Nanopore sequencing of polypeptides with edman degradation

Abstract

Some embodiments of the methods and compositions provided herein relate to nanopore sequencing of a target polypeptide. In some embodiments, a nanopore is embedded in a membrane having a cis and trans surface. Some embodiments include translocating an end of the target polypeptide through the nanopore, measuring a signal generated from the target polypeptide in the nanopore, and removing a terminal residue of the target polypeptide from the target polypeptide with an Edmanase. In some embodiments, a trans solution comprises a blocker capable of attaching to the N-terminal end of the target polypeptide. In some embodiments, a cis solution comprises a leader composition attached to a lock oligonucleotide capable of forming a hairpin structure.

Description

ILLINC.865WO / IP-2907-PCT PATENT NANOPORE SEQUENCING OF POLYPEPTIDES WITH EDMAN DEGRADATIONRELATED APPLICATIONS

[0001] This application claims priority to U. S. Prov. App. No. 63 / 722402 filed November 19, 2024 entitled “NANOPORE SEQUENCING OF POLYPEPTIDES WITH EDMAN DEGRADATION” which is incorporated by reference herein in its entirety.FIELD OF THE INVENTION

[0002] Some embodiments of the methods and compositions provided herein relate to nanopore sequencing of a target polypeptide. In some embodiments, a nanopore is embedded in a membrane having a cis and trans surface. Some embodiments include translocating an end of the target polypeptide through the nanopore, measuring a signal generated from the target polypeptide in the nanopore, and removing a terminal residue of the target polypeptide from the target polypeptide with an Edmanase. In some embodiments, a trans solution comprises a blocker capable of attaching to the N-terminal end of the target polypeptide. In some embodiments, a cis solution comprises a leader composition attached to a lock oligonucleotide capable of forming a hairpin structure.BACKGROUND OF THE INVENTION

[0003] Nanopore-based sensing is an emerging technology with great potential for the detection of diverse organic molecules, sequencing of nucleic acids, and single-molecule analyses of enzymatic reactions and protein folding. Conceptually, nanopore biosensing belongs to the so-called resistive-pulse methods. The use of biological nanopores for resistive- pulse detection of molecular analytes was made possible by the development of planar bilayer recording and the development of single-channel current measurements. Mueller, P. et al., “Reconstitution of cell membrane structure in vitro and its transformation into an excitable system.” Nature 1962, 194, 979-980; Hladky, S B. etal., “Discreteness of conductance change in bimolecular lipid membranes in the presence of certain antibiotics.” Nature 1970, 225, 451- 453. Early studies used channels generated by alamethicin, an antibiotic peptide, Staphylococcus aureus a-hemolysin, and cholera toxins. Krasilnikov, O. V.tV al., “A simple method for the determination of the pore radius of ion channels in planar lipid bilayermembranes.” FEMS Microbiol. Immunol. 1992, 5, 93-100; Korchev, Y. E.et al., “Low conductance states of a single ion channel are not ’closed’.” J. Menibr. Biol. 1995, 147, 233-239. Possibly the biggest push for the development of nanopore biosensing came after it was found that single-stranded DNA and RNA could be threaded through a nanopore, making it a potential technology for nucleic acid sequencing. Kasianowicz, J. J. etal., “Characterization of individual polynucleotide molecules using a membrane channel.” Proc. Natl. Acad. Sci. USA 1996, 93, 13770-13773.SUMMARY OF THE INVENTION

[0004] Some embodiments of the compositions and methods provided herein include a system for characterizing a target polypeptide, comprising: (a) a membrane having a cis and trans surface; (b) a nanopore embedded in the membrane; (c) a target polypeptide tethered or immobilized to the nanopore or to the membrane via a tether at the cis surface; and (d) a trans solution comprising an Edmanase, wherein the trans solution is in contact with the trans surface. In some embodiments, the Edmanase is tethered or immobilized to the membrane or to the nanopore.

[0005] In some embodiments, the trans solution further comprises a blocker attached via a linker to a reactive moiety capable of coupling to a terminal residue of the target polypeptide. In some embodiments, the reactive moiety is an isothiocyanate moiety. In some embodiments, the reactive moiety is a phenyl isothiocyanate (PITC) moiety. In some embodiments, the blocker comprises a branched oligopeptide or a nanoparticle. In some embodiments, the linker comprises a polyethylene glycol (PEG), a 2-6 benzobisoxazole, or a bifunctional oligothiophene. In some embodiments, at least a portion of the target polypeptide is located within the nanopore. In some embodiments, the blocker is attached to the target polypeptide via the linker and the reactive moiety. In some embodiments, the blocker is attached to an N -terminus of the target polypeptide, or a C-terminus of the target polypeptide.

[0006] Some embodiments also include a cis solution comprising a leader composition comprising a reactive moiety capable of coupling to a terminal residue of the target polypeptide. In some embodiments, the reactive moiety is attached to a lock oligonucleotide capable of forming a hairpin structure. In some embodiments, the cis solution is in contact with the cis surface. In some embodiments, the reactive moiety is anisothiocyanate moiety. In some embodiments, the reactive moiety is a phenyl isothiocyanate (PITC) moiety. In some embodiments, the reactive moiety is attached to the lock oligonucleotide via a spacer. In some embodiments, the spacer comprises a first affinity moiety, and the Edmanase comprises a second affinity moiety, wherein the first and second affinity moieties are capable of binding to one another, optionally wherein the binding is specific binding. In some embodiments, the first and second affinity moieties are selected from biotin, a biotin derivative, streptavidin, a streptavidin derivative, tetrazine, or bicyclononyne (BCN). In some embodiments, the spacer comprises (i) a polynucleotide, optionally, wherein the polynucleotide has a length in a range from 1 nucleotide to 50 nucleotides, optionally, in a range from 3 nucleotides to 10 nucleotides; (ii) a homopolymer; (iii) a polyethylene glycol (PEG); or (iv) a polypeptide spacer, optionally having a length less than 10 consecutive residues, and not less than 1 residue. In some embodiments, the spacer has the nucleotide sequence (TTT). In some embodiments, the leader composition is attached to an N-terminus of the target polypeptide via the reactive moiety. In some embodiments, the lock oligonucleotide is capable of hybridizing to an inhibitor oligonucleotide which inhibits formation of the hairpin structure. In some embodiments, the lock oligonucleotide is hybridized to the inhibitor oligonucleotide. In some embodiments, at least a portion of the target polypeptide is located within the nanopore, such that the leader composition is in contact with the trans solution. In some embodiments, the lock oligonucleotide has a hairpin structure. In some embodiments, the spacer of the leader composition has a length such that a signal indicative of the identity of a terminal residue at the non-tethered terminal end of the target polypeptide is increased compared to a leader sequence having a longer spacer or lacking the spacer.

[0007] In some embodiments, the membrane comprises a lipid bilayer or block copolymer.

[0008] In some embodiments, the nanopore comprises a protein nanopore. In some embodiments, the protein nanopore is selected from OmpF, OmpG, CsgG, MspA, a-HL, FhuA, AeL, FraC, Lys, φ29p, ClyA, or Ply AB. In some embodiments, the protein nanopore is MspA or CsgG.

[0009] In some embodiments, the target polypeptide is tethered or immobilized to the nanopore or to the membrane via a C -terminus of the target polypeptide or an N-terminusof the target polypeptide; optionally, wherein the target polypeptide is tethered or immobilized to the nanopore or to the membrane via the C-terminus of the target polypeptide. In some embodiments, the tether comprises a first cleavable linker or the immobilization is reversible. In some embodiments, the Edmanase is tethered or immobilized to the membrane or to the nanopore via a second cleavable linker or the immobilization is reversible. In some embodiments, the first or second cleavable linker comprises a disulfide bond. In some embodiments, the tether comprises an oligonucleotide.

[0010] In some embodiments, the tether has a length greater than a length of a pore of the nanopore through the membrane.

[0011] In some embodiments, a type of residue of the target polypeptide is specifically labelled with a reporter moiety. In some embodiments, at least two, three, four, five, six, or seven different types of residues of the target polypeptide are specifically labeled. In some embodiments, the type of residue is selected from cysteine, lysine, methionine, tryptophan, arginine, tyrosine, serine, or threonine; optionally, wherein the type of residue is selected from cysteine, lysine, or methionine.

[0012] In some embodiments, the target polypeptide has a length greater than 5, 10, 15, 20, 25, 30, 50, 100, 200, 500, 5000, 30,000 consecutive amino acid residues. In some embodiments, the target polypeptide has a length in a range from 5 to 1000, 5 to 500, 5 to 200, or 5 to 100 consecutive amino acid residues.

[0013] In some embodiments, the target polypeptide is linearized.

[0014] Some embodiments also include a source for applying potential difference over the membrane. In some embodiments, the source is capable of modulating and / or reversing the potential difference over the membrane.

[0015] Some embodiments also include a detector for measuring a signal generated while the target polypeptide is within the nanopore. In some embodiments, the detector is capable of measuring a signal generated while the target polypeptide is translocated through the nanopore.

[0016] In some embodiments, the detector is capable of measuring a signal indicative of the identity of a terminal residue at the non-tethered terminal end of the target polypeptide while the terminal residue is located within the nanopore; optionally, wherein the terminal residue is located within a read region within the nanopore.

[0017] Some embodiments of the compositions and methods provided herein include a method of characterizing a target polypeptide, comprising: (a) obtaining any one of the systems provided herein; (b) translocating a non-tethered end of the target polypeptide through the nanopore to the trans surface; (c) measuring a signal generated by the non-tethered end of the target polypeptide located in the nanopore; and (d) removing the terminal residue from the target polypeptide. Some embodiments also include (e) measuring a signal generated by the removing. Some embodiments also include repeating step (b) to step (d) Some embodiments also include repeating steps (b) to step (e). In some embodiments, step (b) comprises applying over the membrane: (i) a potential difference, and / or (ii) an electroosmotic force. In some embodiments, step (c) comprises measuring a signal generated by a terminal residue of the non-tethered end of the target polypeptide located in a read region of the nanopore. In some embodiments, step (c) further comprises cycling the non-tethered end of the target polypeptide located in the nanopore in a repeated movement towards the trans surface and to the cis surface. In some embodiments, step (c) comprises measuring a repeated signal. In some embodiments, step (d) comprises contacting the non-tethered end of the target polypeptide with an Edmanase. In some embodiments, step (d) comprises translocating the non-tethered end of the target polypeptide to the cis surface. In some embodiments, step (e) comprises measuring a signal indicative of the absence of a residue of the target polypeptide in the read region of the nanopore. In some embodiments, step (e) comprises measuring a signal indicative of the absence of the target polypeptide in the nanopore. Some embodiments also include determining the identity of one or more amino acid residues of the target polypeptide based on the signal obtained in step (c).

[0018] In some embodiments, step (a) comprises obtaining any one of the systems provided herein.

[0019] In some embodiments, step (a) further comprises contacting the non-tethered end of the target polypeptide with the reactive moiety such that the blocker attaches to the non-tethered end of the target polypeptide via the linker and the reactive moiety.

[0020] In some embodiments, step (a) further comprises contacting the non- tethered end of the target polypeptide with the leader composition such that the lock oligonucleotide attaches to the non-tethered end of the target polypeptide via the reactivemoiety. In some embodiments, step (b) comprises threading the lock oligonucleotide through the nanopore to the trans surface.

[0021] Some embodiments of the compositions and methods provided herein include a method for preparing a system for characterizing a target polypeptide, comprising: (a) obtaining a nanopore embedded in a membrane, wherein the membrane has a cis and trans surface; (b) tethering or immobilizing a target polypeptide to the nanopore or to the membrane via a tether at the cis surface; and (d) contacting the trans surface with a trans solution comprising an Edmanase.

[0022] In some embodiments, the trans solution further comprises a blocker attached via a linker to a reactive moiety capable of coupling to a terminal residue of the target polypeptide. In some embodiments, the reactive moiety is an isothiocyanate moiety. In some embodiments, the reactive moiety is a phenyl isothiocyanate (PITC) moiety. In some embodiments, the blocker comprises a branched oligopeptide or a nanoparticle. In some embodiments, the linker comprises a polyethylene glycol (PEG), a 2-6 benzobisoxazole, or a bifunctional oligothiophene.

[0023] Some embodiments also include (e) contacting the cis surface with a cis solution comprising a leader composition comprising a reactive moiety capable of coupling to a terminal residue of the target polypeptide, wherein the reactive moiety is attached to a lock oligonucleotide capable of forming a hairpin structure. In some embodiments, the reactive moiety is an isothiocyanate moiety. In some embodiments, the reactive moiety is a phenyl isothiocyanate (PITC) moiety. In some embodiments, the reactive moiety is attached to the lock oligonucleotide via a spacer. In some embodiments, the spacer comprises a first affinity moiety, and the Edmanase comprises a second affinity moiety, wherein the first and second affinity moieties are capable of binding to one another. In some embodiments, the binding is specific binding; In some embodiments, the first and second affinity moieties are selected from biotin, a biotin derivative, streptavidin, a streptavidin derivative, tetrazine, or bicyclononyne (BCN). In some embodiments, the spacer comprises (i) a polynucleotide, optionally, wherein the polynucleotide has a length in a range from 1 nucleotide to 50 nucleotides, optionally, in a range from 3 nucleotides to 10 nucleotides; (ii) a homopolymer; (iii) a polyethylene glycol (PEG); or (iv) a polypeptide spacer, optionally having a length less than 10 consecutive residues, and not less than 1 residue. In some embodiments, the spacer has the nucleotidesequence (TTT). In some embodiments, the lock oligonucleotide is capable of hybridizing to an inhibitor oligonucleotide which inhibits formation of the hairpin structure. In some embodiments, the lock oligonucleotide is hybridized to the inhibitor oligonucleotide.

[0024] In some embodiments, the membrane comprises a lipid bilayer or a block copolymer.

[0025] In some embodiments, the nanopore comprises a protein nanopore. In some embodiments, the protein nanopore is selected from OmpF, OmpG, CsgG, MspA, a-HL, FhuA, AeL, FraC, Lys, φ29p, ClyA, Ply AB, In some embodiments, the protein nanopore is MspA or CsgG.

[0026] In some embodiments, step (b) further comprises obtaining the target polypeptide.

[0027] In some embodiments, step (b) comprises linearizing a native polypeptide. In some embodiments, the linearizing comprises treating the native polypeptide with (i) a denaturation agent, optionally wherein the denaturation agent is selected from sodium dodecyl sulfate (SDS), or guanidinium HCl, or (li) an agent to acetylate or succinylate amino residues of the target polypeptide.

[0028] In some embodiments, step (b) comprises specifically labelling a type of residue of the target polypeptide with a reporter moiety. In some embodiments, the reporter moiety is attached to the residue via a cleavable linker. In some embodiments, step (b) comprises specifically labelling at least two, three, four, five, six or seven different types of residues of the target polypeptide. In some embodiments, the type of residue is selected from cysteine, lysine, methionine, tryptophan, arginine, tyrosine, serine, or threonine. In some embodiments, the type of residue is selected from cysteine, lysine, or methionine.

[0029] In some embodiments, step (b) comprises tethering or immobilizing the target polypeptide to the nanopore or to the membrane via a C -terminus of the target polypeptide or an N -terminus of the target polypeptide. In some embodiments, step (b) comprises tethering or immobilizing the target polypeptide to the nanopore or to the membrane via the C-terminus of the target polypeptide. In some embodiments, step (d) comprises tethering or immobilizing the Edmanase to the nanopore or to the membrane. In some embodiments, the tethering or immobilizing is reversible. In some embodiments, the tether comprises a cleavable linker or the immobilization is reversible. In some embodiments, thecleavable linker comprises a disulfide bond. In some embodiments, the tether comprises an oligonucleotide. In some embodiments, the tether has a length greater than a length of a pore of the nanopore through the membrane.

[0030] In some embodiments, the target polypeptide has a length greater than 5, 10, 15, 20, 25, 30, 50, 100, 200, 500, 5000, 30,000 consecutive amino acid residues; optionally, wherein the target polypeptide has a length in a range from 5 to 1000, 5 to 500, 5 to 200, or 5 to 100 consecutive amino acid residues.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 depicts a schematic of an embodiment of a method for nanopore sequencing of a polypeptide,

[0032] FIG. 2 depicts a schematic of various protein nanopores having different pore diameters. In some embodiments, a pore may be selected such that a pore diameter decreases a contribution to a detectable signal from the amino acids, while emphasizing the signal from labels on reactive groups.

[0033] FIG. 3 depicts an embodiment of a blocker attached to a PITC moiety. The right side is a schematic with a blocker (larger filled oval) linked to the PITC moiety (smaller filled oval); and the left side depicts an example chemical structure corresponding to the same. Such embodiments can prevent pre-mature degradation of peptides in solution.

[0034] FIG. 4 depicts an overview of the Edman degradation cycle in which a free N-terminal residue of a polypeptide reacts with phenyl isothiocyanate (PITC) to form a phenylthiocarbamyl (PTC) intermediate, the PTC intermediate is removed from the polypeptide via an Edmanase-catalyzed reaction to expose a new N-terminal residue of the polypeptide (NHz-peptide).

[0035] FIG. 5 depicts a schematic including a MspA nanopore within a lipid bilayer. A polypeptide having residues (open circles) is attached at one end to the nanopore via a first linker; a blocker (black oval) is attached via a second linker to the other end of the polypeptide. The MspA nanopore includes a read region (box with broken lines) in which the blocker and its linker contribute a constant signal, and terminal amino acid residues of the polypeptide contribute variable signals.

[0036] FIG. 6A depicts an example embodiment of a labeling reagent to specifically label a N-terminus of a polypeptide.

[0037] FIG. 6B depicts a schematic of polypeptides attached to a labeling reagent in which the labeling reagent lacks a spacer (upper portion of FIG. 6B), or includes a spacer (lower portion of FIG. 6B)

[0038] FIG. 7 depicts an example workflow for a method of sequencing a target polypeptide.

[0039] FIG. 8A depicts a schematic of an embodiment in which an Edmanase is tethered or immobilized to the membrane.

[0040] FIG. 8B depicts a schematic of an embodiment of a workflow in which the spacer of the leader oligonucleotide or lock can include a first affinity moiety, and the Edmanase can include a second affinity moiety (left portion); the first and second affinity moieties bind one another (middle portion); and the Edmanase causes cleavage of the terminal residue of the target polypeptide (right portion).

[0041] FIG. 9 depicts an example reaction scheme comparing conventional Edman degradation with a DNA-compatible BF3. Et2O -mediated protocol.

[0042] FIG. 10 depicts an example mechanism of Edmanase-assisted Edman degradation.

[0043] FIG. 11 depicts an example scheme for enhancement of amino acid cleavage efficiency using a bifunctional PITC reagent and a chimeric Edmanase. Diagrams (A) and (B) depict a peptide attached to a solid-phase substrate is modified with a bifunctional NTAA modifier, such as biotin-phenyl isothiocyanate (PITC). Diagram (C) depicts a low affinity Edmanase (> pMKd) is recruited to biotin-PITC labeled NTAAs using a streptavidin-Edmanase chimeric protein. Diagram (D) depicts the efficiency of Edmanase cleavage is greatly improved due to the increase in effective local concentration as a result of the biotin-streptavidin interaction. Diagram (E) depicts the cleaved biotin-PITC labeled NTAA and associated streptavidin-Edmanase chimeric protein diffuse away after cleavage. A number of other bioconjugation recruitment strategies can also be employed. An azide modified PITC is commercially available (4- Azidophenyl isothiocyanate, Sigma), allowing a number of simple transformations of azide-PITC into other bioconjugates of PITC, such as biotin-PITC via a click chemistry reaction with alkyne-biotin.

[0044] FIG. 12A depicts an example of conjugation between a target polypeptide and a nanopore.

[0045] FIG. 12B depicts examples for types of reversible conjugation chemistries.

[0046] FIG. 13A depicts a schematic for conjugating a target polypeptide via an oligonucleotide to a nanopore.

[0047] FIG. 13B depicts example nucleotide modifications.

[0048] FIG. 14 depicts a schematic in which a nanopore includes a mutation to further enhance efficiency of conjugation between the nanopore and a target polypeptide.

[0049] FIG, 15 depicts a schematic in which an oligonucleotide lock includes a fixed length spacer.

[0050] FIG, 16 depicts example nanopore adapter-substrate pairings such as reversible interactions between boronic acids with 1,2-diols, amines or carboxylic acids,

[0051] FIG, 17 depicts a schematic for an embodiment of de novo sequencing via a hybrid on-pore Edman degradation and direct amino acid sensing.DETAILED DESCRIPTION

[0052] Some embodiments of the methods and compositions provided herein relate to nanopore sequencing of a target polypeptide. In some embodiments, a nanopore is embedded in a membrane having a cis and trans surface. Some embodiments include translocating an end of the target polypeptide through the nanopore, measuring a signal generated from the target polypeptide in the nanopore, and removing a terminal residue of the target polypeptide from the target polypeptide with an Edmanase enzyme. In some embodiments, a trans solution comprises a blocker capable of attaching to the N-terminal end of the target polypeptide. In some embodiments, a cis solution comprises a leader composition attached to a lock oligonucleotide capable of forming a hairpin structure.

[0053] Nanopore biosensing is based on naturally occurring protein pores. In a typical experiment, the pores are embedded in a lipid bilayer, which separates two chambers, cis and trans, filled w’ith an electrolyte solution. An applied voltage causes ions to move through the pore and create an electrical field. An analyte can be captured and transported across the pore by different mechanisms. Chinappi, M. et al., “Analytical model for particle capture in nanopores elucidates competition among electrophoresis, electroosmosis, anddielectrophoresis.” ACS Nano 2020, 14, 15816-15828. For charged analytes, electrophoresis may be the dominant form of transport, carrying the analyte toward the electrode of opposite polarity. Carson, S. et al., “Challenges in DNA motion control and sequence readout using nanopore devices.” Nanotechnology 2015, 26, 074004. For neutral or less-charged molecules, electroosmotic flow may be the dominant force directing capture and / or transport. Piguet, F. et al., “Electroosmosis through a- hemolysin that depends on alkali cation type. ” J. Phys. Chem. Lett. 2014, 5, 4362-4367; Gu, L. Q. et al., “Electroosmotic enhancement of the binding of a neutral molecule to a transmembrane pore.” Proc. Natl. Acad. Sci, USA 2003, 100, 15498- 15503, Furthermore, an analyte of appropriate size can enter the pore and alter the ionic current,

[0054] An analyte may change the ionic current by (i) producing a change in the electric field within the pore or through (ii) volume exclusion and binding of ions to the traversing analyte, which reduces the ionic current. Bezrukov, S. M. et al., “Current noise reveals protonation kinetics and number of ionizable sites in an open protein ion channel.” Phys. Rev, Lett. 1993, 70, 2352-2355; Remer, J. E, et al., “Theory for polymer analysis using nanopore-based single-molecule mass spectrometry.” Proc. Natl. Acad. Sci. USA 2010, 107, 12080-12085. Importantly, because numerous ions accompany the passage of a single analyte, a large electrical amplification occurs during a single-molecule translocation. Gurnev, P. A. et al., “Channel-forming bacterial toxins in biosensing and macromolecule delivery.” Toxins 2014, 6, 2483-2540. In a simplified scenario, the duration and amplitude of the current alteration is unique to each analyte or monomeric unit, in the case of polymer analytes. Nanopore-based sensing can employ both naturally occurring, protein pores and manufactured, solid-state nanopores. Xue, L. et al., “Solid-state nanopore sensors.” Nat. Rev. Mater. 2020, 5, 931-951. Biological nanopores, although not as robust as solid-state nanopores, offer better reproducibility due to their defined channel sizes. Wang, S. et al.. Engineering of protein nanopores for sequencing, chemical or protein sensing and disease diagnosis. Curr. Opin. Biotechnol. 2018, 51, 80-89. In addition, lipid bilayers exhibit much lower electrical noise than the materials used to manufacture solid-state nanopores. Fragasso, A. etal., “Comparing current noise in biological and solid-state nanopores.” ACS Nano 2020, 14, 1338-1349.

[0055] For sensing purposes, nanopore technology generally exploits two distinct concepts: (i) direct sensing, in which changes in the current arise as the analyte traverses and directly interacts with the nanopore; and (ii) indirect sensing, in which translocation of anadapter molecule that specifically interacts with the analyte is monitored). Ayub, M. et al., “Engineered transmembrane pores.” Curr. Opin. Chem. Biol. 2016, 34, 117-126; Reynaud, L. et al., “Sensing with Nanopores and Aptamers: A Way Forward.” Sensor 2020, 20, 4495.

[0056] As used herein “peptide” and “polypeptide” can be used interchangeably and include a polymer comprising more than one amino acid residues linked via a peptide bond.

[0057] Next generation sequencing has revolutionized genomics but genetic information alone cannot predict protein abundance, post translational modifications or protein-driven biological processes. Proteomics on the other hand offers a direct insight into protein function, regulation and interactions. Combining proteomics with genomics, transcriptomics and metabolomics will bridge the genotype-phenotype gap and shed more information on novel biomarkers for disease diagnosis and precision medicine.

[0058] Mass spectroscopy has traditi onally been the workhorse for high throughput proteomics profiling. The process begins with the digestion of protein samples followed by the introduction of peptide fragments to a mass spectrometer with quadrupole selectivity and high resolution accurate mass capability. Bottom-up proteomics identifies proteins by the sum of their parts but since the connectivity between peptides is lost, single peptides may be mapped to multiple entries in the protein database. This is especially prevalent with databases of higher eukaryotes due to the presence of related protein family members, alternative splice forms and partial sequences.

[0059] Nanopore-mediated protein sequencing enables the direct, real-time analysis of proteins at the single molecule level. This approach utilizes nanoscale pores to thread individual proteins and detect ionic current changes that occur as each amino acid passes. The protein sequence, including post-translational modifications, are then reconstructed from the unique current signatures generated. Given multiple amino acids reside in the read head concurrently, complex data analysis and nanopore signal interpretation are typically associated with direct strand sequencing. None of the currently available biological nanopores are sensitive enough to differentiate between amino acids with similar steric volumes. In addition, unlike DNA, protein backbones are neutral and amino acid side chains are not uniformly charged, making translocation of proteins in a unidirectional and controlled manner difficult.

[0060] Edman degradation is a chemical reaction that enables the release of N-terminal amino acids, one residue at a time, from a protein / peptide. First described in 1950, the 3 -step process begins with the selective modification of the N-terminal amine with phenyl isothiocyanate (PITC) in aqueous pyridine (FIG. 4). In the second step, the phenylthiocarbamoyl (PTC) intermediate is treated with neat trifluoroacetic acid. This yields a cyclic 2-anilino-5(4)-thiozolinone (ATZ)-modified amino acid and a truncated protein / peptide intermediate that is available for derivisation in a subsequent Edman reaction cycle. In the final step, the cleaved amino acid rearranges into a more stable phenylthiohydantoin (PTH) form in the presence of 20% aqueous TFA. This entire process is repeated in a cyclical manner until all or a defined portion of the protein / peptide sequence has been identifi ed.

[0061] Since conventional Edman degradation employs harsh acidic conditions to initiate the cleavage step, applications involving DNA may be at risk of depurination. To obviate this, dA and dG may be substituted with 7-deazapunne deoxy nucleotides (c7dA and c7dG) and N-terminal PITC-modified amino acids cleaved using boron trifluoride etherate as a milder alternative (FIG. 9). ATZ ammo acids are subsequently transformed to the more stable PTC amino acids under alkaline reducing conditions. Mild cleavage may also be achieved usinga 1:1 mixture of TEAA:acetonitrile at 75°C for 10 min (See e.g., Tetrahedron Lett. 1985, 26, 4375).

[0062] Alternatively, Edmanases may be engineered to catalyse the cleavage step in aqueous buffer at neutral pH. Edmanase is an engineered cysteine protease with a C25G active site mutation that selectively acts on PITC-modified peptides (See e.g., Protein Sci.2015, 24, 571). The reported catalytic efficiency, however, varies from amino acid to ammo acid, with proline cleavage being the least efficient. An example mechanism of Edmanase-assisted Edman degradation and the associated catalytic efficiency for various amino acid substrates is depicted in FIG. 10. Example kinetic parameters of Edmanase for PTC-Xaa substrates are listed in TABLE 1.TABLE 1Substrate kcat (s1) KM (pM) kcat / Kvi (s1M1) PTC-Ala-AMC 0.55 (±0.013) 21.3 (±2.7) 2.6 x 104PTC-Asp-AMC 3.6 (±0.41) 124.5 (±35.0) 2.9 x 104Substrate kcat (S 1) KM (jiM) kcat / Kvi (s’1M’1)PTC-Phe-AMC 0.47 (±0.060) 122.8 (±29.8) 3.8 x 103PTC-Met-AMC 0.54 (±0.083) 271.8 (±67.6) 2.0 x 103PTC-Pro-AMC 0.0014 (±0.0011) | 252.0 (±184.1) 5.7 x 1011PTC-Arg-AMC 0.087 (±0.017) 167.8 (±43.8) 5.2 x 102

[0063] To enhance catalytic efficiency, peptides may be modified with a bifunctional biotin-PITC label followed by cleavage with a streptavidin-Edmanase chimeric protein. FIG. 11 depicts a schematic for enhancement of amino acid cleavage efficiency using a bifunctional PITC reagent and a chimeric Edmanase (See e.g., WO 2019089851 Al; US 20210355483 ). This effectively increases the local concentration as well as the affinity of the enzyme.Edman degradation based membrane nanopore peptide sequencing

[0064] Some embodiments provided herein include a technique for interrogating a protein sequence at the single molecule level using a membrane nanopore setup. By leveraging the cis / trans separation through the presence of the membrane, isolated reactions are implemented to control molecular movement at the level of a nanopore, to allow for accurate reading of discreet peptide residues.

[0065] When a molecule is docked within a membrane nanopore, creating a blockade m the nanopore, and a voltage bias is applied across the membrane, a current signal characteristic to the blockade is produced. This feature can be exploited for interrogating proteins by reading discrete sequences of very short peptides within the constriction region of the nanopore. For example, in an analogous system in which single-stranded DNA is positioned in a MspA pore, the signal generated will mainly be a result of about 4 to 5 nucleobases that are present in the constriction region. By having a linearized peptide docked within the nanopore, the signal generated will be specific to the residues that occupy the constriction.

[0066] If the peptide can be docked by one terminal end in a way that only a few amino acids contribute to the signal, and the sequence being read can be processed one residue at a time, a signal map can be generated that describes the sequence and composition of thepeptide. Within the framework of a membrane nanopore, an embodiment is depicted in FIG. 1 and includes:

[0067] (1) A membrane nanopore system is obtained which includes a MspA protein nanopore embedded in a membrane, such as a lipid bilayer having cis and trans surfaces. A linearized polypeptide with residues (open circles) and N-terminal residues (filled circles) is attached via its C-terminal end to the nanopore exposed at the cis surface. The trans solution contains an “Edmanase” enzyme, and a PITC-blocker which is a blocker (larger filled oval) linked to a phenyl isothiocyanate (PITC) moiety7(smaller filled oval).

[0068] (2) The N-terminal end of the polypeptide is threaded or translocated through the nanopore, and the PITC-blocker reacts with the N-terminus of the polypeptide, forming a covalent PITC-peptide adduct.

[0069] (3) Cycling or flossing the polypeptide back and forth through the nanopore, brings the PITC blocker and the N-termmal amino acid residues into a constriction (read zone / read region) of the nanopore to generate a repeating specific signal which is detected.

[0070] (4) Edamase cleaves the PITC-peptide adduct from the remaining portion of the polypeptide.

[0071] (5) The cycle is repeated.

[0072] Some embodiments include attaching the polypeptide to the nanopore. Single attachment points on nanopores have previously been demonstrated and can be obtained by7having one mutated monomer of the octameric MspA nanopore. See e.g., Wang, K., et al. “LTnambiguous discrimination of all 20 proteinogenic amino acids and their modifications by nanopore.” Nat Methods 21, 92-101 (2024). To be able to process several strands with the same nanopore, the attachment point can be reusable. Attaching a short oligonucleotide strand to the nanopore, and a complementary sequence on the C-terminal of the peptide can facilitate peptide capture, and release through stripping via high voltage bias.

[0073] Some embodiments include threading or translocating the polypeptide through the nanopore. To have better control over the peptide strand using voltage bias, the peptides can be acetylated or succmylated on the ammo residues, turning the peptides into heavily negatively charged strands that become more responsive to positive voltage bias. Another option is to pre-coat the protein with SDS detergent during library’ prep, and thenremove the excess detergent before introduction to the nanopore, otherwise the detergent will likely disrupt the membrane. SDS will impart a negative charge along the protein backbone, while also denaturing the protein so as to avoid a secondary structure that could impede flossing. This allows for the driving of the strands towards the nanopore and gives a better responsiveness to voltage during flossing. Another option is to use electroosmosis to translocate the protein through the pore. The potential advantage of electroosmosis over electrophoresis is that one may not need to coat the protein with a negative charge that must overcome the inherent amino acid charges. One method uses guanidinium HC1 to unfold the protein and permit electroosmosis through an alpha hemolysin pore, while the other modified the CytA pore internal charges to enhance electroosmosis. See e g., Yu L, et al., “Unidirectional single-file transport of full-length proteins through a nanopore.” Nat Biotechnol, 2023 Aug;41(8):l 130-1139; Erratum in: Nat Biotechnol. 2023 Oct;41(10): 1483; and Sauciuc, A., et al. “Translocation of linearized full-length proteins through an engineered nanopore under opposing electrophoretic force.” Nat Biotechnol 42, 1275-1281 (2024).

[0074] Some embodiments include attaching labels, such as reporter moieties, to specific amino acid residues of the polypeptide. In some embodiments, custom design groups can be attached onto amino residues to allow for the generation of specific signals when these residues are in the read zone. Indeed, since an amino acid occupies about 50% of the space in the nanopore read head compared to DN / X, it is conceivable that 10 or more amino acids contribute to the nanopore signal, although as highlighted below the PTC linker should decrease this somewhat. Since there are 20 ammo acids (not including those that have post-translational modifications, PTMs), there are too many possible signals (2010) to unambiguously determine the specific amino acids that contribute to a signal. Therefore, it would be advantageous, and perhaps preferred, to modify the protein during library prep so as to attach ammo-acid specific labels to certain reactive amino acid side chains, for example Cys, Lys and other residues.

[0075] A nanopore of suitable dimensions, such as mutated MspA or ClyA, that has a ~3.3 nm constriction vs the 1.2 nm constriction of M2 MspA or CsgG, can be selected such that the signal is mainly determined not by the amino acids, but rather by the label on the reactive amino acids. Examples of nanopores with various sized constrictions are depicted in FIG. 2. See e.g., Crnkovic A, et al.,. “Biological Nanopores: Engineering on Demand.” Life.2021; 11(1):27. A nanopore can have a read head that is larger than that which would be suitable for DNA or for reading amino acids directly.

[0076] A potential issue with reading labels attached to amino acid residues in a linear polypeptide is that labels which are very close together, such as those attached to adjacent amino acid residues in a linear polypeptide, may be difficult to distinguish from one another, especially in polypeptides that include homopolymer sequences. However, embodiments of the step-by-step degradation provided herein can include obtaining a unique fingerprint because there is potentially a “tick mark” between each amino acid read.

[0077] As depicted in an example embodiment in FIG, 3, the design of the blocker can include three functional components: (1) a negatively charged bulky component that acts as an arresting construct (right portion of figure, larger filled oval); (2) a PITC headgroup (right portion of figure, smaller filled oval); and (3) a linker between the blocker and the PITC. Blockers or ‘arresting constructs’ can be designed bearing branched oligopeptides that can be used as brakes for Ratchet strands. Alternatively, rigid arresting constructs, like nanoparticles, would be an attractive option as they reduce the flexibility of the construct, limiting the variability in signal contribution from the blocker. Polyethylene glycols of varying lengths can be used as linkers. Alternatively, more rigid linkers such as 2-6 benzobisoxazoles or bifunctional oligothiophenes, can be used to limit signal contribution variation. The PITC reactive head would be the covalently attached analogue of the conventional reagent used within the Edman degradation process.

[0078] A standard Edman degradation protocol involves the reaction of a free amino on an N-terminal residue with PITC under mild basic conditions to form a phenylthiocarbamyl (PTC) intermediate. This intermediate is then removed under acidolysis, freeing up the anime on the next N-terminal residue (FIG. 4). However, an Edmanase enzymatic catalyst can process the PTC intermediate without the need to change the pH of the solution to acidic. See e g, Borgo B, Havranek JJ. “Computer-aided design of a catalyst for Edman degradation utilizing substrate-assisted catalysis.” Protein Sci. 2015 Apr;24(4): 571-9. This allows for the Edman degradation to take place within mild buffer conditions that allow the process to be used in the context of a protein nanopore system.

[0079] While the PTC blocker intermediate is on the peptide strands, precise positioning of the final few amino acid residues within the read zone (read region) of thenanopore can be achieved when the polypeptide is cycled or flossed back and forth (moved cis and trans) through the nanopore by reversing an applied voltage bias. The flossing of the strand from cis to trans allows for consensus reads to be achieved on the same residues. Since the degradation biochemistry is sequestered from the N-terminal polypeptide end during the read step, the read can be performed for as long as desired to obtain a satisfactory signal to noise (S: N) ratio, for example a signal sufficient to identify a reporter moiety. Since the arresting construct is enzymatically removed, there is no need for a voltage pulse to move the PTC arresting construct through the pore. Eventually, the Edmanase will release the blocker together with the N-terminal residue, allowing for the cycle to restart.

[0080] It is advantageous to know when the Edmanase reaction has occurred because successive k-mers that give the same or highly similar signals would be difficult to distinguish. Since addition of the PTC linker is not instantaneous, when the current is read upon reversing the voltage, in the absence of the PTC blocker, an open pore current will be read. This open pore current that follows a blocked current is readily distinguishable, and can serve as a “tick mark” indicating that the Edmanase has removed an ammo acid.

[0081] While normally the read region would capture 8-10 amino acids, the PTC linker will take up a significant proportion of this. If the length of the linker is adjusted accordingly, the signal contribution can be set to include only 1-3 amino acid residues. (FIG.5) This significantly reduces the complexity of the signal map that will be obtained when interrogating all the ammo acid combinations. Since the Edmanase will process the peptide strand one residue at a time, the signal generated from consecutive reads will be correlated, allowing for a De Bruijn map to be generated for higher signal calling accuracy.

[0082] The speed or the sequencing will depend mainly on the rate of the enzymatic hydrolysis. Rates for different residues are expected to change, however mutations on the Edmanase enzyme can be directed so that specificity is directed by the PTC-linker adduct, reducing the binding rates dependent on individual amino acid residues.

[0083] In some embodiments, a portion of the polypeptide may not be sequenced because there can be an inaccessible length that is determined by the distance from the cB-side attachment point to the solvent-accessible trans side of the pore. This length of peptide may not be accessible to the trans side chemistry. This length is expected to be on the order of 30 amino acids for MspA.On pore Edman degradation and sequencing

[0084] Some embodiments of the compositions and methods provided herein include a hybrid Edman degradation nanopore approach to enable de novo protein sequencing. Briefly, the N-terminal amino acid of an immobilised protein of interest is labeled with a charged species. The modified amino acid residue is threaded through the pore and identified based on specific changes in the ionic current readings. The analyzed terminal amino acid is then cleaved under mild chemical or enzymatic conditions and the entire process is repeated in a cyclic manner. In this set up, post-translational modifications are detectable with single ammo acid accuracy.

[0085] For example, some such embodiments include obtaining a nanopore embedded in a membrane, in which the membrane has a cis and a trans surface. In some embodiments, a target polypeptide is immobilized on a nanopore or on a membrane. In some embodiments, the C-terminal terminus of the target polypeptide is immobilized on the nanopore at the cis surface. The target polypeptide can be immobilized via a tether. The tether can be negatively charged. The tether can have a length greater than the length of the pore of the nanopore through the membrane.

[0086] In some embodiments, the N-terminus of the target polypeptide can be labeled with a labeling reagent comprising a PITC moiety linked to an oligonucleotide capable of forming a hairpin structure via a spacer. An example embodiment of a labeling regent is depicted in FIG. 6A. The labeling reagent includes a PITC moiety capable of specifically attaching to an N-terminus of a target polypeptide, a ‘TIT spacer, a self-lock oligonucleotide capable of forming a hairpin structure, and an inhibitor oligonucleotide hybridized to a portion of the self-lock oligonucleotide. The spacer can include a nucleotide sequence, such as ‘TFT’. FIG. 6B depicts a function of the spacer in which the spacer reduces K-mer complexity such that a read region of a nanopore detects the spacer and a terminal residue. For example, a target polypeptide labeled with a label lacking a spacer, the portion of the target polypeptide which can be located in the read region of a nanopore can include about 10 residues. In such a case, more than 2O10(about 20 different types of amino acids) signals would need to be convoluted. However, when using a target polypeptide labeled with a label including the spacer, the portionof the target polypeptide which can be located in the read region of a nanopore can just be a terminal residue. In such a case, about 20 signals can be convoluted to identify the residue.

[0087] In some embodiments, a protein sequencing method includes: (1) immobilization of a target polypeptide; (2) selective labeling of the N-terminal amine with an Edman reagent covalently attached to a self-lock; (3) insertion of the oligo-peptide conjugate into the pore and amino acid identification; (4) cleavage of the N-terminal modified amino acid; and (5) ejection of the peptide sequence back to the cis well / surface. An example embodiment of de novo sequencing via a hybrid on-pore Edman degradation assay is depicted in FIG. 7 in a series of five steps. In step (1), a target polypeptide is immobilized on a nanopore, such as an Msp nanopore, embedded in a lipid bilayer. In step (2) the N-terminus of the target polypeptide is selectively labeled with an Edman reagent linked to a self-lock moiety. The self-lock moiety includes an oligonucleotide capable of forming a hairpin structure. The oligonucleotide is hybridized to an inhibitor oligonucleotide which inhibits formation of the hairpin structure. The Edman reagent includes a fixed length ‘TTT’ spacer to reduce K-mer complexity. The Edman reagent is attached to the target polypeptide via a PITC moiety. In step (3), the negatively charged self-lock moiety facilitates the threading of the non-uniformly charged target polypeptide through the pore. Stripping of the inhibitor oligonucleotide triggers the formation of the hairpin structure. The nanopore records the signal derived from the fixed length spacer and the terminal residue of the target polypeptide. In step (4) the terminal residue and self-lock moiety are cleaved from the target polypeptide by an Edmanase present in a solution contacting the trans surface. In step (5), the target polypeptide is ejected from the nanopore and step (2) to step (5) are repeated.

[0088] (1) Immobilization. The C-terminal end of the target polypeptide is modified with an appropriate conjugation handle followed by protein immobilization directly to a nanopore or to the membrane adjacent to the pore opening. Alternatively, the target polypeptide is digested into shorter peptide fragments using LysK (or other appropriate proteases) followed by the chemoselective tagging of the side chain with a conjugation handle before immobilization.

[0089] An example embodiment is depicted in FIG. 12A in which the nanopore or the membrane may be modified with ‘conjugation handle A’ and the target polypeptide ‘conjugation handle B’. Depending on the identity of the reactive handles, the conjugation maybe irreversible or reversible. Reversible atachment of the analyte would allow pore recycling, hence increasing the throughput of the nanopore assay. Examples of reversible conjugation chemistries useful as conjugation handles are depicted in FIG. 12B. Examples of covalent conjugation chemistries useful as conjugation handles include: amine-NHS ester, amine-imidoester, amine-pentofluorophenyl ester, amine-hydroxymethyl phosphine, carboxyl¬ carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyi disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azide¬ alkyne, azide-phosphine, transcyclooctene-tetrazine, norborene-tetrazine, azide-cyclooctyne, azide-norborene. Examples of haptens and binding partners useful as conjugation handles include: biotin-avidin; halotag-alkyl chloride; SNAP-tag-benzyl guanine derivatives; CLIP-tag-benzylcytosine derivatives; and SpyTag-SpyCatcher system (See e.g., Zaken, B,, et al., (2012) PNAS 109 (12) E690-E697, The tether connecting the analyte to the pore or the membrane can be negatively charged and longer than the length of the nanopore to ensure the amino acid closest to the C-terminus can also be suspended in the read head of the nanopore for analysis.

[0090] Oligo hybridization approaches may also be considered to immobilize the analyte to the pore or the membrane. (FIG. 13 A). The oligo can be longer than the length of the nanopore and the Tmof the duplex strong enough to withstand repeated Edman degradation cycles whilst still be ejectable. Nucleotide modifications can be used to tune the strength of peptide-oligo capture (FIG. 13B). Alternatively, noncanonical amino acids, such as click handles such as TCO, BCN, azide, tetrazine, may be expressed on the pore to further enhance the efficiency of the peptide-oligo capture step (FIG. 14).

[0091] (2) Chemoselective end labeling, and (3) nanopore insertion and sequencing. The N-terminal amine reacts exclusively with an Edman reagent (PITC) linked to a self-lock / blocking oligo complex under slightly basic conditions (pH 8). Since peptides and proteins are non-uniformly charged, attachment of a negatively charged oligo facilitates the threading of the target polypeptide strand through the pore. Stripping of the blocking oligo then triggers the formation of a hairpin lock in the trans well, which positions the peptide k-mer in the read head. The MspA read head is roughly 2 nm in length and can accommodate 4-5 nucleotides or 5-6 amino acids in the constriction zone (See e.g., Yan, S., et al.. Nano. Lett 2021, 21, 6703). A single peptide measurement via strand sequencing thus corresponds to >205possible k-mers. Target polypeptides that harbour post translational modifications can further increase the number of unique signal levels and deconvolution complexity. One way to tackle the problem is to introduce a fixed length spacer next to the PITC group to effectively reduce the k-mer size (FIG. 15). Examples of a fixed length spacer include homooligomeric polyethylene glycol (PEG), amino acid units, or nucleotide units.

[0092] (4) Edmanase cleavage, and (5) ejection of the target polypeptide strand. The oligo-peptide strand is further driven into the trans well where the Edmanase resides. Protein engineering may be required to effect cleavage of the PITC moiety linked to an oligo strand. Additionally, the Edmanase can be covalently bound to the nanopore or membrane for better proximity to reactants and thus higher cleavage efficiencies. After cleavage, the voltage is reversed, the peptide strand is ejected and the current returns to the open pore level. This in and of itself acts as a tick-mark to signal the end of the current sequencing cycle and the beginning of the next labeling cycle. The steps are repeated until all or a defined portion of the protein / peptide sequence has been identified.

[0093] Some embodiments also include localizing the Edmanase to the nanopore to enhance cleavage efficiency. For example, the Edmanase can be tethered or immobilized to the membrane to increase its proximity to the nanopore and its substrate. FIG. 8A depicts an embodiment in which an Edmanase is tethered or immobilized to the membrane. In some embodiments, the spacer of the leader oligonucleotide or lock can include a first affinity moiety, and the Edmanase can include a second affinity moiety. For example, the spacer of the leader oligonucleotide or lock moiety can be modified with a biorthogonal reactive group such as biotin, BCN or tetrazine. Following ammo acid identification and translocation of the oligo-peptide strand into the trans well / solution, biotin-streptavidin or BCN-tetrazine reactions take place, effectively increasing the local concentration and affinity of the enzyme. The biorthogonal reactive group may be attached to the spacer or the hairpin lock. FIG. 8B depicts an embodiment of a workflow in which the spacer of the leader oligonucleotide or lock moiety can include a first affinity moiety, and the Edmanase can include a second affinity moiety (left portion); the first and second affinity moieties bind one another (middle portion); and the Edmanase causes cleavage of the terminal residue of the target polypeptide (right portion).

[0094] Some embodiments of the methods and compositions provided herein relate to interaction between a modified amino acid and a pore that has been engineered with an adapter. Briefly, the N-terminal amino acid of an immobilised target polypeptide is labeled with a charged species. Following cleavage, the modified amino acid residue enters the pore and forms transient interactions with the nanoreactor. Individual residues, including post- translational modifications, are then identified based on specific changes in the ionic current readings.

[0095] Whilst single amino acids have been identified using metal nanogaps via recognition tunneling (See e.g,, Nat. Nanotechnol. 2014, 9, 466; Nat. Nanotechnol. 2014, 9, 835), nanopore- assisted protein sequencing by hydrolysis is expected to be more challenging as successful analyte identification relies on the dimensions of the narrowest region of the pore. The single constriction of MspA has dimensions similar to that of a single nucleotide, making it an idea pore for DNA sequencing. Amino acids on the other hand are ~2-3 times smaller than nucleotides and may pass through the pore in multiple orientations, thus making current blockade changes harder to distinguish. To enable direct amino acid sensing m a fixed orientation, the MspA pore can first be modified with a single Ni-NTA complex as carboxylic acids and amino groups are known to react reversibly with metal ions to form transient ternary complexes. / Accordingly, the pore is assembled using one monomeric subunit containing the N90C modification and seven monomeric subunits lacking any cysteine residues (See e.g., Nat. Methods 2024, 21, 92).

[0096] FIG. 16 depicts example embodiments for nanopore adapter-substrate pairings such as reversible interactions between boronic acids with 1,2-diols, amines or carboxylic acids. The reversibility of the reaction may be further tuned by a combination of the solution pH and the pKa of the boronic acids. Similar reversible covalent interactions can be achieved using aldehydes with alcohols or amines as well as host-guest interactions between phenylalanine and cucurbit- 7-uril.

[0097] Some embodiments include methods comprising: (1) immobilisation of the target polypeptide; (2) selective labeling of the N-terminal amine with an Edman reagent covalently attached to a charged species; (3) cleavage of the N-terminal modified amino acid; and (4) chelation to nanopore adapter and identification (FIG. 17).

[0098] (1) Embodiments for immobilization are described above.

[0099] (2) Chemoselective N-terminal labeling. The N-terminal amine reacts exclusively with an Edman reagent (PITC) linked to a charged species capable of interacting with the chosen nanopore adapter. The charged group may be an arresting construct, a single amino acid, a short peptide sequence, a nucleoside mono- or triphosphate or a short oligonucleotide sequence. Examples of adapter pairings include carboxylic acids and / or amines with a Ni-NTA complex; vicinal alcohols with boronic acids; alcohols and / or amines with aldehydes; or phenylalanine with cucurbit-7-uril.

[0100] (3) Edmanase cleavage, and (4) amino acid identification. Sequential release of the N-terminal modified amino acid can be effected by enzymatic or mild chemical cleavage conditions. To ensure the cleaved amino acid enters the capture region of a nanopore, the Edmanase can be covalently bound to the nanopore. Once the negatively charged modified amino acid diffuses close to the nanopore opening, a strong electrophoretic force drives it into the read head and docks it onto the adapter in a fixed orientation, allowing identification in their intrinsic order,

[0101] For Ni-NTA type adapters, provided the non-covalent interaction between the modified ammo acid and Ni2’ is weaker than that of the tetradentate NTA and Ni2+, the chelated substrate will dissociate from the metal complex without triggering the dissociation of Ni2+from the NTA adapter. This effectively reforms open chelation sites for subsequent rounds of amino acid sensing. For reversible covalent type sensors such as boronic acid or aldehyde modified nanopores, release of the PITC-amino acid residue can be activated by a change in solution pH.Certain systems and kits

[0102] Some embodiments of the methods and compositions provided herein include a system or kit for characterizing a target polypeptide, comprising: (a) a membrane having a cis and trans surface; (b) a nanopore embedded in the membrane; (c) a target polypeptide tethered or immobilized to the nanopore or to the membrane via a tether at the cis surface; and (d) a trans solution comprising an Edmanase, wherein the trans solution is in contact with the trans surface. In some embodiments, the Edmanase is tethered or immobilized to the membrane or to the nanopore.

[0103] In some embodiments, the membrane comprises a lipid bilayer or a block co-polymer. In some embodiments, the block co-polymer comprises a deblock copolymer or a triblock copolymer. Each molecule of a block copolymer may include one or more hydrophilic blocks and one or more hydrophobic blocks. The hydrophilic blocks may form outer surfaces of the barrier and the hydrophobic blocks may be located within the barrier. The hydrophobic blocks may include a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydiene, fluormated polyethylene, polypeptide, and poly(isobutylene) (PIB). See e.g., U. S. 20230312856 which is incorporated by reference in its entirety.

[0104] In some embodiments, the nanopore comprises a protein nanopore. In some embodiments, the protein nanopore is selected from OmpF, OmpG, CsgG, MspA, a- HL, FhuA, AeL, FraC, Lys, φ29p, ClyA, Ply AB, In some embodiments, the protein nanopore is MspA or CsgG.

[0105] In some embodiments, the target polypeptide is tethered or immobilized to the nanopore or to the membrane via a C-terminus of the target polypeptide or an N-terminus of the target polypeptide. In some embodiments, the target polypeptide is tethered or immobilized to the nanopore or to the membrane via the C-terminus of the target polypeptide. In some embodiments, the tether comprises a first cleavable linker or the immobilization is reversible. In some embodiments, the Edmanase is tethered or immobilized to the membrane or to the nanopore via a second cleavable linker or the immobilization is reversible. In some embodiments, the first or second cleavable linker comprises a disulfide bond. In some embodiments, the tether comprises an oligonucleotide. In some embodiments, the tether has a length greater than the length of a pore of the nanopore through the membrane.

[0106] In some embodiments, a type of residue of the target polypeptide is specifically labelled with a reporter moiety. In some embodiments, at least two, three, four, five, six, or seven different types of residues of the target polypeptide are specifically labeled. In some embodiments, the type of residue is selected from cysteine, lysine, methionine, tryptophan, arginine, tyrosine, serine, or threonine. In some embodiments, the type of residue is selected from cysteine, lysine, or methionine.

[0107] In some embodiments, the target polypeptide has a length greater than 5, 10, 15, 20, 25, 30, 50, 100, 200, 500, 5000, 30,000 consecutive amino acid residues; optionally, wherein the target polypeptide has a length in a range from 5 to 1000, 5 to 500, 5 to 200, or 5 to 100 consecutive amino acid residues. In some embodiments, the target polypeptide is linearized.

[0108] Some embodiments also include a source for applying potential differences over the membrane. In some embodiments, the source is capable of modulating and / or reversing the potential difference over the membrane. Some embodiments also include a detector for measuring a signal generated while the target polypeptide is within the nanopore. In some embodiments, the detector is capable of measuring a signal generated while the target polypeptide is translocated through the nanopore. In some embodiments, the detector is capable of measuring a signal indicative of the identity of a terminal residue at the non-tethered terminal end of the target polypeptide while the terminal residue is located within the nanopore. In some embodiments, the terminal residue is located within a read region within the nanopore.

[0109] Some embodiments of the kits and systems provided herein include “trans blocker” systems and kits. In some such embodiments, the trans solution further comprises a blocker attached via a linker to a reactive moiety capable of coupling to a terminal residue of the target polypeptide. In some such embodiments, the reactive moiety is an isothiocyanate moiety. In some such embodiments, the reactive moiety is a phenyl isothiocyanate (PITC) moiety. The blocker can include a moiety having a size sufficient to inhibit the blocker translocating through the nanopore. In some embodiments, the blocker comprises a branched oligopeptide or a nanoparticle. In some embodiments, the linker comprises a polyethylene glycol (PEG), a 2-6 benzobisoxazole, or a bifunctional oligothiophene. In some embodiments, at least a portion of the target polypeptide is located within the nanopore. In some embodiments, the blocker is attached to the target polypeptide via the linker and the PITC moiety. In some embodiments, the blocker is attached to an N-termmus of the target polypeptide, or a C-terminus of the target polypeptide.

[0110] Some embodiments of the kits and systems provided herein include “cis leader” systems and kits. Some such embodiments include a cis solution comprising a leader composition comprising a reactive moiety capable of coupling to a terminal residue of the target polypeptide. In some embodiments, the reactive moiety is attached to a lockoligonucleotide capable of forming a hairpin structure. In some embodiments, the cis solution is in contact with the cis surface. In some embodiments, the reactive moiety is an isothiocyanate moiety, optionally, wherein the reactive moiety is a phenyl isothiocyanate (PITC) moiety. In some embodiments, the reactive moiety is attached to the lock oligonucleotide via a spacer. In some embodiments, the spacer comprises a first affinity moiety, and the Edmanase comprises a second affinity moiety. In some embodiments, the first and second affinity moieties are capable of binding to one another. In some embodiments, the binding is specific binding. In some embodiments, the first and second affinity moieties are selected from biotin, a biotin derivative, streptavidin, a streptavidin derivative, tetrazine, or bicyclononyne (BCN). In some embodiments, the spacer comprises (i) a polynucleotide, optionally, wherein the polynucleotide has a length in a range from 1 nucleotide to 50 nucleotides, optionally, in a range from 3 nucleotides to 10 nucleotides; (ii) a homopolymer; (iii) a polyethylene glycol (PEG); or (iv) a polypeptide spacer, optionally having a length less than 10 consecutive residues, and not less than 1 residue. In some embodiments, the spacer has the nucleotide sequence (TTT). In some embodiments, the leader composition is attached to an N-terminus of the target polypeptide via the reactive moiety. In some embodiments, the lock oligonucleotide is capable of hybridizing to an inhibitor oligonucleotide which inhibits formation of the hairpin structure. In some embodiments, the lock oligonucleotide is hybridized to the inhibitor oligonucleotide. In some embodiments, at least a portion of the target polypeptide is located within the nanopore, such that the leader composition is in contact with the trans solution. In some embodiments, the lock oligonucleotide has a hairpin structure. In some embodiments, the spacer of the leader composition has a length such that a signal indicative of the identity of a terminal residue at the non-tethered terminal end of the target polypeptide is increased compared to a leader sequence having a longer spacer or lacking the spacer.Certain methods for characterizing a target polypeptide

[0111] Some embodiments of the methods and compositions provided herein include a method of characterizing a target polypeptide, comprising: (a) obtaining any one of the systems provided herein; (b) translocating a non-tethered end of the target polypeptide through the nanopore to the trans surface; (c) measuring a signal generated by the non-tetheredend of the target polypeptide located in the nanopore; (d) removing the terminal residue from the target polypeptide. Some embodiments also include (e) measuring a signal generated by the removing step (d). Some embodiments also include repeating step (b) to step (d). Some embodiments also include repeating steps (b) to step (e).

[0112] In some embodiments, step (b) comprises applying over the membrane: (i) a potential difference, and / or (ii) an electroosmotic force.

[0113] In some embodiments, step (c) comprises measuring a signal generated by a terminal residue of the non-tethered end of the target polypepti de l ocated in a read region of the nanopore. In some embodiments, step (c) further comprises cycling the non-tethered end of the target polypeptide located in the nanopore in a repeated movement towards the trans surface and to the cis surface. In some embodiments, step (c) comprises measuring a repeated signal.

[0114] In some embodiments, step (d) comprises contacting the non-tethered end of the target polypeptide with an Edmanase.

[0115] In some embodiments, step (d) comprises translocating the non-tethered end of the target polypeptide to the cis surface.

[0116] In some embodiments, step (e) comprises measuring a signal indicative of the absence of a residue of the target polypeptide in the read region of the nanopore. In some embodiments, step (e) comprises measuring a signal indicative of the absence of the target polypeptide in the nanopore. Some embodiments also include determining the identity of one or more ammo acid residues of the target polypeptide based on the signal obtained in step (c).

[0117] Some embodiments of the methods provided herein include obtaining any one of the “trans blocker” systems provided herein. In some such embodiments, step (a) comprises obtaining any one of the systems provided herein in which the trans solution further comprises a blocker attached via a linker to a reactive moiety. In some embodiments, step (a) further comprises contacting the non-tethered end of the target polypeptide with the reactive moiety such that the blocker attaches to the non-tethered end of the target polypeptide via the linker and the reactive moiety.

[0118] Some embodiments of the methods provided herein include obtaining any one of the “cis leader” systems provided herein. In some such embodiments, step (a) comprises obtaining any one of the systems provided herein which also include a cis solution comprisinga leader composition comprising a reactive moiety attached to a lock oligonucleotide capable of forming a hairpin structure, in which the cis solution is in contact with the cis surface. In some embodiments, step (a) further comprises contacting the non-tethered end of the target polypeptide with the leader composition such that the lock oligonucleotide attaches to the non¬ tethered end of the target polypeptide via the reactive moiety. In some embodiments, step (b) comprises threading the lock oligonucleotide through the nanopore to the trans surface.Certain methods for preparing a system

[0119] Some embodiments of the methods and compositions provided herein include a method for preparing a system for characterizing a target polypeptide, comprising: (a) obtaining a nanopore embedded in a membrane, wherein the membrane has a cis and trans surface; (b) tethering or immobilizing a target polypeptide to the nanopore via a tether at the cis surface; and (d) contacting the trans surface with a trans solution comprising an Edmanase,

[0120] In some embodiments, the membrane comprises a lipid bi layer or a block copolymer. In some embodiments, the block co-polymer comprises a deblock copolymer or a triblock copolymer. Each molecule of a block copolymer may include one or more hydrophilic blocks and one or more hydrophobic blocks. The hydrophilic blocks may form outer surfaces of the barrier and the hydrophobic blocks may be located within the barrier. The hydrophobic blocks may include a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydiene, fluormated polyethylene, polypeptide, and poly(isobutylene) (PIB). See e.g., U. S.20230312856 which is incorporated by reference in its entirety.

[0121] In some embodiments, the nanopore comprises a protein nanopore. In some embodiments, the protein nanopore is selected from OmpF, OmpG, CsgG, MspA, a-HL, FhuA, AeL, FraC, Lys, φ29p, ClyA, Ply AB. In some embodiments, the protein nanopore is MspA.

[0122] In some embodiments, step (b) further comprises obtaining the target polypeptide. In some embodiments, step (b) comprises linearizing a native polypeptide. In some embodiments, the linearizing comprises treating the native polypeptide with (i) a denaturation agent, in some embodiments the denaturation agent is selected from sodium dodecyl sulfate (SDS), or guanidinium HC1, or (ii) an agent to acetylate or succinylate aminoresidues of the target polypeptide. In some embodiments, step (b) comprises specifically labelling a type of residue of the target polypeptide with a reporter moiety. In some embodiments, the reporter moiety is attached to the residue via a cleavable linker. In some embodiments, step (b) comprises specifically labelling at least two, three, four, five, six or seven different types of residues of the target polypeptide. In some embodiments, the type of residue is selected from cysteine, lysine, methionine, tryptophan, arginine, tyrosine, serine, or threonine. In some embodiments, the type of residue is selected from cysteine, lysine, or methionine.

[0123] In some embodiments, step (b) comprises tethering or immobilizing the target polypeptide to the nanopore via a C -terminus of the target polypeptide or an N-terminus of the target polypeptide, In some embodiments, step (b) comprises tethering or immobilizing comprises the target polypeptide to the nanopore via the C-terminus of the target polypeptide. In some embodiments, step (d) comprises tethering or immobilizing the Edmanase to the nanopore or to the membrane. In some embodiments, the tethering or immobilizing of the Edmanase and / or of the target polypeptide is reversible. In some embodiments, the tether comprises a cleavable linker or the immobilization is reversible. In some embodiments, the cleavable linker comprises a disulfide bond. In some embodiments, the tether comprises an oligonucleotide. In some embodiments, the tether has a length greater than the length of a pore of the nanopore through the membrane.

[0124] In some embodiments, the target polypeptide has a length greater than 5, 10, 15, 20, 25, 30, 50, 100, 200, 500, 5000, 30,000 consecutive amino acid residues; optionally, wherein the target polypeptide has a length in a range from 5 to 1000, 5 to 500, 5 to 200, or 5 to 100 consecutive amino acid residues.

[0125] Some embodiments for preparing a system for characterizing a target polypeptide include preparing a '‘trans blocker” system provided herein. In some such embodiments, the trans solution further comprises a blocker attached via a linker to a reactive moiety capable of coupling to a terminal residue of the target polypeptide. In some embodiments, the reactive moiety is an isothiocyanate moiety. In some embodiments, the reactive moiety is a phenyl isothiocyanate (PITC) moiety. In some embodiments, the blocker comprises a branched oligopeptide or a nanoparticle. In some embodiments, the linkercomprises a polyethylene glycol (PEG), a 2-6 benzobisoxazole, or a bifunctional oligothiophene.

[0126] Some embodiments for preparing a system for characterizing a target polypeptide include preparing a “ s leader” system provided herein. In some such embodiments also include a step (e) comprising contacting the cis surface with a cis solution comprising a leader composition comprising a reactive moiety attached to a lock oligonucleotide capable of forming a hairpin structure. In some embodiments, the reactive moiety is capable of coupling to a terminal residue of the target polypeptide. In some embodiments, the reactive moiety is an isothiocyanate moiety. In some embodiments, the reactive moiety is a phenyl isothiocyanate (PITC) moiety. In some embodiments, the reactive moiety is attached to the lock oligonucleotide via a spacer. In some embodiments, the spacer comprises a first affinity moiety, and the Edmanase comprises a second affinity moiety, wherein the first and second affinity moieties are capable of binding to one another. In some embodiments, the binding is specific binding. In some embodiments, the first and second affinity moieties are selected from biotin, a biotin derivative, streptavidin, a streptavidin derivative, tetrazine, or bicyclononyne (BCN). In some embodiments, the spacer comprises (i) a polynucleotide, optionally, wherein the polynucleotide has a length in a range from I nucleotide to 50 nucleotides, optionally, in a range from 3 nucleotides to 10 nucleotides; (ii) a homopolymer; (in) a polyethylene glycol (PEG); or (iv) a polypeptide spacer, optionally having a length less than 10 consecutive residues, and not less than 1 residue. In some embodiments, the spacer has the nucleotide sequence (TTT). In some embodiments, the lock oligonucleotide is capable of hybridizing to an inhibitor oligonucleotide which inhibits formation of the hairpin structure. In some embodiments, the lock oligonucleotide is hybridized to the inhibitor oligonucleotide.

[0127] The term “comprising” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0128] The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications willbecome apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention.

[0129] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety' and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

Claims

WHAT IS CLAIMED IS:

1. A system for characterizing a target polypeptide, comprising:(a) a membrane having a cis and trans surface;(b) a nanopore embedded in the membrane;(c) a target polypeptide tethered or immobilized to the nanopore or to the membrane via a tether at the cis surface; and(d) a trans solution comprising an Edmanase, wherein the trans solution is in contact with the trans surface,2. The system of claim 1, wherein the Edmanase is tethered or immobilized to the membrane or to the nanopore.

3. The system of claim 1 or 2, wherein the trans solution further comprises a blocker attached via a linker to a reactive moiety capable of coupling to a terminal residue of the target polypeptide.

4. The system of claim 3, wherein reactive moiety is an isothiocyanate moiety, 5. The system of claim 4, wherein the reactive moiety is a phenyl isothiocyanate (P1TC) moiety6. The system of any one of claims 3-5, wherein the blocker comprises a branched oligopeptide or a nanoparticle.

7. The system of any one of claims 3-6, wherein the linker comprises a polyethylene glycol (PEG), a 2-6 benzobisoxazole, or a bifunctional oligothiophene.

8. The system of any one of claims 3-7, wherein at least a portion of the target polypeptide is located within the nanopore.

9. The system of any one of claims 3-8, wherein the blocker is attached to the target polypeptide via the linker and the reactive moiety.

10. The system of any one of claims 3-9, wherein the blocker is attached to an N-terminus of the target polypeptide, or a C-temiinus of the target polypeptide.

11. The system of claim 1 or 2, further comprising a cis solution comprising a leader composition comprising a reactive moiety capable of coupling to a terminal residue of the target polypeptide, wherein the reactive moiety is attached to a lock oligonucleotide capable of forming a hairpin structure, wherein the cis solution is in contact with the cis surface.

12. The system of claim 11, wherein the reactive moiety is an isothiocyanate moiety.

13. The system of claim 12, wherein the reactive moiety is a phenyl isothiocyanate (PITC) moiety.

14. The system of any one of claims 11-13, wherein the reactive moiety is attached to the lock oligonucleotide via a spacer.

15. The system of claim 14, w’herein the spacer comprises a first affinity moiety, and the Edmanase comprises a second affinity moiety, wherein the first and second affinity moieties are capable of binding to one another.

16. The system of claim 15, wherein the first and second affinity moieties are selected from biotin, a biotin derivative, streptavidin, a streptavidin derivative, tetrazine, or bi cyclononyne (BCN).

17. The system of any one of claims 14-16, wherein the spacer comprises (i) a polynucleotide; (ii) a homopolymer; (iii) a polyethylene glycol (PEG); or (iv) a polypeptide spacer.

18. The system of any one of claims 14-17, wherein the spacer has the nucleotide sequence (TTT).

19. The system of any one of claims 11-18, wherein the leader composition is attached to an N-terminus of the target polypeptide via the reactive moiety.

20. The system of any one of claim 11-19, wherein the lock oligonucleotide is capable of hybridizing to an inhibitor oligonucleotide which inhibits formation of the hairpin structure.

21. The system of claim 20, wherein the lock oligonucleotide is hybridized to the inhibitor oligonucleotide.

22. The system of any one of claim 11-21, wherein at least a portion of the target polypeptide is located within the nanopore, such that the leader composition is in contact with the trans solution.

23. The system of claim 22, wherein the lock oligonucleotide has a hairpin structure.

24. The system of any one of claims 14-23, wherein the spacer of the leader composition has a length such that a signal indicative of the identity of a terminal residue attlie non-tethered terminal end of the target polypeptide is increased compared to a leader sequence having a longer spacer or lacking the spacer.

25. The system of any one of claims 1 -24, wherein the membrane comprises a lipid bilayer or block copolymer.

26. The system of any one of claims 1-25, wherein the nanopore comprises a protein nanopore.

27. The system of claim 26, wherein the protein nanopore is selected from OmpF, OmpG, CsgG, MspA, a-HL, FhuA, AeL, FraC, Lys, <|φ29p, ClyA, Ply AB.

28. The system of claim 27, wherein the protein nanopore is MspA or CsgG, 29. The system of any one of claims 1 -28, wherein the target polypeptide is tethered or immobilized to the nanopore or to the membrane via a C-terminus of the target polypeptide or an N-terminus of the target polypeptide.

30. The system of claim 29, wherein the target polypeptide is tethered or immobilized to the nanopore or to the membrane via the C-terminus of the target polypeptide.

31. The system of any one of claims 1-30, wherein (i) the tether comprises a first cleavable linker or the immobilization is reversible; and / or (ii) the Edmanase is tethered or immobilized to the membrane or to the nanopore via a second cleavable linker or the immobilization is reversible.

32. The system of claim 31, wherein the first or second cleavable linker comprises a disulfide bond.

33. The system of any one of claims 1-32, wherein the tether comprises an oligonucleotide.

34. The system of any one of claims 1-33, wherein the tether has a length greater than a length of a pore of the nanopore through the membrane.

35. The system of any one of claims 1-34, wherein a type of residue of the target polypeptide is specifically labelled with a reporter moiety.

36. The system of claim 35, wherein the reporter moiety is cleavable from the specifically labeled residue.

37. The system of any one of claims 1-36, wherein at least two, three, four, five, six, or seven different types of residues of the target polypeptide are specifically labeled.

38. The system of any one of claims 35-37, wherein the type of residue is selected from cysteine, lysine, methionine, tryptophan, arginine, tyrosine, serine, or threonine.

39. The system of claim 38, wherein the type of residue is selected from cysteine, lysine, or methionine.

40. The system of any one of claims 1-39, wherein the target polypeptide has a length greater than 5, 10, 15, 20, 25, 30, 50, 100 200, 500, 5000, 30,000 consecutive amino acid residues.

41. The system of claim 40, wherein the target polypeptide has a length in a range from 5 to 1000, 5 to 500, 5 to 200, or 5 to 100 consecutive amino acid residues.

42. The system of any one of claims 1-41, wherein the target polypeptide is linearized.

43. The system of any one of claims 1-42, further comprising a source for applying potential difference over the membrane.

44. The system of claim 43, wherein the source is capable of modulating and / or reversing the potential difference over the membrane.

45. The system of any one of claims 1-44, further comprising a detector for measuring a signal generated while the target polypeptide is within the nanopore.

46. The system of claim 45, wherein the detector is capable of measuring a signal generated while the target polypeptide is translocated through the nanopore.

47. The system of claim 45 or 46, wherein the detector is capable of measuring a signal indicative of the identity of a terminal residue at the non-tethered terminal end of the target polypeptide while the terminal residue is located within the nanopore.

48. The system of claim 47, wherein the terminal residue is located within a read region within the nanopore.

49. A method of characterizing a target polypeptide, comprising:(a) obtaining the system of any one of claims 1-48;(b) translocating a non-tethered end of the target polypeptide through the nanopore to the trans surface;(c) measuring a signal generated by the non-tethered end of the target polypeptide located in the nanopore;(d) removing the terminal residue from the target polypeptide; and(e) measuring a signal generated by the removing.

50. The method of claim 49, further comprising repeating step (b) to step (d); optionally repeating steps (b) to step (e).

51. The method of claim 49 or 50, wherein step (b) comprises applying over the membrane: (i) a potential difference, and / or (ii) an electroosmotic force.

52. The method of any one of claims 49-51, wherein step (c) comprises measuring a signal generated by a terminal residue of the non-tethered end of the target polypeptide located in a read region of the nanopore,53. The method of any one of claims 49-52, wherein step (c) further comprises cycling the non-tethered end of the target polypeptide located in the nanopore in a repeated movement towards the trans surface and to the cis surface.

54. The method of claim 53, wherein step (c) comprises measuring a repeated signal.

55. The method of any one of claims 49-54, wherein step (d) comprises contacting the non-tethered end of the target polypeptide with an Edmanase.

56. The method of any one of claims 49-55, wherein step (d) comprises translocating the non-tethered end of the target polypeptide to the cis surface.

57. The method of any one of claims 49-56, wherein step (e) comprises measuring a signal indicative of the absence of a residue of the target polypeptide in the read region of the nanopore.

58. The method of claim 57, wherein step (e) comprises measuring a signal indicative of the absence of the target polypeptide in the nanopore.

59. The method of any one of claims 49-58, further comprising determining the identity of one or more amino acid residues of the target polypeptide based on the signal obtained in step (c).

60. The method of any one of claims 49-59, wherein:step (a) comprises obtaining the system of any one of claims 2-10 and 25-48.

61. The method of claim 60, wherein step (a) further comprises contacting the non- tethered end of the target polypeptide with the reactive moiety such that the blocker attaches to the non-tethered end of the target polypeptide via the linker and the reactive moiety.

62. The method of any one of claims 49-59, wherein:step (a) comprises obtaining the system of any one of claims 11-48.

63. The method of claim 62, wherein step (a) further comprises contacting the non¬ tethered end of the target polypeptide with the leader composition such that the lock oligonucleotide attaches to the non-tethered end of the target polypeptide via the reactive moiety.

64. The method of claim 62 or 63, wherein step (b) comprises threading the lock oligonucleotide through the nanopore to the trans surface.

65. A method for preparing a system for characterizing a target polypeptide, comprising:(a) obtaining a nanopore embedded in a membrane, wherein the membrane has a cis and trans surface;(b) tethering or immobilizing a target polypeptide to the nanopore or to the membrane via a tether at the cis surface; and(d) contacting the trans surface with a trans solution comprising an Edmanase.

66. The method of claim 65, wherein the trans solution further comprises a blocker attached via a linker to a reactive moiety capable of coupling to a terminal residue of the target polypeptide.

67. The method of claim 66, wherein the reactive moiety is an isothiocyanate moiety.

68. The method of claim 67, wherein the reactive moiety is a phenyl isothiocyanate (PITC) moiety.

69. The method of any one of claims 66-68, wherein the blocker comprises a branched oligopeptide or a nanoparticle.

70. The method of any one of claims 66-69, wherein the linker comprises a polyethylene glycol (PEG), a 2-6 benzobisoxazole, or a bifunctional oligothiophene.

71. The method of claim 65, further comprising (e) contacting the cis surface with a cis solution comprising a leader composition comprising a reactive moiety capable of coupling to a terminal residue of the target polypeptide, wherein the reactive moiety is attached to a lock oligonucleotide capable of forming a hairpin structure.

72. The method of claim 71, wherein the reactive moiety is an isothiocyanate moiety.

73. The method of claim 72, wherein the reactive moiety is a phenyl isothiocyanate (PITC) moiety.

74. The method of any one of claims 71-73, wherein the reactive moiety' is attached to the lock oligonucleotide via a spacer.

75. The method of claim 74, wherein the spacer comprises a first affinity moiety, and the Edmanase comprises a second affinity moiety, wherein the first and second affinity moieties are capable of binding to one another, optionally wherein the binding is specific binding;76. The method of claim 75, wherein the first and second affinity moieties are selected from biotin, a biotin derivative, streptavidin, a streptavidin derivative, tetrazine, or bicyclononyne (BCN).

77. The method of any one of claims 74-76, wherein the spacer comprises (i) a polynucleotide; (ii) a homopolymer; (iii) a polyethylene glycol (PEG); or (iv) a polypeptide spacer.

78. The method of any one of claims 74-77, wherein the spacer has the nucleotide sequence (TTT).

79. The method of any one of claim 71-78, wherein the lock oligonucleotide is capable of hybridizing to an inhibitor oligonucleotide which inhibits formation of the hairpin structure.

80. The method of claim 79, wherein the lock oligonucleotide is hybridized to the inhibitor oligonucleotide.

81. The method of any one of claims 65-80, wherein the membrane comprises a lipid bilayer or a block copolymer.

82. The method of any one of claims 65-81, wherein the nanopore comprises a protein nanopore.

83. The method of claim 82, wherein the protein nanopore is selected from OmpF, OmpG, CsgG, MspA, a-HL, FhuA, AeL, FraC, Lys, (|)29p, ClyA, Ply AB.

84. The method of claim 83, wherein the protein nanopore is MspA.

85. The method of any one of claims 65-84, wherein step (b) further comprises obtaining the target polypeptide.

86. The method of claim 85, wherein step (b) comprises linearizing a native polypeptide.

87. The method of claim 86, wherein the linearizing comprises treating the native polypeptide with (i) a denaturation agent, optionally wherein the denaturation agent is selected from sodium dodecyl sulfate (SDS), or guanidinium HC1, or (ii) an agent to acetylate or succinylate amino residues of the target polypeptide.

88. The method of any one of claims 85-87, wherein step (b) comprises specifically labelling a type of residue of the target polypeptide with a reporter moiety,89. The method of claim 88, wherein the reporter moiety is attached to the residue via a cleavable linker,90. The method of any one of claims 85-89, wherein step (b) comprises specifically labelling at least two, three, four, five, six, or seven different types of residues of the target polypeptide.

91. The method of any one of claims 88-90, wherein the type of residue is selected from cysteine, lysine, methionine, tryptophan, arginine, tyrosine, serine, or threonine.

92. The method of claim 91, wherein the type of residue is selected from cysteine, lysine, or methionine.

93. The method of any one of claims 65-92, wherein:(i) step (b) comprises tethering or immobilizing the target polypeptide to the nanopore or to the membrane via a C -terminus of the target polypeptide or an N- terminus of the target polypeptide; and / or(ii) step (d) comprises tethering or immobilizing the Edmanase to the nanopore or to the membrane.

94. The method of claim 93, wherein (b) comprises tethering or immobilizing the target polypeptide to the nanopore or to the membrane via the C -terminus of the target polypeptide.

95. The method of claim 94, wherein the tethering or immobilizing is reversible.

96. The method of any one of claims 65-95, wherein the tether comprises a cleavable linker or the immobilization is reversible.

97. The method of claim 96, wherein the cleavable linker comprises a disulfide bond.

98. The method of any one of claims 65-97, wherein the tether comprises an oligonucleotide.

99. The method of any one of claims 65-98, wherein the tether has a length greater than a length of a pore of the nanopore through the membrane.

100. The method of any one of claims 65-99, wherein the target polypeptide has a length greater than 5, 10, 15, 20, 25, 30, 50, 100 200, 500, 5000, 30,000 consecutive amino acid residues.

101. The method of claim 100, wherein the target polypeptide has a length in a range from 5 to 1000, 5 to 500, 5 to 200, or 5 to 100 consecutive amino acid residues.

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