Methods

Abstract

Provided herein are methods of generating sequencing libraries, as well as associated systems and methods. The sequencing libraries can be used to characterize polynucleotides and / or polypeptides using nanopores.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to GB 2317028.5, filed on November 6, 2023, the entire contents of which are hereby incorporated by reference. Technical Field

[0003] This disclosure relates to methods for generating peptide sequencing libraries containing DNA-peptide hybrid chains. This disclosure also relates to libraries containing DNA-peptide hybrid chains. Such libraries are useful in sequencing methods, such as in methods for generating data for peptide characterization. Related systems and analytical methods are also provided. Background Technology

[0004] The characterization of biomolecules is becoming increasingly important in biomedical and biotechnological applications. For example, nucleic acid sequencing allows the study of genomes and the proteins they encode, and, for instance, allows for the correlation between nucleic acid mutations and observable phenomena such as disease indications. Nucleic acid sequencing can be used in evolutionary biology to study relationships between organisms. Metagenomics involves identifying organisms present in a sample, such as microorganisms in the microbiome, where nucleic acid sequencing allows for the identification of such organisms.

[0005] While techniques for characterizing polynucleotides (e.g., sequencing polynucleotides) have been extensively developed, techniques for characterizing peptides are less sophisticated, despite their significant biotechnological importance. For example, understanding protein sequences allows for the establishment of structure-activity relationships and influences rational drug development strategies for developing ligands specific to receptors. The identification of post-translational modifications is also crucial for understanding the functional properties of many proteins. For instance, in eukaryotes, typically 30–50% of protein species are phosphorylated. Some proteins may possess multiple phosphorylation sites, which can be used to activate or inactivate the protein, promote its degradation, or regulate interactions with protein-coupled proteins.

[0006] Known methods for characterizing peptides include mass spectrometry and Edman degradation.

[0007] Protein mass spectrometry involves characterizing whole proteins or fragments thereof in ionized form. Known methods of protein mass spectrometry include electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI). While mass spectrometry offers several advantages, the results obtained can be affected by the presence of contaminants and can be difficult to process without breaking down fragile molecules. Furthermore, mass spectrometry is not a single-molecule technique and provides only a limited amount of information about the sample being queried. Mass spectrometry is not well-suited for characterizing differences within a population of peptide samples and is cumbersome when attempting to distinguish adjacent residues.

[0008] Edman degradation is an alternative to mass spectrometry that allows for residue-by-residue sequencing of peptides. Edman degradation sequences peptides by sequentially cleaving the N-terminal amino acids and then characterizing the individually cleaved residues using chromatography or electrophoresis. However, edman sequencing is slow, involves the use of expensive reagents, and, like mass spectrometry, is not a single-molecule technique.

[0009] Therefore, there remains a pressing need for new technologies to characterize peptides, especially at the single-molecule level. Single-molecule techniques for characterizing biomolecules such as polynucleotides have proven particularly attractive because they offer high fidelity and avoid amplification bias. Summary of the Invention

[0010] One attractive approach for single-molecule characterization of biomolecules such as peptides is nanopore sensing. Nanopore sensing is an analyte detection and characterization method that relies on the observation of individual binding or interaction events between analyte molecules and ion conduction channels. Nanopore sensors can be generated by placing nanoscale single pores in an electrically insulating membrane and measuring the voltage-driven ion current flowing through the pore in the presence of analyte molecules. The presence of an analyte inside or near the nanopore will alter the ion flow through the pore, resulting in changes in the ions or current measured on the channel. The identity of the analyte is revealed by its unique current characteristics, particularly the duration and extent of the current block and the changes in current level during interaction with the pore. Nanopore sensing has the potential to allow for rapid and inexpensive peptide characterization.

[0011] Nanopore sensing and characterization of peptides have been proposed in the art. For example, WO 2013 / 123379 discloses the use of NTP-driven protein processing unfolding enzymes for processing proteins to be transported through nanopores. WO 2021 / 111125 discloses a method for conjugating a target peptide with a polynucleotide to form a single-chain peptide-polynucleotide conjugate, wherein the protein is treated with the polynucleotide to cause the conjugate to move through the nanopore. WO 2021 / 133168 discloses protein and peptide fingerprinting and sequencing via nanopore translocation of peptide-oligonucleotide complexes. PCT / GB2023 / 052838 discloses a method for characterizing a target peptide as it moves relative to a nanopore. Each of these documents is incorporated herein by reference in its entirety.

[0012] These methods provide useful techniques for characterizing peptides using nanopores. However, there is still a need to improve the quality of data obtained from such methods.

[0013] The inventors have recognized that data quality from the methods described above can be improved if suitable sequencing libraries can be generated. Such libraries can be used to generate exemplary signals from polypeptide chains as they move relative to nanopores. These exemplary signals can then be used to train algorithms for characterizing unknown polypeptide chains, such as residue interpretation algorithms. The methods for characterizing polypeptides will be aided by providing advanced sequencing libraries that can be used to generate datasets for model training. This is described in more detail herein.

[0014] Generating suitable libraries is not straightforward. While solid-phase peptide synthesis can theoretically be used because the peptide sequence can be predetermined, libraries produced by such methods may contain defects, such as impurities introduced during the synthesis reaction (including incomplete chains where one or more amino acid units may be unintentionally omitted). These defects can degrade the quality of the resulting dataset if incorporated into the sequencing library. Furthermore, generating large numbers of sequencing libraries via solid-phase synthesis is expensive, time-consuming, and requires costly reagents and equipment. The maximum length of peptides that can be produced is also typically limited. Producing large numbers of diverse peptides using such methods is often impractical due to cost, complexity, and synthesis timelines.

[0015] The methods presented in this paper address some or all of these problems.

[0016] This disclosure relates to a method for generating a polypeptide sequencing library. The polypeptide sequencing library comprises multiple DNA-peptide hybrid chains. The method comprises translating multiple RNA chains to form multiple polypeptide chains. The translation step is performed under conditions that cause each polypeptide chain to be linked to the RNA chain from which it is translated. Therefore, the product of the translation step is multiple RNA-peptide hybrid chains.

[0017] The method further includes a step of reverse transcription of the RNA portion of the RNA-peptide hybrid chain. The reverse transcription step produces a DNA sequence complementary to the RNA portion of the RNA-peptide hybrid chain. Therefore, the reverse transcription step forms multiple DNA-peptide hybrid chains.

[0018] In some embodiments, the RNA strand translated to form multiple polypeptide chains encodes a polypeptide test sequence, a purification tag, and a cleavage site between the polypeptide test sequence and the purification tag. As explained in more detail below, this can help ensure high purity of the DNA-peptide hybrid chains in the resulting polypeptide sequencing library.

[0019] In some embodiments, the method further includes the step of contacting the plurality of DNA-peptide hybrid chains with a ribonuclease capable of digesting RNA in the DNA-peptide hybrid chains. In some embodiments, the method further includes generating a DNA chain complementary to at least a portion of the DNA portion of the chain generated in the RNA digestion step. In such embodiments, the complementary DNA chain hybridizes with the DNA portion of the chain, such that the resulting DNA-peptide hybrid chain contains at least partially double-stranded DNA portions. As explained in more detail below, in some embodiments, such chains are ideal for characterization using nanopores.

[0020] Furthermore, a library containing multiple polynucleotide-peptide hybrid chains was provided.

[0021] Therefore, this paper provides a method for generating peptide sequencing libraries;

[0022] The polypeptide sequencing library contained multiple DNA-peptide hybrid chains;

[0023] The method includes:

[0024] i) Translating multiple RNA strands to form multiple polypeptide strands, wherein the translation is performed under conditions that link each polypeptide strand to the RNA strand from which it is translated, thereby forming multiple RNA-polypeptide hybrid chains; and

[0025] ii) Reverse transcription of the RNA portion of the RNA-peptide hybrid chain, thereby forming multiple DNA-peptide hybrid chains;

[0026] The RNA strand translated in step (i) encodes a polypeptide test sequence, a purification tag, and a cleavage site between the polypeptide test sequence and the purification tag.

[0027] In some embodiments, the method includes:

[0028] - The DNA-peptide hybrid chain is purified using the purification tag; and

[0029] - The purification tag is removed from the purified DNA-peptide hybrid chain by contacting the purified DNA-peptide hybrid chain with one or more conditions capable of cleaving the cleavage site.

[0030] In some embodiments, the cleavage site can be cleaved by photolysis, enzymatic reaction, or by contacting the DNA-peptide hybrid chain with one or more chemical reagents. In some embodiments, the cleavage site is a protease cleavage site.

[0031] In some embodiments, removing the purification tag involves contacting the purified DNA-peptide hybrid strand with a protease capable of cleaving the cleavage site.

[0032] In some embodiments, the method further includes:

[0033] iii) Contact the plurality of DNA-peptide hybrid chains with a ribonuclease capable of digesting the RNA in the DNA-peptide hybrid chains.

[0034] In some embodiments, the method further includes:

[0035] iv) Generate a DNA strand complementary to at least a portion of the DNA portion of the strand generated in step (iii), such that the complementary DNA strand hybridizes with the DNA portion of the strand; such that the resulting DNA-peptide hybrid strand contains at least a partially double-stranded DNA portion.

[0036] A method for generating peptide sequencing libraries is also provided;

[0037] The polypeptide sequencing library contained multiple DNA-peptide hybrid chains;

[0038] The method includes:

[0039] i) Translating multiple RNA strands to form multiple polypeptide strands, wherein the translation is performed such that each polypeptide probe strand is linked to the RNA strand from which it is translated, thereby forming multiple RNA-polypeptide hybrid strands;

[0040] ii) Reverse transcription of the RNA portion of the RNA-peptide hybrid chain, thereby forming multiple DNA-peptide hybrid chains;

[0041] iii) Contacting the plurality of DNA-peptide hybrid chains with a ribonuclease capable of digesting the RNA in the DNA-peptide hybrid chains; and

[0042] iv) Generate a DNA strand complementary to at least a portion of the DNA portion of the strand generated in step (iii), such that the complementary DNA strand hybridizes with the DNA portion of the strand; such that the resulting DNA-peptide hybrid strand contains at least a partially double-stranded DNA portion.

[0043] In some embodiments, the ribonuclease is an endonuclease. In some embodiments, the ribonuclease is a ribonuclease H family endonuclease.

[0044] In some embodiments, step (i) comprises linking a peptide-reactive nucleotide or oligonucleotide to the RNA strand, thereby forming a peptide-reactive RNA conjugate; and translating the peptide-reactive RNA conjugate. In some embodiments, the peptide-reactive nucleotide or oligonucleotide comprises a moiety of puromycin.

[0045] In some embodiments, step (ii) comprises contacting the peptide-reactive RNA conjugate with the polypeptide chain generated in the translation reaction, provided that stabilizing conditions are present to stabilize the interaction between the peptide-reactive RNA conjugate and the polypeptide chain. In some embodiments, the stabilizing conditions comprise one or more metal salts, optionally one or more potassium salts and / or magnesium salts.

[0046] In some embodiments, step (ii) comprises hybridizing a DNA primer to a polynucleotide portion of the RNA-peptide hybrid chain. In some embodiments, the method comprises chemically stabilizing the bond between the DNA primer and a complementary portion of the RNA-peptide hybrid chain.

[0047] In some embodiments, the method further comprises linking one or more sequencing adaptors to the DNA and / or polypeptide portions of the DNA-peptide hybrid chain.

[0048] In some embodiments, the method further includes:

[0049] - To bring the DNA-peptide hybrid chain into contact with the nanopore; and

[0050] - As the DNA-peptide hybrid chain moves relative to the nanopore, one or more measurements specific to the DNA-peptide hybrid chain are performed; thereby characterizing the DNA-peptide hybrid chain.

[0051] In some embodiments, the method includes contacting the DNA-peptide hybrid chain with a polynucleotide-treated protein capable of controlling the movement of the DNA-peptide hybrid chain relative to the nanopore.

[0052] In some embodiments, the one or more measurements are specific to one or more of the following: (i) the length of the DNA portion of the DNA-peptide hybrid chain; (ii) the identity of the DNA portion of the DNA-peptide hybrid chain; (iii) the sequence of the DNA portion of the DNA-peptide hybrid chain; (iv) the secondary structure of the DNA portion of the DNA-peptide hybrid chain; (v) whether the DNA portion of the DNA-peptide hybrid chain is modified; (vi) the length of the polypeptide portion of the DNA-peptide hybrid chain; (vii) the identity of the polypeptide portion of the DNA-peptide hybrid chain; (viii) the sequence of the polypeptide portion of the DNA-peptide hybrid chain; (ix) the secondary structure of the polypeptide portion of the DNA-peptide hybrid chain; and (x) whether the polypeptide portion of the DNA-peptide hybrid chain is modified.

[0053] In some embodiments, performing one or more measurements specific to the DNA-peptide hybrid chain includes

[0054] - As the DNA-peptide hybrid chain moves relative to the nanopore, one or more electrical and / or optical measurements specific to the DNA portion of the DNA-peptide hybrid chain are performed, thereby determining one or more properties of the DNA portion of the DNA-peptide hybrid chain;

[0055] - As the DNA-peptide hybrid chain moves relative to the nanopore, perform one or more electrical and / or optical measurements specific to the polypeptide portion of the DNA-peptide hybrid chain; and

[0056] - To correlate the output of one or more electrical and / or optical measurements specific to the polypeptide portion of the DNA-peptide hybrid chain with one or more properties of the DNA portion of the DNA-peptide hybrid chain.

[0057] In some embodiments, the method includes

[0058] Determine the sequence of the DNA portion of the DNA-peptide hybrid chain; and

[0059] The electrical or optical signals recorded when the polypeptide portion of the DNA-polypeptide hybrid chain moves relative to the nanopore are correlated with the sequence.

[0060] In some embodiments, the nanopore is a transmembrane protein pore.

[0061] In some embodiments, the method includes a step of transcribing multiple DNA strands to form the multiple RNA strands prior to step (i).

[0062] A sequencing library is also provided, which can be obtained by the method according to any one of the preceding claims.

[0063] A library is also provided comprising multiple RNA-peptide hybrid chains, wherein each RNA-peptide hybrid chain comprises (i) a peptide comprising a peptide test sequence, a purification tag, and a cleavage site between the peptide test sequence and the purification tag; and (ii) an RNA polynucleotide encoding the peptide.

[0064] A sequencing library is also provided comprising multiple DNA-peptide hybrid chains, wherein the DNA-peptide hybrid chains comprise (i) a peptide moiety containing a peptide test sequence; (ii) a DNA polynucleotide moiety containing (a) a sequence encoding the peptide test sequence and / or (b) a sequence complementary to the sequence encoding the peptide test sequence; and (c) a sequencing adaptor. In some embodiments, the sequencing library comprises (i) a DNA polynucleotide moiety containing a sequence complementary to the sequence encoding the peptide test sequence; and (ii) an RNA or DNA polynucleotide moiety containing a sequence encoding the peptide test sequence. In some embodiments, the DNA-peptide hybrid chains are generated as described herein.

[0065] A system is also provided comprising a library as described herein, and nanopores. In some embodiments, the nanopores are as described herein.

[0066] In some embodiments, the system further includes a polynucleotide processing protein capable of controlling the movement of the DNA-peptide hybrid strand in the library relative to the nanopore. In some embodiments, the system further includes a computing device configured to detect information specific to the polynucleotide and / or peptide portions of the DNA-peptide hybrid strand in the library and to selectively process signals obtained when the DNA-peptide hybrid strand moves relative to the nanopore.

[0067] A method is also provided for analyzing measurement signals acquired from each of a plurality of polynucleotide-peptide hybrid chains, the measurement signals being acquired as the polynucleotide-peptide hybrid chains move relative to a nanopore, wherein each of the polynucleotide-peptide hybrid chains comprises (i) a peptide portion containing a peptide test sequence and (ii) a polynucleotide portion containing a sequence encoding the peptide test sequence, the method comprising:

[0068] Identify in each measurement signal:

[0069] (i) the polypeptide signal portion of the measured signal corresponding to the polypeptide portion of the polynucleotide-peptide hybrid chain; and

[0070] (ii) The polynucleotide signal portion of the measured signal corresponding to the polynucleotide portion of the polynucleotide-peptide hybrid chain;

[0071] For each polynucleotide signal portion, one or more characteristics of the polynucleotide signal portion obtained from the polynucleotide portion are obtained;

[0072] Based on the encoding of the polynucleotide moiety, one or more characteristics of the polypeptide moiety of the polynucleotide-polypeptide hybrid chain containing the polynucleotide moiety are obtained from one or more characteristics obtained from each polynucleotide moiety; and

[0073] The one or more properties of each polypeptide moiety are associated with the polypeptide signal portion of the measurement signal obtained from the polynucleotide-polypeptide hybrid chain containing the polynucleotide moiety.

[0074] In some embodiments, the one or more properties of each polynucleotide moiety comprise the polynucleotide sequence of the polynucleotide moiety. In some embodiments, the one or more properties of each polypeptide moiety comprise the amino acid sequence of the polypeptide moiety.

[0075] In some embodiments, the polynucleotide-peptide hybrid chain is generated as described herein.

[0076] In some embodiments, the method further comprises acquiring the measurement signal from each of the plurality of polynucleotide-peptide hybrid chains. In some embodiments, the measurement signal comprises electrical and / or optical measurements.

[0077] In some embodiments, the polynucleotide portion of the polynucleotide-peptide hybrid chain comprises a DNA chain. In some embodiments, the DNA chain comprises single-stranded DNA, double-stranded DNA, or a double-stranded DNA:RNA hybrid.

[0078] In some embodiments, the polypeptide test sequence contains at least one atypical and / or non-proteinogenic amino acid.

[0079] A computer program is also provided, comprising instructions executable by a computer system, the instructions being configured to cause the computer system to perform the methods described herein when executed. A computer storage medium is also provided, storing the computer program described herein. A computer system is also provided, configured to perform the methods described herein. Attached Figure Description

[0080] Figure 1 AD. Assembly stages of the polynucleotide-peptide construct used in Example 1: i. RNA; ii. DNA adapter; iii. Reverse transcription of DNA; iv. Peptide; v. Filling into the second DNA strand; vi. dsDNA tail; vii: helicase, where the arrow indicates the direction of helicase movement;

[0081] Circle = Click on the chemical group; Rhombus = Purinomycin group.

[0082] Figure 2 The current-time traces capturing the assembled polynucleotide-peptide hybrid chains as described in Example 1 are shown. The DNA sequence linked to the C-terminus of the peptide encodes the purification tag, protease site, and nucleotide sequence of the peptide (i), followed by the adapter sequence (ii). This DNA signal is followed by the peptide signal (amino acid sequence: SGGSGDDSGSGEEEEEEEEEE; SEQ ID NO: 10) (iii-highlighted), and the DNA 'tail' linked to the N-terminus of the peptide (iv). The homopolymer extends in the adapter, and the 'tail' produces a flat signal on either side of the peptide.

[0083] Figure 3A Measurement and analysis system 1, comprising measurement system 2 and analysis system 3, is illustrated. Measurement system 2 obtains a measurement signal from a polymer (such as a polynucleotide-peptide hybrid chain described herein) containing a series of polymer units during polymer translocation relative to a detector (such as a nanopore). Analysis system 3 performs a method for analyzing measurement signal 10 to obtain an estimate of the series of polymer units.

[0084] Figure 3B This is a flowchart of a method for associating a peptide measurement signal with the properties of the peptide that generated the signal.

[0085] Figure 4 The identification of the test peptide conjugates in the experiment described in Example 2 is shown.

[0086] Figure 5 Representative current-time traces of three exemplary different conjugates, as described in Example 2, are shown. Detailed Implementation

[0087] This invention will be described with reference to specific embodiments and certain accompanying drawings, but is not limited thereto by the claims. No reference numerals in the claims should be construed as limiting the scope. It should be understood, of course, that not all aspects or advantages can be achieved according to any particular embodiment of the invention. Therefore, for example, those skilled in the art will recognize that the invention may be embodied or practiced in a manner that achieves or optimizes one or more advantages as taught herein, without necessarily achieving other aspects or advantages as may be taught or suggested herein.

[0088] The invention (both in terms of organization and method of operation) and its features and advantages can be best understood by referring to the following detailed description when read in conjunction with the accompanying drawings. Aspects and advantages of the invention will become apparent from the embodiments described below, and will be set forth with reference to these embodiments. Throughout the specification, references to “one embodiment” or “an embodiment” mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Therefore, the phrases “in one embodiment” or “in an embodiment” appearing in various places throughout the specification do not necessarily all refer to the same embodiment, but may refer to the same embodiment. Similarly, it should be understood that in the description of exemplary embodiments of the invention, various features of the invention are sometimes combined in a single embodiment, drawing, or description thereof for the purpose of simplifying the disclosure and aiding in the understanding of one or more aspects of the invention. However, the method of this disclosure should not be construed as reflecting an intention to reflect more features required by the claimed invention than expressly recited in each claim. Rather, as reflected in the following claims, the inventive aspect lies in fewer features than all of the individual foregoing disclosed embodiments.

[0089] It should be understood that, unless the context otherwise requires, the “embodiments” of this disclosure can be specifically combined together. Specific combinations of all disclosed embodiments (unless the context otherwise implies) are further disclosed embodiments of the claimed invention.

[0090] Furthermore, as used in this specification and the appended claims, unless otherwise expressly indicated, the singular forms “a,” “an,” and “the” include plural references. Thus, for example, a reference to “polynucleotide” includes two or more polynucleotides, a reference to “motor protein” includes two or more such proteins, a reference to “helicase” includes two or more helicases, a reference to “monomer” refers to two or more monomers, a reference to “pore” includes two or more pores, etc.

[0091] All publications, patents, and patent applications cited in this article, whether above or below, are hereby incorporated in their entirety by reference.

[0092] definition

[0093] When referring to singular nouns (e.g., "an," "a," "the"), the use of indefinite or definite articles includes the plural form of the noun unless otherwise specified. When the term "comprising" is used in this specification and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third, etc., in the specification and claims are used to distinguish similar elements and are not necessarily used to describe order or chronological sequence. It should be understood that the terms thus used are interchangeable where appropriate, and embodiments of the invention described herein can operate in a different order than that described or illustrated herein. The following terms or definitions are provided only to aid in understanding the invention. Unless specifically defined herein, all terms used herein have the same meaning as understood by one of skill in the art. For the definitions and terminology used in this field, practitioners have specifically referred to Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 4th edition, Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., *Current Protocols in Molecular Biology* (Supplement 114), John Wiley & Sons, New York (2016). The definitions provided herein should not be construed as having a scope less than that understood by one of ordinary skill in the art.

[0094] When referring to measurable values ​​such as quantity or duration, the term “about” as used herein means to encompass deviations from the specified value of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, as such deviations are suitable for the disclosed methods.

[0095] As used herein, “nucleotide sequence,” “DNA sequence,” or “nucleic acid molecule” refers to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Therefore, this term includes double-stranded and single-stranded DNA, as well as RNA. As used herein, the term “nucleic acid” is a single-stranded or double-stranded covalently linked sequence of nucleotides, wherein the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond. Polynucleotides may consist of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids can be synthesized in vitro or isolated from natural sources. Nucleic acids may further include modified DNA or RNA, such as DNA or RNA that has been methylated, or RNA that has undergone post-translational modifications, such as 5' capping with 7-methylguanosine, 3' processing such as cleavage and polyadenylation, and splicing. Nucleic acids can also include synthetic nucleic acids (XNAs), such as hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threonine nucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid (LNA), and peptide nucleic acid (PNA). The size of a nucleic acid (also referred to herein as a “polynucleotide”) is typically expressed as the number of base pairs (bp) in a double-stranded polynucleotide, or, in the case of a single-stranded polynucleotide, as the number of nucleotides (nt). One thousand bp or nt equals one thousand bases (kb). Polynucleotides shorter than approximately 40 nucleotides are often referred to as “oligonucleotides” and may contain primers used for manipulating DNA, such as through polymerase chain reaction (PCR).

[0096] In the context of this disclosure, the term “amino acid” is used in its broadest sense and refers to organic compounds containing amine (NH2) and carboxyl (COOH) functional groups and side chains (e.g., R groups) specific to each amino acid. In some embodiments, an amino acid refers to a naturally occurring L α-amino acid or residue. Commonly used single-letter and three-letter abbreviations for naturally occurring amino acids are used herein: A = Ala; C = Cys; D = Asp; E = Glu; F = Phe; G = Gly; H = His; I = Ile; K = Lys; L = Leu; M = Met; N = Asn; P = Pro; Q = Gln; R = Arg; S = Ser; T = Thr; V = Val; W = Trp; and Y = Tyr (Lehninger, AL, (1975) Biochemistry, 2nd ed., pp. 71-92, Worth Publishers, New York). The general term “amino acid” further includes D-amino acids, retro-inverso amino acids, and chemically modified amino acids (such as amino acid analogs), naturally occurring amino acids that are not typically incorporated into proteins (such as ortholeucine), and chemically synthesized compounds that have properties characteristic of amino acids known in the art (such as β-amino acids). For example, analogs or mimics of phenylalanine or proline that allow conformational restrictions to the same peptide compounds as natural Phe or Pro are included within the definition of amino acids. Such analogs and mimics are referred to herein as “functional equivalents” of the corresponding amino acids. Other examples of amino acids are listed by Roberts and Vellaccio, *The Peptides: Analysis, Synthesis, Biology*, edited by Gross and Meiehofer, Vol. 5, p. 341, Academic Press, Inc., NY, 1983, which is incorporated herein by reference.

[0097] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to polymers of amino acid residues, as well as their variants and synthetic analogs. Therefore, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic, non-naturally occurring amino acids (such as chemical analogs of corresponding naturally occurring amino acids), as well as naturally occurring amino acid polymers. Polypeptides may also undergo maturation or post-translational modification processes, which may include, but are not limited to, glycosylation, proteolytic cleavage, lipolysis, signal peptide cleavage, propeptide cleavage, phosphorylation, etc. Peptides can be prepared using recombinant technologies (e.g., by expressing recombinant or synthetic polynucleotides). Recombinant peptides are typically substantially free of culture medium, for example, less than about 20% of the volume of the protein formulation, more typically less than about 10%, and most typically less than about 5%.

[0098] The term "protein" is used to describe folded polypeptides that have secondary or tertiary structures. Proteins can consist of a single polypeptide or can comprise multiple polypeptides that assemble to form a multimer. The multimer can be a homooligomer or a heterooligomer. Proteins can be naturally occurring or wild-type proteins, or modified or non-natural proteins. For example, a protein may differ from a wild-type protein by the addition, substitution, or deletion of one or more amino acids.

[0099] Protein “variants” encompass peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions relative to the unmodified or wild-type protein discussed and possess similar biological and functional activities to the unmodified protein from which they are derived. As used herein, the term “amino acid identity” refers to the degree to which sequences are identical on an amino acid-to-amino acid basis within a comparison window. Thus, the “sequence identity percentage” is calculated by comparing two optimally aligned sequences within a comparison window, determining the number of positions in which identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appear in both sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the sequence identity percentage.

[0100] For all aspects and embodiments of the invention, the "variant" has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% complete sequence identity with the corresponding wild-type protein's amino acid sequence. Sequence identity can also be a fragment or portion of a full-length polynucleotide or polypeptide. Thus, a sequence may have only 50% overall sequence identity with a full-length reference sequence, but sequences of specific regions, domains, or subunits may share 80%, 90%, or up to 99% sequence identity with the reference sequence.

[0101] The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. Wild-type genes are the most frequently observed genes in a population and are therefore arbitrarily engineered to be in a "normal" or "wild-type" form of the gene. Conversely, the terms "modified," "mutant," or "variant" refer to a gene or gene product that exhibits sequence modifications (e.g., substitution, truncation, or insertion), post-translational modifications, and / or functional properties (e.g., altered characteristics) compared to a wild-type gene or gene product. It should be noted that naturally occurring mutants can be isolated; these mutants are identified by the fact that they possess altered characteristics compared to a wild-type gene or gene product. Methods for introducing or substituting naturally occurring amino acids are well known in the art. For example, the codon for methionine (ATG) can be substituted with the codon for arginine (CGT) at the relevant position in the polynucleotide encoding the mutant monomer, while methionine (M) is replaced with arginine (R). Methods for introducing or substituting non-naturally occurring amino acids are also well known in the art. For example, non-naturally occurring amino acids can be introduced by including synthetic aminoacyl-tRNA in the IVTT system used to express mutant monomers. Alternatively, the non-naturally occurring amino acids can be introduced by expressing mutant monomers in *E. coli* that are auxotrophic for the specific amino acid in the presence of synthetic (i.e., non-naturally occurring) analogs of those specific amino acids. If the mutant monomer is produced using partial peptide synthesis, it can also be produced by naked linking. Conservative substitution replaces an amino acid with another amino acid having a similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acid can have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acid it replaces. Alternatively, conservative substitution can introduce another aromatic or aliphatic amino acid to replace a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected based on the properties of the 20 major amino acids as defined in Table 1 below. In the case of amino acids having similar polarity, this can also be determined with reference to the hydrophilicity scale of the amino acid side chains in Table 2.

[0102] Table 1 - Chemical properties of amino acids

[0103]

[0104] Table 2 - Hydrophilicity Scale

[0105]

[0106] Mutants or modified proteins, monomers, or peptides can also be chemically modified in any manner and at any site. Mutants or modified monomers or peptides can be chemically modified by linking the molecule to one or more cysteine ​​residues (cysteine ​​linkages), linking the molecule to one or more lysine residues, linking the molecule to one or more non-natural amino acids, enzymatically modifying the epitope, or modifying the terminal portion. Suitable methods for performing such modifications are well known in the art. Mutants of modified proteins, monomers, or peptides can be chemically modified by linking any molecule. For example, mutants of modified proteins, monomers, or peptides can be chemically modified by linking with dyes or fluorophores.

[0107] The disclosed method

[0108] This article provides a method for generating peptide sequencing libraries;

[0109] The polypeptide sequencing library contains multiple DNA-peptide hybrid chains; the method includes:

[0110] i) Translating multiple RNA strands to form multiple polypeptide strands, said translation being performed such that each polypeptide strand is linked to the RNA strand from which it is translated, thereby forming multiple RNA-polypeptide hybrid chains; and

[0111] ii) Reverse transcribe the RNA portion of the RNA-peptide hybrid chain to form multiple DNA-peptide hybrid chains.

[0112] As explained below, peptide sequencing libraries can be used to generate data that can be used to characterize peptides during nanopore characterization. Specifically, this disclosure relates to methods described in more detail herein, which involve analyzing measurement data from DNA-peptide hybrid chains. This data can be used to infer or otherwise determine the properties of the peptide moiety of the DNA-peptide hybrid chain. The signal of the peptide moiety in the DNA-peptide hybrid chain can be correlated with the properties of the peptide moiety. The resulting data can be used to train computer models, such as neural networks, to characterize unknown peptides.

[0113] In some embodiments, the use of sequencing libraries as described herein is associated with advantages that will be apparent to those skilled in the art. Specifically, the correlation between the polynucleotide portion and the peptide portion of the construct means that each peptide signal can be definitively identified and assigned to an associated polynucleotide sequence, and the polynucleotide sequence can be used to directly or indirectly infer or determine the fundamental physical properties of the peptide.

[0114] As explained herein, the disclosed methods involve translating multiple RNA strands to form multiple polypeptide chains. Any suitable RNA strand can be used in such methods. RNA translation is well known in the art, and some exemplary conditions are described in more detail herein.

[0115] RNA strands can be produced by any suitable method. In some embodiments, suitable RNA strands are produced by transcription of DNA strands. In some embodiments, suitable RNA strands are produced by transcription of portions of genomic DNA (e.g., from microorganisms such as bacteria or viruses). This is described in more detail herein.

[0116] The disclosed method involves translating multiple RNA strands under conditions that cause each polypeptide strand to be linked to the RNA strand from which it is translated, thereby forming multiple RNA-polypeptide hybrids. Any suitable conditions and methods can be used to link the translated polypeptide strand to the RNA strand from which it is translated. Some exemplary conditions and reactions for linking translated polypeptide strands to their respective RNA strands are described in more detail herein.

[0117] The method further comprises reverse transcription of the RNA portion of the RNA-peptide hybrid chain, thereby forming multiple DNA-peptide hybrid chains. RNA reverse transcription is well known in the art, and some exemplary conditions are described in more detail herein.

[0118] The polypeptide sequencing library contains multiple DNA-peptide hybrid chains. The multiple chains may include at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10000, at least 100000, at least 100000, or more DNA-peptide hybrid chains.

[0119] The DNA-peptide hybrid chains in the library may be identical or different. In some embodiments, the library contains multiple types of DNA-peptide hybrid chains, and each type of DNA-peptide hybrid chain contains multiple identical DNA-peptide hybrid chains. In some embodiments, the library contains at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, or at least 1000 types of DNA-peptide hybrid chains, and each type of DNA-peptide hybrid chain contains at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10000, at least 100000, or at least 1000000 or more identical DNA-peptide hybrid chains.

[0120] In some embodiments, the RNA-encoded polypeptide translated in the disclosed method includes a test sequence, a purification tag, and a cleavage site. Therefore, in some embodiments, the polypeptide translated from the RNA strand comprises a test sequence, a purification tag, and a cleavage site. In some embodiments, the cleavage site is located between the test sequence and the purification tag.

[0121] In some embodiments, the disclosed methods include purifying a DNA-peptide hybrid chain using a purification tag. In some embodiments, the disclosed methods include purifying an RNA-peptide hybrid chain using a purification tag. Purification tags are described in more detail herein.

[0122] In some embodiments, the disclosed method includes removing the purification tag. In some embodiments, the purification tag is removed by cleaving a cleavage site located between the test sequence and the purification tag. Suitable cleavage sites are described in more detail herein.

[0123] As explained in more detail below, the multiple DNA-peptide hybrid chains may comprise single-stranded or double-stranded DNA. The DNA-peptide hybrid chains may comprise DNA:RNA hybrid chains. This is discussed in more detail below.

[0124] As explained above, the translation of an RNA strand produces an RNA-peptide hybrid. Therefore, the RNA-peptide hybrid contains an RNA portion.

[0125] Reverse transcription of the RNA-peptide hybrid produces a DNA strand complementary to at least a portion of the RNA moiety. Therefore, the DNA-peptide hybrid produced by reverse transcription of the RNA moiety of the RNA-peptide hybrid typically comprises a double-stranded polynucleotide moiety containing at least one portion comprising an RNA:DNA chain. The RNA moiety of the RNA:DNA chain can be derived from RNA translated in the disclosed methods. The DNA moiety originates from the reverse transcription step. The resulting construct is an example of a DNA-peptide hybrid as used herein.

[0126] In some embodiments, the disclosed method includes contacting the plurality of DNA-peptide hybrid chains with a ribonuclease capable of digesting RNA in the DNA-peptide hybrid chains. Suitable ribonucleases are described in more detail herein.

[0127] Digestion of RNA in a DNA-peptide hybrid chain (i.e., in a DNA-peptide hybrid chain containing a double-stranded polynucleotide moiety comprising both a DNA strand and an RNA strand) can completely or partially remove RNA from the DNA-peptide hybrid chain. Therefore, digestion can be partial or complete. Typically, digestion removes all RNA from the DNA-peptide hybrid chain. In some embodiments, the DNA-peptide hybrid chain therefore does not contain an RNA strand or an RNA moiety.

[0128] In some embodiments, the DNA-peptide hybrid chain therefore comprises a single-stranded DNA portion. In some embodiments, the single-stranded DNA portion comprises a polynucleotide sequence complementary to the translated RNA sequence. In some embodiments, the single-stranded DNA portion comprises a sequence complementary to the RNA sequence encoding the amino acid sequence of the test peptide. In some embodiments, the single-stranded DNA portion comprises a sequence complementary to the RNA sequence encoding the amino acid sequence of the test peptide, the cleavage site, and the purification tag.

[0129] In some embodiments, the DNA-peptide hybrid chain comprises a polypeptide chain and a DNA chain, with the DNA chain linked to the polypeptide chain via a hybridization region. In some embodiments, the DNA-peptide hybrid chain comprises a DNA sequence complementary to an initial RNA sequence, the initial RNA sequence comprising a portion that hybridizes to an oligonucleotide moiety, which may comprise RNA or DNA and is linked to the polypeptide moiety. In some embodiments, the hybridization is stable. Any suitable stabilizer can be used. Several examples are discussed in more detail herein.

[0130] In some embodiments, the disclosed method comprises generating a DNA strand complementary to at least a portion of the DNA portion of the strand produced by ribonuclease digestion of RNA. This is referred to herein as an “infill” or “fill-in” reaction. In some embodiments, the infill reaction effectively replaces the RNA strand translated in the disclosed method with a similar DNA strand.

[0131] In some embodiments, the DNA-peptide hybrid chain therefore comprises a double-stranded DNA portion. In some embodiments, the double-stranded DNA portion comprises a polynucleotide sequence complementary to the translated RNA sequence. In some embodiments, the single-stranded DNA portion comprises a sequence complementary to the RNA sequence encoding the amino acid sequence of the test peptide. In some embodiments, the single-stranded DNA portion comprises a sequence complementary to the RNA sequence encoding the amino acid sequence of the test peptide, the cleavage site, and the purification tag.

[0132] In some embodiments, the disclosed method includes linking one or more sequencing adaptors to a DNA-peptide hybrid strand generated as described in more detail herein. The one or more sequencing adaptors can be used to facilitate measurements specific to the DNA-peptide hybrid strand or a portion thereof as the DNA-peptide hybrid strand moves relative to the nanopore.

[0133] This document also discloses libraries containing multiple RNA-peptide hybrids or DNA-peptide hybrids as described herein. Such libraries can be generated as described herein. In the methods described herein, such libraries can also be used to generate useful data specific to the polynucleotide and / or peptide motifs of the constructs within such libraries.

[0134] As described in this article, such methods can be computer-implemented methods.

[0135] RNA chain

[0136] The disclosed method involves translating multiple RNA strands to form multiple polypeptide chains. As discussed in more detail herein, the translation reaction is carried out under conditions that link each polypeptide chain to the RNA strand from which it is translated. Thus, multiple RNA-polypeptide hybrid chains are formed.

[0137] Any suitable RNA strand can be translated using the disclosed methods. In some embodiments, the RNA strand is a naturally occurring RNA strand. In some embodiments, the RNA strand is a synthetic RNA strand.

[0138] In some embodiments, RNA chains are secreted from cells. Alternatively, RNA chains can be produced intracellularly, making them necessary to be extracted from cells for use in the disclosed methods.

[0139] In some embodiments, the RNA chain is generated by transcribing multiple DNA strands to form the multiple RNA chains. Therefore, in some embodiments, the RNA chain contains the product of the transcription reaction. In some embodiments, the transcription step is performed prior to step (i) of the disclosed method.

[0140] As used herein, the term “transcription” (“trancribing”, “transcribed”, etc.) refers to the synthesis of RNA from a DNA template. RNA is typically synthesized using a DNA-dependent RNA polymerase (also known as a transcriptase).

[0141] In some embodiments, a bacterial transcriptase is used for transcription. In some embodiments, a viral transcriptase is used for transcription. In some embodiments, a bacteriophage transcriptase is used for transcription. Examples include T3 RNA polymerase, T7 RNA polymerase, Hi-T7® RNA polymerase, SP6 RNA polymerase, *E. coli* Poly(A) polymerase, Poly(U) polymerase, *E. coli* RNA polymerase, core enzymes, and *E. coli* RNA polymerase, holoenzymes. These and other suitable enzymes are available from New England Biolabs (Ipswich, MA, USA) and are generally used according to standard procedures known in the art. In some embodiments, T7 RNA polymerase is used for transcription. T7 RNA polymerase is available as HiScribe from New England Biolabs (Ipswich, MA, USA). TM This was obtained as part of the T7 Rapid High-Yield RNA Synthesis Kit.

[0142] In embodiments where RNA chains are generated by transcribing multiple DNA strands to form said multiple RNA chains, any suitable DNA strand is used. In some embodiments, the DNA strand is a naturally occurring DNA strand. In some embodiments, the DNA strand is a synthetic DNA strand.

[0143] In some embodiments, DNA strands are secreted from cells. Alternatively, DNA strands can be produced within cells, making them necessary to be extracted from cells for use in the disclosed methods.

[0144] In some embodiments, the DNA strand comprises genomic DNA. In some embodiments, the DNA strand comprises a DNA segment from the genome of one or more microorganisms. Suitable microorganisms include bacteria, viruses, yeast, and bacteriophages. In some embodiments, the microorganism is a bacterium. In some embodiments, the microorganism is a bacterium, such as *Escherichia coli*. In some embodiments, the microorganism is a bacteriophage, such as bacteriophage T4, T7, or T10.

[0145] Therefore, RNA strands can contain transcripts of genomic DNA. Genomic DNA can be fragmented. DNA fragmentation can be achieved by any suitable method. For example, methods for DNA fragmentation are known in the art. Such methods can use transposases, such as MuA transposase.

[0146] RNA strands, or the DNA strands transcribed from them, can be obtained or extracted from any organism or microorganism. RNA strands, or the DNA strands transcribed from them, can be obtained from humans or animals (e.g., from urine, lymph, saliva, mucus, semen, or amniotic fluid, or from whole blood, plasma, or serum). They can also be obtained from plants (e.g., cereals, legumes, fruits, or vegetables).

[0147] RNA strands, or DNA strands transcribed from RNA strands, can be provided as an impure mixture of one or more polynucleotides and one or more impurities. Impurities may contain polynucleotides in truncated forms that differ from the target polynucleotide. Impurities may contain cellular material such as genomic DNA, fractions of genomic DNA, plasmids, etc.

[0148] An RNA strand, or the DNA strand from which it is transcribed, can contain any combination of any nucleotides. Nucleotides can be naturally occurring or artificial. One or more nucleotides in the RNA strand, or the DNA strand from which it is transcribed, can be oxidized or methylated. One or more nucleotides in the RNA strand, or the DNA strand from which it is transcribed, can be disrupted. For example, an RNA strand, or the DNA strand from which it is transcribed, can contain pyrimidine dimers. Such dimers are commonly associated with UV damage and are a major cause of melanoma.

[0149] One or more nucleotides in an RNA strand, or a DNA strand from which an RNA strand is transcribed, may be modified, for example, by means of a marker or tag, suitable examples of which are known to those skilled in the art.

[0150] This document discloses examples of modified bases, which can be incorporated into an RNA strand or a DNA strand from which an RNA strand is transcribed, by methods known in the art, such as by polymerase incorporation of the modified nucleotide triphosphate during strand replication (e.g., in PCR) or by polymerase-filling methods. In some embodiments, one or more bases may be chemically modified using reagents known in the art.

[0151] Nucleotides typically contain a nucleobase, a sugar, and at least one phosphate group. The nucleobase and sugar form a nucleoside. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, and more specifically include adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. In the disclosed methods, the sugar is typically ribose. Polynucleotides typically contain the following nucleosides: adenosine (A), uridine (U), guanosine (G), and cytosine (C). Target RNA nucleotides typically contain monophosphates, diphosphates, or triphosphates. Nucleotides may contain more than three phosphates, such as four or five phosphates. Phosphates may be attached to the 5' or 3' side of the nucleotide. Nucleotides in target RNA polynucleotides may be linked to each other in any manner. Nucleotides are typically linked by their sugar and phosphate groups, as in nucleic acids. Nucleotides may be linked by their nucleobases, as in pyrimidine dimers. Nucleotides include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate (dCMP), and deoxymethylcytidine monophosphate. Nucleotides are typically selected from AMP, TMP, GMP, CMP, and UMP. Nucleotides can be baseless (i.e., lacking a nucleobase). Nucleotides can also lack both a nucleobase and a sugar (i.e., are C3 spacers).

[0152] In some embodiments, the RNA strand that may be translated in the disclosed method comprises single-stranded RNA. In some embodiments, the RNA strand that may be translated in the disclosed method comprises double-stranded RNA.

[0153] In some embodiments, the DNA transcribed from the RNA strand may contain single-stranded DNA. In some embodiments, the DNA transcribed from the RNA strand may contain double-stranded DNA.

[0154] The RNA strand and / or the DNA strand transcribed from it can be of any length. For example, the length of the RNA strand and / or the DNA strand transcribed from it can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides or nucleotide pairs. The length of the RNA strand and / or the DNA strand transcribed from it can be 1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotides or nucleotide pairs, or 100,000 or more nucleotides or nucleotide pairs.

[0155] More typically, the length of the RNA strand and / or the DNA strand transcribed from it can be from about 1 to about 10,000 nucleotides or nucleotide pairs, such as from about 10 to about 5,000 nucleotides or nucleotide pairs, such as from about 50 to about 1,000 nucleotides or nucleotide pairs, such as from about 100 to about 500 nucleotides or nucleotide pairs.

[0156] Any number of RNA strands may be used in the disclosed method. For example, the method may involve translating at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10000, at least 100000, or at least 1000000 or more RNA strands. The RNA strands may be the same or different.

[0157] When generating RNA strands by transcribing DNA strands, any number of DNA strands can be used. For example, such methods can involve transcribing at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10000, at least 100000, or at least 1,00000 or more DNA strands. The DNA strands can be the same or different.

[0158] Within the scope of the methods provided herein, the RNA strand translated and / or the DNA strand transcribed from the RNA strand can be labeled with molecular markers. Molecular markers can be modifications of polynucleotides that facilitate the detection of polynucleotides in the methods provided herein. For example, a marker can be a modification of a polynucleotide that alters the signal obtained when a construct containing a polynucleotide is characterized. For example, a marker might interfere with the flux of ions through a nanopore. In this way, labeling can improve the sensitivity of the method.

[0159] The RNA strand translated in the disclosed methods typically encodes a polypeptide test sequence. The test sequence is not limited and can be any sequence of interest to the user of the method. The test sequence may be known or unknown prior to the method. The RNA sequence can be characterized as described herein. For example, as explained herein, in some embodiments, the methods disclosed herein comprise determining the polynucleotide sequence of the RNA strand and thereby determining the amino acid sequence of the polypeptide test sequence.

[0160] In some embodiments, the peptide test sequence comprises at least one atypical amino acid, such as a non-proteinogenic amino acid not naturally encoded in the amino acid set of eukaryotic genomes. In some embodiments, the peptide test sequence comprises at least one of the following atypical amino acids: 3-aminotyrosine, acetyl-lysine, dimethyl-lysine, methyl-lysine, arginine phosphate, aspartic acid phosphate, cysteine ​​phosphate, glutamic acid phosphate, histidine phosphate, lysine phosphate, phosphoserine, threonine phosphate, tyrosine phosphate, propargyl-lysine, pyrrolyl-lysine, trimethyl-lysine, AbK (N6-[[2-(3-methyl-3H-bisacryl-3-yl)ethoxy]carbonyl]-L-lysine), DMNB-cage-serine (O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine).

[0161] Methods for incorporating unconventional amino acids into peptides are known in the art. For example, a cell-free protein translation system can be used, which integrates one or more tRNA molecules containing atypical amino acids.

[0162] In some embodiments, the RNA strand and / or the DNA strand from which it is transcribed may contain a promoter sequence of a polymerase, such as RNA polymerase. In some embodiments, the RNA strand contains a promoter of a bacterial RNA polymerase. In some embodiments, the RNA strand contains a promoter of a viral RNA polymerase. In some embodiments, the RNA strand contains a promoter of a bacteriophage RNA polymerase. In some embodiments, the RNA strand contains a promoter of a T3 or T7 RNA polymerase. In some embodiments, the RNA strand contains a promoter of a T7 RNA polymerase. In some embodiments, the promoter contains the sequence 5'-TAATACGACTCACTATAG-3' (SEQ ID NO: 11).

[0163] RNA translation and linkage with translated peptides

[0164] As explained above, the disclosed method involves translating RNA chains to form multiple polypeptide chains.

[0165] The term "translating" (also referred to as "translation," "translated," etc.) refers to the process of synthesizing a polypeptide from RNA. Typically, the RNA is messenger RNA (mRNA). The RNA sequence defines codons corresponding to specific amino acids or termination signals. The correlation between RNA codons and the resulting amino acids is determined by the genetic code. This is referred to herein as "coding"; therefore, in some embodiments, the RNA contains a sequence encoding a polypeptide test sequence. The standard genetic code is shown in Table 3.

[0166]

[0167] Table 3 - Standard RNA genetic code. =Termination symbol. Those skilled in the art will understand that the corresponding DNA genetic code uses T instead of U.

[0168] In some embodiments, the RNA strand contains a ribosome binding site. In some embodiments, the ribosome binding site contains a Shine-Delgarno sequence. In some embodiments, the ribosome binding site contains the sequence 5'-AGGAGG-3' (SEQ ID NO: 12).

[0169] In some embodiments, the disclosed method may include translating an RNA strand using cellular ribosomes.

[0170] In some embodiments, the disclosed methods include a cell-free or in vitro translation step. In some embodiments, in vitro translation is performed using a translation system selected from rabbit reticulocyte lysate, wheat germ extract, and Escherichia coli cell-free systems. Such systems are commercially available, such as those from Thermo Fisher Scientific. In some embodiments, the translation system is a reconstructed cell-free system. In some embodiments, the translation system comprises a reconstructed system including purified cellular translation components. In some embodiments, the purified components are derived from bacteria, such as Escherichia coli. Such systems are commercially available, such as the PURExpress® system from New England Biolabs (Ipswich, Massachusetts, USA).

[0171] In the disclosed method, multiple RNA chains are translated, the translation being carried out under conditions that link each polypeptide chain to the RNA chain from which it is translated, thereby forming multiple RNA-polypeptide hybrid chains.

[0172] RNA strands can be linked to translated polypeptide strands in any suitable manner.

[0173] In some embodiments, the linking comprises linking a peptide-reactive nucleotide or oligonucleotide to an RNA strand. In some embodiments, the peptide-reactive nucleotide or oligonucleotide contains a peptide-reactive functional group. In some embodiments, the reactive functional group is capable of binding to one or more amino acids in the peptide. In some embodiments, the binding is covalent. In some embodiments, the binding is non-covalent. In some embodiments, step (i) of the disclosed method thus comprises linking a peptide-reactive nucleotide or oligonucleotide to the RNA strand, thereby forming a peptide-reactive RNA conjugate; and translating the peptide-reactive RNA conjugate.

[0174] In some embodiments, the splice oligonucleotide is used to facilitate the linking of a peptide-reactive nucleotide or oligonucleotide to an RNA strand. In some embodiments, the splice oligonucleotide comprises or is composed of a DNA polynucleotide. In some embodiments, the splice oligonucleotide comprises a first portion capable of binding to a peptide-reactive nucleotide or oligonucleotide and a second portion capable of binding to an RNA strand. In some embodiments, the first portion is complementary or substantially complementary to the peptide-reactive nucleotide or oligonucleotide. In some embodiments, the second portion is complementary or substantially complementary to the RNA strand. In some embodiments, the length of the splice oligonucleotide is from about 10 to about 50 nucleotides, such as from about 15 to about 30 nucleotides, for example from about 18 to about 25 nucleotides.

[0175] Any suitable linker chemistry can be used to link the RNA chain to the resulting polypeptide.

[0176] An RNA chain can be attached to a polypeptide at any suitable location. For example, the RNA chain can be attached to the polypeptide at either the N-terminus or the C-terminus. The RNA chain can also be conjugated to the polypeptide via side-chain groups of residues (e.g., amino acid residues). Typically, the RNA chain is attached to the polypeptide at the C-terminus.

[0177] In some embodiments, the RNA chain contains codons encoding amino acids, which contain naturally occurring reactive functional groups that can facilitate linkage with the RNA chain. For example, cysteine ​​residues can be used to form disulfide bonds with the RNA chain or modified groups thereon.

[0178] In some embodiments, the polypeptide is modified to facilitate its conjugation to an RNA chain. For example, in some embodiments, the polypeptide is modified by linking a portion containing a reactive functional group for linking to the RNA chain. For example, in some embodiments, the polypeptide may extend one or more residues (e.g., amino acid residues) at an N-terminus or C-terminus, said one or more residues containing one or more reactive functional groups for reacting with corresponding reactive functional groups on the RNA chain. For example, in some embodiments, the polypeptide may extend one or more cysteine ​​residues at an N-terminus and / or C-terminus. Such residues can be used for linking to the polynucleotide portion of the conjugate, for example, through maleimide chemistry (e.g., by reacting cysteine ​​with an azide-maleimide compound (such as azide-[Pol]-maleimide, where [Pol] is typically a short-chain polymer, such as PEG, e.g., PEG2, PEG3, or PEG4); subsequently coupled with a suitably functionalized polynucleotide, such as a polynucleotide with a BCN group, for reaction with the azide). To avoid any doubt, when a polypeptide contains appropriate naturally occurring residues at its N-terminus and / or C-terminus (e.g., naturally occurring cysteine ​​residues at its N-terminus and / or C-terminus), such residues can be used to link with the RNA strand.

[0179] In some embodiments, residues in the polypeptide are modified to facilitate the linkage of the polypeptide to the RNA chain. In some embodiments, residues in the polypeptide (e.g., amino acid residues) are chemically modified for linkage to the RNA chain. In some embodiments, residues in the polypeptide (e.g., amino acid residues) are enzymatically modified for linkage to the RNA chain.

[0180] The conjugation chemistry between RNA strands and polypeptides is not specifically limited. Any suitable combination of reactive functional groups can be used. Many suitable reactive groups and their chemical targets are known in the art. Some exemplary reactive groups and their corresponding targets include aryl azides that can react with amines, carbodiimides that can react with amines and carboxyl groups, acyl hydrazides that can react with carbohydrates, hydroxymethylphosphine that can react with amines, imine esters that can react with amines, isocyanates that can react with hydroxyl groups, carbonyl groups that can react with hydrazine, maleimides that can react with thiol groups, NHS-esters that can react with amines, PFP-esters that can react with amines, psoralen that can react with thymine, pyridyl disulfides that can react with thiol groups, vinyl sulfones that can react with thiol amines and hydroxyl groups, vinyl sulfonamides, etc.

[0181] Other suitable chemistry for conjugating peptides to RNA chains includes click chemistry. Many suitable click chemical reagents are known in the art. Suitable examples of click chemistry include, but are not limited to, the following:

[0182] (a) Copper (I)-catalyzed azide-alkyne cycloaddition (azide-alkyne Huisgen cycloaddition);

[0183] (b) Strain-promoted azide-alkyne cycloadditions; including olefin and azide [3+2] cycloadditions; olefin and tetrazine reverse demand Diels-Alder reactions; and olefin and tetrazolium photoclick reactions;

[0184] (c) Copper-free variants of 1,3-dipolar cycloaddition reactions in which the azide reacts with an alkyne under strain, for example in a cyclooctane ring, such as in a bicyclic [6.1.0]nonyne (BCN);

[0185] (d) The reaction of an oxygen nucleophile at one junction with the reactive moiety of an epoxide or aziridine at the other junction; and

[0186] (e) Staudinger ligation, in which the alkyne moiety can be replaced by arylphosphine, causing a specific reaction with the azide to yield an amide bond.

[0187] Any reactive group can be used in the linking step. Some suitable reactive groups include [1,4-bis[3-(2-pyridyldithio)propamido]butane; 1,1,1-bis-maleimide triethylene glycol; 3,3'-dithiodipropionate di(N-hydroxysuccinimide); ethylene glycol-bis(succinate N-hydroxysuccinimide); 4,4'-diisothiocyanate stilbene-2,2'-disulfonic acid disodium salt; bis[2-(4-azidosalicylic acid amino)ethyl] disulfide; 3-(2-pyridyldithio)propionate N-hydroxysuccinimide; 4-maleimidebutyrate N-hydroxysuccinimide; iodoacetic acid N-hydroxysuccinimide; S-acetylthiolacetic acid N-hydroxysuccinimide; azide-PEG-maleimide; and alkyne-PEG-maleimide. The reactive group can be any of those groups disclosed in WO 2010 / 086602, and particularly in Table 3 of this application.

[0188] In some embodiments, the reactive group is or comprises an aminonucleoside. In some embodiments, the reactive group is or comprises an aminoacylated tRNA analog. In some embodiments, the reactive group is or comprises a puromycin analog. In some embodiments, the reactive group is a puromycin analog modified at the amino acid moiety or nucleoside moiety. In some embodiments, the reactive group is a puromycin analog modified at the nucleoside moiety. In some embodiments, the reactive group is a puromycin analog selected from 3'-N-aminoacylpuromycin (PANS-amino acid) and 3'-N-aminoacyladenosine amino acid nucleoside (AANS-amino acid). In some embodiments, the reactive group is or comprises a puromycin analog disclosed in Ge et al., Angewandte Chemie International Edition, April 11, 2016; 55(16):4933-7, the entire contents of which are incorporated herein by reference.

[0189] In some embodiments, the reactive group is a puromycin group. Therefore, in some embodiments, the peptide-reactive nucleotide or oligonucleotide contains a puromycin moiety. In some embodiments, the method comprises reacting the puromycin group with a polypeptide formed during the translation step.

[0190] In some embodiments, a puromycin group is included in the oligonucleotide. In some embodiments, the oligonucleotide is about 10 to about 50 nucleotides in length, such as about 20 to about 40 nucleotides. In some embodiments, the oligonucleotide includes one or more spacers, such as one or more spacers described herein. For example, in some embodiments, the oligonucleotide includes about 1 to about 10, such as about 3 to about 5 spacers. In some embodiments, the one or more spacers include one or more spacer 3 or spacer 9 spacers.

[0191] In some embodiments, the oligonucleotide contains a label. Suitable labels are described herein.

[0192] An exemplary oligonucleotide containing a puromycin moiety is:

[0193] / Phosphate / AAAAAAAAAAAAAA(dT-Fluor) AAAAAA / spacer 9 / / spacer 9 / / spacer 9 / ACC-puromycin (SEQ ID NO: 13). Another exemplary oligonucleotide containing the puromycin moiety is: / 5PHOS / AAAAAAAAAAAA(T-Fluorine)AAAAAAAAAAAAAAAA(puromycin) (SEQ ID NO: 4). In some embodiments, the oligonucleotide has a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to that of SEQ ID NO: 13 or SEQ ID NO: 4.

[0194] In some embodiments, the reaction of the puromycin group is facilitated by providing stabilizing conditions for stabilizing the interaction between the peptide-reactive RNA conjugate and the polypeptide chain. Therefore, in some embodiments, step (ii) includes contacting the peptide-reactive RNA conjugate with the polypeptide chain generated in the translation reaction, in the presence of stabilizing conditions capable of stabilizing the interaction between the peptide-reactive RNA conjugate and the polypeptide chain.

[0195] In some embodiments, the stabilizing conditions comprise one or more metal salts. In some embodiments, the stabilizing conditions comprise one or more potassium and / or magnesium salts. In some embodiments, the stabilizing conditions comprise MgCl2 or KCl. In some embodiments, one or more potassium and / or magnesium salts are present at a concentration of about 1 mM to about 1 M, such as about 100 mM to about 800 mM, such as about 300 mM to about 600 mM, for example about 500 mM.

[0196] In some embodiments, the translation reaction occurs on modified RNA. In some embodiments, the translation reaction occurs on RNA linked to peptide-reactive nucleotides or oligonucleotides. In some embodiments, the translation reaction is an in vitro translation reaction. The translation reaction can be performed using commercially available kits, such as the PURExpress® In Vitro Protein Synthesis Kit available from New England Biolabs (Ipswich, Massachusetts, USA).

[0197] Reverse transcription of RNA-peptide hybrid chains

[0198] As explained above, the disclosed method involves reverse transcription of the RNA portion of the RNA-peptide hybrid chain, thereby forming multiple DNA-peptide hybrid chains.

[0199] Therefore, in some embodiments, the disclosed method includes generating a DNA strand complementary to the RNA strand translated in the disclosed method.

[0200] In some embodiments, generating a DNA strand complementary to the RNA strand involves reverse transcription of the RNA sequence. Reverse transcription of the RNA sequence produces a DNA strand with a sequence complementary to the RNA strand. The resulting construct containing the synthetic DNA strand is referred to herein as a DNA-peptide hybrid.

[0201] As used herein, the term "reverse transcription" ("reverse transcribing", "reverse transcribed", etc.) refers to the synthesis of DNA from an RNA template. DNA is typically synthesized using an RNA-dependent DNA polymerase (also known as a reverse transcriptase).

[0202] In some embodiments, the reverse transcription reaction is performed using bacterial reverse transcriptase. In some embodiments, the reverse transcription reaction is performed using viral reverse transcriptase. Examples include avian myeloblastomavirus (AMV) reverse transcriptase and Moloney murine leukemia virus (M-MuLV, MMLV) reverse transcriptase. In some embodiments, the reverse transcriptase is MMLV. These and other suitable enzymes are available from New England Biolabs (Ipswich, Massachusetts, USA) and are generally used according to standard procedures known in the art.

[0203] In some embodiments, the disclosed method includes hybridizing a DNA primer with a polynucleotide portion of an RNA-peptide hybrid chain. The DNA primer can be used as a site for initiating a reverse transcription reaction.

[0204] In some embodiments, the disclosed method includes hybridizing a DNA primer with at least a portion of the RNA portion of an RNA-peptide hybrid chain. In some embodiments, the DNA primer hybridizes with the RNA-peptide hybrid chain at the 3' end of the RNA sequence. In some embodiments, the DNA primer hybridizes with the RNA-peptide hybrid chain, wherein the hybrid chain is adjacent to or at the junction of the polypeptide portion of the RNA-peptide hybrid chain.

[0205] In some embodiments, a DNA primer hybridizes to the RNA portion of an RNA-peptide hybrid chain. In some embodiments, the RNA portion of the RNA-peptide hybrid chain is linked to the polypeptide portion of an RNA-peptide hybrid chain as described herein. In some embodiments, the RNA portion of the RNA-peptide hybrid chain is linked to the polypeptide portion of the RNA-peptide hybrid chain via a polynucleotide linker. In some embodiments, the polynucleotide linker comprises RNA or DNA. In some embodiments, a DNA primer hybridizes to a polynucleotide linker.

[0206] In some embodiments, reverse transcriptase is capable of synthesizing DNA from both RNA and DNA templates. In some embodiments, a DNA primer hybridizes with a polynucleotide linker between the RNA and polypeptide portions of the RNA-peptide hybrid chain, and the reverse transcriptase is capable of synthesizing DNA complementary to the sequence of the polynucleotide linker and the sequence of the RNA portion of the RNA-peptide hybrid chain. This can be advantageous because it allows the generation of multiple distinct DNA-peptide hybrid chains in a library using a common linker and a common primer.

[0207] In some embodiments, the DNA primers are about 5 to about 100 nucleotides in length, such as about 10 to about 50 nucleotides, or about 20 to about 40 nucleotides in length.

[0208] In some embodiments, the DNA primer has at least 50%, for example at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, or at least 99% complementary nucleotide sequences to the RNA portion of the RNA-peptide hybrid chain.

[0209] In some embodiments, the DNA primer is about 5 to about 100 nucleotides in length, such as about 10 to about 50 nucleotides, such as about 20 to about 40 nucleotides; and is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, or at least 99% complementary to the 5 to 100 nucleotides (e.g., about 10 to about 50 nucleotides, such as about 20 to about 40 nucleotides) at the 3' end of the RNA sequence.

[0210] In some embodiments, the sequence of the polynucleotide linker between the DNA primer and the RNA portion of the RNA-peptide hybrid chain and the polypeptide portion of the RNA-peptide hybrid chain is at least 50%, for example, at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, or at least 99% complementary.

[0211] Reverse transcription of the RNA portion of the RNA-peptide hybrid chain results in the production of a DNA-peptide hybrid chain as described herein. The production of a DNA chain complementary to the RNA portion of the RNA-peptide hybrid chain means that, in some embodiments, the resulting DNA-peptide hybrid chain comprises at least partially double-stranded DNA portions. In some embodiments, the DNA-peptide hybrid chain comprises a DNA portion comprising a DNA:RNA hybrid chain. Such chains are suitable for characterization in the methods disclosed herein.

[0212] In some embodiments, the binding between the DNA primer and the complementary portion (e.g., the complementary polynucleotide portion) of the RNA-peptide hybrid chain can be stabilized. In some embodiments, the binding between the DNA synthesized by reverse transcription and the RNA-peptide hybrid chain is stable. In some embodiments, the binding between the DNA synthesized by reverse transcription and the polynucleotide linker between the RNA and polypeptide portions of the RNA-peptide hybrid chain is stable.

[0213] Stabilization can be achieved by any suitable method. Many methods for stabilizing double-stranded polynucleotides are known in the art, and such methods are generally applicable to the methods disclosed.

[0214] In some embodiments, stability can be measured by determining the melting temperature of the duplex, which is formed by the connection (e.g., hybridization) of the strands of an RNA-peptide hybrid (e.g., between a primer and a complementary portion (e.g., a complementary polynucleotide portion)). Interactions that stabilize the strand connections (e.g., hybridization between strands) typically increase the melting temperature of the resulting construct.

[0215] In some embodiments, the primers and / or DNA synthesized via a reverse transcription step comprise one or more modified nucleotides. In some embodiments, the primers comprise one or more modified nucleotides. In some embodiments, the primers comprise one or more synthetic nucleic acids (XNAs), such as hexetol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threonine nucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid (LNA), and peptide nucleic acid (PNA). In some embodiments, the primers comprise one or more LNAs. In some embodiments, the primers comprise one or more, such as about 1 to about 20, such as about 2 to about 10, such as about 3, 4, 5, 6, 7, 8, or 9 LNA bases. LNA bases may, for example, comprise LNA-thymine bases, which may be represented as T+, where the "+" symbol indicates LNA. Locked nucleic acids typically comprise modified RNA monomers, wherein a methylene bridge connects the 2' oxygen to the 4' carbon.

[0216] In some embodiments, the presence of one or more modified bases in the primer stabilizes the interaction between the primer and the complementary portion (e.g., the complementary polynucleotide portion) of the RNA-peptide hybrid chain (e.g., the linker portion between the RNA portion of the RNA-peptide hybrid chain and the polypeptide portion of the RNA-peptide hybrid chain).

[0217] For example, in some embodiments, stabilization is achieved by cross-linking the chains.

[0218] In some embodiments, crosslinks are formed between nucleotides in the DNA and RNA-peptide hybrid chains. In some embodiments, crosslinks are formed between nucleotides in the DNA selected from adenine (A), cytosine (C), guanine (G), and thymine (T); and between nucleotides in the RNA-peptide hybrid chain selected from adenine (A), cytosine (C), guanine (G), thymine (T), and uridine (U). In some embodiments, crosslinks are formed between guanine or cytosine residues in the DNA and guanine or cytosine residues in the RNA-peptide hybrid chain. In some embodiments, crosslinks are formed between thymine residues in the DNA and thymine or uridine residues in the RNA-peptide hybrid chain. In some embodiments, crosslinks are formed between glycosyl groups in the DNA and RNA-peptide hybrid chains. For example, crosslinks may be formed between deoxyribose groups in the DNA and ribose or deoxyribose groups in the RNA-peptide hybrid chain. In some embodiments, crosslinks are formed between one or more nucleotides and one or more glycosyl groups. In some embodiments, crosslinks are formed between the DNA backbone and the backbone of the nucleotide, sugar, or RNA-peptide hybrid chain. In some embodiments, crosslinks are formed between the backbone of the RNA-peptide hybrid chain and the nucleotide, sugar, or DNA backbone.

[0219] In some embodiments, cross-linking is formed by contacting the polynucleotide with a cross-linking agent. Any suitable cross-linking agent can be used.

[0220] In some embodiments, the crosslinking agent is or contains a chromosome-breaking chemical agent. In some embodiments, the crosslinking agent is a chemical agent that is a bifunctional reactive group.

[0221] In some embodiments, the crosslinking agent is a bifunctional alkylating agent. Bifunctional alkylating agents include nitrogen mustard and lipid peroxidation products. Nitrogen mustard is a bifunctional alkylating agent. Nitrogen mustard typically contains a reactive N,N-bis(2-chloroethyl)amine functional group with a variable R group. Nitrogen mustard typically reacts with the N7 position of guanine. Non-limiting examples of nitrogen mustard include chlorinated nitrogen mustard, nitrogen mustard, and phosphoramide nitrogen mustard.

[0222] In some embodiments, the crosslinking agent is mitomycin C or an analogue thereof.

[0223] In some embodiments, the crosslinking agent is a lipid peroxidation product (typically an aldehyde). Non-limiting examples of lipid peroxidation products include acrolein, crotonaldehyde, and malondialdehyde. Other aldehydes that may be used include trans-4-hydroxynonenal, acetaldehyde, and formaldehyde.

[0224] In some embodiments, the crosslinking agent is a platinum compound. In some embodiments, the platinum compound is cisplatin or a derivative thereof. In some embodiments, the chemical reagent is cis-diaminedichloroplatinum. In some embodiments, the chemical reagent is trans-diaminedichloroplatinum.

[0225] In some embodiments, the crosslinking agent is chloroethylnitrosourea, such as carmustine or an analogue thereof.

[0226] In some embodiments, the crosslinking agent is psoralen or a derivative thereof, such as methoxypsoralen.

[0227] In some embodiments, the crosslinking agent is nitrous acid.

[0228] In some embodiments, the crosslinking agent is an intercalating dye. Intercalating dyes are well known in the art.

[0229] In some embodiments, the crosslinking agent is a reagent used to promote the formation of free radicals. Such chemical reagents include metals that react with peroxides to form or promote the crosslinking of free radicals.

[0230] In some embodiments, the cross-linking agent is a click chemistry agent. Click chemistry agents are described in more detail herein in the context of the linking of RNA to a translated polypeptide, and any click chemistry agent or reaction disclosed in that context may also be used to achieve cross-linking.

[0231] In some embodiments, the cross-linking agent is a nucleic acid cross-linking enzyme. Suitable enzymes are commercially available. For example, nucleic acid modifying enzymes that include nucleic acid cross-linking enzymes are available from New England Biolabs (NEB, USA). For example, in some embodiments, the nucleic acid cross-linking enzyme is a protease. Nucleic acid cross-linking enzymes can also function by catalyzing the formation of interstrand cross-links using chemical reagents. For example, the DNA interstrand cross-linking agent 5-(aziridin-1-yl)-4-hydroxyamino-2-nitrobenzamide can be formed from 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) via nitroreductase.

[0232] Digestion

[0233] As explained above, in some embodiments, the disclosed method includes removing the RNA strand from the DNA-peptide hybrid chain.

[0234] Any suitable method can be used to remove RNA strands from DNA-peptide hybrid chains. In some embodiments, RNA strands are removed by chemical or enzymatic digestion. In some embodiments, RNA strands are removed by contacting multiple DNA-peptide hybrid chains with a ribonuclease capable of digesting RNA in the DNA-peptide hybrid chains. Therefore, in some embodiments, the method further includes the following steps:

[0235] iii) Contact the plurality of DNA-peptide hybrid chains with a ribonuclease capable of digesting the RNA in the DNA-peptide hybrid chains.

[0236] In these embodiments, any suitable ribonuclease can be used. In some embodiments, the ribonuclease is an endonuclease. In some embodiments, the ribonuclease is an exonuclease. Typically, the ribonuclease is an endonuclease.

[0237] In some embodiments, the ribonuclease is an EC 3.1.26 type ribonuclease. In some embodiments, the ribonuclease is an EC 3.1.26.4 or 3.1.26.13 type ribonuclease.

[0238] In some embodiments, the ribonuclease is a ribonuclease H family endonuclease. Ribonuclease H (abbreviated as RNase H or RNH) is a family of non-sequence-specific endonucleases that catalyze the cleavage of RNA in RNA / DNA substrates through a hydrolytic mechanism.

[0239] In some embodiments, the ribonuclease is or is derived from ribonuclease H1. In some embodiments, the ribonuclease is or is derived from ribonuclease H2. In some embodiments, the ribonuclease comprises one or more RNase H1-like retroviral ribonuclease H domains.

[0240] In some embodiments, the ribonuclease comprises a 5-strand β-sheet surrounded by an α-helical distribution. In some embodiments, the ribonuclease comprises an active site containing a conserved sequence motif consisting of aspartic and glutamic acid residues, commonly referred to as the DEDD motif. In some embodiments, the DEDD motif residues interact with magnesium or manganese ions during nuclease activity, more typically with magnesium ions. Therefore, in some embodiments, the method is carried out in the presence of magnesium or manganese ions, typically magnesium ions. Magnesium ions may be provided, for example, in the form of MgCl2.

[0241] RNase H is commercially available, for example, from New England Biological Laboratories (Ipswich, Massachusetts, USA). In some embodiments, the ribonuclease is a recombinant RNase H. For example, commercially available RNase H includes recombinant RNase H from *Escherichia coli*. Thermostable RNase H can be used, and it is also commercially available. For example, commercially available thermostable RNase H includes recombinant RNase H from *Streptococcus thermophilus*.

[0242] filling

[0243] In some embodiments, the method further comprises performing a filling reaction to replace the digested RNA with DNA. Thus, the resulting DNA-peptide hybrid chain may contain a double-stranded DNA polynucleotide moiety.

[0244] The filling reaction can be carried out by contacting the digested DNA-peptide hybrid strands with DNA polymerase.

[0245] In some embodiments, a bacterial polymerase is used for the filling reaction. In some embodiments, a viral polymerase is used for the filling reaction. In some embodiments, a bacteriophage polymerase is used for the filling reaction. Examples include T4 DNA polymerase, T7 DNA polymerase, Taq polymerase, DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA polymerase IV, DNA polymerase V, Klenow fragment, etc. These and other suitable enzymes are available from New England Biolabs (Ipswich, Massachusetts, USA) and are generally used according to standard procedures known in the art. In one embodiment, the polymerase may be PyroPhage® 3173 DNA polymerase (commercially available from Lucigen®), SD polymerase (commercially available from Bioron®), Klenow from NEB, or a variant thereof. In one embodiment, the enzyme is Phi29 DNA polymerase or a variant thereof. A modified version of the Phi29 polymerase that can be used in the disclosed methods is disclosed in U.S. Patent No. 5,576,204.

[0246] In some embodiments, the polymerase does not have strand displacement activity. As used herein, the term "strand displacement" describes the ability to displace downstream DNA encountered during synthesis. In some embodiments, T4 DNA polymerase and T7 DNA polymerase may lack strand displacement activity.

[0247] In some embodiments, the DNA strand produced by the filling reaction is complementary to at least a portion of the DNA portion of the strand produced by the reverse transcription step. In some, the filling DNA strand is complementary to at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, or at least 99% of the sequence of the DNA portion of the strand produced by the reverse transcription step. In some embodiments, complementarity is evaluated over the full length of the synthesized DNA strand.

[0248] In some embodiments, the DNA strands produced in the filling reaction hybridize with at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, or at least 99%, of the DNA portion of the strand produced by the reverse transcription step, beyond the length of the synthetic DNA strand.

[0249] Hybridization produces a DNA-peptide hybrid chain that is at least partially double-stranded. Therefore, in some embodiments, the DNA-peptide hybrid chain may contain a double-stranded DNA portion.

[0250] Therefore, in some embodiments, the method includes the following steps

[0251] (iv) Generate a DNA strand complementary to at least a portion of the DNA portion of the strand generated in step (iii) (see above), such that the complementary DNA strand hybridizes with the DNA portion of the strand; thereby such that the resulting DNA-peptide hybrid strand contains at least a partially double-stranded DNA portion.

[0252] purification

[0253] In some embodiments, the disclosed method includes purifying the strand. In some embodiments, the method includes purifying RNA-peptide hybrid strands and / or DNA-peptide hybrid strands.

[0254] RNA-peptide hybrid chains and / or DNA-peptide hybrid chains can be purified by encoding a purification tag in the peptide (i.e., wherein the RNA chain or the DNA chain transcribed to produce the RNA chain encodes the purification tag). Therefore, in some embodiments, the disclosed methods include purifying the DNA-peptide hybrid chain using a purification tag. In some embodiments, the disclosed methods include purifying the RNA-peptide hybrid chain using a purification tag.

[0255] In some embodiments, the purification tag is separated from the peptide test sequence via a cleavable cleavage site, although this is not necessary, and the purification tag may be directly linked to the peptide test sequence. Therefore, in some embodiments, the purification tag is a cleavable purification tag.

[0256] In some embodiments, the cleavage site comprises a cleavable linker. Any suitable cleavable linker may be used in the disclosed methods. The linker may comprise a short-chain oligopeptide or oligonucleotide containing, for example, about 1 to about 20 amino acids or nucleotides. The linker may comprise a polymer, such as polyethylene glycol, or a sugar containing about 1 to about 20 repeating units. For example, the linker may comprise PEG2, PEG3, or PEG4.

[0257] In some embodiments, the cleavable site is cut by physical or chemical means. Any suitable means may be used.

[0258] The cleavage site may include a cleavable portion, which may be, for example, a pH-sensitive group; a redox-sensitive group; a photosensitizing group; a temperature-sensitive group; or a chemically sensitive group that is sensitive to cleavage that occurs through the reaction of the group with a specific chemical.

[0259] In some embodiments, the cleavage site can be cleaved by photolysis, enzymatic reaction, or by contacting the DNA-peptide hybrid chain with one or more chemical reagents. In some embodiments, the cleavage site is a protease cleavage site.

[0260] In some embodiments, the cleavage site comprises a cleavable portion that is cleavable by exposure to light; that is, it is photocleavable. In some embodiments, the cleavable site is cleaved by exposing the RNA-peptide hybrid or DNA-peptide hybrid to light, typically UV light. The photocleavable portion includes (optionally substituted) nitrobenzyl moiety. Such groups are cleavable under UV irradiation.

[0261] In some embodiments, the cleavage site comprises a cleavable portion that is cleavable by exposure to pH changes. Therefore, in some embodiments, the cleavable site is cleaved by exposing the RNA-peptide hybrid or DNA-peptide hybrid to pH changes. pH-sensitive cleavable linkers include hydrazones and cis-aconitine.

[0262] In some embodiments, the cleavage site comprises a cleavable portion that is cleavable by exposure to a chemical reagent. Therefore, in some embodiments, the cleavable site is cleaved by exposing the RNA-peptide hybrid or DNA-peptide hybrid to a chemical reagent, such as a reducing agent. Chemically sensitive cleavable linkers include disulfides. Disulfide bonds are readily cleaved by adding reducing agents such as DTT and β-mercaptoethanol.

[0263] In some embodiments, the cleavage site comprises a cleavable adapter that is cleavable by exposure to an enzyme, such as a protease or nuclease. Thus, in some embodiments, the cleavable site is cleaved by exposing the RNA-peptide hybrid or DNA-peptide hybrid to an enzyme, typically a protease.

[0264] Enzyme-sensitive cleavable portions include protease-sensitive peptides containing recognition sequences for one or more endopeptidases and / or exopeptidases. Examples include the sequence DDDDK (SEQ ID NO: 14; cleaved by intestinal peptidases from *E. coli* and *Saccharomyces cerevisiae*); LVPRGS (SEQ ID NO: 15; cleaved by thrombin and factor Xa); ENLYFQG (SEQ ID NO: 16; cleaved by TEV protease); and LEVLFQGP (SEQ ID NO: 17; cleaved by rhinovirus 3C protease). β-glucuronide linkers can be cleaved by lysosomal β-glucuronidase. Another example is a SUMO tag sequence that can be cleaved by the SUMO protease (ULP1). An example of a SUMO tag sequence is MSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (SEQ ID NO: 248).

[0265] In some embodiments, the cleavable site has the form Cl-Lk, Lk-Cl, or Lk-Cl-Lk, where Cl is the cleavable portion and Lk is the connector.

[0266] Therefore, in some embodiments, the disclosed method further includes:

[0267] - The DNA-peptide hybrid chain is purified using the purification tag; and

[0268] - The purification tag is removed from the purified DNA-peptide hybrid chain by contacting the purified DNA-peptide hybrid chain with one or more conditions capable of cleaving the cleavage site.

[0269] In some embodiments, removing the purification tag involves contacting the purified DNA-peptide hybrid strand with a protease capable of cleaving the cleavage site.

[0270] When the disclosed method involves the purification of DNA-peptide hybrid chains or RNA-peptide hybrid chains, any suitable purification label may be used.

[0271] For example, purification labels may contain or consist of biotin. Biotin is particularly suitable for the disclosed methods because it forms strong non-covalent links with streptavidin and related proteins (neutral avidin, avidin, etc.).

[0272] More commonly, purification tags are peptide purification tags suitable for IMAC (Immobilized Metal Affinity Chromatography) chemistry. For example, purification tags may contain poly-His tags (e.g., HHHH, HHHHHH, or HHHHHHHHH; SEQ ID NO: 18-20). Such tags are suitable for binding to purification carriers containing metals such as nickel or cobalt. Other purification tags include peptide tags such as Strep (WSHPQFEK; SEQ ID NO: 21), FLAG (DYKDDDDK; SEQ ID NO: 22), human influenza hemagglutinin (HA) (YPYDVPDYA; SEQ ID NO: 23), Myc (EQKLISEED; SEQ ID NO: 24), and V5 (GKPIPNPLLGLDST; SEQ ID NO: 25), etc.

[0273] Other suitable purification tags include: biotin-carboxyl carrier protein (BCCP); calmodulin-binding peptide (CBP); chitin-binding domain (CBD); histidine affinity tag (HAT); polyarginine (Arg-tag); polyaspartic acid (Asp-tag); polylysine (Lys-tag); polyphenylalanine (Phe-tag); streptavidin-binding peptide (SBP); tetrazine tag; TCO tag; azide tag; and DBCO / alkyne tag.

[0274] When the disclosed method involves purifying a DNA-peptide hybrid or an RNA-peptide hybrid, the method may include contacting a purification tag with a carrier used for purification.

[0275] Any suitable medium can be used.

[0276] In some embodiments, the carrier comprises a chromatographic matrix, such as agarose or agarose resin. Such resins are commercially available from suppliers such as Sigma-Aldrich.

[0277] In some embodiments, the carrier comprises beads (i.e., one or more beads). Magnetic beads are often used because they are easy to purify, for example, by washing with buffer. Functionalized magnetic beads are commercially available from suppliers such as Sigma-Aldrich and Bio-Rad Laboratories, and are available in a variety of functionalizations.

[0278] In some embodiments, the carrier comprises a solid surface. Any suitable material can be used. Suitable materials include glass, silica, polymers such as polyester, and ceramics such as hydroxyapatite.

[0279] In some embodiments, the carrier is functionalized to bind with a purification tag. Those skilled in the art will understand that the carrier can be functionalized depending on the purification tag contained in the multifunctional molecule used. Alternatively, the purification tag can be selected based on the carrier material to be used. Therefore, the selection of the purification tag and the carrier material are operational parameters that can be determined by the user of the disclosed method.

[0280] In some embodiments, the carrier comprises streptavidin, neutral avidin, or avidin, or a derivative of streptavidin, neutral avidin, or avidin, such as traptavidin. Such carriers are particularly useful when the multifunctional molecule contains a purification tag including biotin.

[0281] In some embodiments, the support comprises a metal, such as nickel or cobalt. The metal ion can be provided along with a suitable chelating agent, such as nitroglycerin (NTA) or iminodiacetic acid (IDA). For example, the support can comprise Ni-NTA. Such supports are particularly useful when the multifunctional molecule contains a purification tag including a His tag.

[0282] In some embodiments, the vector contains streptactin. Such vectors are particularly useful when the multifunctional molecule contains a purification tag including a Strep tag.

[0283] In some embodiments, the vector contains an antibody against sequences such as FLAG, HA, Myc, or V5 as discussed above.

[0284] Sequencing adapters

[0285] In some embodiments, one or more adaptors (also referred to herein as sequencing adaptors) may be linked to the DNA and / or polypeptide portions of a DNA-peptide hybrid chain. Such adaptors can aid in the characterization of the chain as it moves relative to the nanopore.

[0286] An adaptor can be attached to only one end of the strand. Polynucleotide adaptors can be added to both ends of the strand. Alternatively, different adaptors can be added to both ends of the strand.

[0287] An adaptor can be added to both strands of a double-stranded polynucleotide region. An adaptor can also be added to a single-stranded polynucleotide region. Methods for adding an adaptor to a polynucleotide are known in the art. The adaptor can be linked to the polynucleotide, for example, by ligation, by click chemistry, by tagging, by topoisomerization, or by any other suitable method.

[0288] In one embodiment, the adaptor or each adaptor is synthetic or human-derived. Typically, the adaptor or each adaptor comprises a polymer as described herein. In some embodiments, the adaptor or each adaptor comprises a spacer as described herein. In some embodiments, the adaptor or each adaptor comprises a polynucleotide. The polynucleotide adaptor or each polynucleotide adaptor may comprise DNA, RNA, modified DNA (e.g., base-free DNA), RNA, PNA, LNA, BNA, and / or PEG. Typically, the adaptor or each adaptor comprises single-stranded and / or double-stranded DNA or RNA. The adaptor may contain a polynucleotide of the same type as the polynucleotide chain it is linked to. The adaptor may contain a polynucleotide of a different type than the polynucleotide chain it is linked to.

[0289] In some embodiments, the adaptor may be a bridging portion. The bridging portion can be used to connect the two strands of a double-stranded polynucleotide. For example, in some embodiments, the bridging portion is used to connect the template strand of the double-stranded polynucleotide to the complement strand of the double-stranded polynucleotide. A bridging portion can be used to connect the two strands when the DNA-peptide hybrid chain contains a double-stranded polynucleotide portion.

[0290] Bridging portions typically covalently link the two strands of a double-stranded polynucleotide. The bridging portion can be any material capable of linking the two strands of a double-stranded polynucleotide, provided that it does not interfere with the movement of the polynucleotide relative to the nanopore. Suitable bridging portions include, but are not limited to, polymeric linkers, chemical linkers, polynucleotides, or peptides. Typically, bridging portions contain DNA, RNA, modified DNA (such as base-free DNA), RNA, PNA, LNA, or PEG. Bridging portions are more commonly DNA or RNA.

[0291] In some embodiments, the bridging portion is a hairpin receptacle. A hairpin receptacle is an receptacle comprising a single polynucleotide chain, wherein the ends of the polynucleotide chains are capable of hybridizing with or being hybridized with each other, and wherein the middle segment of the polynucleotide forms a loop. Suitable hairpin receptacles can be designed using methods known in the art. In some embodiments, the length of the hairpin loop is typically 4 to 100 nucleotides, for example 4 to 50, such as 4 to 20, such as 4 to 8 nucleotides. In some embodiments, the bridging portion (e.g., the hairpin receptacle) is attached to one end of the double-stranded polynucleotide. The bridging portion (e.g., the hairpin receptacle) is typically not attached to both ends of the chain.

[0292] In some embodiments, the adaptor is a linear adaptor. A linear adaptor can bind to either or both ends of a single-stranded polynucleotide. When the polynucleotide is a double-stranded polynucleotide, the linear adaptor can bind to either or both ends of either or both strands of the double-stranded polynucleotide. The linear adaptor can be attached to either or both ends of either or both of the polynucleotide-peptide conjugate chain and the carrier chain (if present).

[0293] Linear adaptors may contain a leader sequence as described herein. Linear adaptors may contain a portion for hybridization with a tag (such as a well tag) as described herein. The length of a linear adaptor may be from 10 to 150 nucleotides, such as 20 to 120, 30 to 100, 40 to 80, or 50 to 70 nucleotides. Linear adaptors may be single-stranded or double-stranded.

[0294] In some embodiments, the adaptor may be a Y-adaptor. A Y-adaptor is typically a polynucleotide adaptor. A Y-adaptor is typically double-stranded and includes (a) a region at one end where the two strands hybridize together and (b) a region at the other end where the two strands are not complementary. The non-complementary portions of the strands typically form overhangs. The presence of the non-complementary regions in the Y-adaptor gives it its Y-shape because, unlike the double-stranded portion, the two strands typically do not hybridize with each other. The two single-stranded portions of the Y-adaptor may be of the same length or may be of different lengths. For example, one single-stranded portion of the Y-adaptor may be 10 to 150 nucleotides long, such as 20 to 120, 30 to 100, 40 to 80, or 50 to 70 nucleotides long, and the other single-stranded portion of the Y-adaptor may independently be 10 to 150 nucleotides long, such as 20 to 120, 30 to 100, 40 to 80, or 50 to 70 nucleotides long. The length of the double-stranded "stem" portion of the Y-adaptor can be, for example, 10 to 150 nucleotides, 20 to 120, 30 to 100, 40 to 80, or 50 to 70 nucleotides. The Y-adaptor can be attached to either end or both ends of the construct described herein.

[0295] The adaptor can be attached to the chain by any suitable means known in the art. The adaptor can be synthesized separately and chemically or enzymatically attached to the chain. Alternatively, the adaptor can be generated during chain processing. In some embodiments, the adaptor is attached to the chain at or near one end of the polynucleotide portion of the chain. In some embodiments, the adaptor is attached to the chain within 50 nucleotides, such as 20 nucleotides, or even 10 nucleotides, of the end of the polynucleotide portion of the chain. In some embodiments, the adaptor is attached to the polynucleotide portion of the chain at the end of the polynucleotide. When the adaptor is attached to the polynucleotide portion of the chain, the adaptor may contain nucleotides of the same type as the polynucleotide or may contain nucleotides different from the polynucleotide.

[0296] In some embodiments, the integrator is attached to the chain at or near one end of the polypeptide portion of the chain. In some embodiments, the integrator is attached to the chain within 50 amino acids, such as within 20 amino acids, or for example within 10 amino acids, at the end of the polypeptide portion of the chain.

[0297] Integrators particularly suitable for the disclosed methods may comprise linear homopolymer regions (e.g., about 5 to about 20 nucleotides, such as about 10 to about 30 nucleotides, e.g., thymine or cytidine) and / or hybridization sites (as described in more detail herein) for hybridization with one or more tandem chains or anchors. Such integrators may also comprise reactive functional groups for binding to the target peptide. Click chemistry groups are particularly suitable in this regard. Exemplary groups for inclusion in the integrator, for example, include groups that can be micronized in copper-free click chemistry, such as groups based on BCN (bicyclo[6.1.0]nonyne) and its derivatives, dibenzocyclooctyne (DBCO) groups, etc. The reactivity of such groups is well known in the art. For example, BCN groups typically react with groups such as azides, tetrazides, and nitrones, which can be incorporated into the peptide, for example. DBCO groups are highly reactive to azide groups. Other particularly suitable chemical groups include 2-pyridinecarboxylaldehyde (2-PCA) groups and their derivatives. For example, 6-(azidomethyl)-2-pyridinecarboxylaldehyde can react with the N-terminal amino group of a peptide.

[0298] Leader sequence

[0299] In some embodiments, the polynucleotide-peptide chain or the adaptor linked thereto described herein may include a leader sequence. As described herein, the leader sequence can be used to assist the nanopore trap chain.

[0300] In some embodiments, the length of the leader sequence can be from about 10 to 150 nucleotides (e.g., DNA and / or RNA nucleotides), such as 20 to 120, such as 30 to 100, such as 40 to 80, such as 50 to 70 nucleotides, or about 10 to about 60 nucleotides, such as about 20 to about 50, such as about 20 to about 40, such as about 30 nucleotides.

[0301] In some embodiments, the leader sequence is a charged polymer, such as a negatively charged polymer. In some embodiments, the leader sequence comprises a polymer, such as PEG or a polysaccharide. In such embodiments, the length of the leader sequence can be from 10 to 150 monomer units (e.g., ethylene glycol or sugar units), or from 20 to 120, 30 to 100, 40 to 80, or 50 to 70 monomer units (e.g., ethylene glycol or sugar units).

[0302] spacer

[0303] In some embodiments of the methods provided herein, polynucleotides or adapters (e.g., adapters between the RNA and polypeptide portions of an RNA-peptide hybrid chain as described herein) or adaptors as described herein may include spacers.

[0304] For example, a polynucleotide adaptor can contain one to approximately 20 spacers, such as approximately one to approximately 10, or one to approximately five spacers, or one, two, three, four, or five spacers. Spacers can contain any suitable number of spacer units. Spacers can provide an energy barrier that impedes the movement of polynucleotide-treated proteins. For example, spacers can halt the movement of polynucleotide-treated proteins by reducing the traction force of the polynucleotide-treated protein on the polynucleotide. This can be achieved, for example, by using base-free spacers, i.e., spacers from which one or more nucleotides in the polynucleotide adaptor have been removed. Spacers can also physically block the movement of polynucleotide-treated proteins, for example, by introducing large chemical groups to physically impede the movement of polynucleotide-treated proteins.

[0305] In some embodiments, one or more spacers include polynucleotides or conjugates or adaptors used in the methods claimed herein, in order to provide a unique signal as the polynucleotide or conjugate or adaptor passes through or across the nanopore, i.e., as the polynucleotide or conjugate or adaptor moves relative to the nanopore.

[0306] In some embodiments, the spacers may comprise linear molecules, such as polymers. Typically, such spacers have a structure different from the polynucleotide used in the conjugate. For example, if the polynucleotide is DNA, the spacers, or each spacer, typically do not contain DNA. In particular, if the polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), the spacers, or each spacer, typically comprise peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threonine nucleic acid (TNA), locked nucleic acid (LNA), or a synthetic polymer with nucleotide side chains. In some embodiments, the spacer may comprise one or more nitroindole, one or more inosine, one or more acridine, one or more 2-aminopurine, one or more 2-6-diaminopurine, one or more 5-bromo-deoxyuridine, one or more trans-thymidine (trans-dT), one or more trans-dideoxythymidine (ddT), one or more dideoxycytidine (ddC), one or more 5-methylcytidine, one or more 5-hydroxymethylcytidine, one or more 2'-O-methylRNA bases, one or more isodeoxycytidine (iso-dC), one or more isodeoxyguanosine (iso-dG), one or more C3 (OC3H6OPO3) groups, one or more photodegradable (PC) [OC3H6-C(O)NHCH2-C6H3NO2-CH(CH3)OPO3] groups, one or more hexanediol groups, one or more spacer 9 (iSp9) [(OCH2CH2)3OPO3] groups, or one or more spacer 18 (iSp18) [(OCH2CH2)6OPO3] groups; or one or more thiol groups. Spacers can contain any combination of these groups. Many of these groups are commercially available from IDT® (Integrated DNA Technologies®). For example, C3, iSp9, and iSp18 spacers are all available from IDT®. Spacers can contain any number of the above groups as spacer units.

[0307] In some embodiments, the spacer may contain one or more chemical groups that cause the polynucleotide-processed protein to stall. In some embodiments, suitable chemical groups are one or more chemical side groups. The one or more chemical groups may be linked to one or more nucleobases in the polynucleotide, construct, or adaptor. The one or more chemical groups may be linked to the backbone of the polynucleotide adaptor. Any number of suitable chemical groups may be present, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more. Suitable groups include, but are not limited to, fluorophores, streptavidin and / or biotin, cholesterol, methylene blue, dinitrophenol (DNP), digoxigenin and / or anti-digoxigenin and dibenzylcyclooctyne groups. In some embodiments, the spacer may contain a polymer. In some embodiments, the spacer may contain a polymer, said polymer being a polypeptide or polyethylene glycol (PEG).

[0308] In some embodiments, the spacer may comprise one or more abasic nucleotides (i.e., nucleotides lacking a nucleobase), such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more abasic nucleotides. In the abasic nucleotides, the nucleobase may be replaced by -H (idSp) or -OH. The abasic spacer can be inserted into the target polynucleotide by removing a nucleobase from one or more adjacent nucleotides. For example, the polynucleotide may be modified to include 3-methyladenine, 7-methylguanine, 1,N6-vinylidene adenine inosine, or hypoxanthine, and the nucleobase may be removed from these nucleotides using human alkyladenine DNA glycosidase (hAAG). Alternatively, the polynucleotide may be modified to include uracil, and the nucleobase may be removed using uracil-DNA glycosylase (UDG). In one embodiment, the one or more spacers do not contain any abasic nucleotides.

[0309] A method for arresting polynucleotide-treated proteins (such as helicases) on polynucleotide adaptors using spacers is described in WO 2014 / 135838, which is hereby incorporated in its entirety by reference.

[0310] anchor

[0311] In some embodiments, the polynucleotide, its conjugate with a polypeptide, or the adaptor linked thereto may include, for example, a membrane anchor or transmembrane pore anchor linked to the adaptor. In one embodiment, the anchor facilitates the characterization of the conjugate according to the methods disclosed herein. For example, the membrane anchor or transmembrane pore anchor can facilitate the localization of the conjugate around nanopores in a membrane.

[0312] The anchor can be a peptide anchor and / or a hydrophobic anchor that can be inserted into the membrane. In one embodiment, the hydrophobic anchor is a lipid, fatty acid, sterol, carbon nanotube, peptide, protein, or amino acid, such as cholesterol, palmitate, or tocopherol. The anchor may contain thiols, biotin, or surfactants.

[0313] In one embodiment, the anchor may be biotin (for binding to streptavidin), amylose (for binding to maltose-binding proteins or fusion proteins), Ni-NTA (for binding to polyhistidine or polyhistidine-labeled proteins), or a peptide (such as an antigen).

[0314] In one embodiment, an anchor comprises one, or two, three, four, or more adapters. Typical adapters include, but are not limited to, polymers such as polynucleotides, polyethylene glycol (PEG), polysaccharides, and peptides. These adapters can be linear, branched, or cyclic. For example, an adapter can be a cyclic polynucleotide. The adapter can hybridize to a complementary sequence on the cyclic polynucleotide adapter. The one or more anchors or adapters may contain components that can be cleaved or broken down, such as restriction sites or photostable groups. The adapters can be functionalized with maleimide groups to link to cysteine ​​residues in a protein. Suitable adapters are described in WO 2010 / 086602.

[0315] In one embodiment, the anchor is a cholesterol or fatty acyl chain. For example, any fatty acyl chain with a length of 6 to 30 carbon atoms, such as hexadecanoic acid, can be used. Examples of suitable anchors and methods for connecting anchors to connectors are disclosed in WO 2012 / 164270 and WO 2015 / 150786.

[0316] Characterization methods

[0317] In some embodiments, the disclosed method includes contacting a DNA-peptide hybrid chain with a nanopore; and performing one or more measurements specific to the DNA-peptide hybrid chain as the DNA-peptide hybrid chain moves relative to the nanopore, thereby characterizing the DNA-peptide hybrid chain. More generally, the disclosed method includes contacting a polynucleotide-peptide hybrid chain with a detector; and performing one or more measurements specific to the polynucleotide-peptide hybrid chain, thereby characterizing the polynucleotide-peptide hybrid chain. As described in more detail herein, one or more measurements may be performed using a suitable detector. In some embodiments, one or more measurements may be performed as the polynucleotide-peptide hybrid chain moves relative to the detector.

[0318] Any suitable measurement may be performed. In some embodiments, the measurement is specific to one or more of the following: (i) the length of the polynucleotide portion of the polynucleotide-peptide hybrid chain; (ii) the identity of the polynucleotide portion of the polynucleotide-peptide hybrid chain; (iii) the sequence of the polynucleotide portion of the polynucleotide-peptide hybrid chain; (iv) the secondary structure of the polynucleotide portion of the polynucleotide-peptide hybrid chain; (v) whether the polynucleotide portion of the polynucleotide-peptide hybrid chain is modified; (vi) the length of the polypeptide portion of the polynucleotide-peptide hybrid chain; (vii) the identity of the polypeptide portion of the polynucleotide-peptide hybrid chain; (viii) the sequence of the polypeptide portion of the polynucleotide-peptide hybrid chain; (ix) the secondary structure of the polypeptide portion of the polynucleotide-peptide hybrid chain; and (x) whether the polypeptide portion of the polynucleotide-peptide hybrid chain is modified.

[0319] As explained in this article, the measurements that can be performed can be understood in more detail.

[0320] Therefore, as explained above, accurately characterizing peptides using known methods is challenging. This is because high-quality sequencing libraries are needed to generate exemplary peptide characterization data. The methods disclosed in this paper provide such libraries.

[0321] Such libraries can be used to correlate peptide measurement signals with the properties of the peptide that produced the signals. This is useful because, as explained above, such data can be used to infer or otherwise determine the properties of the peptide moiety of a peptide analyte. Therefore, in the disclosed method, the signal of the peptide moiety of a polynucleotide-peptide hybrid chain can be correlated with the properties of the peptide moiety, which can be determined or inferred from knowledge of the properties of the polynucleotide moiety that can be determined using methods known in the art. The resulting data can be used to train computer models, such as neural networks, to characterize unknown peptides.

[0322] In one embodiment, a detector and the obtained measurement signal can be used to evaluate a polynucleotide-peptide hybrid chain as described herein. In some embodiments, evaluating a polynucleotide-peptide hybrid chain includes performing one or more measurements specific to the polynucleotide-peptide hybrid chain. In some embodiments, performing said one or more measurements includes...

[0323] - Use a detector to perform one or more electrical and / or optical measurements specific to the polynucleotide portion of the polynucleotide-peptide hybrid chain, thereby determining one or more characteristics of the polynucleotide portion of the polynucleotide-peptide hybrid chain;

[0324] - Perform one or more electrical and / or optical measurements specific to the polypeptide portion of the polynucleotide-peptide hybrid chain using a detector; and

[0325] - Associate the output of one or more electrical and / or optical measurements specific to the polypeptide portion of the polynucleotide-peptide hybrid chain with one or more properties of the polynucleotide portion of the polynucleotide-peptide hybrid chain.

[0326] In some embodiments, the method includes determining the sequence of the polynucleotide portion of the polynucleotide-peptide hybrid chain; and associating an electrical or optical signal recorded as the polypeptide portion of the polynucleotide-peptide hybrid chain moves relative to a nanopore with the sequence.

[0327] In one embodiment, nanopores and the obtained measurement signals can be used to evaluate polynucleotide-peptide hybrid chains as described herein. In some embodiments, evaluating a polynucleotide-peptide hybrid chain includes performing one or more measurements specific to the polynucleotide-peptide hybrid chain. In some embodiments, performing said one or more measurements includes...

[0328] - As the polynucleotide-peptide hybrid chain moves relative to the nanopore, one or more electrical and / or optical measurements specific to the polynucleotide portion of the polynucleotide-peptide hybrid chain are performed, thereby determining one or more properties of the polynucleotide portion of the polynucleotide-peptide hybrid chain;

[0329] - As the polynucleotide-peptide hybrid chain moves relative to the nanopore, one or more electrical and / or optical measurements specific to the peptide portion of the polynucleotide-peptide hybrid chain are performed; and

[0330] - Associate the output of one or more electrical and / or optical measurements specific to the polypeptide portion of the polynucleotide-peptide hybrid chain with one or more properties of the polynucleotide portion of the polynucleotide-peptide hybrid chain.

[0331] In some embodiments, the method includes determining the sequence of the polynucleotide portion of the polynucleotide-peptide hybrid chain; and associating an electrical or optical signal recorded as the polypeptide portion of the polynucleotide-peptide hybrid chain moves relative to a nanopore with the sequence.

[0332] In some embodiments, the polynucleotide portion of the polynucleotide-peptide hybrid chain comprises a DNA chain. In some embodiments, the DNA chain comprises single-stranded DNA, double-stranded DNA, or a double-stranded DNA:RNA hybrid.

[0333] More details and references Figure 3A and3B Using a detector, such as a nanopore, a measurement signal can be obtained from each of the multiple polynucleotide-peptide hybrid chains. In some embodiments, the polynucleotide-peptide hybrid chains are generated as described herein and / or as described herein. In some embodiments, each polynucleotide-peptide hybrid chain comprises (i) a peptide portion containing a peptide test sequence and (ii) a polynucleotide portion containing a sequence encoding said peptide test sequence.

[0334] In some embodiments, the polynucleotide-peptide hybrid chain is a DNA-peptide hybrid chain as described herein. In some embodiments, each DNA-peptide hybrid chain comprises (i) a peptide portion containing a peptide test sequence and (ii) a DNA polynucleotide portion containing a sequence encoding said peptide test sequence.

[0335] In some embodiments, the analysis step includes analyzing such measurement signals.

[0336] In some embodiments, the method includes identifying a polypeptide signal portion of the measurement signal corresponding to the polypeptide portion of the polynucleotide-peptide hybrid chain. In some embodiments, the method includes identifying a polynucleotide (e.g., DNA) signal portion of the measurement signal corresponding to a polynucleotide (e.g., DNA) portion of the polynucleotide-peptide hybrid chain. In some embodiments, the method includes identifying both a polypeptide signal portion of the measurement signal corresponding to the polypeptide portion of the polynucleotide-peptide hybrid chain and a polynucleotide signal portion of the measurement signal corresponding to the polynucleotide portion of the polynucleotide-peptide hybrid chain.

[0337] Therefore, in some embodiments, the identification step S2 includes identifying in each measurement signal:

[0338] (i) the polypeptide signal portion of the measured signal corresponding to the polypeptide portion of the polynucleotide-peptide hybrid chain; and

[0339] (ii) The polynucleotide signal portion of the measurement signal corresponding to the polynucleotide portion of the polynucleotide-peptide hybrid chain.

[0340] In some embodiments, the method includes a first obtaining step S3, which includes obtaining one or more characteristics of the polynucleotide (e.g., DNA) portion from which the polynucleotide signal portion is obtained. The one or more characteristics may be selected, for example, from: (i) the length of the polynucleotide portion; (ii) the identity of the polynucleotide portion; (iii) the sequence of the polynucleotide portion; (iv) the secondary structure of the polynucleotide portion; and (v) whether the polynucleotide portion is modified.

[0341] In some embodiments, the method includes a second obtaining step S4, which includes obtaining one or more properties of the polypeptide moiety of the polynucleotide-peptide hybrid chain. The one or more properties of the polypeptide moiety may be obtained from one or more properties obtained for each polynucleotide polynucleotide moiety. The one or more properties of the polypeptide moiety may be selected, for example, from: (i) the length of the polypeptide moiety, (ii) the identity of the polypeptide moiety, (iii) the sequence of the polypeptide moiety, (iv) the secondary structure of the polypeptide moiety, and (v) whether the polypeptide moiety is modified. The one or more properties of the polypeptide moiety may be obtained based on the encoding of the polynucleotide moiety. For example, the sequence of the polypeptide moiety may be obtained from the sequence of the polynucleotide moiety. In other words, in some embodiments, the one or more properties of each polynucleotide moiety comprise the polynucleotide sequence of the polynucleotide moiety. Other properties of the polypeptide moiety may be similarly obtained or inferred from the determined properties of the polynucleotide moiety. In some embodiments, the polynucleotide moiety is a DNA moiety as described herein. In some embodiments, the DNA moiety comprises single-stranded DNA, double-stranded DNA, or a double-stranded DNA:RNA hybrid.

[0342] In some embodiments, the method includes an association step S5, which includes associating one or more properties of each polypeptide moiety with a polypeptide signal moiety of a measurement signal obtained from a polynucleotide-polypeptide hybrid chain containing a polynucleotide moiety.

[0343] In some embodiments, the association comprises a database that generates peptide properties and associated peptide measurement signals. In some embodiments, the database may contain at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10000, at least 100000, at least 1000000 or more data entries, wherein each data entry contains (i) one or more peptide properties and (ii) associated peptide signals.

[0344] In some embodiments, as the peptide analyte moves relative to the nanopore, associated data (e.g., a database) is used to train an algorithm for characterizing the peptide analyte.

[0345] In some embodiments, the method includes a measurement step S1 of measuring a measurement signal from each of a plurality of polynucleotide-peptide hybrid chains. In some embodiments, the measurement signal may comprise electrical and / or optical measurements. In some embodiments, the measurement signal is obtained as described herein.

[0346] This document also provides a computer program comprising instructions executable by a computer system, the instructions being configured to cause the computer system to perform the methods disclosed herein when executed. A computer storage medium storing the computer program is also provided. A computer system configured to perform the methods described herein is further provided.

[0347] In some embodiments, the analysis steps discussed above are performed on the analysis system 3. In some embodiments, the measurement steps are performed on the measurement system 2. The analysis system 3 may be physically associated with the measurement system 2 and may also provide control signals to the measurement system 2. In this case, the measurement and analysis system 1, which includes the measurement system 2 and the analysis system 3, may be arranged as disclosed in any of WO-2008 / 102210, WO-2009 / 07734, WO-2010 / 122293, WO-2011 / 067559, or WO2014 / 04443.

[0348] Alternatively, the analysis system 3 can be implemented in a separate device, in which case the series of measurement results are transmitted from the measurement system 2 to the analysis system 3 via any suitable means (typically a data network). For example, a convenient cloud-based implementation is to use the analysis system 3 as a server, with input signals supplied to the server via the Internet.

[0349] The analysis system 3 can be implemented by a computer device that executes computer programs, or by a dedicated hardware device or any combination thereof. In either case, the data used by the method is stored in the memory of the analysis system 3.

[0350] When a computer device executes a computer program, the computer device can be any type of computer system, but is typically of conventional architecture. The computer program can be written in any suitable programming language. The computer program can be stored on a computer-readable storage medium, which can be of any type, such as: a recording medium that can be inserted into a computing system and can store information magnetically, optically, or photomagnetically; a fixed recording medium of the computer system, such as a hard disk drive; or computer memory.

[0351] When a computer device is implemented using dedicated hardware, any suitable type of device can be used, such as an FPGA (Field-Programmable Gate Array) or an ASIC (Application-Specific Integrated Circuit). In a typical embodiment, hardware suitable for parallel computing, such as a graphics processing unit (GPU), can be used to implement parts of the computer program.

[0352] Therefore, in some embodiments, this document provides a method for analyzing measurement signals acquired from each of a plurality of polynucleotide-peptide hybrid chains, the measurement signals being acquired using a detector (e.g., as the polynucleotide-peptide hybrid chain moves relative to the detector, wherein the detector is or comprises a nanopore), wherein each of the polynucleotide-peptide hybrid chains comprises (i) a polypeptide portion containing a polypeptide test sequence and (ii) a polynucleotide portion containing a sequence encoding said polypeptide test sequence, the method comprising:

[0353] Identify in each measurement signal:

[0354] (i) the polypeptide signal portion of the measured signal corresponding to the polypeptide portion of the polynucleotide-peptide hybrid chain; and

[0355] (ii) The polynucleotide signal portion of the measured signal corresponding to the polynucleotide portion of the polynucleotide-peptide hybrid chain;

[0356] For each polynucleotide signal portion, one or more characteristics of the polynucleotide signal portion obtained from the polynucleotide portion are obtained;

[0357] Based on the encoding of the polynucleotide moiety, one or more characteristics of the polypeptide moiety of the polynucleotide-polypeptide hybrid chain containing the polynucleotide moiety are obtained from one or more characteristics obtained from each polynucleotide moiety; and

[0358] The one or more properties of each polypeptide moiety are associated with the polypeptide signal portion of the measurement signal obtained from the polynucleotide-polypeptide hybrid chain containing the polynucleotide moiety.

[0359] In some embodiments, this document provides a method for analyzing a measurement signal acquired from each of a plurality of DNA-peptide hybrid chains, the measurement signal being acquired as the DNA-peptide hybrid chain moves relative to a detector (e.g., a nanopore), wherein each of the DNA-peptide hybrid chains comprises (i) a peptide portion containing a peptide test sequence and (ii) a DNA polynucleotide portion containing a sequence encoding the peptide test sequence, the method comprising:

[0360] Identify in each measurement signal:

[0361] (i) the polypeptide signal portion of the measured signal corresponding to the polypeptide portion of the DNA-polypeptide hybrid chain; and

[0362] (ii) The DNA signal portion of the measured signal corresponding to the DNA polynucleotide portion of the DNA-peptide hybrid chain;

[0363] For each DNA signal portion, one or more characteristics of the DNA polynucleotide portion from which the DNA signal portion is derived are obtained;

[0364] Based on the encoding of the DNA polynucleotide moiety, one or more characteristics of the polypeptide moiety of the DNA-polypeptide hybrid chain containing the DNA polynucleotide moiety are obtained from one or more characteristics obtained from each DNA polynucleotide moiety; and

[0365] One or more properties of each polypeptide moiety are associated with the polypeptide signal moiety of the measurement signal obtained from the DNA-polypeptide hybrid chain containing the DNA moiety.

[0366] Controlling the movement of conjugates relative to nanopores

[0367] In some embodiments, a signal specific to the polynucleotide-peptide hybrid chain is measured as the chain moves relative to the nanopore.

[0368] The movement of the chain or a portion thereof relative to the nanopore can be driven by any suitable means. In some embodiments, the movement of the chain is driven by physical or chemical forces (potentials). In some embodiments, the physical forces are provided by potentials (e.g., voltages) or temperature gradients, etc.

[0369] In some embodiments, chain movement comprises mechanically manipulating the chain, thereby moving the chain relative to the nanopore. In some embodiments, chain movement by mechanical manipulation does not involve treating the protein with a polynucleotide.

[0370] In some embodiments, the chain is moved in the opposite direction to the potential applied across the nanopore by mechanical manipulation. In some embodiments, the potential is a voltage potential applied across the nanopore. In some embodiments, as described in WO 2020 / 128517, the chain is moved relative to the nanopore, the entire contents of which are hereby incorporated by reference, particularly with respect to the discussion of the movement of polynucleotides relative to the nanoreactor in the aforementioned document.

[0371] In some embodiments, when a potential is applied across the nanopore, the chain moves relative to the nanopore. Polynucleotides are negatively charged, and therefore applying a voltage potential across the nanopore will cause the polynucleotide to move relative to the nanopore under the influence of the applied voltage potential. For example, if a positive voltage potential is applied relative to the cis side of the nanopore to the trans side, this will induce a negatively charged analyte to move from the cis side to the trans side. Similarly, if a positive voltage potential is applied relative to the cis side of the nanopore to the trans side, this will prevent a negatively charged analyte from moving from the trans side to the cis side. The opposite occurs if a negative voltage potential is applied relative to the cis side of the nanopore to the trans side. Apparatus and methods for applying appropriate voltages are described in more detail herein.

[0372] In some embodiments, the chemical force is provided by a concentration (e.g., pH) gradient.

[0373] In some embodiments, the movement of the chain relative to the nanopore is controlled using a method as described in WO 2020 / 016573, the entire contents of which are incorporated herein by reference.

[0374] In some embodiments, the movement of the chain is controlled using methods disclosed in any of WO 2021 / 111125, WO 2021 / 133168 or WO 2024 / 094986, the entire contents of each of which are incorporated herein by reference.

[0375] In some embodiments, the polynucleotide-treated protein control chain moves in the same direction as a physical or chemical force (potential). For example, in some embodiments, a positive voltage potential is applied relative to the cis side of the nanopore towards the trans side, and the polynucleotide-treated protein control chain moves from the cis side to the trans side of the nanopore. In some embodiments, a positive voltage potential is applied relative to the trans side of the nanopore towards the cis side, and the polynucleotide-treated protein control chain moves from the trans side to the cis side of the nanopore.

[0376] In some embodiments, the polynucleotide-treated protein control chain moves in the opposite direction to a physical or chemical force (electric potential). For example, in some embodiments, a positive voltage potential is applied relative to the cis side of the nanopore towards the trans side, and the polynucleotide-treated protein control chain moves from the trans side to the cis side of the nanopore. In some embodiments, a positive voltage potential is applied relative to the trans side of the nanopore towards the cis side, and the polynucleotide-treated protein control chain moves from the cis side to the trans side of the nanopore.

[0377] In some embodiments, chain movement is driven by a polynucleotide-treated protein in the absence of an applied electrical potential.

[0378] In embodiments incorporating the disclosed method using a polynucleotide-treated protein, the polynucleotide-treated protein is typically capable of controlling the movement of the chain relative to the nanopore. In other words, the polynucleotide-treated protein is capable of controlling the movement of the chain. As the chain moves relative to the nanopore, the polynucleotide and polypeptide portions of the construct move relative to the nanopore and can thus be characterized.

[0379] Suitable polynucleotide processing proteins are also known as motor proteins or polynucleotide processing enzymes. Suitable polynucleotide processing proteins are known in the art, and some exemplary polynucleotide processing proteins are described in more detail below.

[0380] In one embodiment, the motor protein is or is derived from a polynucleotide processing enzyme. A polynucleotide processing enzyme is a polypeptide capable of interacting with and modifying at least one property of a polynucleotide. Enzymes can modify polynucleotides by cleaving them to form individual nucleotides or shorter nucleotide chains (such as dinucleotides or trinucleotides). Enzymes can modify polynucleotides by orienting or moving them to a specific location.

[0381] In some embodiments, the polynucleotide-treated protein may be present on the construct prior to contact between the chain and the nanopore. For example, the polynucleotide-treated protein may be present on the polynucleotide portion of the chain. In some embodiments, the polynucleotide-treated protein may be present on the adaptor.

[0382] In some embodiments, when the portion of the chain contacting the active site of the polynucleotide-treated protein comprises a polypeptide, the polynucleotide-treated protein is able to remain bound to the chain. In other words, in some embodiments, the polynucleotide-treated protein does not dissociate from the chain when it contacts the polypeptide portion of the chain. In some embodiments, the polynucleotide-treated protein moves freely relative to the polypeptide portion until it contacts one or more subsequent polynucleotide portions of the chain.

[0383] In some embodiments, when the polynucleotide-treated protein contacts a portion of a chain containing a polypeptide, the polynucleotide-treated protein is modified to prevent the polynucleotide-treated protein from detaching from the chain (except through the removal construct or the end of the chain). Such modified polynucleotide-treated proteins are particularly suitable for the disclosed methods.

[0384] Polynucleotide-treated proteins can be modified in any suitable manner. For example, a polynucleotide-treated protein can be loaded onto a chain and then modified to prevent its detachment. Alternatively, a polynucleotide-treated protein can be modified to prevent its detachment before being loaded onto the chain. Modifying a polynucleotide-treated protein to prevent its detachment from the chain can be achieved using methods known in the art (such as those discussed in WO 2014 / 013260, which is hereby incorporated in its entirety by reference) and with particular reference to the paragraphs describing the modification of polynucleotide-treated proteins (polynucleotide-binding proteins) such as helicases to prevent the polynucleotide-treated protein from detaching from the polynucleotide chain.

[0385] For example, a polynucleotide-treated protein may have a polynucleotide unbinding opening; for example, a cavity, crack, or gap through which the polynucleotide chain can pass when the polynucleotide-treated protein dissociates from the chain. In some embodiments, the polynucleotide unbinding opening for a given motor protein (polynucleotide-treated protein) can be determined by referring to its structure (e.g., its X-ray crystal structure). The X-ray crystal structure can be obtained with and / or without a polynucleotide substrate. In some embodiments, the location of the polynucleotide unbinding opening in a given polynucleotide-treated protein can be inferred or confirmed by molecular modeling using standard packaging known in the art. In some embodiments, the polynucleotide unbinding opening can be instantaneously generated by the movement of one or more portions (e.g., one or more domains of the polynucleotide-treated protein).

[0386] Polynucleotide-treated proteins (motor proteins) can be modified by closing polynucleotide unbinding openings. Therefore, closing polynucleotide unbinding openings prevents the polynucleotide-treated protein from dissociating from the polypeptide portion of the chain and from dissociating from the polynucleotide or adaptor. For example, motor proteins can be modified by covalently closing polynucleotide unbinding openings. In some embodiments, the motor protein used for addressing in this manner is a helicase as described herein. Thus, in some embodiments of the disclosed methods, the polynucleotide-treated protein is modified to completely or partially close openings present in at least one conformational state of the unmodified protein through which the polynucleotide chain can unbind.

[0387] Polynucleotide processing proteins can be selected or chosen based on the polynucleotides contained in the chain. Alternatively, the polynucleotides of the chain can be selected or chosen based on the polynucleotide processing proteins used to control chain movement. For example, when the polynucleotide is DNA, DNA motor proteins are typically used. When the polynucleotide is RNA, RNA motor proteins can be used. When the polynucleotide is a hybrid of DNA and RNA, motor proteins capable of processing both DNA and RNA can be used.

[0388] In one embodiment, the motor protein is derived from any member of the 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 3.1.31.

[0389] In some embodiments, the motor protein is a helicase, polymerase, exonuclease, topoisomerase, or a variant thereof.

[0390] In one embodiment, the motor protein is an exonuclease. Suitable enzymes include, but are not limited to, exonuclease I (SEQ ID NO: 26) from *E. coli*, exonuclease III (SEQ ID NO: 27) from *E. coli*, RecJ (SEQ ID NO: 28) from *T. Thermophilus*, bacteriophage λ exonuclease (SEQ ID NO: 29), TatD exonuclease, and variants thereof. The three subunits comprising the sequence shown in SEQ ID NO: 28, or variants thereof, interact to form a trimeric exonuclease.

[0391] In one embodiment, the motor protein is a polymerase. The polymerase may be PyroPhage® 3173 DNA polymerase (commercially available from Lucigen®), SD polymerase (commercially available from Bioron®), Klenow from NEB, or a variant thereof. In one embodiment, the enzyme is Phi29 DNA polymerase (SEQ ID NO: 30) or a variant thereof. A modified version of the Phi29 polymerase that can be used in the disclosed methods is disclosed in U.S. Patent No. 5,576,204.

[0392] In the embodiments provided herein that include methods for controlling the movement of constructs, polynucleotide-peptide conjugate chains, and / or polynucleotide carrier chains by synthesizing chains complementary to polynucleotide chains, the polynucleotide-treated protein is typically a polymerase, such as the polymerase described herein.

[0393] In one embodiment, the polynucleotide processing protein is a topoisomerase. In one embodiment, the topoisomerase is a member of either of the partial classification (EC) groups 5.99.1.2 and 5.99.1.3. The topoisomerase can be a reverse transcriptase, which is an enzyme capable of catalyzing the formation of cDNA from an RNA template. The topoisomerase is commercially available from, for example, New England Biolabs® and Invitrogen®.

[0394] In one embodiment, the polynucleotide processing protein is a transloase. Examples include transloases from the FtsK and SpoIII families.

[0395] In one embodiment, the polynucleotide processing protein is a helicase. Any suitable helicase can be used according to the methods provided herein. For example, the motor protein used according to this disclosure, or each motor protein, can be independently selected from Hel308 helicase, RecD helicase, TraI helicase, TrwC helicase, XPD helicase, and Dda helicase, or variants thereof. Monomeric helicases can comprise several domains linked together. For example, TraI helicase and TraI subgroup helicases can contain two RecD helicase domains, a release enzyme domain, and a C-terminal domain. These domains typically form a monomeric helicase capable of functioning without forming oligomers. Specific examples of suitable helicases include Hel308, NS3, Dda, UvrD, Rep, PcrA, Pif1, and TraI. These helicases typically act on single-stranded DNA. Examples of helicases that can move along both strands of double-stranded DNA include FtsK and hexamerase complexes, or multi-subunit complexes such as RecBCD, and are particularly suitable for some of the embodiments disclosed herein. NS3 helicase is particularly suitable for the disclosed methods because it can process both DNA and RNA, and is therefore suitable for embodiments of the disclosed methods in which the target double-stranded nucleic acid is a DNA-RNA hybrid.

[0396] Hel308 helicase is described in publications such as WO 2013 / 057495, the entire contents of which are incorporated herein by reference. RecD helicase is described in publications such as WO 2013 / 098562, the entire contents of which are incorporated herein by reference. XPD helicase is described in publications such as WO 2013 / 098561, the entire contents of which are incorporated herein by reference. Dda helicase is described in publications such as WO 2015 / 055981 and WO 2016 / 055777, the entire contents of each of which are incorporated herein by reference.

[0397] In one embodiment, the helicase comprises the sequence shown in SEQ ID NO: 31 (Trwc Cba) or a variant thereof, the sequence shown in SEQ ID NO: 32 (Hel308 Mbu) or a variant thereof, or the sequence shown in SEQ ID NO: 33 (Dda) or a variant thereof. The variants may differ from the native sequence in any of the ways discussed herein. An example variant of SEQ ID NO: 33 comprises E94C / A360C. Another example variant of SEQ ID NO: 33 comprises E94C / A360C, and then (ΔM1)G1G2 (i.e., the deletion of M1, and then the addition of G1 and G2).

[0398] In some embodiments, motor proteins (e.g., helicases) can operate in at least two modes of activity (when the motor protein has all the necessary components to facilitate movement, such as fuels and cofactors discussed herein, like ATP and Mg). 2+ It controls chain movement in both an inactive mode of operation (when the motor protein does not provide the components required to promote movement) and an inactive mode of operation (when the motor protein does not provide the components required to promote movement).

[0399] When all the necessary components for movement are provided (i.e., in active mode), motor proteins (e.g., helicases) move along polynucleotides in a 5' to 3' or 3' to 5' direction (depending on the motor protein). Motor proteins can be used to move the chain away from (e.g., out of) the pore (e.g., against applied force) or to move the chain towards (e.g., into) the pore (e.g., using applied force). For example, when the motor protein is captured in the pore at the end of the chain it is moving towards, the direction of the force acting on the motor protein pulls the chain through out of the pore (e.g., into the cis chamber). However, when the distant end of the motor protein is captured in the pore, the direction of the force acting on the motor protein pushes the chain through into the pore (e.g., into the trans chamber).

[0400] When a motor protein (e.g., a helicase) does not provide the necessary components to promote movement (i.e., in inactive mode), the motor protein can bind to the chain and act as a brake to slow the chain's movement relative to the nanopore, for example, by pulling it into the pore with force. In inactive mode, it is not important which end of the chain is captured; the applied force determines the movement relative to the pore, and the polynucleotide-binding protein acts as a brake. The movement control of the polynucleotide-binding protein in inactive mode can be described in several ways, including ratcheting, sliding, and braking.

[0401] Motor proteins typically require fuel to process polynucleotides. This fuel is usually a free nucleotide or a free nucleotide analogue. Free nucleotides can be, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and deoxygenated adenosine monophosphate (dAMP). Deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), and deoxycytidine triphosphate (dCTP). Free nucleotides are typically selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, or dCMP. The most common free nucleotide is adenosine triphosphate (ATP).

[0402] Cofactors of motor proteins are factors that allow motor proteins to function. Cofactors are typically divalent metal cations. These divalent metal cations are typically Mg. 2+ Mn 2+ Ca 2+ or Co 2+ The most common cofactor is Mg. 2+ .

[0403] detector

[0404] The embodiments described herein relate to the movement of a chain (e.g., a DNA-peptide hybrid chain) relative to a nanopore. However, while this disclosure provides nanopores as exemplary detectors, the methods provided herein are also applicable to other detectors, including (i) zero-mode waveguides, (ii) field-effect transistors, optionally nanowire field-effect transistors; (iii) AFM tips; (iv) nanotubes, optionally carbon nanotubes; and (v) nanopores. The disclosed methods are particularly suitable for methods in which polynucleotides move through a detector or through a structure containing a detector, such as a pore in a detector chip.

[0405] Nanopores

[0406] As explained above, in some embodiments, the disclosed method includes performing one or more measurements as a library of a chain (e.g., a DNA-peptide hybrid chain as described herein) or such a chain moves relative to a nanopore.

[0407] In the disclosed method, any suitable nanopore can be used. In one embodiment, the nanopore is a transmembrane pore.

[0408] A transmembrane pore is a structure that extends through the membrane to some extent. The transmembrane pore allows hydrated ions, driven by an applied potential, to flow across or within the membrane. Transmembrane pores typically extend across the entire membrane, allowing hydrated ions to flow from one side to the other. However, a transmembrane pore does not necessarily extend through the membrane. The transmembrane pore may close at one end. For example, a pore can be a hole, gap, channel, groove, or slit in the membrane along which hydrated ions can flow or into.

[0409] Any suitable transmembrane pore can be used in the methods presented herein. The pore can be biological or artificial. Suitable pores include, but are not limited to, protein pores, polynucleotide pores, and solid pores.

[0410] In one embodiment, the solid-state pore may comprise nanochannels. In some embodiments, the solid-state pore is the pore disclosed in WO 2003 / 003446, WO 2009 / 020682 or WO 2016 / 187519, each of which is incorporated herein by reference in its entirety.

[0411] In one embodiment, the hole may be a DNA origami hole (Langecker et al., Science, 2012; 338: 932-936). Suitable DNA origami holes are disclosed in WO2013 / 083983, WO 2018 / 011603 and WO 2020 / 025974, each of which is incorporated in its entirety by reference.

[0412] In one embodiment, the nanopore is a scaffold peptide nanopore. In some embodiments, the pore is a scaffold peptide nanopore disclosed in WO 2020 / 025909 or WO 2020 / 074399, each of which is incorporated herein by reference in its entirety.

[0413] In one embodiment, the nanopore is a transmembrane protein pore. A transmembrane protein pore is a polypeptide or aggregate of polypeptides that allows hydrated ions (such as polynucleotides) to flow from one side of a membrane to the other. In the methods provided herein, transmembrane protein pores are capable of forming pores that allow hydrated ions, driven by an applied potential, to flow from one side of a membrane to the other. Transmembrane protein pores typically allow polynucleotides to flow from one side of a membrane (such as a triblock copolymer membrane) to the other. Transmembrane protein pores allow polynucleotides to move through the pore.

[0414] In one embodiment, the nanopore is a transmembrane protein pore, which is a monomer or oligomer. The pore typically consists of several repeating subunits, such as at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 subunits. The pore is typically a hexamer, heptamer, octamer, or nonamer pore. The pore can be a homooligomer or a heterooligomer.

[0415] In one embodiment, a transmembrane protein pore comprises a barrel or channel through which ions can flow. The pore subunits typically revolve around a central axis and contribute chains to either the transmembrane β-barrel or channel, or the transmembrane α-helical bundle or channel.

[0416] Typically, the barrels or channels of transmembrane protein pores contain amino acids that facilitate interaction with analytes, such as target peptides (as described herein). These amino acids are usually located near the constriction of the barrel or channel. Transmembrane protein pores typically contain one or more positively charged amino acids, such as arginine, lysine, or histidine, or aromatic amino acids, such as tyrosine or tryptophan. These amino acids typically facilitate interactions between the pore and nucleotides, polynucleotides, or nucleic acids.

[0417] In one embodiment, the nanopore is a transmembrane protein pore derived from a β-barrel pore or an α-helical bundle pore. A β-barrel pore comprises a barrel or channel formed by β-chains. Suitable β-barrel pores include, but are not limited to, β-toxins such as α-hemolysin, anthrax toxin, and leukocidin, as well as bacterial outer membrane proteins / porins such as Mycobacterium smegmatis porins (Msp), such as MspA, MspB, MspC, or MspD, CsgG, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, and Neisseria autotransporter (NalP), and other pores such as cytolysins. An α-helical bundle pore comprises a barrel or channel formed by α-helices. Suitable α-helical bundle pores include, but are not limited to, inner membrane proteins and α-outer membrane proteins such as WZA and ClyA toxins.

[0418] In one embodiment, the nanopore is a transmembrane pore derived from or based on Msp, α-hemolysin (α-HL), cytolysin, CsgG, ClyA, Sp1, or the hemolysin fragaceatoxin C (FraC).

[0419] In one embodiment, the nanopore is a transmembrane protein pore derived from CsgG (e.g., CsgG derived from the Escherichia coli strain K-12 substrain MC4100). Such pores are oligomeric and typically contain 7, 8, 9, or 10 monomers derived from CsgG. The pore can be a homooligomeric pore derived from CsgG containing the same monomers. Alternatively, the pore can be a heterooligomeric pore derived from CsgG containing at least one monomer different from the others. Examples of suitable pores derived from CsgG are disclosed in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318, and WO 2019 / 002893, each of which is hereby incorporated in its entirety by reference.

[0420] In one embodiment, the nanopore is a transmembrane pore derived from cytosin. Examples of suitable pores derived from cytosin are disclosed in WO 2013 / 153359, which is hereby generally incorporated herein by reference in its entirety.

[0421] In one embodiment, the nanopore is a transmembrane pore derived from or based on α-hemolysin (α-HL). Wild-type α-hemolysin pores are formed by seven identical monomers or subunits (i.e., they are heptamers). α-hemolysin pores can be α-hemolysin-NN or variants thereof. Variants typically contain N residues at positions E111 and K147.

[0422] In one embodiment, the nanopore is a transmembrane protein pore derived from Msp, such as MspA. Examples of suitable pores derived from MspA are disclosed in WO 2012 / 107778.

[0423] In one embodiment, the nanopores are transmembrane pores derived from or based on ClyA. Examples of suitable pores derived from ClyA are disclosed in the following documents: Soskine et al., Nano Letters 2012 12 (9), 4895-4900; WO 2014 / 153625; and WO 2017 / 098322, each of which is hereby incorporated by reference.

[0424] In one embodiment, the nanopore is a transmembrane pore derived from Phi29. Examples of suitable pores derived from Phi29 are disclosed in the following literature: Wendell et al., Nature Nanotech 4, 765-772 (2009), WO 2010 / 062697, WO 2019 / 157365 and WO 2019 / 157424, each of which is hereby incorporated by reference.

[0425] In some embodiments, the nanopores are selected from M-ring proteins, perforin-2, PlyAB (pleurotolysin), SpoIIIAG, VirB7, type II secretion system protein D, GspD, InvG, PilQ, permein, and portal proteins, including T4, T7, P23_45, G20c, and Phi29 nanopores.

[0426] In one embodiment, the nanopore is a transmembrane pore derived from or based on bacteria of the Rhodococcus species, such as Rhodococcus corynebacteroides or Rhodococcus ruber, such as PorARr, PorBRr, or PorARc. Examples of such pores are described in Piselli et al., *EurBiophys J* 51, 309-323 (2022).

[0427] As explained above, in some embodiments, the nanopore includes a constriction. The constriction is typically a narrowing in the channel through the nanopore, which can determine or control the signal received when the conjugate moves relative to the nanopore. As used herein, both proteins and solid nanopores can include a “constriction.”

[0428] In some embodiments, the nanopores are designed, modified, or selected to have constrictions of a size determined according to the diameter of the construct. In some embodiments, the diameter of the constriction of the pore is at least 1 nm, such as at least 1.5 nm, such as at least 2 nm, such as at least 2.5 nm, such as at least 3 nm. In some embodiments, the diameter of the constriction of the pore is from about 1.5 nm to about 2.5 nm. In some embodiments, the pore has a constriction capable of translocating double-stranded DNA. The diameter of the double-stranded DNA is about 2 nm. DNA-peptide chimeras can be narrower or wider than double-stranded DNA, depending on amino acid and strand interactions.

[0429] In some embodiments, the nanopores are modified to extend the distance between the polynucleotide-treated protein and the constricted region of the nanopore. A method for doing so is disclosed in WO 2021 / 111125.

[0430] Label

[0431] In some embodiments of the methods provided herein, labels on nanopores may be used, for example, to facilitate nanopore trapping constructs.

[0432] The interaction between the tag on the nanopore and the binding site on the construct or chain (which may be, for example, a binding site present in the polynucleotide portion of the DNA-peptide hybrid chain or in the chain-connected adaptor, wherein the binding site may be provided by the anchor or leader sequence of the adaptor or by the capture sequence within the double-stranded stem of the adaptor) can be reversible. For example, the polynucleotide may bind to the tag on the nanopore, for example, through its adaptor, and be released at certain points, for example, during the characterization of the polynucleotide through the nanopore and / or during motor protein processing. Strong non-covalent binding (e.g., biotin / avidin) remains reversible and can be used in some embodiments of the methods described herein. For example, a pair of pore tags and polynucleotide adaptors may be designed to provide sufficient interaction between the complement of the double-stranded polynucleotide (or the complement-connected portion of the adaptor) and the nanopore, such that the complement remains close to the nanopore (without dissociating and diffusing from the nanopore) but is able to be released from the nanopore during processing.

[0433] The pore tag and polynucleotide adapter can be configured such that the binding strength or affinity of the binding site on the polynucleotide (e.g., a binding site provided by the anchor or leader sequence of the adapter or by the capture sequence within the double-stranded stem of the adapter) to the tag on the nanopore is sufficient to maintain the connection between the nanopore and the polynucleotide until an applied force is placed thereon to release the bound polynucleotide from the nanopore.

[0434] In some embodiments, the tag or tether is uncharged. This ensures that the tag or tether will not be pulled into the nanopore under the influence of a potential difference (if present).

[0435] One or more molecules that attract or bind to the construct, polynucleotide-peptide conjugate chain, and / or polynucleotide carrier chain can be linked to the nanopore. Any molecule that hybridizes with the conjugate, adaptor, and / or polynucleotide can be used. The molecules linked to the pore can be selected from PNA tags, PEG linkers, short oligonucleotides, positively charged amino acids, and aptamers. Pores that link such molecules to them are known in the art. For example, pores having short oligonucleotides linked to them are disclosed in Howarka et al. (2001) Nature Biotech 19: 636-639 and WO 2010 / 086620, and pores containing PEG linked within the lumen of the pore are disclosed in Howarka et al. (2000) J. Am. Chem. Soc. 122(11): 2411-2416.

[0436] Short oligonucleotides containing sequences complementary to the sequences in the conjugate (e.g., in the leader sequence in the adaptor or in another single-stranded sequence) linked to nanopores can be used to enhance the capture of constructs, polynucleotide-peptide conjugate chains, and / or polynucleotide carrier chains in the methods described herein.

[0437] In some embodiments, the tag or chain may comprise or be an oligonucleotide (e.g., DNA, RNA, LNA, BNA, PNA, or morpholino). The oligonucleotide may be about 10-30 nucleotides or about 10-20 nucleotides in length. In some embodiments, the oligonucleotide may have at least one end modified for conjugation to other modified or solid substrate surfaces (including, for example, beads) (e.g., a 3' or 5' end). The end modifier may add reactive functional groups that can be used for conjugation. Examples of functional groups that can be added include, but are not limited to, amino, carboxyl, thiol, maleimide, aminooxy, and any combination thereof. The functional group may be combined with spacers of different lengths (e.g., C3, C9, C12, spacer 9, and 18) to increase the physical distance between the functional group and the end of the oligonucleotide sequence.

[0438] Examples of modifications to the 3' and / or 5' ends of oligonucleotides include, but are not limited to, 3' affinity tags and functional groups for chemical linkage (including, for example, 3'-biotin, 3'-primary amine, 3'-disulfide amide, 3'-pyridyl disulfide, and any combination thereof); 5' end modifications (including, for example, 5'-primary amine and / or 5'-dabcyl); modifications for click chemistry (including, for example, 3'-azide, 3'-alkynyl, 5'-azide, 5'-alkynyl), and any combination thereof.

[0439] In some embodiments, the tag or tether may further include a polymeric connector, for example, to facilitate coupling with the nanopore. Exemplary polymeric connectors include, but are not limited to, polyethylene glycol (PEG). The molecular weight of the polymeric connector may be from about 500 Da to about 10 kDa (including end values) or from about 1 kDa to about 5 kDa (including end values). The polymeric connector (e.g., PEG) may be functionalized with different functional groups, including, but not limited to, maleimide, NHS ester, dibenzocyclooctylene (DBCO), azide, biotin, amine, alkyne, aldehyde, and any combination thereof.

[0440] Other examples of tags or chains include, but are not limited to, His tags, biotin or streptavidin, antibodies that bind to analytes, aptamers that bind to analytes, analyte-binding domains such as DNA-binding domains (including, for example, peptide zippers, such as leucine zippers, single-stranded DNA-binding proteins (SSBs)) and any combination thereof.

[0441] Tags or tethers can be attached to the outer surface of a nanopore, for example, on the cis side of a membrane, using any method known in the art. For example, one or more tags or tethers can be attached to a nanopore via one or more cysteine ​​residues (cysteine ​​bonds), one or more primary amines (such as lysine), one or more non-natural amino acids, one or more histidine residues (His tags), one or more biotin or streptavidin residues, one or more antibody-based tags, one or more enzymatic modifications of epitopes (including, for example, acetyltransferases), and any combination thereof. Suitable methods for making such modifications are well known in the art. Suitable non-natural amino acids include, but are not limited to, 4-azido-L-phenylalanine (Faz), and Liu CC and Schultz PG, *Annu. Rev. Biochem.*, 2010, 79, 413-444. Figure 1 Any one of the amino acids numbered 1-71 in the list.

[0442] In some embodiments, one or more tags or chains are linked to nanopores via cysteine ​​bonds, one or more cysteines may be introduced into one or more monomers that form nanopores by substitution. In some embodiments, the nanopores may be chemically modified by linking to: (i) maleimides, including dibromomaleimides such as: 4-phenylazomaleimide, 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 Amine, N-methoxycarbonylmaleimide, N-tert-butylmaleimide, N-(2-aminoethyl)maleimide, 3-maleimide-PROXYL, N-(4-chlorophenyl)maleimide, 1-[4-(dimethylamino)-3,5-dinitrophenyl]-1H-pyrrole-2,5-dione, N-[4-(2-benzimidazolyl)phenyl]maleimide, N-[4-(2-benzoxazolyl)phenyl]maleimide, N-( 1-Naphthyl)-maleimide, N-(2,4-dimethyl)maleimide, N-(2,4-difluorophenyl)maleimide, N-(3-chloro-p-tolyl)-maleimide, 1-(2-amino-ethyl)-pyrrole-2,5-dione hydrochloride, 1-cyclopentyl-3-methyl-2,5-dihydro-1H-pyrrole-2,5-dione, 1-(3-aminopropyl)-2,5-dihydro-1H-pyrrole-2,5-dione hydrochloride, 3 -Methyl-1-[2-oxo-2-(piperazin-1-yl)ethyl]-2,5-dihydro-1H-pyrrole-2,5-dione hydrochloride, 1-benzyl-2,5-dihydro-1H-pyrrole-2,5-dione, 3-methyl-1-(3,3,3-trifluoropropyl)-2,5-dihydro-1H-pyrrole-2,5-dione, 1-[4-(methylamino)cyclohexyl]-2,5-dihydro-1H-pyrrole-2,5-dione trifluoroacetic acid, SMILES O=C1C=CC(=O)N1CC=2C=CN=CC2、SMILES O=C1C=CC(=O)N1CN2CCNCC2、1-Benzyl-3-methyl-2,5-dihydro-1H-pyrrole-2,5-dione、1-(2-fluorophenyl)-3-methyl-2,5-dihydro-1H-pyrrole-2,5-dione、N-(4-phenoxyphenyl)maleimide、N-(4-nitrophenyl)maleimide;(ii) Iodoacetamides, such as: 3-(2-iodoacetamide)-propyl, N-(cyclopropylmethyl)-2-iodoacetamide, 2-iodo-N-(2-phenylethyl)acetamide, 2-iodo-N-(2,2,2-trifluoroethyl)acetamide, N-(4-acetylphenyl)-2-iodoacetamide, N-(4-(aminosulfonyl)phenyl)-2-iodoacetamide, N-(1,3-benzothiazolyl-2-yl)-2-iodoacetamide, N-(2,6-diethylphenyl) (iii) Bromoacetamides: such as N-(4-(acetamido)phenyl)-2-bromoacetamide, N-(2-acetylphenyl)-2-bromoacetamide, 2-bromo-n-(2-cyanophenyl)acetamide, 2-bromo-N-(3-(trifluoromethyl)phenyl)acetamide, N-(2-benzoylphenyl)-2-bromoacetamide, 2-bromo-N-(4-fluorophenyl)acetamide (iv) 3-methylbutyramide, N-benzyl-2-bromo-N-phenylpropionamide, N-(2-bromo-butyryl)-4-chloro-benzenesulfonamide, 2-bromo-N-methyl-N-phenylacetamide, 2-bromo-N-phenethyl-acetamide, 2-adamantane-1-yl-2-bromo-N-cyclohexyl-acetamide, 2-bromo-N-(2-methylphenyl)butyramide, monobromoacetanilide; (iv) disulfides, such as aldehyde thiols-2, aldehyde thiols-4, isopropyl disulfide. , 1-(isobutyldithioalkyl)-2-methylpropane, dibenzyl disulfide, 4-aminophenyl disulfide, 3-(2-pyridyldithio)propionic acid, 3-(2-pyridyldithio)propionic acid hydrazide, 3-(2-pyridyldithio)propionic acid N-succinimide ester, am6amPDP1-βCD; and (v) thiols, such as: 4-phenylthiazolyl-2-thiol, Purpald, 5,6,7,8-tetrahydro-quinazoline-2-thiol.

[0443] In some embodiments, the tag or tether can be attached to the nanopore directly or via one or more adapters. The hybridization adapters described in WO 2010 / 086602 can be used to attach the tag or tether to the nanopore. Alternatively, peptide adapters can be used. Peptide adapters are amino acid sequences. The length, flexibility, and hydrophilicity of peptide adapters are typically designed so that they do not interfere with the function of the monomer and the pore. Typical flexible peptide adapters are extensions of 2 to 20, such as 4, 6, 8, 10, or 16 serine and / or glycine amino acids. More typical flexible adapters include (SG)1, (SG)2, (SG)3, (SG)4, (SG)5, and (SG)8, where S is serine and G is glycine. Typical rigid adapters are extensions of 2 to 30, such as 4, 6, 8, 16, or 24 proline amino acids. More typical rigid adapters include (P) 12 , where P is proline.

[0444] membrane

[0445] Typically, in the disclosed methods, nanopores are present within the membrane. Any suitable membrane can be used in the system.

[0446] Membranes are typically amphiphilic layers. An amphiphilic layer is a layer formed by amphiphilic molecules such as phospholipids, possessing both hydrophilic and lipophilic properties. These amphiphilic molecules can be synthetic or naturally occurring. Non-naturally occurring amphiphiles and monolayer-forming amphiphiles are known in the field and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Block copolymers are polymeric materials in which two or more monomer subunits polymerize together to form a single polymer chain. Block copolymers typically possess properties contributed by each monomer subunit. However, block copolymers can possess unique properties not found in polymers formed by individual subunits. Block copolymers can be designed such that one of the monomer subunits is hydrophobic (i.e., lipophilic) in an aqueous medium, while the others are hydrophilic. In this case, the block copolymer can possess amphiphilic properties and can form a structure mimicking a biological membrane. Block copolymers can be diblock (composed of two monomer subunits), but can also be constructed from more than two monomer subunits to form a more complex arrangement exhibiting amphiphilic behavior. Copolymers can be triblock, tetrablock, or pentablock copolymers. Membranes can be triblock copolymer membranes.

[0447] Archaea bipolar tetraether lipids are naturally occurring lipids that are constructed to form monolayer membranes. These lipids are commonly found in extremophiles, thermophiles, halophiles, and acidophiles that survive in harsh biological environments. Their stability is thought to derive from the fusion properties of the final bilayer. Constructing block copolymer materials that mimic these biological entities is straightforward by generating triblock polymers with a general motif of hydrophilic-hydrophobic-hydrophilic properties. This material can form monomeric membranes that exhibit characteristics similar to lipid bilayers and encompass a range of stages from vesicles to lamellar membranes. Membranes formed from these triblock copolymers retain several advantages over biological lipid membranes. Because the triblock copolymers are synthesized, precise construction can be carefully controlled to provide the correct chain lengths and properties required for membrane formation and interaction with pores and other proteins.

[0448] Block copolymers can also be constructed from subunits that are not classified as lipid submaterials; for example, hydrophobic polymers can be made from siloxanes or other non-hydrocarbon-based monomers. The hydrophilic subsegments of the block copolymers can also possess low protein-binding properties, allowing for the creation of highly resistant membranes when exposed to pristine biological samples. This head unit can also be derived from non-classical lipid head units.

[0449] Compared to bio-lipid membranes, triblock copolymer membranes also offer improved mechanical and environmental stability, such as a much higher operating temperature or pH range. The synthetic properties of block copolymers provide a platform for customizing polymer-based membranes for a wide range of applications.

[0450] In some embodiments, the membrane is one of the membranes disclosed in International Application No. WO2014 / 064443 or WO2014 / 064444.

[0451] Amphiphilic molecules can be chemically modified or functionalized to promote polynucleotide coupling. Amphiphilic layers can be monolayers or bilayers. Amphiphilic layers are typically planar. Amphiphilic layers can be curved. Amphiphilic layers can be supported.

[0452] Amphiphilic membranes are typically naturally mobile, essentially with a diameter of approximately 10. -8 cm s -1 The lipid diffusion rate acts as a two-dimensional liquid. This means that pores and coupled polynucleotides can normally move within the amphiphilic membrane.

[0453] Membranes can be lipid bilayers. Lipid bilayers are models of cell membranes and serve as excellent platforms for a range of experimental studies. For example, lipid bilayers can be used for in vivo studies of membrane proteins via single-channel recording. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of substances. Lipid bilayers can be any type of lipid bilayer. Suitable lipid bilayers include, but are not limited to, planar lipid bilayers, supported bilayers, or liposomes. Lipid bilayers are typically planar lipid bilayers. Suitable lipid bilayers are disclosed in WO 2008 / 102121, WO 2009 / 077734, and WO 2006 / 100484.

[0454] Methods for forming lipid bilayers are known in the art. Lipid bilayers are typically formed by the method of Montal and Mueller (Proceedings of the National Academy of Sciences of the United States of America, 1972; 69:3561-3566), in which a lipid monolayer is carried on an aqueous / air interface passing through two sides of an opening perpendicular to the interface. The lipid is typically added to the surface of an aqueous electrolyte solution by first dissolving the lipid in an organic solvent and then allowing the solvent to evaporate onto the surface of an aqueous solution on either side of the pore. Once the organic solvent evaporates, the solution / air interface on either side of the pore physically moves back and forth across the pore until a bilayer is formed. Planar lipid bilayers can be formed across openings in the membrane or across openings into grooves.

[0455] The Montal and Mueller method is popular because it is a cost-effective and relatively simple approach to forming high-quality lipid bilayers suitable for protein pore insertion. Other common methods for bilayer formation include tip impregnation, bilayer brushing, and patch clamping.

[0456] Tip-immersion bilayer formation requires contact between the open-aperture surface (e.g., a pipette tip) and the surface of the test solution carrying the lipid monolayer. Similarly, the lipid monolayer is initially generated at the solution / air interface by allowing a drop of lipid dissolved in an organic solvent to evaporate at the solution surface. The bilayer is then formed using the Langmuir-Schaefer method, requiring mechanical automation to move the open-aperture relative to the solution surface.

[0457] For the brush-coated bilayer, a drop of lipid dissolved in an organic solvent is applied directly to an opening immersed in an aqueous test solution. The lipid solution is then thinly applied to the opening using a brush or equivalent. The solvent is diluted to allow the formation of the lipid bilayer. However, completely removing the solvent from the bilayer is difficult, and therefore the bilayer formed by this method is less stable and more prone to noise during electrochemical measurements.

[0458] Patch clamping is commonly used in the study of biological cell membranes. The cell membrane is clamped to the end of a pipette by aspiration, and the membrane patch becomes attached within an opening. This method is also suitable for generating lipid bilayers by clamping and then bursting liposomes to release the lipid bilayer sealed within an opening of the pipette. This method requires stable, large, monolayered liposomes and the fabrication of small openings in a material with a glass surface.

[0459] Liposomes can be formed by sonication, extrusion or the Mozafari method (Colas et al. (2007) Micron 38:841-847).

[0460] In some embodiments, the lipid bilayer is formed as described in International Application No. WO 2009 / 077734. Advantageously, in this method, the lipid bilayer is formed from dried lipids. In some embodiments, the lipid bilayer is formed across an opening, as described in WO2009 / 077734.

[0461] A lipid bilayer is formed by two opposing lipid layers. The two lipid layers are arranged such that their hydrophobic tail groups face each other to form a hydrophobic interior. The hydrophilic head groups of the lipids face outwards to the aqueous environment on each side of the bilayer. Bilayers can exist in many lipid phases, including but not limited to liquid disordered phases (fluid laminar), liquid ordered phases, solid ordered phases (laminar gel phases, interdigitated gel phases), and planar bilayer crystals (laminar subgel phases, laminar crystalline phases).

[0462] Any lipid composition that forms a lipid bilayer can be used. The lipid composition is selected such that a lipid bilayer with desired properties (such as surface charge, ability to support membrane proteins, packing density, or mechanical properties) is formed. The lipid composition may contain one or more different lipids. For example, a lipid composition may contain up to 100 lipids. A lipid composition typically contains 1 to 10 lipids. The lipid composition may contain naturally occurring lipids and / or artificial lipids.

[0463] Lipids typically comprise a head group, an interfacial portion, and two hydrophobic tail groups that may be the same or different. Suitable head groups include, but are not limited to: neutral head groups, such as diacylglycerol esters (DG) and ceramides (CM); zwitterionic head groups, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM); negatively charged head groups, such as phosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), and cardiolipin (CA); and positively charged head groups, such as trimethylammonium propane (TAP). Suitable interfacial portions include, but are not limited to, naturally occurring interfacial portions, such as glycerol-based or ceramide-based portions. Suitable hydrophobic tail groups include, but are not limited to, saturated hydrocarbon chains such as lauric acid (n-dodecanoic acid), myristic acid (n-tetradecanoic acid), palmitic acid (H-hexadecanoic acid), stearic acid (n-octadecanoic acid), and arachidic acid (n-eicosanoic acid); unsaturated hydrocarbon chains such as oleic acid (cis-9-octadecanoic acid); and branched hydrocarbon chains such as phytanoyl groups. The chain length and the position and number of double bonds in unsaturated hydrocarbon chains can vary. The chain length and the position and number of branches (such as methyl groups) in branched hydrocarbon chains can also vary. Hydrophobic tail groups can act as ethers or esters, connecting to the interfacial portion. Lipids can be mycolic acids.

[0464] Lipids can also be chemically modified. The head or tail group of a lipid can be chemically modified. Suitable lipids with chemically modified head groups include, but are not limited to: PEG-modified lipids, such as 1,2-diacyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000]; functionalized PEG lipids, such as 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[biotinyl(polyethylene glycol)2000]; and lipids for conjugation modification, such as 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine-N-(succinyl) and 1,2-dispalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-(biotinyl). Suitable lipids with chemically modified tails include, but are not limited to: polymerizable lipids, such as 1,2-bis(10,12-tetracarbadiynyl)-sn-glycerol-3-phosphate choline; fluorinated lipids, such as 1-palmitoyl-2-(16-fluoropalmitoyl)-sn-glycerol-3-phosphate choline; deuterated lipids, such as 1,2-dipalmitoyl-D62-sn-glycerol-3-phosphate choline; and ether-linked lipids, such as 1,2-di-O-phytyl-sn-glycerol-3-phosphate choline. Lipids may be chemically modified or functionalized to facilitate coupling with polynucleotides.

[0465] Amphiphilic layers, such as lipid compositions, typically contain one or more additives that will affect the properties of the layer. Suitable additives include, but are not limited to, fatty acids such as palmitic acid, myristic acid, and oleic acid; fatty alcohols such as palmitol, myristicol, and oleyl alcohol; sterols such as cholesterol, ergosterol, lanosterol, sitosterol, and stigmasterol; lysophospholipids such as 1-acyl-2-hydroxy-sn-glycerol-3-phosphocholine; and ceramides.

[0466] In another embodiment, the membrane comprises a solid layer. The solid layer can be formed of both organic and inorganic materials, including but not limited to microelectronic materials, insulating materials (such as Si3N4, Al2O3, and SiO), organic and inorganic polymers (such as polyamides), plastics (such as Teflon®), or elastomers (such as two-component addition-cured silicone rubber), and glass. The solid layer can be formed of graphene. Suitable graphene layers are disclosed in WO 2009 / 035647. If the membrane comprises a solid layer, pores are typically present in the amphiphilic membrane or layer contained within the solid layer, for example, within holes, traps, gaps, channels, trenches, or slits within the solid layer. Those skilled in the art can prepare suitable solid / amphiphilic hybrid systems. Suitable systems are disclosed in WO 2009 / 020682 and WO 2012 / 005857. Any of the amphiphilic membranes or layers discussed above can be used.

[0467] The methods disclosed herein are typically performed using: (i) an artificial amphiphilic layer containing pores, (ii) a separated, naturally occurring lipid bilayer containing pores, or (iii) cells in which pores are inserted. Artificial amphiphilic layers (such as artificial triblock copolymer layers) are typically used to perform the methods. These layers may contain other transmembrane and / or intramembrane proteins, as well as other molecules besides pores. Suitable equipment and conditions are discussed below. The disclosed methods are typically performed in vitro.

[0468] condition

[0469] The disclosed characterization methods can be performed using any apparatus suitable for studying membrane / pore systems in which pores are inserted into the membrane. The characterization methods can be performed using any apparatus suitable for transmembrane pore sensing. For example, the apparatus may comprise a chamber containing an aqueous solution and a barrier dividing the chamber into two sections. The barrier typically has pores in which a membrane containing transmembrane pores is formed. Transmembrane pores are described herein.

[0470] The characterization method can be performed using the apparatus described in WO 2008 / 102120, WO 2010 / 122293 or WO 00 / 28312.

[0471] Characterization methods may include optical measurements, for example, as described in WO 2016 / 009180 and WO 2021 / 198695.

[0472] Characterization methods may involve measuring the ion current flowing through the pore, typically by measuring the current. Alternatively, the ion current flowing through the pore may be measured optically, as disclosed in Heron et al., *Journal of the American Chemical Society*, Vol. 131, No. 5, 2009. Therefore, the device may also include circuitry capable of applying a potential across the membrane and the pore and measuring the electrical signal. Characterization methods can be performed using patch clamps or voltage clamps. Characterization methods typically involve the use of voltage clamps.

[0473] The characterization methods can be performed on silicon-based well arrays, each containing 128, 256, 512, 1024, 2000, 3000, 4000, 6000, 10000, 12000, 15000 or more wells.

[0474] Characterization methods may involve measuring the current flowing through the pore. These methods are typically performed with a voltage applied across the membrane and the pore. The voltage used is typically +2 V to -2 V, and usually -400 mV to +400 mV. The voltage used is preferably within a range having a lower limit selected from -400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, -20 mV, and 0 mV, and the upper limit is independently selected from +10 mV, +20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV, and +400 mV. The voltage used is more typically in the range of 100 mV to 240 mV, and most typically in the range of 120 mV to 220 mV. By using an increased applied potential, the pore's ability to distinguish different nucleotides can be increased.

[0475] Characterization methods are typically performed in the presence of any charge carriers, such as metal salts, e.g., alkali metal salts; halide salts, e.g., chloride salts, such as alkali metal chloride salts. Charge carriers can include ionic liquids or organic salts, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, or 1-ethyl-3-methylimidazolium chloride. In the exemplary apparatus discussed above, the salt is present in an aqueous solution in the chamber. Potassium chloride (KCl), sodium chloride (NaCl), or cesium chloride (CsCl) is typically used. KCl is typical. The salt can be an alkaline earth metal salt, such as calcium chloride (CaCl2). The salt concentration can be saturated. The salt concentration can be 3 M or lower, and is typically 0.1 M to 2.5 M, 0.3 M to 1.9 M, 0.5 M to 1.8 M, 0.7 M to 1.7 M, 0.9 M to 1.6 M, or 1 M to 1.4 M. The salt concentration is typically 150 mM to 1 M. Characterization methods can be performed using salt concentrations of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M, or at least 3.0 M. High salt concentrations provide a high signal-to-noise ratio and allow identification of currents indicating binding / non-binding against a background of normal current fluctuations.

[0476] Characterization methods are typically performed in the presence of a buffer solution. In the exemplary apparatus discussed above, the buffer solution is present in an aqueous solution in the chamber. Any suitable buffer solution can be used. Typically, the buffer solution is HEPES. Another suitable buffer solution is Tris-HCl buffer. The methods are typically performed at the following pH values: 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7, or 7.0 to 8.8, or 7.5 to 8.5. The pH used can be approximately 7.5.

[0477] Characterization methods can be performed at the following temperatures: 0°C to 100°C, 15°C to 95°C, 16°C to 90°C, 17°C to 85°C, 18°C ​​to 80°C, 19°C to 70°C, or 20°C to 60°C. Characterization methods are typically performed at room temperature. Optionally, characterization methods can be performed at temperatures that support enzyme function (e.g., about 37°C).

[0478] Other aspects

[0479] In one embodiment, this document also provides a sequencing library that can be obtained according to or by the disclosed methods.

[0480] More generally, this document provides a library containing multiple polynucleotide-peptide hybrid chains. In some embodiments, the polynucleotide-peptide hybrid chains may be described in more detail herein.

[0481] In some embodiments, each of the polynucleotide-peptide hybrid chains comprises (i) a polypeptide comprising a polypeptide test sequence, a purification tag, and a cleavage site between the polypeptide test sequence and the purification tag; and (ii) a polynucleotide encoding the polypeptide. The polynucleotide may be a DNA or RNA polynucleotide. The polynucleotide may comprise single-stranded RNA, single-stranded DNA, double-stranded DNA, or a double-stranded DNA:RNA hybrid. The polynucleotide may comprise more than one type of polynucleotide. The chain may comprise a linker region between the polynucleotide portion and the polypeptide portion. The linker may be a linker as described herein. The linker may comprise or consist of a polynucleotide. The linker may comprise or consist of a polynucleotide that is the same as or different from the polynucleotide portion of the chain.

[0482] In some embodiments, a library is provided comprising multiple RNA-peptide hybrid chains, each of which comprises (i) a peptide comprising a peptide test sequence, a purification tag, and a cleavage site between the peptide test sequence and the purification tag; and (ii) an RNA polynucleotide encoding the peptide.

[0483] A sequencing library is also provided, comprising multiple DNA-peptide hybrid chains, wherein the DNA-peptide hybrid chains comprise (i) a peptide portion containing a peptide test sequence; (ii) a DNA polynucleotide portion comprising (a) a sequence encoding the peptide test sequence and / or (b) a sequence complementary to the sequence encoding the peptide test sequence; and (c) a sequencing adaptor.

[0484] In some embodiments, the sequencing library comprises (i) a DNA polynucleotide portion containing a sequence complementary to a sequence encoding the polypeptide test sequence; and (ii) an RNA or DNA polynucleotide portion containing a sequence encoding the polypeptide test sequence. In some embodiments, the sequencing library comprises an RNA polynucleotide portion. In some embodiments, the sequencing library comprises a DNA polynucleotide portion.

[0485] system

[0486] In some embodiments, this document provides a system comprising a library as described herein and nanopores.

[0487] In some embodiments, the nanopore is as described herein. In some embodiments, the system further includes a polynucleotide processing protein capable of controlling the movement of strands in the library relative to the nanopore. In some embodiments, the system includes computing means configured to detect information specific to polynucleotide and / or polypeptide moieties of the strands in the library and selectively process signals acquired when the DNA-peptide hybrid strands move relative to the nanopore. In some embodiments, the system includes receiving means for receiving data from the detection of the polypeptide, processing means for processing signals acquired when the conjugate moves relative to the nanopore, and output means for outputting the characterization information thus acquired.

[0488] It should be understood that although specific embodiments, constructions, and materials and / or molecules have been discussed herein with respect to the methods according to the invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of the invention. The foregoing embodiments and the following examples are provided for illustrative purposes only and should not be considered as limiting the scope of this application. This application is limited only by the claims.

[0489] Example

[0490] Example 1

[0491] Component assembly

[0492] The oligonucleotide sequences used are shown in Table 4. The gene fragment (SEQ ID NO: 1) encoding the T7 promoter, Twin-Strep-tag® purification tag, TEV protease site, and test peptide (sequence: SGGSGDDSGSGEEEEEEEEEE; SEQ ID NO: 10) was amplified by PCR using 50 ng of template DNA as input to 50 µL of the reaction at a primer annealing temperature of 72°C using Q5 Master Mix (New England Biolabs, catalog number M0492). The forward primer (SEQ ID NO: 2) was annealed to the start of the T7 promoter, and the reverse primer (SEQ ID NO: 3) was annealed to the end of the peptide variant sequence. The PCR products were purified using AmpureXP magnetic SPRI beads (Beckman Coulter, catalog number A63882) as follows: the PCR reaction solution was brought to 100 µL with nuclease-free water (NFW). Add 0.8 times excess SPRI beads (80 µL) and then incubate on a rotary mixer at room temperature for 10 minutes. The beads precipitate on a magnet and are washed twice with 75% ethanol, and then removed from the magnetic rack. For elution, add NFW (25 µL) to the beads and incubate on a rotary mixer at room temperature for 10 minutes. This purification reaction method will subsequently be referred to as '0.8X SPRI'.

[0493] Then, using the HiScribe® T7 High-Yield RNA Synthesis Kit (New England Biolabs, catalogue E2040S), the amplified DNA was transcribed into RNA via in vitro transcription, following the manufacturer's instructions, using 1 µg of purified PCR product as a template in a 20 µL reaction volume. The template was digested with DNase I (New England Biolabs, catalogue M0303S) and DNase I buffer, following the manufacturer's instructions. The reaction was then purified using the Monarch RNA Cleanup Kit (500 µg) (New England Biolabs, catalogue T2050), following the manufacturer's instructions, and the RNA was eluted in 50 µL of NFW.

[0494] The resulting RNA was then ligated to a DNA adapter with a 3' puromycin group (SEQ ID NO: 4). To bring the RNA and adapter ends together for ligation, a DNA clip (SEQ ID NO: 5) was used; one portion of the clip was designed to anneal to the 3' end of the RNA, and the other portion was designed to anneal to the 5' end of the adapter. The clip ligation reaction was performed as follows: In a total reaction volume of 20 µL, 8 µM RNA, 8 µM clip, and 8 µM puromycin adapter were first added to a test tube, and the volume was brought to 16 µL with NFW. To anneal the strands together, the sample was heated to 65 °C for 2 minutes, and then held at room temperature for another 3 minutes. 2 µL of T4 DNA ligase buffer and 2 µL of T4 DNA ligase (New England Biolabs, catalog number M0202T) were added, and the mixture was incubated at room temperature for 15 minutes. The reaction solution was purified with 0.8X SPRI. The result of these steps was the ligation of RNA to a DNA adapter with a puromycin group (“RNA adapter”), as shown below. Figure 1 As shown in Figure A.

[0495] The RNA adapter was then added to the in vitro translation mixture. Once the purified tag, protease site, and peptide variant were translated, the ribosome stopped at the RNA-DNA ligation point just before the adapter. This allowed the puromycin group on the adapter to interact with the ribosome, resulting in the C-terminus of the peptide being ligated to the adapter. Translation was performed using the PURExpress® In Vitro Protein Synthesis Kit (New England Biolabs, catalog E6800), with 5 μg of RNA-adaptor as input in 25 µL of reaction solution. The reaction solution was incubated at 25 °C with stirring for 2 h. To improve the efficiency of puromycin interaction and peptide ligation, 7.5 µL of coupling buffer (2.05 M KCl and 172 mM Mg(OAc)2) was added, followed by overnight incubation at -20 °C. The reaction solution was purified the next day using 0.8X SPRI.

[0496] The translated RNA-linker peptide is then reverse transcribed to produce a DNA strand complementary to the RNA sequence. This step stabilizes the RNA molecule and provides a template for replacing RNA with DNA. SuperScript is used according to the manufacturer's protocol. TM Reverse transcription was performed using IV reverse transcriptase (Ingenieur, catalog number 18090010), with 5 μg RNA-adaptor-peptide as input per 20 µL reaction solution. The primers used in this step (SEQ ID NO: 6) were complementary to the adaptor sequence and the 3' end of the RNA.

[0497] Then, TEV protease digestion was performed to purify the tag and protease tag from the peptide cleavage, leaving only the linked peptide variant. The reverse transcription reaction mixture (20 µL) was mixed with 4 µL TEV buffer, 14 µL NFW, and 2 µL TEV protease (New England Biolabs, catalog number P8112S) and incubated at 30°C for 1 hour. The digestion was purified using 0.8X SPRI. The result at the end of these steps was the ligation of the RNA-adaptor to the peptide variant (RNA-peptide hybrid chain), such as... Figure 1 As shown in B.

[0498] The RNA-peptide hybrid strand was then treated with RNase H, an RNase endonuclease. This digests the RNA in the double-stranded RNA-DNA duplex. In this step, the DNA remains linked to the peptide via adapter sequences. The TEV digest (25 µL) was mixed with 5 µL RNase H buffer, 18 µL NFW, and 2 µL RNase H (New England Biolabs, catalog number M0274) and incubated at 37°C for 20 minutes. The digestion was purified using 0.8X SPRI.

[0499] A filling reaction was performed using T7 DNA polymerase to replace the digested RNA with a second DNA strand. T7 DNA polymerase lacks strand displacement activity and therefore does not replace the ligation linkers during synthesis. The reaction was performed in two parts: primer annealing and second-strand synthesis. For primer annealing, 1 µg (or a maximum of 7.8 µL) of the RNase-treated product was mixed with 2.4 µM primer (SEQ ID NO: 7) and 1 µL of annealing buffer (50 mM NaCl, 100 mM Tris pH 7.5). The reaction mixture was brought to a final volume of 10 µL with water and incubated at 37°C for 5 minutes, followed by an incubation on ice for another 2 minutes. The primers were engineered to anneal to the start of the RNA transcript and modified with a tetrazine group at the 5' end. For second-strand synthesis, 5 µL of T7 DNA polymerase buffer and 1 µL of T7 DNA polymerase (New England Biolabs, catalog number M0274) were added. The reaction mixture was brought to a final volume of 50 µL with NFW and incubated at 37°C for 2 minutes. To stop the reaction, transfer the test tube to ice and add EDTA to a final concentration of 20 mM. Purify the packed reaction using 0.8X SPRI.

[0500] Gap ligation is used to close the gap between the newly synthesized DNA strand and the adapter. The filling reaction product is mixed with 5 µL of T4 DNA ligase buffer, 5 µL of T4 DNA ligase (New England Biolabs, catalog number M0202T), and brought to a final volume of 50 µL with NFW. Ligation is allowed to proceed at room temperature for 15 minutes. The ligation solution is then purified with 0.8X SPRI. The result at the end of these steps is a continuous DNA-DNA duplex linked to a peptide at the C-terminus (DNA-peptide hybrid chain), such as… Figure 1 As shown in C.

[0501] The N-terminus of the peptide was then partially activated with an azide via a 2-pyridinecarboxaldehyde (2-PCA) reaction to allow for ligation of the dsDNA 'tail' in subsequent steps. The DNA-peptide hybrid strand was reacted overnight at 25°C with 10 mM 2-PCA-azide in reaction buffer (100 mM NaCl, 25 mM HEPES, pH 8.5) with shaking. The reaction was activated by purification with 0.8X SPRI without eluting the construct from the beads.

[0502] The dsDNA 'tail' and sequencing adaptor were sequentially ligated to the hybridization strand bound to SPRI beads. The sequencing adaptor contained a barcode adaptor (Oxford Nanopore Technologies plc) ligated to the dsDNA strand (SEQ ID NO: 8a, 8b), with the top strand containing a 3' TCO click group. The adaptor was first ligated by adding a 10-fold molar excess of the adaptor to the activated DNA-peptide hybridization strand and a 3.7-fold volume excess of 28% PEG buffer (2.5 M NaCl, 25 mM HEPES pH 7.5, 28% (w / v) PEG 8K) to the adaptor volume. The reaction mixture was incubated at 25°C with shaking at 2000 RPM for 2 hours, during which time the TCO click group reacted with the tetrazine click group present at the 5' end of the filling primer (SEQ ID NO: 7). Unreacted linkers were removed by depositing SPRI beads on a magnetic rack, removing the supernatant, and washing twice with 9.6% PEG buffer (10 mM Tris, 1 M NaCl, 100 μM EDTA, 9.6% (w / v) PEG8000). After the second wash, the 'tail' was then joined by adding a 10-fold molar excess of 'tail' to the activated DNA-peptide hybrid chain and adding a 3.7-fold volume excess of 28% PEG buffer to the 'tail' volume (SEQ ID NO: 9a, 9b). The reaction mixture was incubated at 25°C with shaking at 2000 RPM for 2 hours, during which time the click group reacted with the azide click group present at the N-terminus of the peptide. SPRI beads were deposited on a magnetic rack, the supernatant was removed, and unreacted tails were removed by washing twice with 9% PEG buffer (10 mM Tris, 1 M NaCl, 100 μM EDTA, 9.6% (w / v) PEG8000). Finally, the construct was eluted in 15 µL of elution buffer (50 mM NaCl, 25 mM HEPES, pH 7.5).

[0503] Fully assembled DNA-peptide sequencing constructs such as Figure 1 As shown in D.

[0504] Data collection

[0505] Electrical data were collected using a custom MinION flow cell (Oxford Nanopore Technologies Ltd.) containing protein nanopores. Samples were prepared by mixing 15 µL of a polynucleotide-peptide conjugate in sequencing buffer. The flow cell was first rinsed with 1 mL of rinse buffer. The sample (75 µL) was then introduced into the flow cell through the SpotON port. Electrical data were obtained at a sampling rate of 4 kHz and an applied potential of 180 mV.

[0506] Results and discussion

[0507] Controlled movement of DNA-peptide hybrid chains through nanopores using helicases, as described in WO 2021 / 111125. Figure 2 An example current-time trace obtained using the above-described construct is shown.

[0508] The nucleotide sequence of the DNA portion of the DNA-peptide hybrid chain was determined. This nucleotide sequence can be used to obtain the amino acid sequence of the peptide portion of the same chain. The nucleotide sequence confirms that the peptide portion of the chain has the polypeptide sequence SGGSGDDSGSGEEEEEEEEEE.

[0509] The peptide sequence in the current-time trace can be correlated with the measured peptide signal. Such correlation data allows for the interpretation of peptide measurement signals from peptide analytes (e.g., unknown sequences) based on the peptide analyte sequence.

[0510]

[0511]

[0512] Table 4: Oligonucleotides used in Example 1. T+ indicates locked nucleic acid (LNA) bases. All oligonucleotides were ordered from Integrated DNA Technologies or ATDBio.

[0513] Example 2

[0514] Test peptide sequence

[0515] Fifty different test peptide sequences were created. Each contained the amino acid sequence SGSGSXXXXXXXSGSGSG (SEQ ID NO: 47), where XXXXXXX represents a variable region of seven amino acids in length. The variable region contained random sequences of three different amino acids, D, W, and T. These three amino acids have different properties (aspartic acid (D) = acidic / negatively charged; tryptophan (W) = aromatic / hydrophobic; threonine (T) = small, aliphatic) and were selected to provide different peptide signals with various properties on a nanopore sequencing device.

[0516] Component assembly

[0517] The oligonucleotide sequences used are shown in Table 5. The sequences of the gene fragments used are shown in Table 6. Gene fragments encoding the T7 promoter, Twin-Strep-tag® purification tag, SUMO tag, and test peptide (sequence: SGSGSXXXXXXXSGSGSG (SEQ ID NO: 47), as described above) were all combined in equimolar ratios and amplified by PCR using Q5 High Fidelity Master Mix (New England Biolabs, catalog number M0492). The forward primer (SEQ ID NO: 37) was annealed to the initiation of the T7 promoter, and the reverse primer (SEQ ID NO: 38) was annealed to the end of the peptide variant sequence. The PCR products were purified using AmpureXP magnetic SPRI beads (Beckman Coulter, catalog number A63882).

[0518] Then, using the HiScribe® T7 High-Yield RNA Synthesis Kit (New England Biolabs, catalog number E2040S), the amplified DNA was transcribed into RNA via in vitro transcription, following the manufacturer's instructions. The DNA template was then digested with DNase I (New England Biolabs, catalog number M0303S) and DNase I buffer, following the manufacturer's instructions. The RNA was then eluted in NFW using the Monarch RNA Cleanup Kit (New England Biolabs, catalog number T2050), following the manufacturer's instructions.

[0519] The resulting RNA was then ligated to a DNA adapter (SEQ ID NO: 39) with a 3' puromycin group. A DNA clip (SEQ ID NO: 40) was used to bring the RNA and adapter ends together for ligation; one portion of the clip was designed to anneal to the 3' end of the RNA, and another portion was designed to anneal to the 5' end of the adapter. The reaction solution was purified using SPRI beads. The result of these steps was the ligation of RNA to a DNA adapter with a puromycin group (“RNA adapter”), as shown below. Figure 1 As shown in Figure A.

[0520] The RNA adapter was then added to the in vitro translation mixture. Once the purified tag, protease site, and peptide variant were translated, the ribosome stopped precisely at the RNA-DNA junction preceding the adapter. This allowed the puromycin group on the adapter to interact with the ribosome, resulting in the C-terminus of the peptide attaching to the adapter. Translation was performed using the PURExpress® In Vitro Protein Synthesis Kit (New England Biolabs, catalog number E6800). The reaction mixture was purified using SPRI beads.

[0521] The translated RNA-linker peptide is then reverse transcribed to produce a DNA strand complementary to the RNA sequence. This step stabilizes the RNA molecule and provides a template for replacing RNA with DNA. SuperScript is used according to the manufacturer's protocol. TM Reverse transcription is performed using IV reverse transcriptase (Ingenieur, catalog number 18090010). The primer used in this step (SEQ ID NO: 41) is complementary to the adapter sequence and the 3' end of the RNA.

[0522] Then, SUMO digestion is performed to cleave the tag and protease tag from the peptide, leaving only the linked peptide variant. The result of these steps is the linkage of the RNA-adaptor to the peptide variant (RNA-peptide hybrid chain), such as... Figure 1 As shown in B.

[0523] The RNA-peptide hybrid strand is then treated with an RNA endonuclease, RNase H. This digests the RNA in the double-stranded RNA-DNA duplex. During this step, the DNA remains linked to the peptide via adapter sequences.

[0524] A filling reaction is performed using T7 DNA polymerase to replace the digested RNA with a second DNA strand. T7 DNA polymerase lacks strand displacement activity, therefore it does not replace the ligation linkers during synthesis.

[0525] Gap ligation is used to close the gap between the newly synthesized DNA strand and the adapter. The result of these steps is a continuous DNA-DNA duplex linked to a peptide at the C-terminus (DNA-peptide hybrid chain), such as... Figure 1 As shown in C.

[0526] The N-terminus of the peptide is then activated by an azide reaction via 2-pyridinecarboxaldehyde (2-PCA) to allow ligation of the dsDNA 'tail' in subsequent steps.

[0527] The sequencing adaptor and the dsDNA tail were sequentially ligated to the hybridization strand. The sequencing adaptor contained a barcode adaptor (Oxford Nanopore Technologies Ltd.) ligated to the dsDNA strand (SEQ ID NO: 43, 44), wherein the top strand contained a 3' TCO click group. The adaptor was first ligated by adding excess adaptor to the activated DNA-peptide hybridization strand. The reaction solution was incubated, during which time the TCO click group reacted with the tetrazine click group present at the 5' end of the filling primer (SEQ ID NO: 42). Unreacted adaptors were removed. The 'tail' was then ligated by adding excess 'tail' to the activated DNA-peptide hybridization strand (SEQ ID NO: 45, 46). The reaction solution was incubated, during which time the click group reacted with the azide click group present at the N-terminus of the peptide. Unreacted 'tails' were removed, and the construct was eluted in elution buffer.

[0528] Fully assembled DNA-peptide sequencing constructs such as Figure 1 As shown in D.

[0529] Data collection

[0530] Electrical data were collected using a custom MinION flow cell (Oxford Nanopore Technologies Ltd.) containing protein nanopores. Controlled movement of the test DNA-peptide conjugates was performed using helicases, as described in WO 2021 / 111125. All 50 different test peptide conjugates were analyzed simultaneously in the same flow cell. Samples were prepared by mixing the polynucleotide-peptide conjugates with sequencing buffer and NFW. The flow cell was rinsed with a rinse buffer containing the rinsing chain. The sample was then introduced into the flow cell through the SpotON port. Electrical data were acquired at 21 °C with a sampling rate of 1.5 kHz and an applied potential of 200 mV.

[0531] Results and discussion

[0532] Of the 50 peptide conjugates tested, all but three were represented in the sequencing run; this is in Figure 4 The data is shown in the figure. Data were obtained from all the conjugates represented; representative current-time traces for the three different conjugates are shown in the figure. Figure 5 As shown in the diagram. In each case, the nucleotide sequence of the DNA portion of the DNA-peptide hybrid strand was determined. The nucleotide sequence can be used to obtain the amino acid sequence of the peptide portion of the same strand, thereby allowing the peptide measurement signal to be interpreted based on the obtained amino acid sequence.

[0533] The results demonstrate that a variety of DNA-peptide conjugates can be prepared and sequenced in multiplex mode to create peptide sequencing libraries.

[0534] Table 5:

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542]

Claims

1. A method for generating peptide sequencing libraries; The polypeptide sequencing library contained multiple DNA-peptide hybrid chains; The method includes: i) Translating multiple RNA strands to form multiple polypeptide strands, said translation being performed such that each polypeptide strand is linked to the RNA strand from which it is translated, thereby forming multiple RNA-polypeptide hybrid chains; and ii) Reverse transcription of the RNA portion of the RNA-peptide hybrid chain, thereby forming multiple DNA-peptide hybrid chains; The RNA strand translated in step (i) encodes a polypeptide test sequence, a purification tag, and a cleavage site between the polypeptide test sequence and the purification tag.

2. The method according to claim 1, comprising: - The DNA-peptide hybrid chain is purified using the purification tag; and - The purification tag is removed from the purified DNA-peptide hybrid chain by contacting the purified DNA-peptide hybrid chain with one or more conditions capable of cleaving the cleavage site.

3. The method according to claim 1 or 2, wherein the cleavage site is capable of being cleaved by photolysis, enzymatic reaction, or by contacting the DNA-peptide hybrid chain with one or more chemical reagents; Optionally, the cleavage site is a protease cleavage site.

4. The method according to any one of claims 2 to 3, wherein removing the purification tag comprises contacting the purified DNA-peptide hybrid strand with a protease capable of cleaving the cleavage site.

5. The method according to any one of the preceding claims, further comprising: iii) Contact the plurality of DNA-peptide hybrid chains with a ribonuclease capable of digesting the RNA in the DNA-peptide hybrid chains.

6. The method of claim 5, further comprising: iv) Generate a DNA strand complementary to at least a portion of the DNA portion of the strand generated in step (iii), such that the complementary DNA strand hybridizes with the DNA portion of the strand; such that the resulting DNA-peptide hybrid strand contains at least a partially double-stranded DNA portion.

7. A method for generating peptide sequencing libraries; The polypeptide sequencing library contained multiple DNA-peptide hybrid chains; The method includes: i) Translating multiple RNA strands to form multiple polypeptide strands, wherein the translation is performed such that each polypeptide probe strand is linked to the RNA strand from which it is translated, thereby forming multiple RNA-polypeptide hybrid strands; ii) Reverse transcription of the RNA portion of the RNA-peptide hybrid chain, thereby forming multiple DNA-peptide hybrid chains; iii) Contacting the plurality of DNA-peptide hybrid chains with a ribonuclease capable of digesting the RNA in the DNA-peptide hybrid chains; and iv) Generate a DNA strand complementary to at least a portion of the DNA portion of the strand generated in step (iii), such that the complementary DNA strand hybridizes with the DNA portion of the strand; such that the resulting DNA-peptide hybrid strand contains at least a partially double-stranded DNA portion.

8. The method according to any one of claims 5 to 7, wherein the ribonuclease is an endonuclease; Optionally, the ribonuclease described therein is an endonuclease of the ribonuclease H family.

9. The method according to any one of the preceding claims, wherein step (i) comprises linking a peptide-reactive nucleotide or oligonucleotide to the RNA strand, thereby forming a peptide-reactive RNA conjugate; and translating the peptide-reactive RNA conjugate.

10. The method of claim 9, wherein the peptide-reactive nucleotide or oligonucleotide comprises a moiety of puromycin.

11. The method according to any one of the preceding claims, wherein step (ii) comprises contacting the peptide-reactive RNA conjugate with the polypeptide chain generated in the translation reaction under stable conditions capable of stabilizing the interaction between the peptide-reactive RNA conjugate and the polypeptide chain.

12. The method of claim 11, wherein the stabilizing conditions comprise one or more metal salts, optionally one or more potassium salts and / or magnesium salts.

13. The method according to any one of the preceding claims, wherein step (ii) comprises hybridizing a DNA primer with a polynucleotide portion of the RNA-peptide hybrid chain.

14. The method of claim 13, further comprising stabilizing the bonding between the DNA primer and the complementary portion of the RNA-peptide hybrid chain.

15. The method according to any one of the preceding claims, wherein the method further comprises linking one or more sequencing adaptors to the DNA and / or polypeptide portion of the DNA-peptide hybrid chain.

16. The method according to any one of the preceding claims, further comprising: - To bring the DNA-peptide hybrid chain into contact with the nanopore; and - As the DNA-peptide hybrid chain moves relative to the nanopore, one or more measurements specific to the DNA-peptide hybrid chain are performed; thereby characterizing the DNA-peptide hybrid chain.

17. The method of claim 16, further comprising contacting the DNA-peptide hybrid chain with a polynucleotide-treated protein capable of controlling the movement of the DNA-peptide hybrid chain relative to the nanopore.

18. The method of claim 16 or claim 17, wherein said one or more measurements are specific to one or more of the following: (i) the length of the DNA portion of the DNA-peptide hybrid chain; (ii) the identity of the DNA portion of the DNA-peptide hybrid chain; (iii) the sequence of the DNA portion of the DNA-peptide hybrid chain; (iv) the secondary structure of the DNA portion of the DNA-peptide hybrid chain; (v) whether the DNA portion of the DNA portion of the DNA-peptide hybrid chain is modified; (vi) the length of the polypeptide portion of the DNA-peptide hybrid chain; (vii) the identity of the polypeptide portion of the DNA-peptide hybrid chain; (viii) the sequence of the polypeptide portion of the DNA-peptide hybrid chain; (ix) the secondary structure of the polypeptide portion of the DNA-peptide hybrid chain; and (x) whether the polypeptide portion of the DNA portion of the DNA-peptide hybrid chain is modified.

19. The method according to any one of claims 16 to 18, wherein performing one or more measurements specific to the DNA-peptide hybrid chain comprises - As the DNA-peptide hybrid chain moves relative to the nanopore, one or more electrical and / or optical measurements specific to the DNA portion of the DNA-peptide hybrid chain are performed, thereby determining one or more properties of the DNA portion of the DNA-peptide hybrid chain; - As the DNA-peptide hybrid chain moves relative to the nanopore, perform one or more electrical and / or optical measurements specific to the polypeptide portion of the DNA-peptide hybrid chain; and - To correlate the output of one or more electrical and / or optical measurements specific to the polypeptide portion of the DNA-peptide hybrid chain with one or more properties of the DNA portion of the DNA-peptide hybrid chain.

20. The method according to any one of claims 16 to 19, comprising: Determine the sequence of the DNA portion of the DNA-peptide hybrid chain; and The electrical or optical signals recorded when the polypeptide portion of the DNA-polypeptide hybrid chain moves relative to the nanopore are correlated with the sequence.

21. The method according to any one of claims 16 to 20, wherein the nanopore is a transmembrane protein pore.

22. The method according to any one of the preceding claims, comprising the step of transcribing multiple DNA strands to form said multiple RNA strands prior to step (i).

23. A sequencing library that can be obtained by the method according to any one of the preceding claims.

24. A library comprising a plurality of RNA-peptide hybrid chains, wherein each of the RNA-peptide hybrid chains comprises (i) a polypeptide comprising a polypeptide test sequence, a purification tag, and a cleavage site between the polypeptide test sequence and the purification tag; and (ii) an RNA polynucleotide encoding the polypeptide.

25. A sequencing library comprising a plurality of DNA-peptide hybrid chains, wherein the DNA-peptide hybrid chains comprise (i) a peptide portion comprising a peptide test sequence; (ii) a DNA polynucleotide portion comprising (a) a sequence encoding the peptide test sequence and / or (b) a sequence complementary to the sequence encoding the peptide test sequence; and (c) a sequencing adaptor.

26. The sequencing library of claim 25, comprising (i) a DNA polynucleotide portion comprising a sequence complementary to a sequence encoding the polypeptide test sequence; and (ii) an RNA or DNA polynucleotide portion comprising a sequence encoding the polypeptide test sequence.

27. The sequencing library according to claim 25 or claim 26, wherein the DNA-peptide hybrid strand is generated according to any one of claims 1 to 22.

28. A system comprising - The library according to any one of claims 23 to 27; and -Nanopores; Optionally, the nanopores are as defined in claim 21.

29. The system of claim 28, further comprising: - A polynucleotide-processing protein capable of controlling the movement of the DNA-peptide hybrid chain relative to the nanopore in the library; and / or - A computing device configured to detect information specific to the polynucleotide and / or polypeptide portions of the DNA-peptide hybrid chain in the library, and to selectively process signals obtained when the DNA-peptide hybrid chain moves relative to the nanopore.

30. A method for analyzing a measurement signal acquired from each of a plurality of polynucleotide-peptide hybrid chains, the measurement signal being acquired as the polynucleotide-peptide hybrid chain moves relative to a nanopore, wherein each of the polynucleotide-peptide hybrid chains comprises (i) a polypeptide portion comprising a polypeptide test sequence and (ii) a polynucleotide portion comprising a sequence encoding the polypeptide test sequence, the method comprising: Identify in each measurement signal: (i) the polypeptide signal portion of the measured signal corresponding to the polypeptide portion of the polynucleotide-peptide hybrid chain; and (ii) The polynucleotide signal portion of the measured signal corresponding to the polynucleotide portion of the polynucleotide-peptide hybrid chain; For each polynucleotide signal portion, one or more characteristics of the polynucleotide signal portion obtained from the polynucleotide portion are obtained; Based on the encoding of the polynucleotide moiety, one or more characteristics of the polypeptide moiety of the polynucleotide-polypeptide hybrid chain containing the polynucleotide moiety are obtained from one or more characteristics obtained from each polynucleotide moiety. as well as The one or more properties of each polypeptide moiety are associated with the polypeptide signal portion of the measurement signal obtained from the polynucleotide-polypeptide hybrid chain containing the polynucleotide moiety.

31. The method of claim 30, wherein the one or more characteristics of each polynucleotide moiety comprise the polynucleotide sequence of the polynucleotide moiety.

32. The method of claim 31, wherein the one or more properties of each polypeptide moiety comprise the amino acid sequence of the polypeptide moiety.

33. The method according to any one of claims 30 to 32, wherein the polynucleotide-peptide hybrid chain is generated in the method according to any one of claims 1 to 22.

34. The method according to any one of claims 30 to 33, further comprising acquiring the measurement signal from each of the plurality of polynucleotide-peptide hybrid chains.

35. The method of claim 34, wherein the measurement signal comprises electrical and / or optical measurements.

36. The method according to any one of claims 30 to 35, wherein the polynucleotide portion of the polynucleotide-peptide hybrid chain comprises a DNA strand; Optionally, the DNA strand comprises single-stranded DNA, double-stranded DNA, or a double-stranded DNA:RNA hybrid.

37. The method according to any one of claims 1 to 22 or 30 to 36; or the sequencing library according to any one of claims 23 or 25 to 27; or the library according to claim 24; or the system according to any one of claims 28 to 29, wherein the polypeptide test sequence comprises at least one atypical and / or non-proteogenous amino acid.

38. A computer program comprising instructions executable by a computer system, the instructions being configured to cause the computer system to perform the method according to any one of claims 30 to 33 or 36 to 37 when executed.

39. A computer storage medium storing a computer program according to claim 38.

40. A computer system configured to perform the method according to any one of claims 30 to 33 or 36 to 37.

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