otherwise specified, all terms used herein are understood by those skilled in the art. It has the same meaning as the meaning of [something]. Experts will refer to definitions and technical terms, especially Sambro. ok et al.,Molecular Cloning: A Laboratory Manual, 4 th ed., Cold Spring Harbor Press Plainsview, New York (2012), and Ausubel et al. al.,Current Protocols in Molecular Biol ogy(Supplement 114),John Wiley&Sons,New See York (2016). The definitions provided herein should be understood by those skilled in the art. It should not be interpreted as being narrower than the range that can be included.

[0037] As used herein, "approximately" refers to measurable values ​​such as quantity and duration. ±20% or ±10% from the specified value, more preferably ±5%, even more preferably It is intended to include variations of ±1%, more preferably ±0.1%, and The fluctuations are appropriate for carrying out the disclosed methods.

[0038] The terms "nucleotide sequence," "DNA sequence," or "nucleic acid molecule" used herein and This is a nucleus of any length, either a ribonucleotide or a deoxyribonucleotide. This refers to the polymer form of ocide. This term refers only to the primary structure of the molecule. Therefore This term includes double-stranded and single-stranded DNA and RNA. The term "nucleic acid" refers to the term in which the 3' and 5' ends of each nucleotide are phosphodiester. A single-stranded or double-stranded covalent nucleotide sequence linked by a single bond. Yes. Polynucleotides are composed of deoxyribonucleotide bases, but ribonucleated It can be composed of rheotide bases. Nucleic acids can be produced synthetically in vitro or naturally. Nucleic acids can be isolated from natural sources. Nucleic acids include modified DNA or RNA, for example, methylated DNA or RNA that has undergone post-translational modification, for example, 7-methylguanosine 3'-processing such as 5'-capping, cleavage and polyadenylation, as well as Further RNA being used for splicing may be included. Nucleic acids include hexitol nucleic acids. (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol Synthesis of nucleotides (GNA), locked nucleotides (LNA), and peptide nucleotides (PNA). Nucleic acids (XNA) may also be included. Nucleic acids, also referred to herein as "polynucleotides," are cytonucleotides. The number of base pairs (bp) in a double-stranded polynucleotide, or a single-stranded polynucleotide, is typically the number of base pairs (bp) in a double-stranded polynucleotide. In the case of nucleotides, the number of nucleotides (nt) is expressed as 1000 bp or nt. This corresponds to kilobases (kb). Polynucleotides less than approximately 40 nucleotides in length are: Typically called "oligonucleotides," they are used in polymerase chain reactions (PCR), etc. It may include primers for use in manipulating DNA.

[0039] In connection with this disclosure, the term “amino acid” is used in its broadest sense, and each ami Along with a side chain specific to ano acids (e.g., an R group), amines (NH2) and carboxyls ( In some embodiments, the invention is intended to include organic compounds containing a COOH functional group. Amino acids refer to naturally occurring L-α-amino acids or residues. Commonly used one- and three-letter abbreviations for ano acids: A=Ala, C=Cys, D= Asp, E=Glu, F=Phe, G=Gly, H=His, I=Ile, K=Lys, L=Leu, M=Met, N=Asn, P=Pro, Q=Gln, R=Arg, S=Se r, T=Thr, V=Val, W=Trp, and Y=Tyr are used herein ( Lehninger, AL, (1975) Biochemistry, 2d ed. pp.71-92, Worth Publishers, New York). "Ami The general term "amino acid" refers to D-amino acids, retroinversoamino acids, and amino acids. Chemically modified amino acids such as acid analogs, and proteins such as norleucine, are typically incorporated into these proteins. This text describes the characteristics of naturally occurring amino acids that are not included in the diet, as well as amino acids such as β-amino acids. The invention further includes chemically synthesized compounds having properties known in the art. For example, natural Phenylalamine allows for the same structural limitations as Phe or Pro for peptide compounds. Analogues or mimics of nin or proline are included within the definition of an amino acid. The bodies and mimics are referred to herein as “functional equivalents” of their respective amino acids. Other examples of ano acids are incorporated herein by reference, Roberts and Vel. laccio,The Peptides:Analysis,Synthesis,B iology,Gross and Meiehofer,eds.,Vol.5 p. Listed by 341, Academic Press, Inc., NY 1983. .

[0040] The terms "polypeptide" and "peptide" refer to polymers of amino acid residues, and The terms are used interchangeably herein to refer to their variants and synthetic analogues. These terms refer to the chemistry of naturally occurring amino acids, where one or more amino acid residues correspond to each other. Amino acid polymers, which are synthetic amino acids that do not exist in nature, such as analogs, and naturally occurring amino acids This applies to existing amino acid polymers. Polypeptides undergo glycosylation and protein degradation. This may include target cleavage, lipidization, signal peptide cleavage, propeptide cleavage, phosphorylation, etc. Peptides may also undergo maturation or post-translational modification processes, which are not limited to these. These can be produced, for example, by the expression of recombinant or synthetic polynucleotides using the following techniques. Recombinant peptides typically contain substantially no culture medium, for example. Furthermore, the culture medium should be less than approximately 20% of the volume of the protein preparation, more preferably less than approximately 10%. Most preferably, this corresponds to less than approximately 5%.

[0041] The term "protein" refers to a folded polypeptide that has a secondary or tertiary structure. It is used to explain that a protein may consist of a single polypeptide, Alternatively, it may contain multiple polypeptides that aggregate to form a multimer. The multimer is homooligo It can be a mer or a heterooligomer. Proteins are naturally occurring proteins. It may be a natural or wild-type protein, or a modified or natural protein. It may be a protein that does not exist in the original. A protein is, for example, one or more amino acids Additions, substitutions, or deletions may result in a protein that differs from the wild-type protein.

[0042] A "mutant" of a protein is compared to the unmodified or wild-type protein in question. unmodified proteins that have amino acid substitutions, deletions, and / or insertions, and from which they are derived Peptides, oligopeptides, and polypeptides that have biological and functional activity similar to that of cereals. This includes amino acids, proteins, and enzymes. The term "amino acid identity" as used herein is used in this specification. The term refers to the degree to which a sequence is identical amino acid by amino acid across a comparison window. Therefore, the "percentage of sequence identity" is best divided into two categories across the comparison window. The aligned sequences are compared, and identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met occur in both sequences. Determine the number of positions and calculate the number of matching positions, then compare the number of matching positions in the comparison window. Divide by the total number of positions (i.e., window size), and multiply the result by 100 to get the same array size. It is calculated by determining the percentage of one sex.

[0043] In all aspects and embodiments of the present invention, "mutant" is the corresponding wild-type protein. At least 50%, 60%, 70%, 80%, 90%, and 95% of the amino acid sequence of the protein. It has 99% or complete sequence identity. Sequence identity is defined as full-length polynucleotides. Or it may be a fragment or part of a polypeptide. Therefore, the sequence is While it may have only 50% sequence identity overall with the full-length reference sequence, certain regions, domains, The sequence of the subunits is 80%, 90%, or 99% identical to the reference sequence. They can share a common gender.

[0044] The term "wild type" refers to a gene or gene product isolated from a naturally occurring source. It refers to the wild-type gene, which is most frequently observed in a given population, and therefore, any of the genes. This is the "normal" or "wild-type" form designed for this purpose. In contrast, the "modified" or "mutant" form is a variant. The terms "variant" or "mutant" refer to a variant of a gene or gene product compared to its sequence. Modifications (e.g., substitution, truncation, or insertion), post-translational modifications, and / or functional characteristics. This refers to a gene or gene product that exhibits (for example, a modified characteristic). It should be noted that allogenes can be isolated, and these are wild-type genes or genes. Identified by the fact that it has modified characteristics compared to the product. Naturally occurring Methods for introducing or substituting amino acids are well known in the art. For example, methio Nin (M) is methyl at the relevant position in the polynucleotide encoding the mutant monomer. By replacing the nin codon (ATG) with the arginine codon (CGT), It can be substituted with ginine(R). A method for introducing or substituting amino acids that do not exist in nature. The relevant laws are also well known in the field of technology. For example, amino acids that do not exist in nature are mutant monomers. The IVTT system used to express [the gene] includes synthetic aminoacyl-tRNA. They can be introduced by... Alternatively, they can be introduced by... the synthesis of specific amino acids (i.e., naturally... In the presence of (non-existent) analogues, E. has nutritional requirements for those specific amino acids. They can be introduced by expressing mutant monomers in coli. When monomers are produced using partial peptide synthesis, naked ligation ( It can also be produced by (naked ligation). Conservative substitutions are amino Replace the acid with other amino acids that have a similar chemical structure, similar chemical properties, or similar side chain volume. Replace. The introduced amino acids have similar polarity, hydrophilicity, and polarity to the amino acids they replace. It may be aqueous, basic, acidic, neutral, or charged. Alternatively, a conservative substitution may be an existing By introducing another amino acid that is aromatic or aliphatic in place of an aromatic or aliphatic amino acid Obtain. Conservative amino acid changes are well known in the art and are defined in Table 1 below. They can be selected according to the characteristics of the main amino acids. If the amino acids have similar polarity, This can also be determined by referring to the hydrophobicity scale of the amino acid side chains in Table 2. [Table 1] [Table 2]

[0045] Mutant or modified proteins, monomers, or peptides may be used in any way and any way. Site-specific chemical modification is also possible. Mutants or modified monomers or peptides are preferred. In addition, it involves the attachment of molecules to one or more cysteine ​​molecules (cysteine ​​bond) and one or more lysine molecules. Molecular attachment to, molecular attachment to one or more non-natural amino acids, enzymatic modification of epitopes, Alternatively, it is chemically modified by terminal modifications. A suitable method for carrying out such modifications is This is well known in the art. Modified protein, monomer, or peptide variants are available. It can be chemically modified by the attachment of certain molecules. For example, modified proteins, monomers, Alternatively, peptide variants may be chemically modified by the attachment of dyes or fluorophores. ru.

[0046] Method of disclosure This disclosure relates to the formation of a conjugate with polynucleotides and polynucleotide handlings. By using protein to control the movement of conjugates to nanopores, This concerns methods for characterizing polypeptides.

[0047] Using polypeptide handling enzymes to control polypeptide transfer to nanopores. In contrast to the methods that attempt to achieve this, the method of the present disclosure uses a polynucleotide handling enzyme This allows for the control of polypeptide migration to nanopores.

[0048] The methods disclosed herein involve conjugates that do not consist solely of polynucleotides. It utilizes the ability of polynucleotide handling proteins to control the movement of polynucleotides. Conjugates containing lipeptides are polynucleotides as described herein. It can be transported in a controlled manner using a sizing protein. Polynucleotide handling proteins suitable for this purpose are described in more detail herein. It is.

[0049] Therefore, what is provided herein is a method for characterizing a target polypeptide. , -Conjugate the target polypeptide into a polynucleotide, and then conjugate the polynucleotide- Forming polypeptide conjugates, - The conjugate can control the movement of polynucleotides into the nanopore. By bringing it into contact with a polynucleotide handling protein, - When the conjugate moves relative to the nanopore, one characteristic of polypeptides This includes performing the above measurements, This is a method for characterizing polypeptides.

[0050] Any suitable polypeptide can be characterized using the methods disclosed herein. Yes, it is possible. In some embodiments, the target polypeptide is a protein or naturally occurring It is a polypeptide. In some embodiments, the polypeptide is a synthetic polypeptide. It is a polypeptide that can be characterized according to the method disclosed. It is described in more detail in the book.

[0051] When forming a conjugate for use in the manner disclosed herein, any appropriate A specific polynucleotide can be used. In some embodiments, the polynucleotide The rheotide has a length of at least the same length as the portion of the target polypeptide to be characterized. In some embodiments, the polynucleotide is the target polynucleotide to be characterized. It has a length longer than the tide portion. This will be discussed in more detail below. Polynucleotides suitable for use in the method are disclosed in more detail herein.

[0052] In the disclosed method, the target polypeptide is polynucleotide using any suitable means. It can be conjugated with rheotide. Several exemplary methods are described in more detail herein. It is stated.

[0053] The conjugate formed by the disclosed method is a polynucleotide relative to the nanopore. It is brought into contact with a polynucleotide handling protein that can control its movement. Exemplary polynucleotide handling proteins are described in more detail herein. It is.

[0054] Polynucleotide handling proteins transfer polynucleotides to nanopores. It controls movement. Therefore, polynucleotide handling proteins are used in nanopores. The movement of the conjugate is controlled. The disclosed method uses any suitable nanopore. It can be used. Nanopores suitable for use in the disclosed method are described in more detail herein. It is described there.

[0055] The disclosed method involves the polypeptide moving relative to the nanopore as the conjugate moves. This includes performing one or more characteristic measurements. One or more measurements may be any suitable measurement. It is possible. Typically, one or more measurements are electrical measurements, e.g., current measurements, and / or 1 It is an optical measurement of more than one degree. Apparatus for recording appropriate measurements, and such measurements The information we can provide is described in more detail in this specification.

[0056] Characterization of target polypeptides As disclosed herein, polynucleotides are polypeptides for nanopores It can be used to control movement. The movement of polynucleotides is controlled by polynucleotide hands. It is controlled by a drinking protein. Polynucleotides are polynucleotides in the conjugate. Because it is conjugated to a peptide, the movement of polynucleotides is similar to the movement of polypeptides. To drive motion.

[0057] Polynucleotides for controlling the movement of polynucleotides, and therefore polypeptides. The use of oxidative handling proteins features polypeptides known in the art. This may relate to the advantages compared to other methods of attachment. For example, polynucleotide handling. Proteins have a higher turnover rate compared to polypeptide handling enzymes. The handling of creotides can be processed. This is previously known. Compared to the method described, the polypeptide characterized according to the disclosed method is characterized This means that the data can be obtained more quickly.

[0058] These and other benefits will become apparent through this disclosure.

[0059] In developing the method disclosed herein, the inventors have a shorter barrel or channel When nanopores with longer barrels or channels are used compared to nanopores. We have found that the length of the polypeptides that can be characterized is typically improved. Without being bound by the argument, the inventors believe that this is a polynucleotide as disclosed. When used in combination with cytohandling proteins, longer barrels or char Pores with a flannel are polynucleotides more polynucleotides than pores with a shorter barrel or channel. A longer distance between the active site of the cytohandling protein and the constriction in the nanopore It is thought that this may be for the purpose of [something]. This distance is called RED (Leader-Enzyme Distance). It can be discovered. A person skilled in the art would know that the morphology of the nanopore is not limited (as will be discussed below). This will be understood. Nanopores can be protein nanopores or solid nanopores. If the nanopore does not have a constriction within the nanopore channel, the constriction as used herein is For example, in one embodiment, it can be identified by the opening of a nanopore.

[0060] Without being constrained by theory, the particles within the conjugate that can be characterized by nanopores The length of the lipeptide portion may correspond to or be determined by RED. It is presumed that, in other words, the nanopore may include a reading head, and one or more measurements are performed by the nanopore. The "read portion" of the lipeptide is distinctive, and the length of the read portion is determined by the reading head. This corresponds to the distance between the active site of the polynucleotide handling protein, or That will be determined.

[0061] With this in mind, in some embodiments of the disclosed method, polynucleotides , having at least the same length as the portion of the target polypeptide to be characterized. In this embodiment, the polynucleotide is a portion of the target polypeptide to be characterized. It has a longer length than [this]. This means that the length of the polypeptide portion that can be characterized is [this]. Renucleotide handling proteins control the amount of polynucleotides needed to move Therefore, it is guaranteed that there will be no restrictions.

[0062] This method is illustrated by referring to Figure 1, which shows one non-limiting example of the disclosed method. It can be understood. Conjugates may include polynucleotides and polypeptides, poly Peptides thread nanopores, polynucleotide handling proteins It is brought into contact with a substance. In the illustrated embodiment, further polynucleotides are used , facilitating the threading of polypeptides through nanopores. Such use is disclosed. This is within the scope of possible methods, but it is not mandatory.

[0063] Polynucleotide handling proteins are conjugated into polypeptides. It processes polynucleotides. Polynucleotide handling proteins are poly When processing nucleotides, the conjugate passes through the nanopore, therefore Polypeptides pass through nanopores. When polypeptides pass through nanopores, the polypeptides Chido is characterized.

[0064] In the example shown in Figure 1, the polynucleotide handling protein is polynucleotide handling From the "perspective" of the doping protein, move the conjugate "inside" the pore. Example For example, as shown, polynucleotide handling proteins are used in nanopores. It is located on the side of the transformer and moves the conjugate into the pore, i.e., from the cis side to the transformer side. The opposite setting can also be used.

[0065] In other words, in some embodiments, polynucleotide handling proteins The quality is located on the cis side of the nanopore, and the polynucleotide handling protein is located on the nanopore. This controls the movement of the conjugate from the cis side to the trans side of the nanopore. Therefore, In some embodiments, the polynucleotide handling protein is located on the cis side of the nanopore. Located at this position, the polynucleotide handling protein moves from the cis side of the nanopore to the nanopore. Controlling the movement of polynucleotides to the trans side, thereby controlling the movement of polynucleotides through nanopores. Control the movement of the cydoid.

[0066] In another embodiment, a polynucleotide handling protein is used in nanopores. Located on the trans side, the polynucleotide handling protein is located from the trans side of the nanopore. Controlling the movement of the conjugate to the cis side of the nanopore. Therefore, several implementations In this state, the polynucleotide handling protein is located on the trans side of the nanopore, The renucleotide handling protein moves from the trans side of the nanopore to the cis side of the nanopore. Control the movement of polynucleotides to and thereby the movement of polypeptides through nanopores. To control.

[0067] As described herein, a conjugate may include a leader. Any suitable leader can be used to achieve this. In some cases, the leader is It can be a polynucleotide. The leader is the same as the polynucleotide in the conjugate. It may be, or it may be different. As explained above, the leader passes through the nanopore This can facilitate threading of the denjugate.

[0068] In other words, in some embodiments, the conjugate is L-{PN}-P m of It includes one or more structures of form, -L is the leader, and L is optionally part N. -P is a polypeptide, -N contains a polynucleotide, -m is 0 or 1, The method involves threading the leader (L) through a nanopore, thereby creating polypeptides. This may include bringing (P) into contact with the nanopore.

[0069] In some such embodiments, polynucleotide handling proteins Located on the cis side of the nanopore, the method involves a polynucleotide handling protein, which is then used in the nano Controlling the movement of the polynucleotide moiety (N) from the cis side of the pore to the trans side of the nanopore. This makes it possible to control the movement of polypeptides (P) through nanopores. Includes. In other embodiments, the polynucleotide handling protein is a nanopore Located on the trans side, the method involves polynucleotide handling proteins in nanopores. Controlling the movement of the polynucleotide (N) from the trans side to the cis side of the nanopore. This enables and thereby controls the movement of polypeptides (P) through nanopores. .

[0070] As will be described in more detail herein, a conjugate is one or more adapters This may include a call / or anchor. A non-restrictive example of such a setting is shown in Figure 2.

[0071] As will be described in more detail herein, in some embodiments, the conjugate is , comprising multiple polynucleotides and polypeptides. In such embodiments, polynu Cleotide handling proteins facilitate the continuous transfer of polynucleotides to nanopores. This precisely controls and, therefore, allows for the continuous movement of polypeptides into nanopores. Thus, each polypeptide within the conjugate is characterized sequentially in the disclosed manner. It can be attached.

[0072] For example, the conjugate is L-P1-N-{PN} n -P m One or more forms of It may include construction, -n is a positive integer, -L is the leader, and L is optionally part N. -Each P may be the same or different; -Each N, which may be the same or different, contains a polynucleotide, and -m is 0 or 1, The method involves threading the leader (L) through a nanopore, thereby creating polypeptides. This may include bringing (P1) into contact with the nanopore.

[0073] Typically, in such embodiments, n is 1 to about 1000, for example, 2 to about 100, e.g. For example, approximately 3 to approximately 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0074] In some such embodiments, polynucleotide handling proteins Located on the cis side of the nanopore, the method involves a polynucleotide handling protein, which is then used in the nano The continuous movement of each polynucleotide (N) from the cis side of the pore to the trans side of the nanopore. This allows for control, thereby enabling each polypeptide (P) to pass through the nanopore continuously. This includes controlling the movement. In other such embodiments, polynucleotides The doring protein is located on the trans side of the nanopore, and the method involves polynucleotide hands. The ring protein moves from the trans side of the nanopore to the cis side of the nanopore, and each polynucleotide This makes it possible to continuously control the movement of do (N), thereby allowing each polypeptide (P) to This includes controlling the movement of a material as it continuously passes through a nanopore.

[0075] Those skilled in the art will know that if the conjugate contains more than one polypeptide, (more than specified herein) (As described in detail) Polynucleotide handling proteins and polypeptides It can be advantageous that the conjugate remains bound without dissociating upon contact. You will understand that, for example, as shown in Figure 3, this allows for polynucleotide Handling proteins control the movement of conjugates relative to nanopores. Because the polypeptide portion in the conjugate moves along the contiguous portion of the polynucleotide, As you come into contact with the minutes, it becomes possible to pass through them.

[0076] A non-limiting example of a more complex configuration according to the embodiment shown in Figure 3 is shown in Figure 4. Therefore, various adapters and tethers are used to facilitate polypeptide characterization. As schematically shown in Figure 5, a person skilled in the art can incorporate multiple such sections. You will understand that this is possible, but only one polypeptide section is shown in Figure 4. It is being done.

[0077] Another non-limiting embodiment of the disclosed method is schematically shown in Figure 6. The compound may contain polynucleotides and polypeptides, with the polypeptides forming nanopores. It is brought into contact with polynucleotide handling proteins in a loading manner. (Figure) In the shown embodiment, a leader (which is optionally a further polynucleotide) is used , facilitating the threading of polypeptides through nanopores. Such use is disclosed. This is within the scope of possible methods, but it is not mandatory.

[0078] Polynucleotide handling proteins are conjugated into polypeptides. It processes polynucleotides. Polynucleotide handling proteins are poly When processing nucleotides, the conjugate passes through the nanopore, therefore Polypeptides pass through nanopores. When polypeptides pass through nanopores, the polypeptides Chido is characterized.

[0079] In the example shown in Figure 6, the polynucleotide handling protein is polynucleotide handling From the "perspective" of the binding protein, the conjugate is moved "outside" the pore. Example For example, as shown, polynucleotide handling proteins are cis nanopores It is positioned on the side and moves the conjugate into the pore, i.e., from the transformer side to the cis side. The opposite setting can also be used.

[0080] In other words, in some embodiments, polynucleotide handling proteins The quality is located on the cis side of the nanopore, and the polynucleotide handling protein is located on the nanopore. This controls the movement of the conjugate from the transformer side to the cis side of the nanopore. Therefore, In some embodiments, polynucleotide handling proteins are used in nanopores. Located on the trans side, the polynucleotide handling protein is located from the trans side of the nanopore. Controlling the movement of polynucleotides to the cis side of the nanopore, thereby controlling the passage of polynucleotides through the nanopore. It controls the movement of peptides.

[0081] In other embodiments, the polynucleotide handling protein is located on the trans side of the nanopore, and the polynucleotide handling protein controls movement of the conjugate from the cis side of the nanopore to the trans side of the nanopore. Accordingly, in some embodiments thereof, the polynucleotide handling protein is located on the trans side of the nanopore , wherein the polynucleotide handling protein controls movement of the polynucleotide from the cis side of the nanopore to the trans side of the nanopore, thereby controlling movement of the polypeptide through the nanopore .

[0082] Using the same notation as above, in some embodiments, the conjugate comprises L-{ P-N}-P m comprising one or more structures of the form -L is a leader, L is optionally an N moiety, -P is a polypeptide, -N comprises a polynucleotide, -m is 0 or 1, the method may comprise threading the leader (L) through the nanopore, thereby bringing the polypeptide (P) into contact with the nanopore.

[0083] 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 (N) from the trans side of the nanopore to the cis side of the nanopore , thereby controlling movement of the polypeptide (P) through the nanopore . In other such embodiments, the polynucleotide handling protein is a nano Located on the trans side of the pore, the method involves polynucleotide handling proteins, nano This controls the movement of polynucleotides (N) from the cis side of a pore to the trans side of a nanopore. This enables the control of polypeptide (P) movement through nanopores. nothing.

[0084] Several embodiments, in particular as discussed above, polynucleotide handling proteins In an embodiment in which the substance controls the movement of the conjugate "outside" the nanopore, the conjugate The linker may include a blocking portion attached to the polypeptide via an optional linker. The blocking portion is typically too large to pass through the nanopore. When the movement of the conjugate relative to A brings the blocking portion into contact with the nanopore, the nanopore Further movement of the conjugate through A is prevented. In such a case, the polynucleotide Dehandling proteins can potentially temporarily break the bond from the conjugate. It has properties. Under a conjugate applied force (e.g., electric potential or chemical potential) In embodiments of the disclosed method, the conjugate moves toward the nanopore, and the poly The pore is opened in the opposite direction to the movement controlled by nucleotide handling proteins. And it can "go back". The conjugate can move back through the pore, The polypeptide portion of the code can be characterized again.

[0085] This process sequentially conjugates polynucleotide handling proteins By combining and recombining, this can be repeated multiple times. In this way, The conjugate can vibrate through the pore (i.e., through the nanopore "f can be lost). This "flossing" allows the polypeptide of the conjugate portion to be repeatedly characterized by the nanopore. In some embodiments , this allows for improved accuracy of the characterization information.

[0086] In such embodiments, any suitable blocking moiety may be used. For example, the conjugate may be modified with biotin, and the blocking moiety may be, for example, strep travidin, avidin or neutravidin. The blocking moiety may be a large chemical group such as a dendrimer. The blocking moiety may be a nanoparticle or bea d. Other suitable blocking moieties will be apparent to those skilled in the art.

[0087] A non-limiting example of such a method is shown in Figure 7.

[0088] Accordingly, in some embodiments, the method comprises: i) contacting the conjugate with the nanopore such that the blocking moiety is on the opposite side of the nanopore relative to the polynucleotide handling protein; ii) contacting the polynucleotide of the conjugate with the polynucleotide handling protein ; iii) allowing the polynucleotide handling protein to control movement of the polynucleotide with respect to the nanopore , thereby controlling movement of the polypeptide through the nanopore; ; iv) wherein when the blocking moiety contacts the nanopore and thereby prevents further movement of the conjuga te through the nanopore, the polynucleotide handling protein is allowed to process the polynucleo The bond is temporarily broken from the nucleotide, thereby, under the applied force, the polynucleotide The conjugate moves in the opposite direction to the direction of movement controlled by the doring protein in the nanopore. To enable movement through, v) Optionally, repeat steps (ii) to (iv) to pass the polypeptide through the nanopore. This includes causing the cyd to vibrate.

[0089] Displacer unit As described above, without being bound by theory, the inventors have found a shorter barrel or a shorter choke. Nanopores with longer barrels or channels are used compared to nanopores with a flannel. When used, it has been found that the length of the characteristic polypeptide is typically improved. As explained above, this is schematically shown in Figure 8A(A), "R It is thought that the distance is correlated with or determined by the "ED" distance.

[0090] Therefore, in some embodiments, the nanopore is a polynucleotide handling line. The nanopore is modified to extend the distance between the protein and the narrowed region of the nanopore. Typically, polynucleotide handling proteins conjugate nanopores. Polynucleotides are determined when used to control the movement of the gate. The endodontic protein is modified to extend the distance between it and the constricted region of the nanopore. In some embodiments, to control the movement of the conjugate relative to the nanopore When used, polynucleotide handling proteins come into contact with nanopores, for example. For example, it is in a "seated position" that is in contact with the cis or trans opening of the nanopore. This is explained in more detail in the specification and is schematically shown in Figure 8A(B).

[0091] In some embodiments, the active site of a polynucleotide handling protein and The distance to the nanopore can be extended by using a displacer unit. The usage of the displacer unit is schematically shown in Figure 8A(C). Therefore, In some embodiments, the method provided herein provides a displacer unit. This includes doing so. In some embodiments, the displacer unit is polyp The nucleotide handling protein was isolated from the nanopore, thereby enabling polynucleotide handling. This is intended to extend the distance between the ring protein and the nanopore.

[0092] In such embodiments, any suitable displacer unit can be used. Yes, it is possible. For example, the displacer unit can be provided as a protein.

[0093] Any suitable protein can be used as a displacer unit. Example Typical proteins employ ring-shaped conformations such as polymers, Therefore, it contains a protein that can be easily positioned at the entrance of the nanopore. Includes the nanopores, helicases (e.g., T7 helicase) and their variants as described above. Many suitable ring-shaped proteins are known in this field. Displacers are, It does not need to have activity of its own. In some embodiments, the displacer Knit contains any polynucleotides or peptides in the conjugate. It does not provide any meaningful identification.

[0094] In some embodiments, the displacer unit comprises one or more polynucleo tide handling proteins or inactive variants thereof. This is schematically shown in FIG. 8B. As shown, the polynucleotide handling protein (E1) is used to control movement of the conjugate relative to the nanopore. The polynucleo tide handling proteins E2…E n do not control movement of the conjugate relative to the nanopore because they are initially associated with the polypeptide portion of the conjugate. However, they displace the polynucleotide handling protein E1 from the nanopore, increasing RED .

[0095] In such embodiments, the polynucleoti de handling protein used as the displacer unit may be the same as or different from the polynu cleotide handling protein used to control movement of the conjugate. In some embodi ments, the polynucleotide handling protei n used as the displacer unit is the same polynucleo tide handling protein formed from an inactive variant thereof.

[0096] One or more displacer units, for example one or more displacer units described herein, can be attached (e.g., covalently or non-covalently) to the nanopore. Alternatively, one or more displacer units can be associated with the nanopore, for example by polynucleotide control of their position relative to the nanopore .

[0097] In other embodiments, the polynucleotide handling protein is polynucleotide Modified to extend the distance from the active site of the handling protein to the nanopore. Polynucleotide handling proteins are typically polynucleotide handling proteins. Ring proteins are used to control the movement of conjugates to nanopores. Sometimes, as is determined, the active site of polynucleotide handling proteins and nanoparticles The distance between A and B is modified to be extended. This is described in more detail herein. .

[0098] polypeptide As explained above, the disclosed method involves the conjugate moving relative to the nanopore. This sometimes involves characterizing the target polypeptide within the conjugate.

[0099] Any suitable polypeptide may be characterized in the manner disclosed.

[0100] In some embodiments, the target polypeptide is an unmodified protein or a portion thereof. It is a molecule, or a naturally occurring polypeptide, or a portion thereof.

[0101] In some embodiments, the target polypeptide is secreted from the cell. Alternatively, The target polypeptide must be extracted from the cell for characterization by the disclosed method. To prevent this, it can be produced within the cell. Polypeptides are plasmids, for example, Sam brook et al.,Molecular Cloning: A Laborat Ory Manual, 4 th ed., Cold Spring Harbor Pr ess, Plainsview, New York (2012), and Ausubel et al.,Current Protocols in Molecular B iology(Supplement 114),John Wiley&Sons,N To clone the protein according to the method described in New York (2016) It may contain the cellular expression product of the plasmid used.

[0102] Polypeptides can be obtained from or extracted from any organism or microorganism. Polypeptides can be obtained from humans or animals, for example, from urine, lymph, saliva, mucus, and semen. Polypeptides can be obtained from liquid or amniotic fluid, or from whole blood, plasma, or serum. It can be obtained from plants, such as grains, legumes, fruits, or vegetables.

[0103] The target polypeptide is an impure mixture of one or more polypeptides and one or more impurities. It may be provided as a “target polyp This may include a truncated form of the target polypeptide, which is different from "Petido." For example, the target polypeptide may be a full-length protein, and the impurities may contain a protein fraction. It is possible. Impurities may also contain proteins other than the target protein, which are, for example, fine It can be co-purified from the cell culture or obtained from the sample.

[0104] Polypeptides are any amino acid, amino acid analogues, and modified amino acids (i.e., It may contain any combination of amino acid derivatives in the polypeptide. Conductors (and analogues, etc.) can be distinguished by their physical size and electric charge.

[0105] Amino acids / derivatives / analogs, whether naturally occurring or artificial, good.

[0106] In some embodiments, the polypeptide comprises any naturally occurring amino acids. Obtained. 20 amino acids are coded by the universal genetic code. These are Alani N(A), Arginine(R), Asparagine(N), Aspartic acid(D), Cysteine (C), glutamic acid / glutamate (E), glutamine (Q), glycine (G), histamine Thidine (H), Isoleucine (I), Leucine (L), Lysine (K), Methionine (M) Phenylalanine (F), proline (P), serine (S), threonine (T), trip These are tophan (W), tyrosine (Y), and valine (V). Other naturally occurring mysids No acids include selenocysteine ​​and pyrrolicin.

[0107] In some embodiments, the polypeptide is modified. The polypeptide is then modified for detection using the disclosed method. In terms of application, the disclosed method is for characterizing the modification in the target polypeptide. It is.

[0108] In some embodiments, one or more amino acids / derivatives / analogs in the polypeptide are modified Decorated. In some embodiments, amino acids / derivatives / analogous compounds in polypeptides. One or more of the bodies are post-translation modified. Therefore, the method disclosed herein is Used to detect the presence, absence, and number of positions of post-translational modifications in lipeptides. This can be done. The disclosed method characterizes the degree to which a polypeptide is post-translationally modified. It can be used for that purpose.

[0109] Any one or more post-translational modifications can be present in a polypeptide. Typical post-translational modifications include: Modification by hydrophobic groups, modification by cofactors, addition of chemical groups, saccharification (non-enzymatic bonding of sugars), This includes otinization and pegylation. Post-translational modifications are used for bioengineering or biomedical purposes. In some cases, the chemical modifications performed in the laboratory are unnatural. This can lead to the opposite of natural countermeasures. In contrast to the actual product, the peptides, polypeptides, or proteins produced in the laboratory are It may become possible to monitor the bell.

[0110] Examples of post-translational modifications by hydrophobic groups include myristoylation, myristic acid, and C 14 saturated acid Adhesion; palmitoylation, palmitic acid, C 16 Adhesion of saturated acid; isoprenylation or pre Nylation, attachment of isoprenoid group; Farnesylation, attachment of farnesol group; Geranyl Geranylation, attachment of geranylgeraniol groups; and glycation and amide bonding. This includes glycosylphosphatidylinositol (GPI) anchor formation via synthesis.

[0111] Examples of post-translational modifications by cofactors include lipoylation and attachment of lipoate (C8) functional groups; Rabinization, flavin moiety (e.g., flavin mononucleotide (FMN) or flavin a Attachment of denine dinucleotide (FAD); for example, via a thioether bond with cysteine. Attachment of heme C; phosphopantheteinylation, attachment of 4'-phosphopantheteinyl group; This includes the formation of retynlidene schiff bases.

[0112] Examples of post-translational modifications by the addition of chemical groups include acylation, for example, O-acylation (esterification). ), N-acylation (amide) or S-acylation (thioester); acetylation, for example Attachment of an acetyl group to the N-terminus or lysine; formylation; alkylation, methyl or ethyl Addition of alkyl groups such as lysine; methylation, for example, the addition of a methyl group to lysine or arginine. Addition; amidation; butyric oxidation; gamma carboxylation; glycosylation, e.g., arginine, as Paragine, cysteine, hydroxylysine, serine, threonine, tyrosine, or trip Enzymatic attachment of glycosyl groups to tophan; polysialylation, attachment of polysialic acid; mackerel Hydroxylation; iodization; bromination; citrullination; nucleotide addition, discussed above. Attachment of any nucleotide, such as one of those being attached, ADP-ribosylation; oxidation Phosphorylation, e.g., serine, threonine, or tyrosine (O-linked) or histidine Attachment of a phosphate group to an N-bond; adenylylation, e.g., tyrosine (O-bonded form) or Attachment of the adenylyl moiety to histidine or lysine (N-linked); propionation; pi Formation of logglutamic acid; S-glutathione; smoylation; S-nitrosylation; succinylation, e.g., attachment of a succinyl group to lysine; selenoylation , selenium uptake; and ubiquitination, pursuit of ubiquitin subunits (N-linked type) It includes additions.

[0113] Labeling of polypeptides with molecular labels is within the scope of the methods provided herein. Molecular labeling facilitates the detection of polypeptides in the methods provided herein. This could be a modification of the peptide. For example, labeling may occur when the conjugate is characterized. This could involve modifying the polypeptide to alter the resulting signal. For example, the labeling could be done on a nanopoly The label can interfere with the flow of ions through A. In this way, the label can improve the sensitivity of the method. .

[0114] In some embodiments, the polypeptide is one or more cross-linked sections, e.g. For example, including a CC bridge. In some embodiments, the polypeptide is disclosed. It is not cross-linked before being characterized using the method.

[0115] In some embodiments, the polypeptide comprises sulfide-containing amino acids, and therefore Therefore, there is a possibility of forming a disulfide bond. Typically, in such embodiments Before the polypeptide is characterized using the disclosed method, DTT (dithio Trial (such as sreitol) or TCEP (tris(2-carboxyethyl)phosphine) It is reduced using medicine.

[0116] In some embodiments, polypeptides are full-length proteins or naturally occurring proteins. It is a polypeptide. In some embodiments, it is a protein or a naturally occurring polypeptide. Lipeptides are fragmented before they are conjugated into polynucleotides. In the embodiment, the protein or polypeptide is chemically or enzymatically fragmented. In some embodiments, the polypeptide or polypeptide fragment is longer It can be conjugated to form a target polypeptide.

[0117] The polypeptide can be any appropriate polypeptide of any length. In some embodiments... In this case, the polypeptide has a length of approximately 2 to 300 peptide units. Several implementations Morphologically, polypeptides consist of approximately 2 to 100 peptide units, for example, approximately 2 to 50 peptide units. Peptide units, for example, approximately 2 to approximately 40 peptide units, for example, approximately 2 to approximately 30 peptide units, For example, approximately 2 to approximately 25 peptide units, for example, approximately 2 to approximately 20 peptide units; or approximately 3 to Approximately 50 peptide units, for example, approximately 3 to approximately 40 peptide units, for example, approximately 3 to approximately 30 peptides units, for example, approximately 3 to approximately 25 peptide units, for example, approximately 3 to approximately 20 peptide units; also This is approximately 5 to approximately 50 peptide units, for example, approximately 5 to approximately 40 peptide units, for example, approximately 5 to approximately 30 peptide units, for example, approximately 5 to approximately 25 peptide units, for example, approximately 5 to approximately 20 peptides Units; for example, about 7 to about 16 peptide units, for example, about 9 to about 12 peptide units; also It has a length of approximately 16 to 25 peptide units, for example, approximately 18 to 22 peptide units. .

[0118] Any number of polypeptides can be characterized in the disclosed manner. For example, by is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, or more The above may relate to the characterization of polynucleotides. When two or more polypeptides are used... These could be two or more examples of different polypeptides or the same polypeptide.

[0119] Therefore, the measurements performed by the disclosed method are typically (i) the length of the polypeptide. (ii) polypeptide identity, (iii) polypeptide sequence, (iv) polypeptide The secondary structure of the cytoside and whether the (v) polypeptide is modified are selected from the available options. It is clear that the lipeptide is characterized by one or more properties. In a typical embodiment, The measurement involves the sequence of the polypeptide, or whether the polypeptide has, for example, one or more post-translational modifications. It is characterized by whether or not it is modified by. In some embodiments, the measurement is, This is a characteristic of the lipeptide sequence.

[0120] In some embodiments, the polypeptide is in a relaxed form. Morphologically, polypeptides are maintained in a linearized form. Retaining lipeptides prevents polypeptide "bunching" within the nanopore. Therefore, this can facilitate the characterization of polypeptides at the residue level.

[0121] Polypeptides can be held in a linearized form using any suitable means.

[0122] For example, if a polypeptide is charged, applying a voltage will cause the polypeptide to become charged. It can be held in a linearized form.

[0123] If the polypeptide is uncharged or only slightly charged, adjust the pH. By doing so, the charge can be changed or controlled. For example, by increasing the relative negative charge of a polypeptide... By using a high pH to maintain the polypeptide in a linearized form, This can be achieved. Increasing the negative charge of the polypeptide results in a linearized shape, for example, under a positive voltage. It can be maintained in that state. Alternatively, to increase the relative positive charge of the polypeptide. By using a low pH, polypeptides can be retained in a linearized form. Increasing the positive charge of the polypeptide allows it to be retained in a linearized form, for example, under a negative voltage. This is possible. In the disclosed method, polynucleotide handling protein This is used to control the movement of polynucleotides into nanopores. Otides are typically negatively charged, and like polynucleotides, they can increase pH. This enhances the linearization of the polypeptide, thereby making the polypeptide more negatively charged. This is generally the most appropriate approach. In this way, the conjugate maintains a negative charge overall. Therefore, it can be easily moved under applied voltage.

[0124] Polypeptides are retained in a linearized form by using appropriate denaturation conditions. To obtain. Appropriate denaturation conditions include, for example, appropriate guanidine HCl and / or urea. This includes the presence of a denaturing agent of a certain concentration. The concentration of such a denaturing agent used in the disclosed method is Depending on the target polypeptide characterized by the method, it can be easily selected by those skilled in the art. can.

[0125] Polypeptides can be retained in a linearized form by using an appropriate surfactant. It may be. Detergents suitable for use in the disclosed manner include SDS (Sodium Dodecyl Sulfate) It includes (mu).

[0126] The polypeptide is preserved in a linearized form by carrying out the disclosed method at high temperature. It can be maintained. When the temperature is increased, the intrachain bonds are overcome, and the polypeptide takes on a linearized form. It is possible.

[0127] Polypeptides are linearized by performing the method disclosed under strong electroosmosis. Such a force can be maintained in that form. By using the asymmetric salt condition, and / or can be provided by providing an appropriate charge to the channel of the nanopore. The charge of protein nanopore channels can be modified, for example, by mutagenesis. Changing the charge of a nanopore is within the capabilities of those skilled in the art. When the potential is changed, when an electric potential is applied across the nanopore, the cations and anions passing through the nanopore... A strong electroosmotic force is generated from an unbalanced flow of ON energy.

[0128] Polypeptides pass through structures such as nanopillar arrays, nanoslits, or nanogangs. By crossing the top, it can be held in a linearized form. In some embodiments Therefore, the physical constraints of such a structure force the polypeptide to adopt a linearized form. It is possible.

[0129] Conjugate formation As will be described in more detail herein, the conjugate conjugates the target polypeptide. Contains jugated polynucleotides.

[0130] The target polypeptide can be conjugated to a polynucleotide at any appropriate position. For example, polypeptides have polynucleotides at their N-terminus or C-terminus. Polypeptides can be conjugated. The polypeptide consists of residues (e.g., amino acids) within the polypeptide. It can be conjugated into polynucleotides via the side chain groups of the residues.

[0131] In some embodiments, the target polypeptide is conjugated to a polynucleotide. It has naturally occurring reactive functional groups that can be used to promote methylation. For example, Stain residues are used to create disulfide bonds to polynucleotides or modifying groups thereon. It is possible to form this.

[0132] In some embodiments, the target polypeptide is its conduit to a polynucleotide. Modified to facilitate polypegation. For example, in some embodiments, polypeg Butido involves attaching a portion containing a reactive functional group for attachment to a polynucleotide. Modified by, for example, in some embodiments, the polypeptide is polynucleotide One containing one or more reactive functional groups to react with the corresponding reactive functional group on the rheotide The above residues (e.g., amino acid residues) can be extended at the N-terminus or C-terminus. For example, In some embodiments, the polypeptide is N by one or more cysteine ​​residues. Such residues can be extended at the terminal and / or C-terminus. (For example, azido-maleimide conversion of cysteine, such as azido-[Pol]-maleimide) Reaction with a compound, where [Pol] is typically PEG, e.g., PEG2, PEG3, or short-chain polymers such as PEG4; followed by appropriately functionalized polynucleotides For example, coupling to polynucleotides possessing a BCN group for reaction with azide It can be used (by G) to attach to the polynucleotide portion of the conjugate. The chemical properties are described in Example 2. To avoid misunderstanding, the polypeptide is N-terminus. and / or a suitable naturally occurring residue at the C-terminus (e.g., N-terminus and / or C-terminus) If the end contains a naturally occurring cysteine ​​residue, such residue is considered a polynucleotide. It can be used for adhesion to [a certain surface].

[0133] In some embodiments, the residue in the target polypeptide is the poly It is modified to promote attachment to nucleotides. In some embodiments, poly Residues in peptides (e.g., amino acid residues) are chemically attached to polynucleotides. It is modified in some way. Mino acid residues are enzymatically modified to attach to polynucleotides.

[0134] The conjugation chemistry between polynucleotides and polypeptides in a conjugate is However, it 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. Exemplary reactive groups and their corresponding targets include aryl amides that can react with amines. Carbodiimides that can react with amines and carboxyl groups, and hyaluronic acid that can react with carbohydrates. Drazide, hydroxymethylphosphine that can react with amines, imide that can react with amines Esters, isocyanates that can react with hydroxyl groups, and carbohydrates that can react with hydrazines. NHS esters that can react with nyl, sulfhydryl groups, maleimides, and amines. PFP esters that can react with amines, psoralens and sulfhydryls that can react with thymines. Pyridyl disulfide, sulfhydrylamine, and hydroxyl groups that can react with the group This includes vinyl sulfones, vinyl sulfonamides, and other suitable materials.

[0135] Other suitable chemistry for conjugating polypeptides into polynucleotides includes This includes click chemistry. Many suitable click chemistry reagents are included in this technology. Known in the field. Good examples of click chemistry include, but these include Not limited. (a) Azide-alkyne cycloaddition by copper(I) catalyst (azide-alkyne hysgen cycloaddition) (Additional), (b) Strain-accelerated azide-alkyne cycloaddition; [3+2] cycloaddition of alkenes and azides; The reverse demand Diels-Alder reaction between alkenes and tetrazine; and the reaction between alkenes and tetrazole. Including the photoclick reaction, (c) Azide, for example, in a cyclooctane ring, for example, bicyclic [6.1.0]nonine (BC A copper-free barrier reacts with strained alkynes in N in a 1,3-dipolar cycloaddition reaction. Ant, (d) Reactivity of the oxygen nucleophile on one linker with the epoxide or aziridine on the other. Reaction with the part, and (e) Substitution of the alkyne moiety with an arylphosphine to induce a specific reaction with the azide. A Staudinger linkage can be formed by adding an amide bond.

[0136] Any reactive group can be used to form the conjugate. A suitable reactive group is [1,4-bis[3-(2-pyridyldithio)propionamide] Butane; 1,1-1-Bis-maleimidetriethylene glycol; 3,3'-Dithiodipropyl Di(N-hydroxysuccinimide) ropionic acid; ethylene glycol-bis( N-hydroxysuccinimide succinate; 4,4'-diisothiocyanatosty Ruben-2,2'-disulfonate disodium salt; bis[2-(4-azidosalicylamine] [Ethyl] disulfide; N-hydroxy 3-(2-pyridyldithio)propionate cucinimide ester; 4-maleimidobutyrate N-hydroxysuccinimide ester; yo N-hydroxysuccinimide acetate; N-acetylthioglycolate Droxysuccinimide esters; azide-PEG-maleimide; and alkyne-PE It contains G-maleimide. The reactive group is described in WO2010 / 086602, in particular in the table of its application. Any of those disclosed in Section 3 may be used.

[0137] In some embodiments, the reactive functional group is polynucleotide prior to the conjugation step. The target functional group is contained in the polypeptide, which is contained in the rheotide. In other embodiments, the conjugate Before the gated step, the reactive functional group is contained in the polypeptide, and the target functional group is contained in the polynucleotide. It is contained in the rheotide. In some embodiments, the reactive functional group is directly attached to the polypeptide. They are in contact with each other. In some embodiments, the reactive functional group is connected to the polymer via a spacer. It is attached to the petroleum. Any suitable spacer can be used. SAM includes, for example, alkyldiamines such as ethyldiamine.

[0138] As is clear from the above considerations, in some embodiments, the conjugate is multiple Includes polypeptide sections and / or multiple polynucleotide sections. For example In a conjugate, P is a polypeptide and N is a polynucleotide, morphologically... - May include the structure PNPNPN…. In such embodiments, polynucle The rheotide handling protein continuously applies the N portion of the conjugate to the nanopore. This controls the movement of the P section relative to the nanopore, and therefore continuously controls it. This enables the continuous characterization of the P section. In such embodiments, multiple A number of polynucleotides and polypeptides are combined together by the same or different chemicals. It can be jugated.

[0139] As described herein, a conjugate may include a leader. Any suitable reader can be used to achieve this. In some embodiments In embodiments where the leader is a polynucleotide, The leader is the same type of polynucleotide used in the conjugate. The leader could be a creotide, or it could be a different type of polynucleotide. For example, the polynucleotides in the conjugate could be DNA, and the leader could be RNA. It is possible, or the opposite is possible.

[0140] In some embodiments, the leader is a charged polymer, for example, a negatively charged polymer. It is a polymer. In some embodiments, the leader uses a polymer such as PEG or a polysaccharide. Includes. In such embodiments, the leader has 10 to 150 monomer units (e.g., E Length of ethylene glycol or sugar units, e.g., 20-120, e.g., 30-100 For example, 40-80 monomer units, for example, 50-70 monomer units (for example, ethylene glycoside) It can be the length of a scalar or sugar unit.

[0141] Polynucleotides As will be described in more detail herein, the method provided herein is a polypeptide The polynucleotides are conjugated into polynucleotides, and polynucleotide handling proteins are created. This includes using it to control the movement of the conjugate relative to the nanopore.

[0142] Any suitable polynucleotide can be used in the disclosed method.

[0143] In some embodiments, polynucleotides are secreted from cells. Alternatively, polynucleotides Cleotides must be extracted from cells for use in the disclosed method. It can be produced within cells.

[0144] A polynucleotide is an impure mixture of one or more polynucleotides and one or more impurities. It may be supplied as a compound. Impurities are polynucleotides used for the formation of the conjugate. It may contain partially excised polynucleotides, different from otidols. For example, conjugate The polynucleotides used for the formation of the genome can be genomic DNA, and impurities are genome It may contain fractions such as DNA and plasmids. Target polynucleotides are a subset of genomic DNA. Undesirable polynucleotides, which may be coding regions, can include non-coding regions of DNA. .

[0145] Examples of polynucleotides include DNA and RNA. Bases can sometimes be identified by their physical size.

[0146] Polynucleotides, or nucleic acids, may contain any combination of any nucleotides. Nucleotides may be naturally occurring or artificial. One or more nucleotides in a polynucleotide. The nucleotides may be oxidized or methylated. One or more of these nucleotides in a polynucleotide. The nucleotides of a polynucleotide can be damaged. For example, polynucleotides are pyrimidine It may contain dimers. Such dimers are typically associated with UV damage. It is the main cause of cutaneous melanoma.

[0147] One or more nucleotides in a polynucleotide are modified, for example, with a label or tag. A suitable example thereof is known to those skilled in the art. A polynucleotide is a spacer of one or more spacers. It may include adapters, such as sequencing adapters, which may be included in polynucleotides. The adapters, tags, and spacers are described in more detail herein.

[0148] Examples of modified bases are disclosed herein and are obtained by means known in the art. For example, polymerase incorporation of modified nucleotide triphosphates in a strand copy (e.g., P Can be incorporated into polynucleotides by CR or polymerase packing method. In some embodiments, one or more bases are reagents known in the art. It can be modified by the chemical means used.

[0149] Nucleotides typically consist of a nucleic acid base, a sugar, and at least one phosphate group. Nucleic acid bases and sugars form nucleosides. Nucleic acid bases are typically heterocyclic. Nucleic acid bases include purines and pyrimidines, more specifically adenine (A) and guanine. (G), thymine (T), uracil (U), and cytosine (C) are included, but these include Not limited to sugars. Sugars are typically pentoses. Nucleotide sugars include ribose and This includes, but is not limited to, deoxyribose. The sugar is preferably deoxyribose. It is Bose. The polynucleotide is preferably the following nucleoside:deoxyadeno Syn (dA), deoxyuridine (dU) and / or thymidine (dT), deoxyg It contains anosine (dG) and deoxycytidine (dC). Nucleotides are typical These are ribonucleotides or deoxyribonucleotides. Nucleotides are typical Typically, a nucleotide contains monophosphate, diphosphate, or triphosphate. A nucleotide contains more than three phosphates. It may contain phosphate, for example, four or five phosphate groups. The phosphate group is located at the 5' end of the nucleotide. Alternatively, they may be attached to the 3' side. Nucleotides in a polynucleotide can be attached to each other in any manner. They can adhere. Nucleotides, typically like nucleic acids, have their sugar and phosphate groups attached. Therefore, they adhere. Nucleotides, like pyrimidine dimers, are formed by their nucleic acid bases. It can be connected in this way.

[0150] Polynucleotides can be double-stranded or single-stranded.

[0151] In some embodiments, polynucleotides are single-stranded DNA. In the application form, the polynucleotide is single-stranded RNA. In some embodiments, Polynucleotides are single-stranded DNA-RNA hybrids. Brids perform the ligation of single-stranded DNA into RNA, or vice versa. Polynucleotides can be prepared. Polynucleotides are most typically single-stranded deoxyribonucleic acid (DNA). Alternatively, it is single-stranded ribonucleic acid (RNA).

[0152] In some embodiments, the polynucleotide is double-stranded DNA. In this state, polynucleotides are double-stranded RNA. In some embodiments, A nucleotide is a double-stranded DNA-RNA hybrid. Hybrids can be prepared from single-stranded RNA by reverse transcription of cDNA complement.

[0153] Polynucleotides can be of any length. For example, polynucleotides can be at least Also 10, at least 50, at least 100, at least 150, at least 200, less At least 250, at least 300, at least 400, or at least 500 Nucles It can be the length of an octide or a pair of nucleotides. A polynucleotide is 1,000 nucleos Length of nucleotide or longer than the length of a nucleotide pair, or 5000 nucleotides or longer than a nucleotide pair It may be longer than 100,000 nucleotides or longer than the length of a nucleotide pair.

[0154] More typically, polynucleotides consist of approximately 1 to 10,000 nucleotides or nucleotides. It has the length of a rheotide pair. For example, about 1 to about 1000 nucleotides or nucleotides. pairs (for example, about 10 to about 1000 nucleotides or nucleotide pairs), for example, about 5 to Approximately 500 nucleotides or nucleotide pairs, for example, about 10 to about 100 nucleotides. or nucleotide pairs, for example, about 20 to about 80 nucleotides or nucleotide pairs, for example Approximately 30 to 50 nucleotides or nucleotide pairs.

[0155] Any number of polynucleotides can be used in the disclosed manner. For example, is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, or more This may include using the polynucleotides listed above. It may also include using two or more polynucleotides. In that case, they are either different polynucleotides or two of the same polynucleotide. One possible example is that polynucleotides may be naturally occurring or artificially created.

[0156] Nucleotides can have any identity, and nucleotides include adenosine monophosphate ( AMP, guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine Monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine Monophosphates, 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) This includes, but is not limited to, deoxymethylcytidine monophosphate. The rheotide is preferably AMP, TMP, GMP, CMP, UMP, dAMP, dTM Selected from P, dGMP, dCMP, and dUMP. Nucleotides are debased. Nucleotides can be (i.e., they can lack nucleic acid bases). Nucleotides can also lack nucleic acid bases and sugars. (That is, it is a C3 spacer.)

[0157] Polynucleotides are products of PCR reactions, genomic DNA, and endonucleases. May include substances and / or DNA libraries. Polynucleotides are found in any organism. Alternatively, they can be obtained from or extracted from microorganisms. Polynucleotides are hy From a vegetarian or animal, for example, from urine, lymph, saliva, mucus, semen, or amniotic fluid, It can be obtained from whole blood, plasma, or serum. Polynucleotides are found in plants, such as grains. Polynucleotides can be obtained from legumes, fruits, or vegetables. It may include. Genomic DNA can be fragmented. DNA can be fragmented by any preferred method. It is possible. For example, methods for fragmenting DNA are known in the art, and such methods A transposase such as MuA transposase can be used. Preferably Genomic DNA is not fragmented.

[0158] The labeling of polynucleotides with molecular labels is within the scope of the methods provided herein. The molecular label is a polynucleotide or condyl in the method provided herein. This could be a modification of polynucleotides that facilitates the detection of the nucleotide. For example, the label could be a nucleotide. Modify the signal obtained when the jugate is characterized, to the polynucleotide. Modification is possible. For example, a label can interfere with the flow of ions through a nanopore. Therefore, markings may improve the sensitivity of the method.

[0159] adapter In some embodiments of the methods provided herein, a polynucleotide is attached thereto It has a polynucleotide adapter attached. The adapter is typically a polynucleotide It contains polynucleotide chains that can be attached to the ends of the tide.

[0160] In some embodiments, the adapter is conjugated with the polypeptide. Before being attached to the polynucleotide, in some embodiments, an adapter It attaches to polynucleotide and polypeptide conjugates.

[0161] Therefore, in some embodiments, the method is an adapter (for example, as described herein). The adapter is attached to the polynucleotide, and the polynucleotide / adapter construct is marked. This includes forming a conjugate by conjugating it to a target polypeptide. In some embodiments, the conjugate is an adapter (for example, as described herein). The adapter is attached to the polynucleotide, and the adapter is attached to the target polypeptide. It is formed by causing a conjugate to form.

[0162] In some embodiments, the adapter conjugates to polynucleotides. It may be selected or modified to provide a specific part for a particular purpose.

[0163] The adapter can be attached to only one end of the polynucleotide or conjugate. Yes, it is possible. A polynucleotide adapter can handle both ends of a polynucleotide or conjugate. It can be attached to the end. Alternatively, a different adapter can be a polynucleotide or conjugate. It can be attached to the two ends of the character.

[0164] Adapters can be added to both strands of a double-stranded polynucleotide. Adapters can be attached to nucleotides. Adapters can be attached to polynucleotides. The method of adding is known in the art. The adapter is, for example, by ligation Therefore, through click chemistry, through tagmentation, topoisomerase It may be attached to the polynucleotide by conversion or by any other suitable method.

[0165] In one embodiment, the adapter or each adapter is synthetic or artificial. Typically, The adapter or each adapter contains the polymer described herein. In some embodiments, Each adapter includes a spacer as described herein. In that embodiment, the adapter or each adapter comprises a polynucleotide. Polynucleotide adapters can handle DNA, RNA, modified DNA (e.g., debased DNA), May include RNA, PNA, LNA, BNA, and / or PEG. Typically, such or Each adapter contains single-stranded and / or double-stranded DNA or RNA. It may contain polynucleotides of the same type as the polynucleotide chain to which it is attached. The adapter is a different type of polynucleotide than the polynucleotide chain it is attached to. It may contain tides. Polynucleotides used in some embodiments as disclosed. The octido strand is a single-stranded DNA strand, and the adapter is DNA or RNA, typically a single strand. It contains stranded DNA. In some embodiments, the polynucleotide is a double-stranded DNA strand. The adapter contains DNA or RNA, such as double-stranded or single-stranded DNA.

[0166] In some embodiments, the adapter may be a crosslinking portion. Double-stranded polynucleotide A bridging section can be used to connect two chains of ocid. For example, how many In that embodiment, the crosslinking portion is a double-stranded polynucleotide template strand. It is used to connect to the complementary strand of polynucleotides.

[0167] A crosslink typically covalently bonds two strands of a double-stranded polynucleotide. A fraction can be any one that can link two strands of a double-stranded polynucleotide. However, this is conditional on the cross-linking portion not interfering with the movement of polynucleotides to the nanopore. The number of items shall be determined. Suitable crosslinking portions include polymer linkers, chemical linkers, polynucleotides, The crosslinked portion may include, but is not limited to, polypeptides. Preferably, the crosslinked portion is D NA, RNA, modified DNA (e.g., debased DNA), RNA, PNA, LNA, or Contains PEG. The crosslinking portion is more preferably DNA or RNA.

[0168] In some embodiments, the bridging portion is a hairpin adapter. It is an adapter containing a single polynucleotide chain, and the ends of the polynucleotide chain are mutual They can hybridize to each other, or hybridize to each other and polynucleotides The central part of the octave forms a loop. A suitable hairpin loop adapter is for this technology. It can be designed using methods known in the field. In some embodiments, a hairpin loop These are typically 4-100 nucleotides in length, e.g., 4-50, e.g., 4-20 For example, the length is 4 to 8 nucleotides. In some embodiments, the crosslinking portion ( For example, a hairpin adapter is attached to one end of a double-stranded polynucleotide. The components (e.g., hairpin adapters) are typically attached to both ends of a double-stranded polynucleotide. I haven't done that.

[0169] In some embodiments, the adapter is a linear adapter. Polynucleotides can be bound to either or both ends of this chain of polynucleotides. If the otinode is a double-stranded polynucleotide, the linear adapter is a double-stranded polynucleotide. It can be attached to either or both ends of either or both chains. The adapter may include a leader sequence as described herein. A linear adapter is For hybridization with tags such as those described herein (e.g., pore tags) It may include the portion. A linear adapter is 10 to 150 nucleotides long, for example, 20 ~120, for example, 30~100, for example, 40~80, for example, 50~70 Nukureochi It can be any length. The linear adapter may be single-stranded. The linear adapter may also be double-stranded. stomach.

[0170] In some embodiments, the adapter may be a Y-adapter. Typically, it is a polynucleotide adapter. The Y adapter is typically double-stranded. Yes, (a) a region at one end where the two strands are hybridized together, and (b) the other end The ends contain two non-complementary regions of the chain. The non-complementary parts of the chain are typically, It forms a bar hang. The presence of a non-complementary region in the Y adapter is different from the double-stranded portion. Since the strands of the books typically do not hybridize with each other, the adapter is given a Y shape. The two single-chain sections of the adapter may be the same length or different lengths. For example, one single-stranded portion of a Y-adapter is 10 to 150 nucleotides long, for instance. 20-120, for example, 30-100, for example, 40-80, for example, 50-70 Nukure The length of the Otid can be such that the other single strand of the Y adapter is independently 10-15 Length of nucleotide, e.g., 20-120, e.g., 30-100, e.g., 40- 80, for example, it can be 50-70 nucleotides long. The double-stranded "ste" of the Y adapter. The "mu" part is, for example, a length of 10 to 150 nucleotides, for example, 20 to 120, for example They can be 30-100 nucleotides long, for example, 40-80 nucleotides long, or for example, 50-70 nucleotides long. ru.

[0171] The adapter targets the polynucleotide by any suitable means known in the art. It can be linked to the target polynucleotide. The adapter is synthesized separately and chemically attached to the target polynucleotide. Alternatively, the adapter can be used as a standard. They may be generated during the processing of the target polynucleotide. In some embodiments, The adapter is located at one end of the target polynucleotide, or near it. It is linked to the target polynucleotide. In some embodiments, the adapter is linked to the target polynucleotide. Within the last 50 nucleotides of the creotide, for example, within 20 nucleotides, for example, within 10 nucleotides, It is linked to the target polynucleotide. In some embodiments, the adapter is linked to the target polynucleotide. The target polynucleotide is linked at the end of the polynucleotide. The adapter is attached to the target polynucleotide. When linked to a nucleotide, the adapter is of the same type as the target polynucleotide. It may contain nucleotides of or different nucleotides from the target polynucleotide. It may include.

[0172] Adapters particularly suitable for use in the disclosed manner include linear homopolymer regions (e.g., about 5 to about 20 nucleotides, for example, about 10 to about 30 nucleotides, for example, thymine or To hybridize cytidine and / or one or more tethers or anchors It may include hybridization sites (such as those described in more detail herein). Such adapters also contain reactive functional groups for binding to the target polypeptide. It is possible. Click chemistry groups are particularly suitable in this respect. For example, included in adapters. Examples of groups used for this purpose include copper-free click chemistry that become particulate ( (particulate) a group that can be obtained, for example, BCN (bicyclo[6.1.0]nonine) This includes groups based on and its derivatives, such as the dibenzocyclooctin (DBCO) group. Reactions of such groups are well known in the art. For example, the BCN group is typically, for example Reacts with groups such as azides, tetrazines, and nitrones that can be incorporated into polypeptides. The DBCO group exhibits high reactivity with azide groups. In particular, other suitable chemical groups include 2 -Includes pyridine carboxyaldehyde (2-PCA) groups and their derivatives. For example, 6-(azidomethyl)-2-pyridinecarboxyaldehyde is the N-terminus of a peptide. It can react with amino groups.

[0173] Spacer In some embodiments of the methods provided herein, polynucleotides, polypeptides The conjugate formed by the reaction with the , or the adapter described herein, Spacers may be included. For example, one or more spacers may be included in the polynucleotide adapter. It is possible. For example, a polynucleotide adapter has 1 to about 20 spacers, for example , 1 to approximately 10, for example, 1 to approximately 5 spacers, for example, 1, 2, 3, 4, or 5 spacers It may include a spacer. The spacer may include any suitable number of spacer units. The ser proposes an energy barrier that hinders the movement of polynucleotide handling proteins. It can be used for polynucleotide handling on polynucleotides. By reducing the traction force of proteins, polynucleotide handling proteins This can cause quality to stall. For example, a debase spacer, i.e., a base that Use a spacer from which one or more nucleotides have been removed in the renucleotide adapter. This can be achieved by introducing a larger chemical group into the polyn. By physically hindering the movement of creotide handling proteins, polynucleotides The movement of tide-handling proteins can be physically blocked.

[0174] In some embodiments, one or more spacers pass through the nanopore. When they cross a nanopore, that is, when they move relative to a nanopore, Polynucleotides used in the manner claimed herein to provide a specific signal It is included in Otid or Conjugate, or in Adapter.

[0175] In some embodiments, the spacer may include linear molecules such as polymers. Typical In terms of the spacers, such spacers are different from the polynucleotides used in the conjugate. It has a structure such that, for example, if the polynucleotide is DNA, then the spacer - Typically, it does not contain DNA. In particular, polynucleotides are deoxyribonucleic acid. If it is (DNA) or ribonucleic acid (RNA), then the spacer or each spacer is preferably These are peptide nucleic acids (PNA), glycerol nucleic acids (GNA), and threose nucleic acids (TNA). This includes locked nucleic acids (LNA) or synthetic polymers having nucleotide side chains. In one embodiment, the spacer comprises one or more nitroindoles and one or more inosine. , one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines Phosphorus, one or more 5-bromodoxyuridines, one or more inverted thymidines (inverted dT) , one or more inverted dideoxythymidines (ddT), one or more dideoxycytidines ( ddC), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, One or more 2'-O-methylRNA bases, one or more iso-deoxycytidine (Iso- dC), one or more iso-deoxyguanosine (Iso-dG), one or more C3 (OC) 3H6OPO3) group, one or more light-cuttable (PC)[OC3H6-C(O)NHCH 2-C6H3NO2-CH(CH3)OPO3] group, one or more hexanediol groups, 1 One or more spacers 9(iSp9)[(OCH2CH2)3OPO3] or one The above spacers 18 (iSp18)[(OCH2CH2)6OPO3], or one The above thiol bonds may be included. The spacer may include any combination of these groups. Many of these groups are IDT (registered trademark) (Integrated DNA). It is commercially available from Technologies (registered trademark). For example, C3, iSp9 The iSp18 spacers are all available from IDT®. The spacer unit may include any number of the above-mentioned bases.

[0176] In some embodiments, the spacer is used to handle polynucleotide handling proteins. It may contain one or more chemical groups that cause stalling. In some embodiments, a preferred chemical group is 1 It is a pendant chemical group of one or more chemical groups. One or more chemical groups are polynucleotides, constructs or It can attach to one or more nucleic acid bases in the adapter. One or more chemical groups are polynucleo It can adhere to the frame of the Chido adapter. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1 There may be any number of suitable chemical groups, such as two or more. Suitable groups include full Orofoa, streptavidin and / or biotin, cholesterol, methylene blue Lu, dinitrophenol (DNP), digoxigenin and / or anti-digoxigenin , and dibenzylcyclooctin groups, but not limited to these. In some embodiments, the spacer may contain a polymer. In some embodiments, the spacer - may include polymers that are polypeptides or polyethylene glycol (PEG). .

[0177] In some embodiments, the spacer is one or more debaset nucleotides (i.e., Nucleotides lacking nucleic acid bases, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 It may contain 12 or more debasalized nucleotides. Nucleic acid bases are debasalized nucleotides. It can be replaced by -H(idSp) or -OH in the ocide. The process removes nucleic acid bases from one or more adjacent nucleotides, thereby targeting the target nucleotide. It can be inserted into a polynucleotide. For example, a polynucleotide can be 3-methyladenine, Contains 7-methylguanine, 1,N6-ethenoadenine inosine, or hypoxanthine Nucleic acid bases can be modified in such a way that human alkyladenine DNA glycosylase (hAAG) These nucleotides can be removed using ). Alternatively, polynucleotides It may be modified to contain uracil, and the nucleic acid base is uracil DNA glycosylase (UD It can be removed by G). In one embodiment, one or more spacers are removed from any of the debases. It does not contain nucleotides.

[0178] Using a spacer, polynucleotides such as helicases are placed on the polynucleotide adapter. Methods for stalling drug handling proteins are incorporated in their entirety herein by reference. It is described in WO2014 / 135838.

[0179] anchor In some embodiments, polynucleotides, polypeptides and their conjugates, Or, an adapter attached thereto, for example, a membrane anchor or membrane penetration attached to the adapter. This may include through-pore anchors. In one embodiment, the anchor is provided in the manner disclosed herein. Helps characterize conjugates, for example, membrane anchors or transmembrane pore anchors. This may promote the localization of conjugates around nanopores in the film.

[0180] The anchor is a polypeptide anchor and / or hydrophobic anchor that can be inserted into the membrane. It can be an anchor. In one embodiment, the hydrophobic anchor is a lipid, fatty acid, sterol, This is a fluorocarbon nanotube, polypeptide, protein, or amino acid, for example, These are sterols, palmitates, or tocopherols. The anchors are thiols, bicarbonates. It may contain otin or a surfactant.

[0181] In one embodiment, the anchor is biotin (for binding to streptavidin), (mal Amylose (for binding to tose-binding proteins or fusion proteins), (poly-hybrid) Ni-NTA (for binding to stidine or polyhistidine-tagged proteins), Alternatively, it may be a peptide (such as an antigen).

[0182] In one embodiment, the anchor consists of one linker, or two, three, four, or more. The linker may include a polymer, such as a polynucleotide. It contains polyethylene glycol (PEG), polysaccharides, and polypeptides, but these These linkers are not limited to linear, branched, or annular in shape. For example, The linker may be a cyclic polynucleotide. The adapter is a cyclic polynucleotide. It can hybridize to complementary sequences on the drinker. One or more anchors or one or more The linker is composed of components that can be cleaved or broken down, such as limiting sites or photodissociable groups. It may contain. The linker is functionalized with a maleimide group and is attached to the cysteine ​​residue of the protein. It adheres. Suitable linkers are described in WO2010 / 086602.

[0183] In one embodiment, the anchor is cholesterol or a fatty acyl chain. For example, Any fatty acyl chain having a carbon atom length of 6 to 30, such as xadecanoic acid, can be used. Examples of suitable anchors and methods for attaching anchors to adapters are given in WO2012 / 1 Disclosed in 64270 and WO2015 / 150786.

[0184] Control of conjugate migration in nanopores As will be explained in more detail above, the method provided herein involves conjugating, Polynucleotide handheld device that can control the movement of polynucleotides to nanopores When the druging protein comes into contact with the conjugate, and the conjugate moves relative to the nanopore, This includes performing one or more measurements characteristic of polypeptides.

[0185] 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 due to physical or chemical forces (electric potential). It is driven by... In some embodiments, the physical force is driven by the electrical (e.g., voltage) potential... It is provided by a temperature gradient, etc.

[0186] In some embodiments, when a potential is applied across the nanopore, the conjugate It moves relative to the nanopore. Because polynucleotides are negatively charged, they cross the nanopore. When an electrical potential is applied, the polynucleotides, under the influence of the applied potential, move toward the nanopore. It moves. For example, a positive potential is applied to the transformer side of the nanopore relative to the cis side of the nanopore. As a result, the negatively charged analyte moves from the cis side of the nanopore to the trans side of the nanopore. It is induced in this way. Similarly, a positive potential is induced on the transform side of the nanopore relative to the cis side of the nanopore. When applied, this negatively charges the nanopore from the transform side to the cis side. The movement of the analyte is hindered. A negative potential is present on the transformer side of the nanopore relative to the cis side of the nanopore. When applied, the opposite occurs. Apparatus and methods for applying the appropriate voltage are described herein. It is described in more detail.

[0187] In some embodiments, the chemical force is provided by a concentration (e.g., pH) gradient. .

[0188] In some embodiments, polynucleotide handling proteins are physically Alternatively, it controls the movement of conjugates in the same direction as the chemical force (electric potential). For example, how many In that embodiment, a positive potential is applied to the transformer side of the nanopore relative to the cis side of the nanopore. Therefore, the polynucleotide handling protein moves from the cis side of the nanopore to the t of the nanopore. Controls the movement of the conjugate toward the lance. In some embodiments, a positive potential It is applied to the cis side of the nanopore relative to the trans side of the nanopore, and polynucleotide compounds The drinking protein is a conjugate from the trans side of the nanopore to the cis side of the nanopore. Controls its movement.

[0189] In some embodiments, polynucleotide handling proteins are physically Alternatively, it controls the movement of conjugates in the opposite direction to the chemical force (electric potential). For example, In some embodiments, a positive potential is present on the transformer side of the nanopore relative to the cis side of the nanopore. When applied, the polynucleotide handling protein moves from the trans side of the nanopore to the nano Controls the movement of the conjugate to the cis side of the pore. In some embodiments, a positive potential However, when applied to the cis side of the nanopore relative to the trans side of the nanopore, polynucleotide compounds are applied. The drinking protein is a conjugate from the cis side of the nanopore to the trans side of the nanopore. Controls its movement.

[0190] In some embodiments, the movement of the conjugate occurs when no potential is applied. It is driven by polynucleotide handling proteins.

[0191] In the disclosed method, the polynucleotide handling protein is applied to the nanopore. It is possible to control the movement of polynucleotides. In other words, polynucleotides Handling proteins can control the movement of conjugates. In the embodiment, the polynucleotide handling protein is a polynucleotide and The movement of polypeptides can be controlled.

[0192] Suitable polynucleotide handling proteins are motor proteins or polynucleotides. Also known as a cleotide handling enzyme. Appropriate polynucleotide handling enzymes G proteins are known in this field, and several exemplary polynucleotide proteins The ring protein is described in more detail below.

[0193] In one embodiment, the motor protein is a polynucleotide handling enzyme. , or derived therefrom. Polynucleotide handling enzymes interact with polynucleotides. It is a polypeptide that interacts with and can modify at least one of its properties. Enzymes can process individual nucleotides or shorter chains of nucleotides, such as dinucleotides or trinucleotides. To form creotides, polynucleotides are cleaved by the polynucleotides. The 'do' may be modified. The enzyme either orients it or moves it to a specific position. Polynucleotides may be modified in this way.

[0194] In some embodiments, the polynucleotide handling protein is used in nanopores It can exist on the conjugate before contact with it. For example, polynucleotides Ending proteins can reside on polynucleotides within a conjugate. In some embodiments, the polynucleotide handling protein is condyloma. It exists on the adapter which includes part of the conjugate, or on part of the conjugate. It is possible.

[0195] In some embodiments, the polynucleotide handling protein is polynucleotide The conjugate portion in contact with the active site of the rheotide handling protein is poly If it contains peptides, they can remain bound to the conjugate. In other words, In some embodiments, the polynucleotide handling protein is polynucleotide When the rheotide handling protein comes into contact with the polypeptide portion of the conjugate, It does not dissociate from the conjugate. In some embodiments, polynucleotide handlings The protein is in contact with one or more subsequent polynucleotide portions of the conjugate. Then, you can move freely within the polypeptide portion.

[0196] In some embodiments, the polynucleotide handling protein is polynucleotide The rheotide handling protein comes into contact with the conjugate portion containing the polypeptide. Sometimes, from conjugates, polynucleotides or adapters (conjugates, poly Other than by detaching from the end of a renucleotide or adapter (pass-off). They are modified to prevent detachment. Such modified polynucleotide hands The ring protein is particularly well suited for use in the disclosed manner.

[0197] Polynucleotide handling proteins can be adapted in any preferred manner. For example, polynucleotide handling proteins are polynucleotides, conjugates Loaded onto the gate or adapter, and then modified to prevent it from detaching It is possible to load it onto a polynucleotide, conjugate, or adapter. Previously, the polynucleotide handling protein was modified to prevent the protein from detaching. It can be prevented. Polynucleotide handling proteins handle polynucleotides, con Polynucleotide handling to prevent detachment from the jugate or adapter. Protein modification is performed by methods known in the art, for example, by reference, which are incorporated herein in whole. Using the method described in WO2014 / 013260, and To prevent renucleotide handling proteins from detaching from the polynucleotide chain. Polynucleotide handling proteins such as helicases (polynucleotide binding proteins) This can be achieved by referring specifically to the section describing the modification of protein.

[0198] For example, polynucleotide handling proteins are polynucleotide handling When a protein detaches from a polynucleotide chain, the polynucleotides that the chain can pass through... Nucleotide-unbound openings may have, for example, cavities, grooves, or voids. Several implementations Morphologically, the po of a given motor protein (polynucleotide handling protein) The non-binding opening of a nucleotide can be identified by referring to its structure, for example, its X-ray crystal. This can be determined by referring to the structure. The X-ray crystal structure is polynucleotide group It can be obtained in the presence and / or absence of the quality. In some embodiments, given Location of polynucleotide-unbound openings in polynucleotide handling proteins This is estimated by molecular modeling using standard packages known in the relevant art or This can be confirmed. In some embodiments, the polynucleotide unbound opening is a polynucleotide. For the movement of one or more parts of an oxidative handling protein, for example, one or more domains Therefore, it can be generated temporarily.

[0199] Polynucleotide handling proteins (motor proteins) are polynucleotides It can be modified by closing the non-binding openings of polynucleotides. Closing the binding opening is the role of polynucleotide handling proteins in conjugating Not only does it prevent the polypeptide portion from detaching, but it also prevents it from becoming a polynucleotide or This can prevent the motor protein from detaching from the adapter. For example, a motor protein can be prevented from detaching from the adapter. The cleotide unbound openings can be modified by covalently closing them. In embodiments, preferred motor proteins for this purpose are described herein. It is a helicase that does so. Therefore, in some embodiments of the disclosed method Therefore, polynucleotide handling proteins are responsible for unbinding polynucleotide chains. An opening present in at least one conformational state of the unmodified protein. It is modified to close completely or partially.

[0200] Polynucleotide handling proteins are characterized in the manner disclosed herein. The polynucleotides used in the conjugate may be selected or chosen according to the polynucleotides used. Alternatively, polynucleotides are used to control the movement of conjugates. Polynucleotide handling proteins are selected or may be selected according to their specific characteristics. If the polynucleotide is DNA, then typically DNA motor proteins are used. It can be used. If the polynucleotide is RNA, the RNA motor protein can be used. It can be used. If a polynucleotide is a hybrid of DNA and RNA, then DNA and RN A motor protein capable of processing both A can be used.

[0201] In one embodiment, the motor protein is more preferably enzyme classification (EC) group 3.1 .11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21, 3 .1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30, and 3.1 It originates from one of the members of .31.

[0202] In some embodiments of the claimed method, the motor protein is a helicase, These are polymerases, exonucleases, topoisomerases, or their variants.

[0203] In one embodiment, the motor protein is an exonuclease. Preferred enzymes include , exonuclease I (SEQ ID NO: 1) derived from E. coli, exo Nuclease III enzyme (SEQ ID NO: 2), derived from T. thermophilus RecJ (SEQ ID NO: 3) and bacteriophage lambda exonuclease (SEQ ID NO: 4), T This includes, but is not limited to, atD exonuclease and its variants. Three subunits, including the sequence shown in Sequence ID No. 3 or its variants, interact. Then, trimer exonuclease is formed.

[0204] In one embodiment, the motor protein is a polymerase. The polymerase is Py roPhage(registered trademark)3173 DNA polymerase (Lucigen(registered trademark) (Commercially available from Corporation), SD polymerase (Bioron ( (Registered trademark) Commercially available Klenow manufactured by NEB, or variants thereof. Obtain. In one embodiment, the enzyme is Phi29 DNA polymerase (SEQ ID NO: 5) or It is a variant of the Phi29 polymerase that can be used in the disclosed method. The version is disclosed in U.S. Patent No. 5,576,204.

[0205] The movement of conjugates is controlled by synthesizing a chain complementary to the polynucleotide. Embodiments of the methods provided herein, which include the action of handling polynucleotides Proteins are typically polymerases, such as the polymerases described herein. .

[0206] In one embodiment, the polynucleotide handling protein is a topoisomerase. In one embodiment, the topoisomerase is preferably from subgroup (EC) 5.99. It is a member of either 1.2 or 5.99.1.3. Topoisomerase is It is a reverse transcriptase, an enzyme that can catalyze the formation of cDNA from an RNA template. They can be obtained, for example, from New England Biolabs® and It is commercially available from Invitrogen (registered trademark).

[0207] In one embodiment, the polynucleotide handling protein is a helicase. A suitable helicase can be used according to the method provided herein. For example If so, the motor proteins used in accordance with this disclosure are independently Hel30 8-helicase, RecD helicase, TraI helicase, TrwC helicase, XPD Helicase and Dda helicase, or their variants, may be selected. Monomer Helicases can contain several domains attached together. For example, TraI helicase Helicases and TraI subgroup helicases have two RecD helicase domains, It may include the luxation domain and the C-terminal domain. These domains are typically It forms monomeric helicases that can function without forming oligomers. Specific examples of suitable helicases include Hel308, NS3, Dda, UvrD, and Rep Examples include PcrA, Pif1, and TraI. These helicases are typically It acts on single-stranded DNA. It can move along both strands of double-stranded DNA. Examples of licases include FtfK and hexameric enzyme complexes, or multi-enzymes such as RecBCD. A subunit complex is one example. NS3 helicase processes both DNA and RNA. It can be singled, and therefore the target double-stranded nucleic acid is a DNA-RNA hybrid. Because it can be used in embodiments of the disclosed method, it is particularly useful for use in the disclosed method. Suitable.

[0208] Hel308 helicase is incorporated by reference in its entirety in WO2013 / 0574. It is described in publications such as 95. RecD helicase incorporates all content by reference. It is described in publications such as WO2013 / 098562. XPD helicase is The full content is incorporated by reference in publications such as WO2013 / 098561. Dda helicase incorporates all of its contents by reference in WO2015 / 0. It is documented in publications such as 55981 and WO2016 / 055777.

[0209] In one embodiment, the helicase is the sequence shown in Sequence ID No. 6 (Trwc Cba) The variant of this sequence, the sequence shown in Sequence ID No. 7 (Hel308 Mbu), or its variant The form, or the sequence (Dda) shown in Sequence ID No. 8, or its variants. The variants are, The sequence may differ from the natural sequence in any of the methods discussed herein. Example of Sequence ID 8 Exemplary variants include E94C / A360C. Further exemplary variants of Sequence ID No. 8 are , E94C / A360C, then (ΔM1)G1G2 (i.e., deletion of M1, then G Includes additions of 1 and G2.

[0210] In some embodiments, a motor protein (e.g., helicase) is at least Two active operating modes (all the components necessary for motor proteins to facilitate movement, For example, ATP and Mg considered herein 2+ Equipped with fuels and cofactors such as (when necessary) and one inactive operating mode (when the motor protein is needed to facilitate movement) (If it does not have the necessary components) the movement of the conjugate can be controlled.

[0211] If it has all the necessary components to facilitate movement (i.e., in active mode) Motor proteins (egericases) are located along polynucleotides from 5' to 3' or It moves in the 3' to 5' direction (depending on the motor protein). Using protein, move the conjugate away from the pore (for example, against the applied force) (For example, to release it outside), or to pore the conjugate (for example, with the applied force) It can be moved toward (for example, into) a motor protein. When the end of the conjugate, which the substance is moving toward, is captured by the pore, the motor tan Proteins act against the direction of force, forming threaded conjugates. It is withdrawn from the pore (for example, into the cis chamber). However, the motor protein When the detached end is trapped within the pore, the motor protein moves according to the direction of the force. The screw-shaped conjugate is then pushed into the pore (for example, into the transformer chamber).

[0212] Motor proteins (e.g., helicases) possess the components necessary to facilitate movement. If not present (i.e., in inactive mode), the motor protein binds to the conjugate. For example, it moves relative to the nanopore by being pulled into the pore by force. It can function as a brake to delay the movement of structures when inactive. In this case, it doesn't matter which end of the conjugate is captured, the conjugate The applied force that determines the movement of the polynucleotide-binding protein relative to the pore is important. The protein functions as a brake. In inactive mode, polynucleotide-binding protein Control of conjugate movement by ratcheting, sliding, and swaying It can be explained in several ways, including by working.

[0213] Motor proteins typically handle the processing of polynucleotides. It requires fuel to do so. The fuel is typically free nucleotides or free nuclei. It is an othiocyan analog. The free nucleotides are adenosine monophosphate (AMP) and adenosine. Diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), Guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (T MP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), Uridine diphosphate (UDP), Uridine triphosphate (UTP), Cytidine monophosphate Cytidine acid (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenophosphate Nosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine Deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine Deoxyguanosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine Deoxyguanosine triphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine Deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate Deoxyuridine monophosphate (dTTP), deoxyuridine diphosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (d CMP), deoxycytidine diphosphate (dCDP), and deoxycytidine triphosphate ( Free nucleotides may be one or more of the following (dCTP): , normally, AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, Or selected from dCMP. Free nucleotides are typically adenosine triphosphate ( It is ATP.

[0214] Motor protein cofactors are factors that enable motor proteins to function. The cofactor is preferably a divalent metal cation. Mg 2+ Mn 2+ Ca 2+ , or Co 2+ The cofactor is most preferably Mg 2+ That is the case.

[0215] As described herein, in some embodiments, polynucleotide handlers The ing protein is used to control the movement of the conjugate to the nanopore. In this case, to extend the distance between the polynucleotide handling protein and the nanopore Then, polynucleotide handling proteins are added.

[0216] Polynucleotide handling proteins can be modified in any appropriate manner. Modification of proteins such as nucleotide handling proteins is within the scope of the knowledge of a person skilled in the art. That is the case.

[0217] Polynucleotide handling proteins introduce additional amino acids into their protein structure. It can be modified by the following: In some embodiments, polynucleotide hands Ring proteins are characterized by the development of one or more loop regions that extend beyond the protein's natural range. Modified by insertion. Polynucleotide handling proteins have multiple sub In embodiments including a unit, one or more loop regions are polynucleotide handlers It can be introduced into one or more subunits of a genoprotein.

[0218] Polynucleotide handling proteins are polynucleotide handling proteins When the quality is in a "seated position" relative to the nanopore, one or more are needed to replace the nanopore. It can be modified by the fusion of additional domains.

[0219] nanopore As described above, the method disclosed herein is a polynucleotide handling method This involves using an protein to control the movement of the conjugate relative to the nanopore.

[0220] The disclosed method can use any suitable nanopore. In one embodiment, Nanopores are transmembrane pores.

[0221] Transmembrane pores are structures that extend to some extent across the membrane. Therefore, they are driven by the applied potential. The hydrated ions can then flow across or within the membrane. Typically, A traverses the entire membrane, thereby allowing hydrated ions to move from one side of the membrane to the other. This allows fluid to flow through the membrane. However, transmembrane pores do not need to cross the membrane. One end may be closed. For example, a pore is a well, gap, or channel in a membrane. It can be a groove or slit through which hydrated ions can flow. Cut.

[0222] Any transmembrane pore can be used in the manner provided herein. The pore is biological and It can be artificial or natural. Suitable pores include protein pores, polynucleotide pores, and This includes, but is not limited to, solid pores. In one embodiment, the solid pore is a nanochar. It may contain flannel. The pores may be DNA origami pores (Langecker et al. l., Science, 2012;338:932-936). Suitable DNA origami pore This is disclosed in WO2013 / 083983.

[0223] In one embodiment, the nanopore is a transmembrane protein pore. This allows hydrated ions such as polynucleotides to flow from one side of the membrane to the other. A polypeptide or an aggregate of polypeptides. In the method provided herein, a membrane Transpermeable protein pores allow hydrated ions, driven by the applied potential, to move from one side of the membrane to the other. It can form pores that allow flow. Transmembrane protein pores are preferred. In this case, polynucleotides flow from one side of a membrane, such as a triblock copolymer membrane, to the other side. This makes it possible. Transmembrane protein pores allow polynucleotides to move through the pore. It makes it possible to do so.

[0224] In one embodiment, the nanopore is a transmembrane protein pore that is a monomer or oligomer. The pores are preferably at least 6, at least 7, at least 8, and at 9, at least 10, at least 11, at least 12, at least 13, at least 1 4. Several repeating subunits such as at least 15 or at least 16 subunits It is composed of units. The pore is preferably a hexamer, heptamer, octamer, or It is a notameric pore. The pore can be either a homo-oligomer or a hetero-oligomer. .

[0225] In one embodiment, the transmembrane protein pore is a barrel through which ions can pass and flow. Or it contains channels. The pore subunits typically surround a central axis and membrane the chain. In a transmembrane β-barrel or channel or a transmembrane α-helix bundle or channel To contribute.

[0226] Typically, the barrel or channel of a transmembrane protein pore is a target polynucleotide. It contains amino acids that promote interaction with analytes such as those described herein. The amino acids are preferably located near the barrel or channel constriction. Protein pores typically contain one or more of the following: arginine, lysine, or histidine. It contains positively charged amino acids, or aromatic amino acids such as tyrosine or tryptophan. These amino acids are typically pores, nucleotides, polynucleotides, or nuclei. It promotes interaction with acids.

[0227] In one embodiment, the nanopores originate from β-barrel pores or α-helix bundle pores. It is a transmembrane protein pore. The β-barrel pore is a barrel or chain formed from the β chain. Contains channels. Suitable β-barrel pores contain β-toxins, such as α-hemolytic toxins, anthrax toxins, and leucosidines, as well as bacterial outer membrane proteins / porins, e.g., Mycobacter terium smegmatis porin (Msp), e.g., MspA, MspB, M spC, or MspD, CsgG, outer membrane porin F (OmpF), outer membrane porin G (Omp G) Outer membrane phospholipase A, and Neisseria autotransporter lipota It contains protein (NalP) and other pores, such as lysenine, but is not limited to these. It is not determined. The α-helix bundle pore is a barrel formed from the α-helix. It contains channels. Suitable α-helix bundle pores are inner membrane proteins and α-outer membrane proteins. This includes, but is not limited to, proteins such as WZA and ClyA toxins.

[0228] In one embodiment, the nanopore contains Msp, α-hemolysin (α-HL), lysenin, and CsgG. , ClyA, Sp1, or hemolytic protein fragaceatoxin C (FraC) These are transmembrane pores that result from or are based on them.

[0229] In one embodiment, the nanopore is CsgG, for example, E. coli strain K-12 sub-strain MC4 It originates from CsgG derived from 100. Such pores are oligomers, and typically Csg Contains 7, 8, 9, or 10 monomers derived from G. Pore contains the same monomer. It could be a homo-oligomeric pore derived from CsgG. Alternatively, the pore is different from others. It may be a hetero-oligomeric pore derived from CsgG containing at least one monomer. A suitable example of a pore derived from CsgG is W, which is incorporated in whole by reference herein. This information is disclosed in O2016 / 034591.

[0230] In one embodiment, the nanopore is a transmembrane pore derived from lysenine. A suitable example of a pore is WO2013 / 1, which is incorporated in its entirety herein by reference. It is disclosed in 53359.

[0231] In one embodiment, the nanopores are derived from or based on α-hemolysin (α-HL). It is a transmembrane pore. The wild-type α-hemolytic toxin pore consists of seven identical monomers or subunits. It is formed from (i.e., it is a heptamer). The α-hemolysin pore is α-hemolysin -NN or a variant thereof. The variant is preferably at position E111 and K147 It contains an N residue at position 1.

[0232] In one embodiment, the nanopore is a transmembrane derived from Msp, for example, MspA. This is a protein pore. An example of a suitable pore derived from MspA is WO2012 / 1077. It is disclosed in 78.

[0233] In one embodiment, the nanopore is derived from or based on ClyA. That is the case.

[0234] As described above, in some embodiments, the nanopore includes a constriction. A typical constriction is In essence, this is the narrowing of the channel through the nanopore, which means that the conjugate enters the nanopore. In contrast, the signals obtained when moving can be determined or controlled. In this case, both protein and solid nanopores typically contain "constrictions."

[0235] In some embodiments, nanopores are used for polynucleotide handling proteins. The distance between the nanopore and the constricted region is modified to be extended. In some embodiments, In this process, polynucleotide handling proteins act as conjugates to nanopores. When used to control the movement of polynucleotides, nanopores are used for polynucleotide handling. The protein is modified to extend the distance between it and the narrowed region of the nanopore. In this embodiment, the nanopore is a polynucleotide handling protein. When in contact with the polynucleotide handling protein, the narrowed region of the nanopore It is modified to extend the distance between them.

[0236] In some embodiments, nanopores are used for polynucleotide handling proteins. It is modified to extend the distance between the active site and the narrowed region of the nanopore. In the embodiment, the distance is such that the polynucleotide handling protein is able to reach the nanopore. When used to control the movement of a jugate, and / or polynucleo When the nucleotide handling protein is in contact with the nanopore, polynucleotide handling This could be the distance between the active site of the ing protein and the narrowing of the nanopore.

[0237] Nanopores can be modified in any suitable way. Nanopores such as protein nanopores Modification is within the scope of the knowledge of those skilled in the art. Modification of solid nanopores is routine, and nanopores Control the substrate on which the nanopores are formed (e.g., thickness) or control the components on which the nanopores are formed. It can be achieved through control.

[0238] For example, nanopores can be modified to extend the length of the channel passing through the pore.

[0239] Protein nanopores can be modified by introducing additional amino acids into the pore structure. In some embodiments, protein nanopores extend beyond the natural range of nanopores. Modified by introducing one or more loop regions that extend along the surface. Nanopores are modified by introducing multiple loop regions. In embodiments including a b unit, one or more loop regions are defined as one or more subunits of a nanopore. It can be introduced into knitting. The loop region extends beyond the cis entrance of the nanopore, for example. It is possible to do so.

[0240] Protein nanopores are modified to extend the length of the barrel or channel passing through the pore. It is possible. For example, the pores of a beta barrel follow the protein sequence of the part that forms the barrel. The barrel can be modified by introducing additional amino acids, thereby extending its length. The rational design of the sites associated with such modifications is, for example, in proteins and / or so This can be done by referring to the structure (e.g., X-ray) of the monomer subunits. can.

[0241] Protein nanopores are modified by the fusion of one or more additional domains, resulting in nanopores. This can increase the "situation" of polynucleotide handling proteins. .

[0242] In some embodiments, one protein nanopore is fused to another protein nanopore. It is possible to modify protein nanopores in this way. The chain is constructed including a single channel through which it passes, and the channel narrowing and polynucleotide The distance between the handling protein and the other proteins can be increased. In such cases, multiple Nanopores may be the same or different.

[0243] tag In some embodiments of the methods provided herein, for example, condyloma by nanopore Tags can be used on nanopores to facilitate the capture of the cephalopods.

[0244] Interaction between the tag on the nanopore and the binding site on the polynucleotide (e.g., conjugate) Present in the polynucleotide portion of the gate, or in the adapter attached to the conjugate. The connection site, where the connection site is determined by the adapter's anchor or leader arrangement, (or can be provided by a capture array within the double-stranded stem of the adapter) is reversible It may also be the case that polynucleotides are nanopores via their adapters. Combine with the tag above, for example, during the characterization of polynucleotides by nanopore and / or It can be released at some point during processing by motor proteins. The binding (e.g., biotin / avidin) remains reversible, as described herein. It is useful in several embodiments of the method. For example, the complementary material (separated from the nanopore) It is retained in the vicinity of the nanopore (without diffusion), but when processed, the nanopore So that it can be released from, the complement of the double-stranded polynucleotide (or attached to the complement) To provide sufficient interaction between the adapter part and the nanopore, a pore tag And a pair of polynucleotide adapters may be designed.

[0245] The pore tag and polynucleotide adapter are attached to the tag on the nanopore. The connection point (for example, by the anchor or leader array of the adapter, or by the adapter The binding strength or after of the binding site (provided by the capture sequence within the double-stranded stem of the pteropod) The initiation releases bound polynucleotides from the nanopore when an applied force is exerted. Until then, sufficient to maintain the coupling between the nanopore and the polynucleotide. It can be constructed as follows.

[0246] In some embodiments, the tag or tether is uncharged. This results in a potential difference If such an effect exists, it is important to ensure that the tag or tether is not drawn into the nanopore under its influence. It can be done for sure.

[0247] Attracting conjugates, polynucleotides, or adapters or these One or more molecules to be bonded may be linked to the nanopore. Conjugate, Adapt Any molecule that hybridizes to a nucleotide and / or polynucleotide may be used. The molecules attached to the pore include PNA tags, PEG linkers, short oligonucleotides, and positive loads. Electroamino acids and aptamers may be selected. Such molecules are bound to the pore. This is known in the field. For example, a pore to which a short oligonucleotide is attached is , Howarka et al (2001) Nature Biotech.19:63 Disclosed in 6-639 and WO2010 / 086620, attached within the lumen of the pore Pores containing attached PEG are described in Howarka et al (2000) J.Am.Che This is disclosed in m.Soc.122(11):2411-2416.

[0248] The distribution during conjugate (for example, in the leader array in an adapter or in another single-stranded array) Using short oligonucleotides attached to nanopores, which contain complementary sequences in the column, the present invention In the method described in the details, the capture of conjugates may be enhanced.

[0249] In some embodiments, the tag or tether is an oligonucleotide (e.g., DNA). Even if it contains RNA, LNA, BNA, PNA, or morpholino, or if it is It is also acceptable. Oligonucleotides are approximately 10 to 30 nucleotides in length or approximately 10 to 2 It can have a length of 0 nucleotides. In some embodiments, oligonucleotides Chido is used for conjugation to other modification sites or, for example, to the surface of a solid substrate containing beads. Having at least one modified terminal (e.g., a 3'- or 5'-terminal) This is possible. Terminal modifiers may also be added to reactive functional groups that can be used for bonding. Examples of functional groups that can be added include amino, carboxyl, thiol, and maleic. Examples include, but are not limited to, dihydrogenated compounds, aminooxygenated compounds, and any combination thereof. No. Functional groups are added to the physical distance from the end of the oligonucleotide sequence to the functional group. , spacers of different lengths (e.g., C3, C9, C12, spacers 9 and 18) They can be combined.

[0250] Examples of 3' and / or 5' terminal modifications of oligonucleotides include those due to chemical bonding. 3' affinity tag and functional group (e.g., 3'-biotin, 3'-primary amine, 3') Includes '-disulfideamides, 3'-pyridyldithio, and any combination thereof. (mu); 5' terminal modification (e.g., including 5'-first ammine and / or 5'-dapsyl) Mu); Modification for click chemistry (e.g., 3'-azide, 3'-alkyne, 5 Examples include '-azid, 5'-alkyn, and any combination thereof. , but not limited to these.

[0251] In some embodiments, the tag or tether is used for coupling to, for example, a nanopore. To facilitate the process, a polymer linker may be further included. An exemplary polymer linker is Polymer linker This ranges from approximately 500Da to approximately 10kDa (including both ends), or from approximately 1kDa to approximately 5kDa (including both ends). It may have a molecular weight of (including). The polymer linker (e.g., PEG) is, for example, These are not limited to, but include maleimide, NHS ester, and dibenzocyclooctin (DBC O), azides, biotins, amines, alkynes, aldehydes, and any combination thereof It can be functionalized with different functional groups, including wasabi.

[0252] Other examples of tags or tethers include His tags, biotin or streptavidin, and tests. Antibodies that bind to the body, aptamers that bind to samples, and sample-binding domains such as DNA-binding domains In (for example, peptide zippers like leucine zippers, single-stranded DNA binding proteins) This includes, but is not limited to, quality (SSB), and any combination thereof. I can't.

[0253] Using any method known in the art, a tag or tether may be attached to the outer surface of a nanopore, for example. Alternatively, one or more tags or tethers may be attached to the cis side of the membrane. The above cysteine ​​(cysteine ​​bond), one or more primary amines such as lysine, one or more non- Natural amino acids, one or more histidines (His-tagged), one or more biotins or streptococci Putavidin, tag based on one or more antibodies, one or more enzymatic modifications of an epitope (for example) (For example, acetyltransferases, and any combination thereof, can be used to transfer na It can adhere to the pores. A preferred method for carrying out such modification is the present technology. It is well known in the field. Suitable non-natural amino acids include 4-azido-L-phenylalanine (F az), and Liu CCand Schultz PG, Annu.Rev The numbers 1 to 71 in Figure 1 of Biochem., 2010, 79, 413-444 are numbered as follows: It contains, but is not limited to, any of the following amino acids.

[0254] How many tags or tethers are attached to the nanopore via cysteine ​​bonds? In that embodiment, one or more cysteine ​​molecules are added to one or more monomers that form nanopores. It can be introduced by substitution. In some embodiments, the nanopore is attached to the following It can be chemically modified by: (i) 4-phenylazomareinanyl, 1,N-(2 -Hydroxyethyl)maleimide, N-cyclohexylmaleimide, 1,3-maleimide Propionic acid, 1,1-4-aminophenyl-1H-pyrrole,2,5,dione,1.1 -4-hydroxyphenyl-1H-pyrrole,2,5,dione,N-ethylmaleimide, N-methoxycarbonylmaleimide, N-tert-butylmaleimide, N-(2-amide Noethyl)maleimide, 3-maleimide-proxyl, N-(4-chlorophenyl)male Imide, 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 Imide, N-(2,4-xylyl)maleimide, N-(2,4-difluorophenyl)ma Reimide, N-(3-chloro-para-tolyl)-maleimide, 1-(2-amino-ethyl )-Pyrrole-2,5-dione hydrochloride, 1-cyclopentyl-3-methyl-2, 5-dihydro-1H-pyrrole-2,5-dione, 1-(3-aminopropyl)-2,5 -Dihydro-1H-pyrrole-2,5-dione hydrochloride, 3-methyl-1-[2- Oxo-2-(piperazin-1-yl)ethyl]-2,5-dihydro-1H-pyrrole- 2,5-Dione hydrochloride, 1-benzyl-2,5-dihydro-1H-pyrrole-2 ,5-dione,3-methyl-1-(3,3,3-trifluoropropyl)-2,5-dihy Dro-1H-pyrrole-2,5-dione, 1-[4-(methylamino)cyclohexyl] -2,5-dihydro-1H-pyrrole-2,5-dionetrifluoroacetic 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)maley Maleimides containing diabromomaleimides such as mido and N-(4-nitrophenyl)maleimide Mido, (ii)3-(2-iodoacetamide)-proxyl, N-(cyclopropyl me (Tyl)-2-iodoacetamide, 2-iodo-N-(2-phenylethyl)acetamide D, 2-iodo-N-(2,2,2-trifluoroethyl)acetamide, N-(4- Cetylphenyl)-2-iodoacetamide, N-(4-(aminosulfonyl)phenyl )-2-iodoacetamide, N-(1,3-benzothiazole-2-yl)-2-io Iodoacetamide, N-(2,6-(diethylphenyl)-2-iodoacetamide, N- iodoacetamide such as (2-benzoyl-4-chlorophenyl)-2-iodoacetamide Amide, (iii)N-(4-(acetylamino)phenyl)-2-bromoacetamide , N-(2-acetylphenyl)-2-bromoacetamide, 2-bromo-n-(2-acetylphenyl) Anophenyl)acetamide, 2-bromo-N-(3((trifluoromethyl)pheni (L) acetamide, N-(2-benzoylphenyl)-2-bromoacetamide, 2- Romo-N-(4-fluorophenyl)-3-methylbutanamide, N-benzyl-2-butanamide Romo-N-phenylpropionamide, N-(2-bromo-butyl)-4-chlorobene Zensulfonamide, 2-bromo-N-methyl-N-phenylacetamide, 2-bromo -N-phenethylacetamide, 2-adamantan-1-yl-2-bromo-N-cycline Rohexyl acetamide, 2-bromo-N-(2-methylphenyl)butanamide, mo Bromoacetamide such as nobromoacetanilide, (iv) aldrithiol-2, a Ludolithiol-4, isopropyl disulfide, 1-(isobutyldisulfanil)- 2-methylpropane, dibenzyl disulfide, 4-aminophenyl disulfide, 3- (2-Pyridyldithio)propionic acid, 3-(2-Pyridyldithio)propionic acid hydra Zide, 3-(2-pyridyldithio)propionate N-succinimidyl ester, am6 Disulfides such as amPDP1-βCD, and (v)4-phenylthiazole-2- Thiols, perpaldos, 5,6,7,8-tetrahydroquinazoline-2-thiol, etc. Thiol.

[0255] In some embodiments, the tag or tether is directly attached to the nanopore or to one or more phosphorus It may be attached via a car. Tags or tethers are described in WO2010 / 086602. A hybrid linker may be used to attach to the nanopores. Alternatively, a peptide linker may be used. It may be used. The peptide linker is an amino acid sequence. Peptide linker length, Flexibility and hydrophilicity are typically designed not to interfere with the function of monomers and pores. The preferred number of flexible peptide linkers is 2 to 20, for example, 4, 6, 8, 10, and It is a stretch of 16 serine and / or glycine amino acids. More preferable The flexible linkers are (SG)1, (SG)2, (SG)3, (SG)4, (SG)5, It contains (SG)8, where S is serine and G is glycine. Preferred rigid linker This is a stretch of 20 to 30 proline amino acids, for example, 4, 6, 8, 16, or 24 proline amino acids. It is. A more preferable rigid linker is one in which P is proline, (P) 12 Includes.

[0256] film Typically, in the disclosed method, nanopores are typically present in a film. A suitable film can be used in the system.

[0257] The membrane is preferably an amphiphilic layer. The amphiphilic layer has both hydrophilic and lipophilic properties. It is a layer formed from amphiphilic molecules such as phospholipids that have [a certain property]. Amphiphilic molecules are synthesized... These may be naturally occurring. They may form amphiphilic substances and monolayers that do not exist naturally. The amphiphilic substances that make up these are known in the art, and they include, for example, block copolymers. Rimmer (Gonzalez-Perez et al., Langmuir, 2009, Block copolymers include 25,10447-10450). - A polymer material in which subunits are polymerized together to form a single polymer chain. A cyclopolymer typically possesses properties contributed by each monomer subunit. However, block copolymers are polymers formed from individual subunits. Block copolymers may possess unique properties that others do not. One of them is hydrophobic (i.e., lipophilic), while the other subunits are lipophilic in aqueous media. It can be manipulated to be aqueous. In this case, the block copolymer may have amphiphilic properties. , it can form structures that mimic biological membranes. Block copolymers are diblocks (two mono It may consist of a monomer subunit, but is constructed from three or more monomer subunits. This can form more complex arrangements that behave as amphiphilic substances. Copolymers are tribro The film may be a tetrablock, tetrablock, or pentablock copolymer. It is a triblock copolymer membrane.

[0258] Archaeal bipolar tetraether lipids are constructed such that the lipids form a monolayer membrane. These are naturally occurring lipids. These lipids are generally the extreme conditions that allow organisms to survive in harsh environments. It can be found in halophilic bacteria, thermophilic bacteria, halophilic bacteria, and acidophilic bacteria. Their stability is determined by the final two molecules. It is thought to be obtained from the fusion properties of the layers. The general motif has hydrophilic-hydrophobic-hydrophilic properties. By creating triblock polymers that mimic these biological entities, Constructing lipid copolymers is straightforward. This material behaves similarly to lipid bilayers. It can form monomer films and encompass a wide range of phase behaviors, from vesicles to layered films. Membranes formed from these triblock copolymers have several advantages over biological lipid membranes. It possesses. Since the triblock copolymer is synthesized, its exact structure is carefully considered. Controlling the correct behavior required to form membranes and interact with pores and other proteins. Chain length and properties can be provided.

[0259] Block copolymers are constructed from subunits that are not classified as lipid submaterials. In some cases, hydrophobic polymers may be siloxanes or other non-hydrocarbon monomers. These can be produced. The hydrophilic subsection of the block copolymer has low protein binding properties. This also allows for the creation of membranes that are highly resistant when exposed to raw biological samples. This head group unit may also originate from an unclassified lipid head group.

[0260] Triblock copolymer membranes exhibit increased mechanical and environmental stability compared to biological lipid membranes. It also has properties such as a much higher operating temperature or pH range. Synthesis of block copolymers. These properties provide a basis for customizing polymer-based films for a wide range of applications.

[0261] In some embodiments, the film is related to International Application No. WO2014 / 064443 or the same. It is one of the membranes disclosed in WO2014 / 064444.

[0262] Amphiphilic molecules are chemically modified to facilitate the coupling of polynucleotides. It may be functionalized or otherwise. The amphiphilic layer may be a monolayer or a dilayer. The amphiphilic layer is typically planar. The amphiphilic layer may also be curved. The sex demographic may be supported.

[0263] Amphiphilic membranes are typically about 10 -8 cm s -1 Secondary lipid diffusion rate It is naturally mobile, acting essentially as a fluid. This is due to pores and couplings. This means that the polynucleotides can typically move within an amphipathic membrane. .

[0264] The membrane can be a lipid bilayer. The lipid bilayer is a model of the cell membrane and is suitable for a wide range of experiments. It serves as an excellent foundation for research. For example, lipid bilayers can be used for single-channel recording. It can be used for in vitro investigations of membrane proteins. Alternatively, lipid bifurcation The sublayer can be used as a biosensor to detect the presence of a wide range of substances. The lipid bilayer can be any lipid bilayer. Suitable lipid bilayers include planar lipids. Lipids include, but are not limited to, a bilayer, a supporting bilayer, or a liposome. The bilayer is preferably a planar lipid bilayer. A suitable lipid bilayer is WO200 8 / 102121, WO2009 / 077734, and WO2006 / 100484 It has been disclosed.

[0265] Methods for forming lipid bilayers are known in the art. Lipid bilayers are, Montal and Mueller(Proc.Natl.Acad.Sci.US Generally formed by the method of A., 1972; 69: 3561-3566), In this case, the lipid monolayer has pores perpendicular to the interface on both sides of the aqueous solution / air interface. It passes through and becomes supported. Lipids usually first dissolve in an organic solvent, and then a small amount of The solvent is added to the interface of the aqueous solution by evaporating it on both sides of the pore. As the organic solvent evaporates, a bilayer is formed at the solution / air interface on both sides of the opening. They move physically up and down across the opening. The planar lipid bilayer crosses the opening within the membrane. Alternatively, it may be formed in a recessed area across the opening.

[0266] The Montal & Mueller method is suitable for protein pore insertion and involves the bifurcation of high-quality lipids. It is popular because it is a cost-effective and relatively simple method for forming a sub-layer. Other common methods for forming molecular layers include tip-dipping and bilayer formation. Painting bilayers and liposome bilayer patch clusters It contains a pump.

[0267] Tip immersion bilayer formation involves placing an open surface (for example) on the surface of a test solution supporting a monolayer of lipids. This involves contacting the pipette tip. In this case as well, the lipid monolayer is the most Initially, a small amount of lipid dissolved in the organic solvent is evaporated at the solution surface, thereby creating a solution / air environment. It is generated as a plane. Subsequently, the bilayer is formed by the Langmuir-Shafer method. Mechanical automation is required to move the opening relative to the solution surface.

[0268] In the case of bilayer coating, a small amount of lipid dissolved in an organic solvent is immersed in the open solution of the test aqueous solution. It is applied directly to the opening. The lipid solution is applied across the opening using a paint brush or equivalent. It can be spread thinly. By thinning the solvent, the formation of a lipid bilayer is brought about. However However, it is difficult to completely remove the solvent from the bilayer, and as a result, The bilayer formed by this method has low stability and is prone to generating noise during electrochemical measurements. stomach.

[0269] Patch clamps are commonly used in the study of biological cell membranes. The cell membrane is pinpointed by suction. The membrane patch is attached to the end of the pet, and adheres across the opening. The liposomes are retained and then ruptured to seal the lipid bilayer across the pipette opening. This method is adapted to produce a lipid bilayer. It is necessary to create small openings in monolayer liposomes and materials having a glass surface. To be essential.

[0270] Liposomes are processed by sonication, extrusion, or the Mozafari method (Colas et al. It can be formed by (l. (2007) Micron 38:841-847) .

[0271] In some embodiments, the lipid bilayer is specified in International Application No. WO2009 / 077734 It is formed as described. This method is advantageous in that the lipid bilayer is formed from dry lipids. In the most preferred embodiment, the lipid bilayer is described in WO2009 / 077734. As shown, it is formed across the opening.

[0272] A lipid bilayer is formed from two opposing layers of lipids. These two lipid layers are These hydrophobic tail groups are arranged to face each other, forming a hydrophobic interior. The hydrophilic head groups of the bilayer are oriented outward toward the aqueous environment on both sides of the bilayer. The layers include a liquid disordered phase (fluid layered), a liquid ordered phase, and a solid ordered phase (layered gel phase, comb-type gel phase). This includes, but is not limited to, planar bilayer crystals (layered subgel phase, lamellar crystal phase). It may not be present in all lipid phases, but could be present in several.

[0273] Any lipid composition that forms a lipid bilayer can be used. Required properties, such as surface charge, ability to support membrane proteins, packing density, or mechanical properties. A lipid bilayer with specific properties is selected. The lipid composition is selected to form one or more different It may contain lipids. For example, a lipid composition may contain up to 100 lipids. Yes, it is possible. The lipid composition preferably contains 1 to 10 lipids. It may contain natural lipids and / or artificial lipids.

[0274] Lipids typically consist of a head group, an interface, and two components that may be the same or different. It contains hydrophobic tail groups. Suitable head groups include neutral head groups, such as diacylglycerides. Lido (DG) and ceramide (CM), zwitterionic head group, for example, phosphatidylco Phosphorus (PC), phosphatidylethanolamine (PE), and sphingomyelin ( SM), negatively charged head groups, for example, phosphatidylglycerol (PG), phosphatidylglycerol Phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (P A), and cardiolipin (CA), and positively charged head groups, for example, trim This includes, but is not limited to, ammonium nitrate-propane (TAP). Suitable environments The surface portion contains naturally occurring interface portions, such as glycerol-based portions or ceramide-based portions. This includes, but is not limited to, saturated hydrocarbon chains. Suitable hydrophobic tail groups include saturated hydrocarbon chains. For example, lauric acid (n-dodecanolic acid) Myristic acid (n-tetradecononinic acid (n-Tetradecononic aci d)), palmitic acid (n-hexadecanoic acid), stearic acid (n-octadecanoic acid), and arachidic acid (n-eicosanoic acid), unsaturated hydrocarbon chains, for example, oleic acid ( This includes s-9-octadecanoic acid, as well as branched hydrocarbon chains, such as phytanoyl. However, it is not limited to these. The length of the chain and the position of the double bond in the unsaturated hydrocarbon chain, and The number of branches can vary. This depends on the length of the chain, such as the methyl group in the branched hydrocarbon chain, and the position of the branches. The number may vary. Hydrophobic tail groups are linked to the interface as ethers or esters. It is possible. The lipids may be mycolic acids.

[0275] Lipids can also be chemically modified. The head group or tail group of a lipid can be chemically modified. It can be decorated. Suitable lipids in which the head group is chemically modified include PEG-modified lipids, for example. For example, 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy] (Polyethylene glycol)-2000], functionalized PEG lipids, e.g., 1,2-diste Aroyl-sn-glycero-3phosphoethanolamine-N-[biotinyl(polyethylene [Lingrycol]2000, as well as lipids modified for conjugation, for example , 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Suc Synyl) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine This includes, but is not limited to, -N-(biothinyl). The tail group is chemically modified. Suitable lipids include polymerizable lipids, such as 1,2-bis(10,12-tricosaglycan (tricosadiynoyl)-sn-glycero-3-phosphocholine, f Fluoropolymers, for example, 1-palmitoyl-2-(16-fluoropalmitoyl)-sn-g Lysero-3-phosphocholine, deuterated lipids, e.g., 1,2-dipalmitoyl-D62- sn-glycero-3-phosphocholine, and ether-bound lipids, such as 1,2-di-O This includes, but is not limited to, -phytanyl-sn-glycero-3-phosphocholine. Lipids can be chemically modified to facilitate polynucleotide coupling, even if they are not functional. It may be made capable.

[0276] An amphiphilic layer, such as a lipid composition, will typically affect the properties of the layer. It contains one or more additives. Preferred additives include fatty acids, such as palmitic acid and myristic acid. Acids, and oleic acid, fatty alcohols, such as palmitic acid, myristic acid Alcohol, and oleic alcohol, sterols, such as cholesterol, ergos Terol, lanosterol, sitosterol, and stigmasterol, lysophospholipids For example, 1-acyl-2-hydroxy-sn-glycero-3-phosphocholine, and This includes, but is not limited to, lamidopropyl bezoars.

[0277] In another embodiment, the film includes a solid layer. The solid layer is a microelectronic material, an insulating material, for example. For example, Si3N4, A12O3, and SiO, organic and inorganic polymers, for example, poly Amides, plastics, for example, Teflon®, or elastomers, for example This includes, but is not limited to, two-component addition-curing silicone rubber and glass, organic It can be formed from both material and inorganic materials. The solid layer is formed from graphene. It is possible. A suitable graphene layer is disclosed in WO2009 / 035647. The film is solid. When a solid layer is included, the pore is typically within the solid layer, for example, a hole, well, or gap within the solid layer. , present in an amphiphilic film or layer contained within a channel, groove, or slit. If available, a suitable solid / amphiphilic hybrid system can be prepared. A suitable system is W This is disclosed in O2009 / 020682 and WO2012 / 005857. Any of the amphiphilic membranes or layers can be used.

[0278] The methods disclosed herein typically include (i) an artificial amphiphilic layer containing a pore, (ii) ) an isolated, naturally occurring lipid bilayer containing a pore, or (iii) a pore inserted inside This is performed using the introduced cells. This method typically involves artificial triblock coping This is carried out using artificial amphiphilic layers such as Mer layers. In addition to pores, the layer also has other transmembrane layers. It may contain proteins and / or intramembrane proteins, as well as other molecules. Suitable apparatus and The conditions are discussed below. The disclosed methods are typically performed in vitro. .

[0279] conditions As explained above, the disclosed method involves a conjugate containing a polypeptide that This involves characterizing the polypeptide as it moves toward the nopore.

[0280] The characterization method uses any suitable instrument for investigating membrane / pore systems in which pores are inserted into the membrane. The characterization method can be performed using any apparatus suitable for transmembrane pore sensing. This can be done. For example, the apparatus includes a chamber containing an aqueous solution, and the chamber is divided into two sections. It may have a barrier. The barrier may have an opening in which a membrane is formed, including a transmembrane pore. The pore is described herein.

[0281] The characterization method is WO2008 / 102120, WO2010 / 122293, or This can be carried out using the apparatus described in WO00 / 28312.

[0282] Characterization methods typically involve measuring the flow of ionic current through the pore by measuring the electric current. This may include doing so. Alternatively, the flow of ions through the pore is, Heron et al: Disclosed in J.Am.Chem.Soc.9 Vol.131, No.5, 2009 It can be measured optically so as to be measured. Therefore, the apparatus applies a potential and the film and It may also include an electrical circuit capable of measuring electrical signals across the pore. The characterization method is: This can be done using patch clamps or voltage clamps. The characterization method is preferably This involves the use of a voltage clamp.

[0283] The characterization method is as follows: Each array has 128, 256, 512, 1024, 2000, and 3000 elements. 4000, 6000, 10000, 12000, 15000, or more wells It can be run on a silicon-based well array equipped with [the necessary components].

[0284] Characterization methods may include measuring the current flowing through the pore. This method is typically This is performed with a voltage applied across the film and pore. The voltage used is typical. The voltage range is typically +2V to -2V, usually -400mV to +400mV. The pressure is preferably -400mV, -300mV, -200mV, -150mV, -10 A lower limit selected from 0mV, -50mV, -20mV, and 0mV, and +10mV, + 20mV, +50mV, +100mV, +150mV, +200mV, +300mV, The voltage used is within a range having an upper limit independently selected from +400mV. The more preferably range is 100mV to 240mV, most preferably 120mV to 22 It is within the range of 0mV. By using an increased applied potential, different nuclei can be obtained for each pore. It is possible to increase the discrimination between creotides.

[0285] Characterization methods typically involve metal salts, e.g., alkali metal salts, halide salts, etc. For example, it can be performed in the presence of any charge carrier such as chloride salts, for instance, alkali metal chloride salts. The charge carrier is an ionic liquid or organic salt, for example, tetramethylammonium chloride. M, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, also This may include 1-ethyl-3-methylimidazolium chloride. Exemplary examples considered above In such apparatuses, salts are present in the aqueous solution within the chamber. Potassium chloride (KCl), sodium chloride NaCl or cesium chloride (CsCl) are typically used. Preferably, the salt may be an alkaline earth metal salt such as calcium chloride (CaCl2). The salt concentration may be at saturation. The salt concentration may be less than 3M, and is typically 0.1 ~2.5M, 0.3~1.9M, 0.5~1.8M, 0.7~1.7M, 0.9~1.6 The concentration is M, or 1M to 1.4M. The salt concentration is preferably 150mM to 1M. The marking method is preferably at least 0.3M, for example at least 0.4M, less Both 0.5M, at least 0.6M, at least 0.8M, at least 1.0M, less All are 1.5M, at least 2.0M, at least 2.5M, or at least 3.0M It is performed using salt concentration. High salt concentration provides a high signal-to-noise ratio and normal This allows for the identification of currents that indicate coupling / uncoupling to the background of current fluctuations.

[0286] Characterization methods are typically performed in the presence of buffers. An example is discussed above. In the apparatus, the buffer solution is present in the aqueous solution within the chamber. Any suitable buffer solution can be used. Typically, the buffer is HEPES. Another preferred buffer is Tris-HCl. This is a buffer solution. This method is typically used for solutions with concentrations of 4.0-12.0, 4.5-10.0, and 5.0- 9.0, 5.5-8.8, 6.0-8.7, or 7.0-8.8, or 7.5-8. The process is carried out at a pH of 5. The pH used is preferably around 7.5.

[0287] The characterization methods are 0°C to 100°C, 15°C to 95°C, 16°C to 90°C, and 17°C to 85°C. It can be carried out at 18°C ​​to 80°C, 19°C to 70°C, or 20°C to 60°C. Characterization The procedure is typically performed at room temperature. Characterization methods are optional and support enzyme function. It is performed at a temperature, for example, around 37°C.

[0288] Modified nanopores Nanopores including a constricted region are also provided, and this nanopore has a constricted region and is in contact with the nanopore. Modified to increase the distance between the polynucleotide handling protein and the other proteins. The nanopore may be as described herein. It can be modified as described.

[0289] system -Nanopores including a constricted region, - Conjugates containing polypeptides conjugated to polynucleotides, -Polynucleotide handling proteins, A system including this is also provided. i) When the polynucleotide handling enzyme is in contact with this nanopore This increases the distance between the constricted region and the active site of the polynucleotide handling protein. Modified to add, and / or ii) This system is connected between the nanopore and the polynucleotide handling protein. They are positioned, thereby connecting the nanopore and the active site of the polynucleotide handling protein. It further includes one or more displacer units that extend the distance between them.

[0290] In some embodiments, nanopores, conjugates and / or polynucleotides Chip handling proteins, and optionally, if present, one or more dispersive The racing unit is as described herein.

[0291] kit -Nanopores including a constricted region, -Polynucleotides containing reactive functional groups for conjugate to target polynucleotides Ochido and, -A kit containing polynucleotide handling proteins is also provided.

[0292] In some embodiments, this nanopore is used by polynucleotide handling enzymes When in contact with the nanopore, the constricted region and the polynucleotide handling protein It is modified to increase the distance between them.

[0293] In some embodiments, this kit includes nanopores and polynucleotide handlings. One or more display units to extend the distance between the active site of the protein and the surrounding area. It also includes a set.

[0294] In some embodiments, nanopores, polynucleotides and / or polynucleotides Oxide handling proteins, and optionally, if present, one or more dissolved proteins. The placer unit is as described herein.

[0295] The kit is adapted to run on a computer system and is also provided herein. It can be configured to be used with the algorithm. The algorithm is polypeptide Characteristic features of the polypeptide (for example, the polypeptide sequence and / or the polypeptide is modified) It detects information (characteristic of whether or not it is present) and polynucleotides conjugated to polynucleotides Select the signal obtained when the peptide-containing conjugate moves toward the nanopore. It can be adapted to process in a specific way. It is characteristic of polypeptides (for example). , characteristic of the polypeptide sequence and / or whether the polypeptide is modified. ) Detects information and contains a conduit containing a polypeptide conjugated to polynucleotides The signal obtained when the vertex moves toward the nanopore is selectively processed. A system including computing means configured in such a way is also provided. In terms of form, the system includes receiving means for receiving data from polypeptide detection, and For processing the signals obtained when the jugate moves toward the nanopore Includes processing means and output means for outputting the characterization information thus obtained. nothing.

[0296] Specific embodiments, specific configurations, and materials and / or molecules may be used in the method according to the present invention. While the present invention has been discussed herein, various changes or modifications to its form and details may be made to the present invention. Please understand that this may be done without deviating from the scope and purpose of the aforementioned implementation. The forms and the following examples are provided for illustrative purposes only and are not intended to limit their use. This application should not be limited by the claims alone. [Examples]

[0297] Example 1 This example involves two polynucleotides; dsDNA Y adapter (DNA1) and dsD Controlled translocation of a conjugate containing a polypeptide adjacent to the NA tail (DNA2). This shows that the polynucleotide handling protein on the cis side of the nanopore is initially D NA1 is unwound, translocated at 5'-3' on ssDNA, and then into the polypeptide section. By sliding across and finally unwinding the DNA2 segment, conjugate Controls the movement of this structure. This structure moves from the cis side to the transform side of the nanopore, passing through RED. As time passes, the polypeptide section can be visualized in a current-versus-time plot, allowing for characterization. This will make it possible.

[0298] The Y adapter is a DNA oligonucleotide (SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 11). Prepared by annealing 13). (Described in WO2014 / 013260) As shown, load the DNA motor (Dda helicase) into the adapter and close it. The material was then purified by HPLC. The Y adapter facilitates capture by nanopores. It includes 30 C3 reader sections and side arms for tethering to the membrane. The DNA tail contains DNA oligonucleotides (SEQ ID NO: 14, SEQ ID NO: 16). It was created by annealing.

[0299] In this example, the model polypeptide analytes (SEQ ID NOs. 20, 21, 22) have an N-terminus and, Pre-synthesis using an ethyldiamine spacer along the peptide backbone, immediately after the C-terminus. The obtained product contained the azide portion. Next, each analyte was divided into azide and BCN (bicyclo[6] [1.0] nonine) via a copper-free click chemistry reaction between the moieties, Y ada The pters and DNA tails were conjugated. A schematic diagram of the resulting construct is shown in Figure 4A. The sample is from Agentcourt AMPure XP (Beckman Coul). Purified using ter) beads, Oxford Nanopore Technol The cells were washed twice with LNB from the Ogies sequencing kit (SQK-LSK109). The jugated substrate was mixed with 10 mM Tris-Cl and 50 mM NaCl (pH 8.0). It eluted into ).

[0300] Electrical measurements were performed by Oxford Nanopore Technologies' Min Using ION Mk1b and a custom MinION flow cell with MspA nanopores. The results were obtained using a flow cell with a 50 nM DNA tether and ATP-less sequencing. It was flushed with a tether mix containing buffer. First, 800 μL of the tether mix was added. Add 5 minutes, then another 200 μL of the mix, with the SpotON port open. The DNA-peptide construct was fed into the system. The DNA-peptide construct was then subjected to a sequencing buffer lacking ATP and O Oxford Nanopore Technologies Sequencing Kit (SQ Prepared with LB of K-LSK109) at a concentration of 0.5 nM, "Sequencing Mix This resulted in 75 μL of sequencing mix being introduced via the SpotON flow cell port. The mixture was then added to the MinION flow cell. The mixture was incubated on the flow cell for 5-10 minutes. By baiting, it enabled tethering of the structure and subsequent capture by nanopores. ATP If it is not present, the DNA motor will remain stuck in the spacer region of the Y adapter, The denjugate is captured in the nanopore but does not translocate. After incubation, ATP Add 200 μL of sequencing buffer containing ATP, and in the presence of ATP, the captured DN A-peptide conjugates move across nanopores by helicase, resulting in reproducible This results in a certain electrical footprint.

[0301] Run a standard sequencing script at 180mV for 30 minutes to 1 hour, and statically update every minute. A flip was performed to remove the expanded nanopore block. The raw data was obtained using MinKNOW software. Using software (Oxford Nanopore Technologies) The data was collected in a FAST5 file.

[0302] Model peptide conjugated to DNA Y adapter and tail (SEQ ID NO: 2) For one of the 0) examples, Figure 9 shows an example of a current-versus-time trace. Y Adapter Section The dsDNA tail is separated from the peptide portion of the "winding curve" (trace). This makes it possible to characterize peptides.

[0303] Multiple translocation events were observed per second, achieving high throughput. Multiple captures. And an example of a current-time trace showing a translocation event is shown using the same structure as in Figure 9 ( Figure 10 shows the results for the peptide region of SEQ ID NO: 20.

[0304] Characterization of other conjugate polynucleotide-polypeptide constructs, The procedure was carried out as described. Figures 11-13 show positively charged amino acids (SEQ ID NO: 21; Figure 11). Aromatic amino acids (SEQ ID NO: 22, Figure 12) and negatively charged amino acids (SEQ ID NO: 20 Reproducible electrical properties enable the characterization of constructs incorporating peptide regions, including (Figure 13). This shows the trace of the flow time.

[0305] For ease of reference, a schematic structure of the construct obtained using the peptide of Sequence ID No. 22. The structure is shown in Figure 14.

[0306] Example 2 This example shows a polymer obtained from a peptide that was not pre-synthesized to contain an attachable group. The usefulness of the disclosed method in characterizing creotide-polypeptide constructs It has been proven.

[0307] In this embodiment, the Y adapter is the same as in Example 1, and the dsDNA tail is DNA By annealing oligonucleotides (SEQ ID NO: 15, SEQ ID NO: 16) It was manufactured. Data collection was performed using the protocol established in Example 1, Oxford MinION Mk1b from Nanopore Technologies and MspA This was performed using a custom MinION flow cell with Nopore.

[0308] The peptide analyte used in this example is the model peptide (G) used in Example 1. GSGDDSGSG (sequence number 20 of Example 1; sequence number 23 of Example 2) is almost identical. However, there was a polynucleotide adapter and a click chemistry conjugate for the tail. It lacked pre-synthesized azide molecules for chemical application. To enable maleimide chemistry, additional It contained a C-terminal cysteine. The N-terminus of the peptide was a tetrazine-NHS ester compound. Sensualized with BroadPharm (product code: BP-22946). Non-conjugated. Tetrazine tetrazine is an amino-functionalized magnetic particle (Sigma Aldrich, product code Removed by :53572).

[0309] Next, the peptides were incubated overnight at 4°C with DNA tails (SEQ ID NO: 15, SEQ ID NO: 16). Incubate the click reaction between tetrazine and TCO (transcyclooctene). The response was accelerated. After incubation, possible disulfides between C-terminal peptide cysteine ​​molecules were observed. The bond was reduced with 5 mM DTT at room temperature for 30 minutes, and the peptide-DNA conjugate was A Using gencourt AMPure XP beads (Beckman Coulter) The solution was purified using a method to remove unreacted peptides and DTT. Next, the exposed cysteine ​​was treated with an amethyst. Zido-PEG3-maleimide (BroadPharm, Product Code: BP-22468) This caused a reaction. Excess maleimide linker was removed by Agentcourt AMPure XP. Removed with a series, and the structure is converted to a Y adapter via click chemistry between BCN and Azid. The reaction was carried out overnight at 4°C. The C-terminus of the peptide was connected to the Y-adapter, and the N-terminus to the DNA tape. The resulting structure, formed by conjugation between the two, is called Agentcourt. Purified using AMPure XP beads, and fully constructed from peptide-DNA tails. The objects were separated.

[0310] The final structure is shown in Example 1 using the illustrative current trace shown in Figure 15. It was evaluated as such. As you can see, the characterization of the peptide was based on the prior combination of the attachment site. It was possible without needing to achieve anything.

[0311] Example 3 This example characterizes a 21-amino acid peptide compared to a 10-amino acid peptide. We will compare the methods of disclosure.

[0312] A polynucleotide-polypeptide conjugate of 21 amino acid peptides was used in Example 1. Prepared and analyzed according to the method described in [reference]. Current vs. time obtained in 21 amino acid constructs. The trace was compared with that obtained from the 10-amino acid construct from Example 2. The petit sequence is GDDDGSASGDDDGSASGDDDG(21aa; Sequence ID 24) ) and GGSGDDSGSG(10aa; Sequence ID 20) were

[0313] Polynucleotide-peptide conjugates of 10-amino acid peptides and 21-amino acid peptides Figure 16 shows data illustrating the current-time trace related to gate transposition. The two traces placed on the kale are current sections of a 21-amino acid polypeptide. This indicates that it is approximately twice the length of a 10-amino acid polypeptide.

[0314] Therefore, this embodiment uses the disclosed method to produce poly of various and extended lengths We will confirm that we can characterize the peptides.

[0315] Explanation of the sequence list Sequence ID 1 is an exonuclease (with hexahistidine tag) derived from E. coli. The amino acid sequence of I (EcoExo I) is shown. Sequence ID 2 shows the amino acid sequence of the exonuclease III enzyme derived from E. coli. vinegar. Sequence ID 3 is a RecJ enzyme derived from T. thermophilus (TthRecJ- The amino acid sequence of cd) is shown. Sequence ID 4 shows the amino acid sequence of bacteriophage lambda exonuclease. This array is one of three identical subunits that make up the trimmer. (ht tp: / / www.neb.com / nebecomm / products / produ ctM0262.asp). Sequence ID 5 is a Phi29 DNA polymer derived from Bacillus subtilis. The amino acid sequence of the enzyme is shown. Sequence ID 6 is Trwc Cba(Citromicrobium bathyma The amino acid sequence of rinum helicase is shown. Sequence ID 7 is Hel308 Mbu(Methanococcoides burt (onii) Shows the amino acid sequence of helicase. Sequence ID 8 is the amino acid sequence of Dda helicase 1993 derived from the intestinal bacterium phage T4. This indicates. Sequence ID 11 is a first used to generate a Y adapter as described in Example 1. Polynucleotide chain (C3[(OC3H6OPO3)) reader with 3' BCN click It has an enzyme stall chemistry; 8=iSp18[(OCH2CH2)6OPO3]) The sequence of DNA1 (top) is shown. Sequence ID 12 is a second used to generate a Y adapter as described in Example 1. This shows the sequence of a polynucleotide chain (DNA1-back with side arms for tethering). . Sequence ID 13 is a third used to generate a Y adapter as described in Example 1. This shows the sequence of a polynucleotide chain (DNA1-bottom). Sequence ID 14 is used for generating a dsDNA tail as described in Example 1. The arrangement of a polynucleotide chain (DNA2-top strand, 5'BCN click chemistry) Show the columns. Sequence ID 15 is used for generating a dsDNA tail as described in Example 1. 2 polynucleotide strands (DNA2-top strand, 5'TCO(orthogonal click chemistry) This shows the array )). Sequence ID 16 is used for generating dsDNA tails as described in Example 2. This shows the sequence of a renucleotide strand (DNA2-bottom strand, without side arms). Sequence ID No. 20 is a first polynucleotide polypeptide as described in Example 1. The amino acid sequence of the first peptide fragment used to generate the construct is shown. Sequence ID No. 21 is a second polynucleotide polypeptide as described in Example 1. The amino acid sequence of the second peptide fragment used to generate the construct is shown. Sequence ID No. 22 is a third polynucleotide polypeptide as described in Example 1. The amino acid sequence of the third peptide fragment used to generate the construct is shown. Sequence ID No. 23 is a polynucleotide-polypeptide construct as described in Example 2. The amino acid sequence of the peptide fragment used to produce it is shown. Sequence ID No. 24 is a polynucleotide-polypeptide construct as described in Example 3. The amino acid sequence of the 21-amino acid peptide fragment used to produce it is shown.

[0316] Sequence List Sequence List 1

[0317] TIFF2026143392000003.tif47141 Sequence Listing 2

[0318] TIFF2026143392000004.tif29141 Sequence Listing 3

[0319] TIFF2026143392000005.tif40141 Sequence Listing 4

[0320] TIFF2026143392000006.tif24141 Sequence Listing 5

[0321] TIFF2026143392000007.tif55141 Sequence Listing 6

[0322] TIFF2026143392000008.tif83141 Sequence Listing 7

[0323] TIFF2026143392000009.tif63141 Sequence Listing 8

[0324] TIFF2026143392000010.tif40141 Sequence Listing 9

[0325] TIFF2026143392000011.tif14141 Sequence Listing 10

[0326] TIFF2026143392000012.tif14118 Sequence Listing 11

[0327] TIFF2026143392000013.tif9120 Sequence Listing 12

[0328] TIFF2026143392000014.tif13136 Sequence Listing 13

[0329] TIFF2026143392000015.tif13138 Sequence Listing 14

[0330] TIFF2026143392000016.tif13132 Sequence Listing 15

[0331] TIFF2026143392000017.tif22104 Sequence Listing 16

[0332] TIFF2026143392000018.tif22104 Sequence Listing 17

[0333] TIFF2026143392000019.tif22104 Sequence Listing 18

[0334] TIFF2026143392000020.tif1432 Sequence Listing 19

[0335] TIFF2026143392000021.tif1363

Claims

1. A method for characterizing a target polypeptide, - The target polypeptide is conjugated into a polynucleotide, and the polynucleotide Forming a do-polypeptide conjugate, - The conjugate controls the movement of the polynucleotide relative to the nanopore. This involves contacting a polynucleotide handling protein that can do this, - When the conjugate moves relative to the nanopore, the polypeptide is particularly This includes performing one or more characteristic measurements, A method for characterizing the polypeptide thereby.

2. The nanopore is constricted between the polynucleotide handling protein and the nanopore. The method according to claim 1, further modified to extend the distance between the region and the region.

3. The displacer unit is used to move the polypeptide handling protein Separation from nanopores, thereby separating the polynucleotide handling protein and the nanopores The method according to claim 1 or 2, comprising extending the distance between the nopore and the nopore.

4. The method according to claim 3, wherein the displacer unit comprises one or more proteins. 。

5. The polynucleotide handling protein The protein is modified to extend the distance from the active site to the nanopore. The method described in any one of the requests 1 to 4.

6. The con If the jugate portion contains a polypeptide, the polynucleotide handling protein The substance can remain bound to the conjugate, according to any of claims 1 to 5. The method described in any one of the items.

7. The polynucleotide handling protein contains the polypeptide conjugate When the polynucleotide handling protein comes into contact with the portion of the con Modified to prevent detachment from the jugate, according to any one of claims 1 to 6 Method of description.

8. The aforementioned polynucleotide handling protein releases the bonds between polynucleotide chains. An opening exists in at least one conformational state of the unmodified protein that can perform this operation. Modified to completely or partially close the part, according to any one of claims 1 to 7 Method of description.

9. The polynucleotide handling protein is a helicase, according to claims 1 to 8. The method described in either of the above terms.

10. The conjugate consists of multiple polypeptide sections and / or multiple polynucleotides. The method according to any one of claims 1 to 9, including an ocid section.

11. The polypeptide has a length of 2 to about 50 peptide units, any one of claims 1 to 10 The method described in item 1.

12. The polypeptide is held in a linearized form, according to any one of claims 1 to 11. Method of description.

13. Claims 1 to 1000, wherein the polynucleotide has a length of about 10 to about 1000 nucleotides. The method described in any one of the twelve items.

14. One or more adapters and / or one or more tethers and / or one or more amplifiers Claims 1 to 13, wherein the ker is attached to the polynucleotide in the conjugate. The method described in any one of the items.

15. i) The polynucleotide handling protein is located on the cis side of the nanopore, The polynucleotide handling protein moves from the cis side of the nanopore to the nano Controlling the movement of the conjugate to the transformer side of the nopore, or ii) The polynucleotide handling protein is on the trans side of the nanopore. The polynucleotide handling protein is located in the terminal of the nanopore. Controlling the movement of the conjugate from the cis side to the cis side of the nanopore, The method described in any one of items 1 to 14.

16. The polynucleotide handling protein is located on the cis side of the nanopore, The polynucleotide handling protein moves from the cis side of the nanopore to the nano Controlling the movement of the polynucleotide to the trans side of the nopore, thereby The method according to claim 15, for controlling the movement of the polypeptide through a nanopore.

17. The polynucleotide handling protein is located on the trans side of the nanopore. Furthermore, the polynucleotide handling protein is on the trans side of the nanopore. The movement of the polynucleotide to the cis side of the nanopore is controlled thereby The method according to claim 15, wherein the movement of the polypeptide through the nanopore is controlled.

18. The aforementioned conjugate is L-{P-N}-P ｍ It includes one or more structures of the form, -L is the leader, and L is optionally part N, -P is a polypeptide, -N contains polynucleotides, and -m is either 0 or 1, The above method involves threading the leader (L) through the nanopore, and thereby This includes contacting the polypeptide (P) with the nanopore, i) The polynucleotide handling protein is located on the cis side of the nanopore. Furthermore, the method is such that the polynucleotide handling protein is the nanopore The transfer of the polynucleotide portion (N) from the cis side to the trans side of the nanopore This enables control of the movement of the polypeptide (P) passing through the nanopore. This includes controlling the aforementioned movement, or ii) The polynucleotide handling protein is on the trans side of the nanopore. The method is such that the polynucleotide handling protein is located in the nanopore The polynucleotide portion (N) from the trans side to the cis side of the nanopore This makes it possible to control the movement, thereby allowing the polypeptide to pass through the nanopore ( The method according to any one of claims 1 to 15, for controlling the movement of P).

19. The aforementioned conjugate is L-P １ -N-{P-N} ｎ -P ｍ One or more structures of the form Including, -n is a positive integer, -L is the leader, and L is optionally part N, - Each P, which may be the same or different, is a polypeptide. - Each N, which may be the same or different, contains a polynucleotide, and -m is either 0 or 1, The above method involves threading the leader (L) through the nanopore, and thereby Polypeptide (P １ This includes bringing the nanopore into contact with the nanopore. i) The polynucleotide handling protein is located on the cis side of the nanopore. Furthermore, the method is such that the polynucleotide handling protein is the nanopore The transfer of each polynucleotide (N) from the cis side to the trans side of the nanopore is linked This enables continuous control, thereby controlling each polypeptide (P) passing through the nanopore. This includes continuously controlling the aforementioned movement, or ii) The polynucleotide handling protein is on the trans side of the nanopore. The method is such that the polynucleotide handling protein is located in the nanopore The transfer of each polynucleotide (N) from the trans side to the cis side of the nanopore This enables continuous control of the movement, thereby allowing each polypeptide to pass through the nanopore ( The method according to claim 18, comprising continuously controlling the movement of P).

20. i) The polynucleotide handling protein is located on the cis side of the nanopore, The polynucleotide handling protein is transmitted from the trans side of the nanopore to the nano Controlling the movement of the conjugate to the cis side of the nopore, or ii) The polynucleotide handling protein is on the trans side of the nanopore. The polynucleotide handling protein is located on the cis side of the nanopore. Controlling the movement of the conjugate from the nanopore to the transformer side, The method described in any one of items 1 to 14.

21. The polynucleotide handling protein is located on the cis side of the nanopore, The polynucleotide handling protein is located in front of the trans side of the nanopore. The movement of the polynucleotide to the cis side of the nanopore is controlled, thereby The method according to claim 20, wherein the movement of the polypeptide through the nanopore is controlled.

22. The polynucleotide handling protein is located on the trans side of the nanopore. Furthermore, the polynucleotide handling protein moves from the cis side of the nanopore to the front The movement of the polynucleotide to the trans side of the nanopore is controlled, thereby The method according to claim 20, wherein the movement of the polypeptide through the nanopore is controlled.

23. The aforementioned conjugate is L-{P-N}-P ｍ It includes one or more structures of the form, -L is the leader, and L is optionally part N, -P is a polypeptide, -N contains polynucleotides, -m is 0 or 1, The above method involves threading the leader (L) through the nanopore, and thereby This includes contacting the polypeptide (P) with the nanopore, i) The polynucleotide handling protein is located on the cis side of the nanopore. Furthermore, the method is such that the polynucleotide handling protein is the nanopore The transfer of the polynucleotide (N) from the trans side to the cis side of the nanopore This makes it possible to control the polypeptide (P) passing through the nanopore. This includes controlling movement, or i) The polynucleotide handling protein is on the trans side of the nanopore. The method involves the polynucleotide handling protein being located in the nanopore. The transfer of the polynucleotide (N) from the cis side to the trans side of the nanopore This enables control of the movement of the polypeptide (P) passing through the nanopore. A method according to any one of claims 1 to 14 or 20, comprising controlling the aforementioned movement. 。

24. The conjugate is attached to the polypeptide via an optional linker. Including the locking portion, the method is i) The blocking portion is in front of the polynucleotide handling protein The conjugate is brought into contact with the nanopore so that it is on the opposite side of the nanopore. and, ii) The polynucleotide of the conjugate is handled by the polynucleotide handling To bring the protein into contact with the protein, iii) The polynucleotide handling protein is the polynucleotide handling protein for the nanopore This makes it possible to control the aforementioned movement of the renucleotide, thereby before it passes through the nanopore. Controlling the movement of the polypeptide, iv) The blocking portion comes into contact with the nanopore, thereby allowing the material to pass through the nanopore. To prevent further movement of the conjugate, the polynucleotide handling tank The protein temporarily breaks its bond from the polynucleotide, thereby under the applied force. And in the opposite direction to the direction of movement controlled by the polynucleotide handling protein To enable the conjugate to move through the nanopore in that direction, v) Optionally repeat steps (ii) to (iv) and pass the nanopore through the Any of claims 1 to 14 or 20 to 23, comprising vibrating a polypeptide. The method described in item 1.

25. The one or more measurements described above determine (i) the length of the polypeptide, and (ii) the length of the polypeptide. Identity, (iii) sequence of the polypeptide, (iv) secondary structure of the polypeptide, (v) whether the polypeptide is modified, selected from (v) the polypeptide The method according to any one of claims 1 to 24, characterized by one or more features of D.

26. A nanopore including a constricted region, wherein the nanopore comprises the constricted region and the nanopore To increase the distance between the contacting polynucleotide handling protein and the protein. Modified nanopores.

27. - Nanopores including a constricted region, - Conjugates containing polypeptides conjugated to polynucleotides, - Polynucleotide handling proteins, A system that includes, i) The nanopore is in contact with the polynucleotide handling enzyme. When this is happening, the constricted region and the active site of the polynucleotide handling protein Modified to increase the distance between and and / or ii) The system comprises the nanopore and the polynucleotide handling protein It is positioned between the nanopore and the polynucleotide handling protein One or more displacer units that extend the distance between the active site of the substance and the displacer unit. A system that further includes this.

28. The aforementioned nanopore, conjugate and / or polynucleotide handling protein Quality, and optionally, if present, one or more of the above-mentioned displacer units, The system according to claim 27, as defined in any one of claims 2 to 14.

29. - Nanopores including a constricted region, - Polynucleotides containing reactive functional groups for conjugation to target polynucleotides Ochido and, - A kit containing polynucleotide handling proteins.

30. (i) The nanopore is in contact with the polynucleotide handling enzyme. At that time, between the constricted region and the polynucleotide handling protein Modified to increase the distance, and / or (ii) the kit and the nanopore To extend the distance between the active site of the polynucleotide handling protein and the other The kit according to claim 29, further comprising one or more displacer units.

31. The aforementioned nanopores, polynucleotides and / or polynucleotide handling tampers The quality, and optionally, if present, one or more of the above-mentioned displacer units or as defined in any one of claims 2 to 14, as described in claim 29 or 30 The kit.

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