Amino acid binding agents and their uses

Description

AMINO ACID BINDING AGENTS AND THEIR USESCROSS REFERENCE

[0001] This application claims benefit of U.S. Provisional Patent Application No. 63 / 584,382 filed September 21, 2023, U.S. Provisional Patent Application No. 63 / 588,867, filed October 9, 2023, and U.S. Provisional Patent Application No. 63 / 589,391, filed October 11, 2023, each of which are incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with U.S. government support under Grant Numbers HG012960 and GM148298, awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 16, 2024, is named 60652-708_601_SL.xml and is 106,896 bytes in size.BACKGROUND

[0004] Characterization of polymeric analytes, such as proteins, is essential for understanding biology and pathology and for development of diagnostics and therapeutics. De novo sequencing of polymeric analytes (e.g., proteins) is necessary for protein discovery, determination of structural -functional relationships, and identification of biomarker targets.

[0005] The use of binding agents that recognize and bind to polymeric analytes is useful in identifying the presence of the polymeric analytes in a sample. However, current binding agents are limited in their ability to sensitively and selectively detect individual monomer types (e.g., amino acid types). Accordingly, new binding agents for detecting each monomer type of a given polymeric analyte are needed.SUMMARY

[0006] Recognized herein is a need for novel binding agents that can recognize and bind to individual monomers of polymeric analytes. In some embodiments, the present disclosure provides binding agents that can recognize and bind to individual amino acids or derivativesthereof. The derivatives thereof may comprise modified amino acids, such as chemically or enzymatically modified amino acids.

[0007] In an aspect, provided herein is a binding agent that binds to one type of proteinogenic amino acid or derivative thereof with greater specificity or with higher affinity than to all other types of proteinogenic amino acids or derivatives thereof, wherein the one type of amino acid is not tryptophan. In some aspects, provided herein is a binding agent that binds to one type of proteinogenic amino acid or derivative thereof of a set of two or more proteinogenic amino acids or derivatives thereof with greater specificity or with higher affinity than to all other types of proteinogenic amino acids or derivatives thereof in the set, wherein the one type of amino acid is not tryptophan.

[0008] In some embodiments, the set of two or more proteinogenic amino acids or derivatives thereof comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different proteinogenic amino acids or derivatives thereof. In some embodiments, the set of two or more proteinogenic amino acids or derivatives thereof comprises naturally occurring proteinogenic amino acids. In some embodiments, the set of two or more proteinogenic amino acids or derivatives thereof comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 standard proteinogenic amino acids or derivatives thereof. In some embodiments, the set of two or more proteinogenic amino acids or derivatives thereof consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 standard proteinogenic amino acids or derivatives thereof.

[0009] In some embodiments, the binding agent is part of a library of binding agents, wherein the library of binding agents comprise a plurality of binding agents that collectively can recognize and specifically bind to at least 5 different types of proteinogenic amino acids. In some embodiments, the binding agent is part of a library of binding agents, wherein the library of binding agents comprise a plurality of binding agents that collectively can recognize and specifically bind to all 20 types of standard proteinogenic amino acids. In some embodiments, a binding agent of the library of binding agents can recognize and specifically bind to at least one post-translationally modified amino acid. In some embodiments, the library of binding agents comprises a plurality of polyclonal antibodies. In some embodiments, the library of binding agents comprises a plurality of monoclonal antibodies.

[0010] In some embodiments, the binding agent comprises an antibody, an antibody fragment, a single-chain variable fragment (scFv), or a nanobody. In some embodiments, the binding agent comprises an antibody. In some embodiments, the antibody is an immunoglobulin G (IgG). In some embodiments, the antibody is generated by immunizing an animal with an immunogenically effective amount of a composition comprising a carrier protein coupled to anamino acid or amino acid derivative. In some embodiments, the composition comprises the carrier protein comprising a polymeric linker that couples to the amino acid or amino acid derivative. In some embodiments, the polymeric linker comprises polyethylene glycol (PEG). In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the binding agent comprises an engineered scFv. In some embodiments, the engineered scFv is derived from an IgG. In some embodiments, the engineered scFv is engineered using directed evolution. In some embodiments, the engineered scFv is affinity matured using yeast surface display, phage display, ribosome display, or continuous evolution. In some embodiments, the engineered scFv has improved specificity to the one type of proteinogenic amino acid or derivative thereof through use of yeast surface display.

[0011] In some embodiments, the derivative or the derivatives thereof comprise a chemically modified amino acid. In some embodiments, the chemically modified amino acid comprises a phenylthiohydantoin, phenylthiocarbamyl, or anilinothiazolinone derivative of the amino acid. In some embodiments, the chemically modified amino acid comprises an amino acid coupled to a linker. In some embodiments, the linker is coupled to the amino acid at an N- terminal end. In some embodiments, the linker coupled to the chemically modified amino acid comprises a phenylthiohydantoin, phenylthiocarbamyl, or anilinothiazolinone moiety. In some embodiments, the linker is coupled to the amino acid at a C-terminal end. In some embodiments, the linker comprises a linking nucleic acid molecule. In some embodiments, the linker comprises an amino acid reactive group and a linking nucleic acid molecule.

[0012] In some embodiments, binding of the binding agent specifically to the one type of proteinogenic amino acid or derivative thereof over all other types of proteinogenic amino acids or derivatives thereof is measured using an indirect enzyme-linked immunosorbent assay (ELISA). In some embodiments, binding of the binding agent specifically to the one type of proteinogenic amino acid or derivative thereof over all other types of proteinogenic amino acids in the set or derivatives thereof is measured using an indirect enzyme-linked immunosorbent assay (ELISA).

[0013] In some embodiments, binding of the binding agent specifically to the one type of proteinogenic amino acid or derivative thereof over all other types of proteinogenic amino acids or derivatives thereof is measured using surface plasmon resonance. In some embodiments, binding of the binding agent specifically to the one type of proteinogenic amino acid or derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in the set is measured using surface plasmon resonance.

[0014] In some embodiments, binding of the binding agent specifically to the one type of proteinogenic amino acid or derivative thereof over all other types of proteinogenic amino acids or derivatives thereof is measured using bio layer interferometry. In some embodiments, binding of the binding agent specifically to the one type of proteinogenic amino acid or derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in the set is measured using bio layer interferometry.

[0015] In some embodiments, the binding agent comprises a detectable label. In some embodiments, the detectable label comprises a nucleic acid molecule, a fluorophore, a mass tag, or a protein. In some embodiments, the detectable label comprises a protein, wherein the protein comprises an additional antibody or additional antibody fragment. In some embodiments, the detectable label comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a barcode sequence. In some embodiments, the detectable label is coupled to the binding agent using a moiety selected from the group consisting of biotin, desthiobiotin, avidin, streptavidin, neutravidin, SpyCatcher, SpyTag, SNAP tag, a click chemistry moiety, and a cysteine tag. In some embodiments, the binding agent comprises a noncanonical amino acid, wherein the detectable label is coupled to the binding agent via the noncanonical amino acid. In some embodiments, the noncanonical amino acid comprises a click chemistry moiety.

[0016] In some embodiments, the binding agent specifically binds to phenylalanine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof. In some embodiments, the binding agent specifically binds to phenylalanine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in the set. In some embodiments, the binding agent specifically binds to leucine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof. In some embodiments, the binding agent specifically binds to leucine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in the set. In some embodiments, the binding agent specifically binds to valine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof. In some embodiments, the binding agent specifically binds to valine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in the set. In some embodiments, the binding agent specifically binds to tyrosine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof. In some embodiments, the binding agent specifically binds to tyrosine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in the set. In some embodiments, the binding agent specifically binds to proline or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof. In some embodiments, thebinding agent specifically binds to proline or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in the set.

[0017] In some embodiments, the binding agent is derived from a mouse.

[0018] In another aspect, provided herein is an antibody fragment that specifically binds to one type of proteinogenic amino acid or derivative thereof of a set of two or more proteinogenic amino acids or derivatives thereof with greater specificity or with higher affinity than to all other types of proteinogenic amino acids or derivatives thereof in the set.

[0019] In some embodiments, the set of two or more proteinogenic amino acids or derivatives thereof comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different proteinogenic amino acids or derivatives thereof. In some embodiments, the set of two or more proteinogenic amino acids or derivatives thereof comprises naturally occurring proteinogenic amino acids. In some embodiments, thet set of two or more proteinogenic amino acids or derivatives thereof comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 standard proteinogenic amino acids or derivatives thereof. In some embodiments, the set of two or more proteinogenic amino acids or derivatives thereof consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 standard proteinogenic amino acids or derivatives thereof.

[0020] In some embodiments, the antibody fragment is part of a library of antibodies or antibody fragments, wherein the library of antibodies or antibody fragments comprise a plurality of antibodies or antibody fragments that collectively can recognize and specifically bind to at least 5 different types of proteinogenic amino acids or derivatives thereof. In some embodiments, the antibody fragment is part of a library of antibodies or antibody fragments, wherein the library of antibodies or antibody fragments comprise a plurality of antibodies or antibody fragments that collectively can recognize and specifically bind to all 20 types of standard proteinogenic amino acids or derivatives thereof. In some embodiments, a binding agent of the library of antibodies or antibody fragments comprises a plurality of polyclonal antibodies. In some embodiments, the library of antibodies or antibody fragments comprises a plurality of monoclonal antibodies.

[0021] In some embodiments, the antibody fragment is derived from an antibody. In some embodiments, the antibody is an immunoglobulin G (IgG). In some embodiments, the antibody is generated by immunizing an animal with an immunogenically effective amount of a composition comprising a carrier protein coupled to an amino acid or amino acid derivative. In some embodiments, the composition comprises the carrier protein comprising a polymeric linker that couples to the amino acid or amino acid derivative. In some embodiments, the polymericlinker comprises polyethylene glycol (PEG). In some embodiments, the antibody is a monoclonal antibody.

[0022] In some embodiments, the antibody fragment comprises an engineered scFv. In some embodiments, the engineered scFv is derived from an IgG. In some embodiments, the engineered scFv is engineered using directed evolution. In some embodiments, the engineered scFv is affinity matured using yeast surface display, phage display, ribosome display, or continuous evolution. In some embodiments, the engineered scFv has improved specificity to the one type of proteinogenic amino acid or derivative thereof through use of yeast surface display.

[0023] In some embodiments, the derivative or the derivatives thereof comprise a chemically modified amino acid. In some embodiments, the chemically modified amino acid comprises a phenylthiohydantoin, phenylthiocarbamyl, or anilinothiazolinone derivative of the amino acid. In some embodiments, the chemically modified amino acid comprises an amino acid coupled to a linker. In some embodiments, the linker is coupled to the amino acid at an N- terminal end. In some embodiments, the linker coupled to the chemically modified amino acid comprises a phenylthiohydantoin, phenylthiocarbamyl, or anilinothiazolinone moiety. In some embodiments, the linker is coupled to the amino acid at a C-terminal end. In some embodiments, the linker comprises a linking nucleic acid molecule. In some embodiments, the linker comprises an amino acid reactive group and a linking nucleic acid molecule. In some embodiments, binding of the antibody fragment specifically to the one type of proteinogenic amino acid or derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in the set is measured using an indirect enzyme-linked immunosorbent assay (ELISA). In some embodiments, binding of the antibody fragment specifically to the one type of proteinogenic amino acid or derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in the set is measured using surface plasmon resonance. In some embodiments, binding of the antibody fragment specifically to the one type of proteinogenic amino acid or derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in the set is measured using bio layer interferometry. In some embodiments, the antibody fragment comprises a detectable label. In some embodiments, the detectable label comprises a nucleic acid molecule, a fluorophore, a mass tag, or a protein. In some embodiments, the detectable label comprises a protein, wherein the protein comprises an additional antibody or additional antibody fragment. In some embodiments, the detectable label comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a barcode sequence. In some embodiments, the detectable label is coupled to the antibody or antibody fragment using amoiety selected from the group consisting of biotin, desthiobiotin, avidin, streptavidin, neutravidin, SpyCatcher, SpyTag, SNAP tag, a click chemistry moiety, and a cysteine tag. In some embodiments, the antibody fragment comprises a noncanonical amino acid, wherein the detectable label is coupled to the antibody or antibody fragment via the noncanonical amino acid. In some embodiments, the noncanonical amino acid comprises a click chemistry moiety.

[0024] In some embodiments, the antibody fragment specifically binds to phenylalanine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in the set. In some embodiments, the antibody fragment specifically binds to leucine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in the set. In some embodiments, the antibody fragment specifically binds to valine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in the set. In some embodiments, the antibody fragment specifically binds to tyrosine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in the set. In some embodiments, the antibody fragment specifically binds to proline or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in the set.

[0025] In another aspect, provided herein is a binding agent that binds to an amino acid or derivative thereof at an equilibrium dissociation constant (Kd) of less than 1 nanomolar (1 nM), as determined by biolayer interferometry (BLI) or surface plasmon resonance (SPR).

[0026] In some embodiments, the binding agent comprises an antibody or antibody fragment.

[0027] In some embodiments, the binding agent binds specifically to one type of amino acid or derivative thereof.

[0028] In some embodiments, the dissociation constant of the binding agent to the amino acid or derivative thereof is less than dissociation constants of the binding agent to other types of amino acids or derivatives thereof.

[0029] In some embodiments, the binding agent is part of a library of binding agents, wherein the library of binding agents comprise a plurality of binding agents that collectively can recognize and specifically bind to at least 5 different types of proteinogenic amino acids. In some embodiments, the binding agent is part of a library of binding agents, wherein the binding agents comprises a plurality of binding agents that collectively can recognize and specifically bind to all 20 types of standard proteinogenic amino acids. In some embodiments, a binding agent of the library of binding agents comprises a plurality of polyclonal antibodies. In some embodiments, the library of binding agents comprises a plurality of monoclonal antibodies.

[0030] In some embodiments, the binding agent comprises an immunoglobulin G (IgG).

[0031] In some embodiments, the binding agent is generated by immunizing an animal with an immunogenically effective amount of a composition comprising a carrier protein coupled to an amino acid or amino acid derivative. In some embodiments, the composition comprises the carrier protein comprising a polymeric linker that couples to the amino acid or amino acid derivative. In some embodiments, the polymeric linker comprises polyethylene glycol (PEG).

[0032] In some embodiments, the binding agent is a monoclonal antibody.

[0033] In some embodiments, the binding agent comprises an engineered scFv. In some embodiments, the engineered scFv is derived from an IgG. In some embodiments, the engineered scFv is engineered using directed evolution. In some embodiments, the engineered scFv is affinity matured using yeast surface display, phage display, ribosome display, or continuous evolution. In some embodiments, the engineered scFv has improved specificity to the one type of proteinogenic amino acid or derivative thereof through use of yeast surface display.

[0034] In some embodiments, the derivative or the derivatives thereof comprise a chemically modified amino acid. In some embodiments, the chemically modified amino acid comprises a phenylthiohydantoin, phenylthiocarbamyl, or anilinothiazolinone derivative of the amino acid. In some embodiments, the chemically modified amino acid comprises an amino acid coupled to a linker. In some embodiments, the linker is coupled to the amino acid at an N- terminal end. In some embodiments, the linker coupled to the chemically modified amino acid comprises a phenylthiohydantoin, phenylthiocarbamyl, or anilinothiazolinone moiety. In some embodiments, the linker is coupled to the amino acid at a C-terminal end. In some embodiments, the linker comprises a linking nucleic acid molecule. In some embodiments, the linker comprises an amino acid reactive group and a linking nucleic acid molecule.

[0035] In some embodiments, binding of the binding agent to the amino acid or derivative thereof is measured using an indirect enzyme-linked immunosorbent assay (ELISA). In some embodiments, binding of the binding agent to the amino acid or derivative thereof is measured using surface plasmon resonance. In some embodiments, binding of the binding agent to the amino acid or derivative thereof is measured using bio layer interferometry.

[0036] In some embodiments, the binding agent comprises a detectable label. In some embodiments, the detectable label comprises a nucleic acid molecule, a fluorophore, a mass tag, or a protein. In some embodiments, the detectable label comprises a protein, wherein the protein comprises an additional antibody or additional antibody fragment. In some embodiments, the detectable label comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a barcode sequence. In some embodiments, the detectable label is coupled to the binding agentusing a moiety selected from the group consisting of biotin, desthiobiotin, avidin, streptavidin, neutravidin, SpyCatcher, SpyTag, SNAP tag, a click chemistry moiety, and a cysteine tag. In some embodiments, the binding agent comprises a noncanonical amino acid, wherein the detectable label is coupled to the binding agent via the noncanonical amino acid. In some embodiments, the noncanonical amino acid comprises a click chemistry moiety.

[0037] In some embodiments, the binding agent specifically binds to phenylalanine or a derivative thereof over all other types of standard proteinogenic amino acids or derivatives thereof. In some embodiments, the binding agent specifically binds to leucine or a derivative thereof over all other types of standard proteinogenic amino acids or derivatives thereof. In some embodiments, the binding agent specifically binds to valine or a derivative thereof over all other types of standard proteinogenic amino acids or derivatives thereof. In some embodiments, the binding agent specifically binds to tyrosine or a derivative thereof over all other types of standard proteinogenic amino acids or derivatives thereof. In some embodiments, the binding agent specifically binds to proline or a derivative thereof over all other types of standard proteinogenic amino acids or derivatives thereof.

[0038] In another aspect, provided herein is an antibody fragment that specifically binds a free amino acid or derivative thereof, wherein the free amino acid is not directly coupled to a peptide.

[0039] In some embodiments, the free amino acid is a cleaved amino acid. In some embodiments, the cleaved amino acid is cleaved using a chemical stimulus. In some embodiments, the chemical stimulus comprises use of an isothiocyanate. In some embodiments, the chemical stimulus comprises use of an acid.

[0040] In some embodiments, the derivative or the derivatives thereof comprise a chemically modified amino acid. In some embodiments, the chemically modified amino acid comprises a phenylthiohydantoin, phenylthiocarbamyl, or anilinothiazolinone derivative of the amino acid. In some embodiments, the chemically modified amino acid comprises an amino acid coupled to a linker. In some embodiments, the linker is coupled to the amino acid at an N- terminal end. In some embodiments, the linker coupled to the chemically modified amino acid comprises a phenylthiohydantoin, phenylthiocarbamyl, or anilinothiazolinone moiety. In some embodiments, the linker is coupled to the amino acid at a C-terminal end. In some embodiments, the linker comprises a linking nucleic acid molecule. In some embodiments, the linker comprises an amino acid reactive group and a linking nucleic acid molecule.

[0041] In some embodiments, the antibody fragment is part of a library of antibodies or antibody fragments, wherein the library of antibodies or antibody fragments comprise a pluralityof antibodies or antibody fragments that collectively can recognize and specifically bind to at least 5 different types of proteinogenic amino acids. In some embodiments, the antibody fragment is part of a library of antibodies or antibody fragments, wherein the library of antibodies or antibody fragments comprise a plurality of antibodies or antibody fragments that collectively can recognize and specifically bind to all 20 types of standard proteinogenic amino acids. In some embodiments, a binding agent of the library of antibodies or antibody fragments comprises a plurality of polyclonal antibodies. In some embodiments, the library of antibodies or antibody fragments comprises a plurality of monoclonal antibodies.

[0042] In some embodiments, the antibody fragment is derived from an antibody. In some embodiments, the antibody is an immunoglobulin G (IgG). In some embodiments, the antibody is generated by immunizing an animal with an immunogenically effective amount of a composition comprising a carrier protein coupled to an amino acid or amino acid derivative. In some embodiments, the composition comprises a carrier protein comprising a polymeric linker that couples to the amino acid or amino acid derivative. In some embodiments, the polymeric linker comprises polyethylene glycol (PEG). In some embodiments, the antibody is a monoclonal antibody.

[0043] In some embodiments, the antibody fragment comprises an engineered scFv. In some embodiments, the engineered scFv is derived from an IgG. In some embodiments, the engineered scFv is engineered using directed evolution. In some embodiments, the engineered scFv is affinity matured using yeast surface display, phage display, ribosome display, or continuous evolution. In some embodiments, the engineered scFv has improved specificity to the free amino acid or derivative thereof through use of yeast surface display.

[0044] In some embodiments, the derivative or the derivatives thereof comprise a chemically modified amino acid. In some embodiments, the chemically modified amino acid comprises a phenylthiohydantoin, phenylthiocarbamyl, or anilinothiazolinone derivative of the amino acid. In some embodiments, the chemically modified amino acid comprises an amino acid coupled to a linker. In some embodiments, the linker is coupled to the amino acid at an N- terminal end. In some embodiments, the linker coupled to the chemically modified amino acid comprises a phenylthiohydantoin, phenylthiocarbamyl, or anilinothiazolinone moiety. In some embodiments, the linker is coupled to the amino acid at a C-terminal end. In some embodiments, the linker comprises a linking nucleic acid molecule. In some embodiments, the linker comprises an amino acid reactive group and a linking nucleic acid molecule.

[0045] In some embodiments, binding of the antibody fragment specifically to the free amino acid or derivative thereof is measured using an indirect enzyme-linked immunosorbentassay (ELISA). In some embodiments, binding of the antibody fragment specifically to free amino acid or derivative thereof is measured using surface plasmon resonance. In some embodiments, binding of the antibody fragment specifically to the free amino acid or derivative thereof is measured using bio layer interferometry.

[0046] In some embodiments, the antibody fragment comprises a detectable label. In some embodiments, the detectable label comprises a nucleic acid molecule, a fluorophore, a mass tag, or a protein. In some embodiments, the detectable label comprises a protein, wherein the protein comprises an additional antibody or additional antibody fragment. In some embodiments, the detectable label comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a barcode sequence. In some embodiments, the detectable label is coupled to the antibody or antibody fragment using a moiety selected from the group consisting of biotin, desthiobiotin, avidin, streptavidin, neutravidin, SpyCatcher, SpyTag, SNAP tag, a click chemistry moiety, and a cysteine tag. In some embodiments, the antibody fragment comprises a noncanonical amino acid, wherein the detectable label is coupled to the antibody or antibody fragment via the noncanonical amino acid. In some embodiments, the noncanonical amino acid comprises a click chemistry moiety.

[0047] In some embodiments, the antibody fragment specifically binds to phenylalanine or a derivative thereof over all other types of standard proteinogenic amino acids or derivatives thereof. In some embodiments, the antibody fragment specifically binds to leucine or a derivative thereof over all other types of standard proteinogenic amino acids or derivatives thereof. In some embodiments, the antibody fragment specifically binds to valine or a derivative thereof over all other types of standard proteinogenic amino acids or derivatives thereof. In some embodiments, the antibody fragment specifically binds to tyrosine or a derivative thereof over all other types of standard proteinogenic amino acids or derivatives thereof. In some embodiments, the antibody fragment specifically binds to proline or a derivative thereof over all other types of standard proteinogenic amino acids or derivatives thereof.

[0048] In another aspect, disclosed herein is an antibody or antibody fragment that binds to isoleucine and valine amino acids or derivatives thereof, wherein the antibody or antibody fragment binds with greater affinity to isoleucine than to valine.

[0049] In some embodiments, the affinity is measured using ELISA.

[0050] In yet another aspect, provided herein is an antibody or antibody fragment that binds to phenylalanine, tyrosine, and tryptophan amino acids or derivatives thereof, wherein the antibody or antibody fragment binds with greater affinity to phenylalanine than to tyrosine or tryptophan.

[0051] In some embodiments, the affinity is measured using ELISA.

[0052] In another aspect, disclosed herein is a method of identifying amino acids, comprising: (a) providing (i) a plurality of peptides comprising a plurality of amino acids, wherein the plurality of amino acids comprises N different types of amino acids and (ii) M different types of antibody or antibody fragments, wherein an antibody or antibody fragment of the M different types of antibody or antibody fragments can bind to more than one amino acid type, wherein M and N are integers greater than 1; (b) recording an interaction between an antibody or antibody fragment of the M different types of antibody or antibody fragments with an amino acid of the plurality of amino acids or derivative thereof; (c) repeating (a) and (b) at least once to generate a binding pattern of the M different types of antibody or antibody fragments; and (d) using the binding pattern to determine an identity of each amino acid of the N different types of amino acids.

[0053] In some embodiments, (d) comprises, determining which antibody or antibody fragments of the M different types of antibody or antibody fragments bind to the amino acid to generate the binding pattern.

[0054] In yet another aspect, provided herein is a method of identifying an amino acid, comprising: (a) providing (i) a peptide comprising a plurality of amino acids, and (ii) an antibody or antibody fragment that can recognize at least two amino acids; (b) recording an interaction between the antibody or antibody fragment with an amino acid or derivative thereof of the peptide; (c) repeating (a) and (b) N times to generate a binding pattern of the antibody or antibody fragment to the amino acid; and (d) using the binding pattern to determine an identity of the amino acid or derivative thereof.

[0055] In some embodiments, during (a), a plurality of peptides is provided, and wherein (b), (c), and (d) are performed on the plurality of peptides.

[0056] In some embodiments, the amino acid is an N-terminal amino acid.

[0057] In some embodiments, the antibody or antibody fragment is part of a library of antibody or antibody fragments.

[0058] In some embodiments, (d) comprises determining a ratio of a number of recorded binding events between the antibody or antibody fragment and the amino acid to N.

[0059] In another aspect, disclosed herein is an antibody, wherein the antibody is produced by a method, comprising: (a) immunizing an animal with an immunogenically effective amount of a composition comprising a carrier protein coupled to a polymeric linker molecule, and an amino acid or derivative thereof; and (b) obtaining the antibody.

[0060] In some embodiments, the polymeric linker comprises polyethylene glycol (PEG).

[0061] In some embodiments, the carrier protein comprises bovine serum albumin (BSA).

[0062] In some embodiments, the polymeric linker is coupled to the carrier protein and the amino acid or derivative thereof.

[0063] In some embodiments, the derivative thereof comprise a chemically modified amino acid. In some embodiments, the chemically modified amino acid comprises a phenylthiohydantoin, phenylthiocarbamyl, or anilinothiazolinone derivative of the amino acid. In some embodiments, the chemically modified amino acid comprises an amino acid coupled to a linker. In some embodiments, the linker is coupled to the amino acid at an N-terminal end. In some embodiments, the linker coupled to the chemically modified amino acid comprises a phenylthiohydantoin, phenylthiocarbamyl, or anilinothiazolinone moiety. In some embodiments, the linker is coupled to the amino acid at a C-terminal end. In some embodiments, the linker comprises a linking nucleic acid molecule. In some embodiments, the linker comprises an amino acid reactive group and a linking nucleic acid molecule.

[0064] In some embodiments, the method further comprises, obtaining a polyclonal mixture of antibodies from the animal, wherein the polyclonal mixture comprises the antibody. In some embodiments, the antibody is a monoclonal antibody, wherein the method further comprises, generating the monoclonal antibody from the polyclonal mixture of antibodies. In some embodiments, the generating comprises using a hybridoma.

[0065] In some embodiments, the method further comprises, using phage panning to generate the antibody. In some embodiments, the method further comprises, obtaining a first antibody from the animal, derivatizing the first antibody to one or more single-chain fragments (scFvs), performing phage panning on the one or more scFvs, and generating the antibody from the one or more scFvs.

[0066] In another aspect, provided herein is an antibody or antibody fragment, comprising a heavy chain (VH) comprising three complementarity determining regions (CDRs) and a light chain (VL) comprising three CDRs, wherein: VH CDR #1 has an amino acid sequence selected from the group consisting of GYAFTSYK (SEQ ID NO: 1); GFNINDYH (SEQ ID NO: 2); GYTFTDYY (SEQ ID NO: 3); DFNIQDYY (SEQ ID NO: 4); GFNIKD (SEQ ID NO: 5); GFTFSSYA (SEQ ID NO: 6); GYSFTDYT (SEQ ID NO: 7); GYTFTIYW (SEQ ID NO: 8); GYTSTIYW (SEQ ID NO: 9); GYYITSGYY (SEQ ID NO: 10); and GFTFSSFG (SEQ ID NO: 11); VH CDR #2 has an amino acid sequence selected from the group consisting of IDPYNGGT (SEQ ID NO: 12); IDPENGVT (SEQ ID NO: 13); INPNNGGT (SEQ ID NO: 14); IDPENGDT (SEQ ID NO: 15); ISSGGSYT (SEQ ID NO: 16); INPYNGFT (SEQ ID NO: 17); IIPTTGYT (SEQ ID NO: 18); INPNTGYS (SEQ ID NO: 19); ISHDGNN (SEQ ID NO: 20); andISSGSTNI (SEQ ID NO: 21); VH CDR #3 has an amino acid sequence selected from the group consisting of ARSGYSFNY (SEQ ID NO: 22); NGINYEGHFDY (SEQ ID NO: 23);ANWRRSGWFAY (SEQ ID NO: 24); NAVRYANYPRAMDY (SEQ ID NO: 25); NAMRYGNYPRPMDY (SEQ ID NO: 26); ARPGDGYYKYFDV (SEQ ID NO: 27); AIWLRRENFDY (SEQ ID NO: 28); ALWLRRENFDY (SEQ ID NO: 29); EMPDYSGYHDY (SEQ ID NO: 30); SMPDYSAFHDY (SEQ ID NO: 31); ESFIRTTYKVPVDY (SEQ ID NO: 32); and ARFDYDRGAFAY (SEQ ID NO: 33); VL CDR #1 has an amino acid sequence selected from the group consisting of QGISGN (SEQ ID NO: 34); QSLLNSRIRKNH (SEQ ID NO: 35); QDINSY (SEQ ID NO: 36); QSLLNSRTRKNH (SEQ ID NO: 37);QSLLNSRTRKNS (SEQ ID NO: 38); SSVSY (SEQ ID NO: 39); QTIVHSNGNTY (SEQ ID NO: 40); QSLLNSRTRKNN (SEQ ID NO: 41); QSLFNSRTRKNY (SEQ ID NO: 42);ETVDYYGTSL (SEQ ID NO: 43); and QSLLKNSNQKNY (SEQ ID NO: 44); VLCDR #2 has an amino acid sequence selected from the group consisting of HGT; WAS; RAN; ATS; KVS; AAS; or VL CDR #3 has an amino acid sequence selected from the group consisting of VQYAQFPYT (SEQ ID NO: 51); KQSYNLRT (SEQ ID NO: 52); LQYDEFPPT (SEQ ID NO: 53); KQSYNLPT (SEQ ID NO: 54); QQWRSNPPT (SEQ ID NO: 55); FQGSHVPYT (SEQ ID NO: 56); KQSFNLLT (SEQ ID NO: 57); KQAYNLQT (SEQ ID NO: 58); QQSRKIPWT (SEQ ID NO: 59); and QQFYNYLT (SEQ ID NO: 60).

[0067] In another aspect, provided herein is an antibody or antibody fragment comprising a heavy chain sequence selected from Table 7.

[0068] In another aspect, provided herein is an antibody or antibody fragment comprising a light chain sequence selected from Table 8.

[0069] In another aspect, provided herein is a nucleic acid composition comprising a first nucleic acid encoding a variable heavy chain region comprising amino acid residues of Table 7 and a second nucleic acid encoding a variable light chain region comprising an amino acid residues of Table 8.

[0070] In another aspect, provided herein is an antibody or antibody fragment comprising a variable heavy chain sequence selected from Table 9.

[0071] In another aspect, provided herein is an antibody or antibody fragment comprising a variable light chain sequence selected from Table 10.

[0072] In another aspect, provided herein is a nucleic acid composition comprising a first nucleic acid encoding a heavy chain region comprising amino acid residues of Table 7 or Table 9 and a second nucleic acid encoding a light chain region comprising amino acid residues of Table 8 or Table 10.

[0073] In another aspect, provided herein is a kit comprising a plurality of antibody or antibody fragments, wherein said plurality of antibody or antibody fragments specifically or semi-specifically bind to at least 5 different types of proteinogenic amino acids or derivatives thereof.

[0074] In another aspect, provided herein is a method for processing a polymeric analyte comprising a plurality of monomers, the method comprising: (a) providing the polymeric analyte, a capture moiety, and a polymerizable molecule; (b) coupling a monomer of the plurality of monomers to the capture moiety to generate a monomer-capture moiety complex; (c) contacting the monomer-capture moiety complex with a binding agent coupled to a barcode molecule; and (d) using a nuclease to couple the barcode molecule to the polymerizable molecule or the capture moiety.

[0075] In some embodiments, the nuclease is a CRISPR-associated (Cas) enzyme or a prime editor enzyme.

[0076] In some embodiments, the monomer-capture moiety complex of (c) comprises a cleaved monomer-capture moiety complex, and further comprising, prior to (c), cleaving the monomer-capture moiety complex of (b) to generate the cleaved monomer-capture moiety complex.

[0077] In some embodiments, the method further comprises, subsequent to (a)-(d), (e) decoupling the monomer from the capture moiety. In some embodiments, the method further comprises repeating (a)-(e), thereby sequencing the polymeric analyte.

[0078] In some embodiments, the method further comprises repeating (a)-(d).

[0079] In some embodiments, the polymerizable molecule is coupled to the polymeric analyte.

[0080] In some embodiments, the polymerizable molecule is associated with the polymeric analyte.

[0081] In some embodiments, the polymerizable molecule comprises a nucleic acid barcode molecule comprising a barcode sequence.

[0082] In some embodiments, the polymeric analyte comprises a peptide.

[0083] In some embodiments, the capture moiety and the polymerizable molecule each comprise a nucleic acid molecule.

[0084] In some embodiments, the polymerizable molecule comprises a tandem array of CRISPR-Cas9 target sites. In some embodiments, the tandem array of CRISPR-Cas9 target sites comprise a plurality of target sites comprising a first target site, wherein the first target site is active and wherein the plurality of target sites except for the first target site is inactive. In someembodiments, the plurality of target sites except for the first target site is truncated and therefore inactive. In some embodiments, the plurality of target sites except for the first target site is truncated at the 5’ ends of each target site of the plurality of target sites. In some embodiments, the barcode molecule comprises a pegRNA molecule comprising a barcode sequence. In some embodiments, the pegRNA further comprises a key sequence, wherein the key sequence is configured to activate a second target site of the plurality of target sites, wherein the second target site is located adjacent to the first target site. In some embodiments, (d) comprises using the Cas or prime editor enzyme to couple the pegRNA to the first target site, thereby inserting the barcode sequence and the key sequence adjacent to the first target site. In some embodiments, the inserting results in deactivation of the first target site and activation of the second target site.

[0085] In some embodiments, the polymerizable molecule comprises a protospacer adjacent motif (PAM) and a first CRISPR-Cas9 target site. In some embodiments, the barcode molecule comprises a pegRNA molecule comprising a barcode sequence and a propagator sequence comprising a second CRISPR-Cas9 target site. In some embodiments, (d) comprises using the Cas or prime editor enzyme to couple the pegRNA between the first CRISPR-Cas9 target site and the PAM, thereby inserting the barcode sequence and the propagator sequence adjacent to the first CRISPR-Cas9 target site. In some embodiments, the inserting results in deactivation of the first CRISPR-Cas9 target site and activation of the second CRISPR-Cas9 target site.

[0086] In some embodiments, the polymeric analyte comprises a peptide and wherein the plurality of monomers comprises a plurality of amino acids of the peptide.

[0087] In some embodiments, the binding agent comprises an antibody or antibody fragment.

[0088] In some embodiments, the capture moiety or the polymerizable molecule is coupled to a substrate.

[0089] In some embodiments, the capture moiety and the polymerizable molecule are coupled to the substrate.

[0090] In another aspect, disclosed herein is a composition, comprising: an antibody or antibody fragment coupled to a guide RNA (gRNA).

[0091] In some embodiments, the gRNA comprises a barcode sequence. In some embodiments, the barcode sequence identifies the antibody or antibody fragment. In some embodiments, the gRNA further comprises a key sequence, wherein the key sequence is configured to insert into a tandem array of CRISPR-Cas9 target sites comprising a first target site and a second target site. In some embodiments, the key sequence is configured to insertbetween the first target site and the second target site. In some embodiments, the gRNA further comprises a propagator sequence comprising a CRISPR-Cas9 target site. In some embodiments, the propagator sequence is configured to insert into or adjacent to a polymerizable molecule comprising an additional CRISPR-Cas9 target site and a PAM. In some embodiments, the propagator sequence is configured to insert between the additional CRISPR-Cas9 target site and the PAM. In some embodiments, the antibody or the antibody fragment specifically binds to a monomer of a polymeric analyte comprising a plurality of monomers. In some embodiments, the polymeric analyte comprises a peptide and wherein the antibody or the antibody fragment binds to an amino acid or derivative thereof. In some embodiments, the antibody or antibody fragment specifically binds to one of the 20 standard proteinogenic amino acids or derivative thereof. In some embodiments, the derivative thereof comprises a chemically modified amino acid. In some embodiments, the chemically modified amino acid comprises a phenylthiocarbamoyl, phenylthiohydantoin, or anilinothiazolinone derivative of the amino acid.

[0092] In another aspect, provided herein is a bispecific binding agent, comprising a first binding moiety and a second binding moiety; wherein the first binding moiety binds to a first small molecule; wherein the second binding moiety binds to a second small molecule; and wherein the first small molecule is different from the second small molecule.

[0093] In some embodiments, the first small molecule comprises an amino acid or derivative thereof. In some embodiments, the derivative thereof comprises a chemically modified amino acid. In some embodiments, the chemically modified amino acid comprises a phenylthiocarbamoyl, phenylthiohydantoin, or anilinothiazolinone derivative of the amino acid.

[0094] In some embodiments, the second small molecule comprises a hapten. In some embodiments, the hapten comprises or is coupled to a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises a nucleic acid barcode molecule. In some embodiments, the nucleic acid barcode molecule identifies the bispecific binding agent. In some embodiments, the nucleic acid barcode molecule comprises temporal information. In some embodiments, the hapten is selected from the group consisting of digoxigenin, 2,4-DNP, and penicillin.

[0095] In some embodiments, the bispecific binding agent comprises an antibody, antibody fragment, nanobody, or aptamer.

[0096] In another aspect, provided herein is a method for processing a polymeric analyte, comprising: (a) providing the polymeric analyte, wherein the polymeric analyte comprises a plurality of monomers; (b) coupling a monomer of the plurality of monomers to a capture moiety; (c) cleaving the monomer from the polymeric analyte, thereby generating a cleavedmonomer; (d) contacting the cleaved monomer with a bispecific binding agent; wherein the bispecific binding agent is configured to recognize and bind to the cleaved monomer and a small molecule; (e) providing the small molecule; and (f) contacting the bispecific binding agent with the small molecule.

[0097] In some embodiments, the polymeric analyte comprises a peptide and wherein the plurality of monomers is a plurality of amino acids.

[0098] In some embodiments, (b) is mediated using a linker. In some embodiments, the linker comprises a monomer-reactive group and a second reactive group. In some embodiments, the second reactive group comprises a click chemistry moiety. In some embodiments, the method further comprises contacting the linker with a linking nucleic acid molecule, wherein the linking nucleic acid molecule comprises a third reactive group capable of reacting with the second reactive group.

[0099] In some embodiments, the cleaved monomer comprises an amino acid or derivative thereof. In some embodiments, the derivative thereof comprises a chemically modified amino acid. In some embodiments, the chemically modified amino acid comprises a phenylthiocarbamoyl, phenylthiohydantoin, or anilinothiazolinone derivative of the amino acid.

[0100] In some embodiments, the second small molecule comprises a hapten. In some embodiments, the hapten comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises a nucleic acid barcode molecule. In some embodiments, the nucleic acid barcode molecule identifies the bispecific binding agent. In some embodiments, the nucleic acid barcode molecule comprises temporal information. In some embodiments, the method further comprises, coupling the nucleic acid barcode molecule to an additional nucleic acid molecule. In some embodiments, the additional nucleic acid molecule is located adjacent to the cleaved monomer on the substrate. In some embodiments, the hapten is selected from the group consisting of digoxigenin, 2,4-DNP, and penicillin.

[0101] In some embodiments, the bispecific binding agent comprises an antibody, antibody fragment, nanobody, or aptamer.

[0102] In some embodiments, the capture moiety comprises a nucleic acid molecule. In some embodiments, the capture moiety is coupled to a substrate. In some embodiments, the substrate comprises a bead.

[0103] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0104] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0105] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0106] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. 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.BRIEF DESCRIPTION OF THE DRAWINGS

[0107] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0108] FIG. 1A schematically shows an example workflow for processing polymeric analytes molecules (e.g., peptides) described herein. FIG. IB schematically shows another example workflow for processing polymeric analytes in solution. FIG. 1C schematically shows another example workflow for processing polymeric analytes and detection. FIG. ID schematically shows another workflow for processing polymeric analytes using a nuclease for barcoding. FIG. IE schematically shows another example workflow for processing polymeric analytes and detection using a nanopore sequencing system. FIG. IF schematically shows another example workflow for processing polymeric analytes and detection using a nanopore sequencing system. FIG. 1G schematically shows another example workflow for processingpolymeric analytes and detection in solution. FIG. 1H schematically shows an example workflow for processing polymeric analytes using a bispecific binding agent.

[0109] FIG. 2 schematically shows an example linker for connecting polymerizable molecules to polymeric analytes.

[0110] FIG. 3 schematically shows a method of using binding patterns of multi-specific binding agents to identify an amino acid.

[0111] FIG. 4 schematically shows an example of a sequencing approach to analyze or characterize polymeric analytes. FIG. 4 discloses SEQ ID NO: 113.

[0112] FIG. 5 schematically shows a computer system that is programmed or otherwise configured to implement methods provided herein.

[0113] FIG. 6 shows data of a plurality of binding agents described herein that specifically or semi-specifically bind to an amino acid derivative.

[0114] FIG. 7 shows ELISA data for an anti-leucine binding agent.

[0115] FIG. 8 shows ELISA data for a plurality of binding agents that selectively bind a single amino acid type or derivative thereof over all other amino acid types or derivatives thereof.

[0116] FIG. 9 shows ELISA data for binding agents that recognize an amino acid derivative and not the native amino acid when attached to a peptide.

[0117] FIG. 10 shows ELISA data exhibiting how bioconjugation using multiple approaches does not deleteriously impact the binding profile of a binding agent.

[0118] FIG. 11A shows example biolayer interferometry (BLI) data for an antiphenylalanine binding agent purified from a mouse monoclone. FIG. 11B shows example BLI data for a recombinant anti -phenylalanine binding agent. FIG. 11C shows example BLI data for an anti-leucine binding agent purified from a mouse monoclone. FIG. 11D shows example BLI data for a recombinant anti-leucine binding agent. FIG. HE shows example BLI data for a recombinant anti -tryptophan binding agent. FIG. HF shows example BLI data for an antivaline binding agent purified from a mouse monoclone.

[0119] FIG. 12A shows example imaging data indicating colocalization of an anti-Phe binding agent with its cognate molecule, a Phe derivative. FIG. 12B shows example imaging data indicating colocalization of an anti-Phe binding agent with its cognate molecule. FIG. 12C shows example imaging data indicating specificity or colocalization efficiency of an anti-Phe binding agent with its cognate molecule and the relative on-target percentage.

[0120] FIG. 13 shows example data for using unique binding patterns to identify a plurality of amino acid types using six different binding agents.

[0121] FIG. 14 shows example data of a directed evolution approach (yeast display) for improving the specificity of a binding agent.DETAILED DESCRIPTION

[0122] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.Definitions

[0123] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0124] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0125] References to “one embodiment,” “an embodiment,” “example embodiment,” “some embodiments,” “certain embodiments,” “various embodiments,” etc., indicate that the embodiment s) of the disclosed technology so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment” does not necessarily refer to the same embodiment, although it may.

[0126] Ranges may be expressed herein as from “about” or “approximately” or “substantially” one particular value and / or to “about” or “approximately” or “substantially” another particular value. When such a range is expressed, other exemplary embodiments include from the one particular value and / or to the other particular value. Further, the term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean arange of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably still up to ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value.

[0127] By “comprising” or “containing” or “including” is meant that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, material, particles, method steps have the same function as what is named.

[0128] Throughout this description, various components may be identified having specific values or parameters, however, these items are provided as exemplary embodiments. Indeed, the exemplary embodiments do not limit the various aspects and concepts of the present disclosure as many comparable parameters, sizes, ranges, and / or values may be implemented. The terms “first,” “second,” and the like, “primary,” “secondary,” and the like, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[0129] As used herein, the term “protein” generally refers to a molecule comprising two or more amino acids joined by a peptide bond. A protein may also be referred to as a “polypeptide”, “oligopeptide”, or “peptide”. A protein can be a naturally occurring molecule, or a synthetic molecule (e.g., an artificial protein, peptide, enzyme). A protein may include one or more non-natural amino acids, modified amino acids, or non-amino acid linkers. A protein may contain D-amino acid enantiomers, L- amino acid enantiomers or both. Amino acids of a protein may be modified naturally or synthetically, such as by post-translational modifications or by chemical modification. In some circumstances, different proteins may be distinguished from each other based on different genes from which they are expressed in an organism, different primary sequence length or different primary sequence composition. Proteins expressed from the same gene may nonetheless be different proteoforms, for example, being distinguished based on non-identical length, non-identical amino acid sequence or non-identical post-translational modifications. Different proteins can be distinguished based on one or both of gene of origin and proteoform state.

[0130] As used herein, the term “peptide” may refer to any short, single peptide chain. A peptide may be no more than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less than about 5 amino acids in length. A peptide may have a known or unknownbiological function or activity. Peptides can include natural, synthetic, modified, or degraded proteins or peptides, or a combination thereof. Peptides can include proteinogenic, natural, synthetic, or modified amino acids or amino acid residues, or a combination thereof.

[0131] As used herein, the term “single analyte” may refer to an analyte that is individually manipulated or distinguished from other analytes. A single analyte may comprise a biomolecule or a synthetic molecule. A single analyte may comprise a small molecule. A single analyte can be a single molecule (e.g., a single biomolecule such as a single protein, nucleic acid molecule, affinity reagent, lipid, carbohydrate, etc.), a single complex of two or more molecules (e.g., a multimeric protein having two or more separable subunits, a single protein attached to a nucleic acid molecule or a single protein attached to an affinity reagent), a single particle, or the like. Reference herein to a “single analyte” in the context of a composition, system or method herein does not necessarily exclude application of the composition, system or method to multiple single analytes that are manipulated or distinguished individually, unless indicated contextually or explicitly to the contrary.

[0132] As used herein, “polypeptide” refers to two or more amino acids linked together by a peptide bond. The term “polypeptide” includes proteins that have a C-terminal end and an N- terminal end as generally known in the art and may be synthetic in origin or naturally occurring. As used herein “at least a portion of the polypeptide” refers to 2 or more amino acids of the polypeptide. A polypeptide may comprise one or more peptides. Optionally, a portion of the polypeptide includes at least: 1, 5, 10, 20, 30 or 50 amino acids, either consecutive or with gaps, of the complete amino acid sequence of the polypeptide, or the full amino acid sequence of the polypeptide.

[0133] As used herein, the term “sample” refers to a collected substance or material that comprises or is suspected to comprise one or more analytes of interest (e.g., biomolecules, e.g., polypeptides). A sample may be modified for purposes such as storage or stability. A sample may be naturally occurring or synthetic. A sample may be processed to separate or remove unwanted fractions or impurities from the analyte(s) of interest. A sample may be enriched or purified. For example, a sample may comprise a fraction of a separation process (e.g., chromatography, fractionation, electrophoresis, etc.). Alternatively, a sample may not be subjected to processing that separates or removes any unwanted fractions or impurities from the analyte(s) of interest. A sample may be obtained from any suitable source or location, including from organisms, cells, tissues, cell preparations, cell-free compositions, the environment (e.g., air, water, dirt, soil, agriculture, soil, dust, sewage). A sample may be obtained from an organism or part of an organism, such as from a fluid, tissue, or cell. A sample may include biological and / or non-biological components. As used herein, the terms “biological sample” or “biological source” refer to a sample that is derived from a predominantly biological system or organism, such as one or more viral particles, cells (e.g. individualized cells), organelles (e.g. individualized organelles), tissues, bodily fluids, bone, cartilage, and exoskeleton. A biological sample may comprise a prokaryotic cell (e.g., bacteria) or eukaryotic cell (e.g., fungus, protist, algae, plant, animal). A biological sample may comprise a majority of biological material on a mass basis, excluding the weight of fluid within the sample. Biological samples may comprise one or more proteins, referred to herein as protein samples. Biological samples can be acquired from various sources, e.g., from a clinical patient sample, such as blood, serum, plasma, Cerebral Spinal Fluid (CSF), saliva, mucosal secretions, sputum, urine, lymph, perspiration, vaginal fluid, semen, fecal matter, amniotic fluid, perspiration, synovial fluid, fine needle aspirates, a tissue biopsy, a tumor biopsy, etc. diabodies, monobodies, single domain antibodies (sdAb), as well as bi-functional (i.e., bispecific, e.g., bi-specific T-cell engager) etc. A biological sample may be processed to purify and retain one or more biomolecules (e.g., proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, metabolites, etc.) from the biological sample. A biological sample (e.g., a protein sample) may be derived from cultured cells, which may be treated or untreated. A biological sample (e.g., a protein sample) can also result from tissue specimens, such as biopsy samples, which may optionally be processed to liberate biomolecules (e.g., proteins) contained therein. Tissue samples may also be derived from in vivo specimens, including fresh, frozen, acute, and fixed tissues.

[0134] As used herein, the terms “antibody” and “immunoglobulin” may generally refer to proteins that can recognize and bind to a specific antigen. An antibody or immunoglobulin may refer to an antibody isotype, a full-length antibody (e.g., comprising two identical light chains and two identical heavy chains), fragments of antibodies including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, linear antibodies, and fusion proteins including an antigen-binding portion of an antibody and a nonantibody protein. The antibodies may be detectably labeled, e.g., with a fluorophore, radioisotope, enzyme (e.g., a peroxidase) which generates a detectable product, fluorescent protein, nucleic acid barcode sequence, and the like. The antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair) or analogs thereof (e.g., desthiobiotin, neutravidin, streptavidin, etc.), SpyCatcher and SpyTag, SNAP -tag® and the like. The antibodies may comprise a fusion protein, e.g., an antibody and a detectable protein such as a fluorescent protein, e.g., green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), mVenus, tdTomato, and the like.Also encompassed by the terms are nanobodies, Fab', Fv, F(ab')2, scFv, bis-scFv, diabodies, triabodies, tetrabodies, minibodies, and other antibody fragments that retain specific binding to antigen. Antibodies may exist in a variety of other forms including, for example, Fv, Fab, and (Fab)2, diabodies, monobodies, single domain antibodies (sdAb), as well as bi-functional (i.e., bi- specific, e.g., bi-specific T-cell engager) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), which are incorporated herein by reference). (See, generally, Hood et al., Immunology, Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are herein incorporated by reference). Naturally occurring immunoglobulins or antibody types include immunoglobulin A, immunoglobulin G, immunoglobulin D, immunoglobulin E, immunoglobulin M, or other immunoreactive components. An antibody may comprise complementarity determining regions (CDRs) in both the light chain and heavy chain, while the more highly conserved portions of the variable domains are referred to as the framework (FR).

[0135] The term “antibody fragment” may generally be used to refer to fragments of antibodies including, but not limited to, Fab, Fv, scFv, and Fd fragments, nanobodies, Fab', F(ab')2, bis-scFv, diabodies, triabodies, tetrabodies, minibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins including an antigen-binding portion of an antibody. A Fab fragment generally refers to an antibody fragment that contains the constant domain of the light chain, the first constant domain (CHI) of the heavy chain and the variable domains of the light chain and heavy chain (VL and VH, respectively). A Fab' fragment generally comprise additional residues at the carboxyl terminus of the heavy chain CHI domain as compared to Fab fragments, including one or more cysteine residues from the antibody hinge region. Fab' fragments are produced by cleavage of the disulfide bond at the hinge cysteine residues of the F(ab')2 pepsin digestion product. Fab and F(ab')2 fragments lack a fragment crystallizable (Fc) region of an intact antibody. A single-chain antibody (or single-domain antibody) is composed of a single VH or VL domain.

[0136] As used herein, “Fc” region generally refers to the fragment crystallizable constant region of an antibody. The Fc region generally does not comprise an antigen-specific binding region. In IgG, IgA, and IgD antibody isotypes, the Fc region contains two identical protein fragments, derived from the second and third constant domains (CH2 and CH3) of the two heavy chains of an antibody. IgM and IgE Fc regions contain three heavy chain constant domains (CH2, CH3, and CH4) in each polypeptide chain. A variant Fc region comprises an amino acid sequence that differs from that of a native sequence Fc region. A “native Fc region” or “native Fc sequence”comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. A non-naturally occurring Fc region or variant sequence Fc region may refer to an Fc region that has one or more amino acid mutations on a position, an Fc region or variant sequence that has a percent homology or identity to a native Fc region.

[0137] As used herein, “Fv” fragment generally refers to the minimum fragment of an antibody that contains a complete target recognition and binding site. The Fv contains a dimer of one heavy chain variable region (VH) and one light chain variable region (VL) in association with one another. The three complementarity-determining regions (CDRs) of each variable domain (VH or VL) may interact to define a target binding site on the surface of the VH-VL dimer. In some cases, the six CDRs confer target binding specificity; however, in some instances, a single variable domain (e.g., just the VH or VL) having only three CDRs, e.g., a single-domain antibody (sdAb), may specifically recognize and bind the target. A single-chain Fv (“scFv”) comprises the VH and VL domains. The VH and VL domains of an scFv may be comprised within a single polypeptide chain, often with a polypeptide linker between the VH and VL domains. A bispecific scFv (bis- scFv) may comprise two VH and VL domains and may be homodimeric (same VH and VL) or heterodimeric (different VH and VL).

[0138] As used herein, the term “diabodies” generally refers to antibody fragments that comprise two sets of VH and VL domains that are joined in an inter-chain fashion. Diabodies typically are bivalent (have two antigen-binding sites). Diabodies can be heterodimers of two scFv fragments, in which the VH and VL domains of the two scFv fragments are present on different polypeptide chains.

[0139] As used herein, the term “variable region” generally refers to the domain of an antibody heavy chain or light chain that is involved in binding to the antigen. A variable domain typically comprises four conserved framework regions and three complementarity determining regions. In some instances, a single VH or VL domain is sufficient to bind the antigen; in other instances, an Fv containing VH and VL domains is required to recognize and bind to an antigen.

[0140] The term “complementarity determining region (CDR)” as used herein generally refers to the hypervariable region of an antibody variable domain which is hypervariable in sequence and may form structurally defined loops or contain the antigen-contacting residues. Antibodies generally comprise six CDRs, three in the VH and three in the VL. The CDRs in each chain may be in interspersed between the four FR regions.

[0141] The term “monoclonal antibody,” as used herein, generally refers to an antibody that is derived from a single clone. A monoclonal antibody is not limited to antibodies produced through hybridoma technology. A monoclonal antibody may be derived from a single clone,including any eukaryotic, prokaryotic, archaeal, or viral (e.g., phage) clone. Monoclonal antibodies may be prepared using any useful technique or combination of techniques such as hybridoma, recombinant, and directed evolution approaches such as yeast display, yeast surface display, phage display, bacterial display, mammalian display, mammalian surface display, ribosome display, or mRNA display technologies.

[0142] The term “epitope” or “antigenic determinant” generally refers to a site on an antigen to which an antibody specifically binds, e.g., as defined by the specific method used to identify it. Epitopes may comprise a portion of a peptide, e.g., several amino acids, which can be formed from contiguous amino acids or noncontiguous amino acids (which may be spatially juxtaposed by tertiary folding of the peptide). As described herein, an epitope may comprise a single amino acid or a derivative thereof (e.g., a chemically or biologically modified amino acid), which may not be a part or portion of a peptide (e.g., a physically isolated amino acid or derivative thereof).

[0143] As referred to herein, a “chimeric antibody” refers to an antibody in which a portion of the heavy chain, the light chain, or both is derived from a first species or antibody class immunoglobulin and the remainder regions is derived from a second species or antibody class that is different from the first species or antibody class.

[0144] The antibodies of the present disclosure may be derived from an animal including but not limited to a mouse, rat, rabbit, non-human primate, llama, chicken, or other animal. Alternatively, or in addition to, the antibodies may be engineered to be non-animal or chimeric antibodies, e.g., recombinant antibodies, in vitro-derived antibodies, e.g., antibodies grown in useful cell lines such as monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells); baby hamster kidney cells (BEK); mouse sertoli cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, myeloma cells such as YO, NS0 and Sp2 / 0. Additional examples of mammalian cell lines for use in generating antibodies can be found in Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003), which is incorporated by reference herein.

[0145] The antibodies of the present disclosure may be polyclonal, monoclonal, genetically engineered, or otherwise modified, including but not limited to chimeric antibodies, humanized antibodies, phenotypically engineered, etc.

[0146] The antibodies and nucleic acid molecules described herein may be described by the polypeptide or nucleic acid sequences. Unless otherwise indicated, polypeptide sequences are provided in the N-to-C orientation and nucleic acid sequences are provided in the 5’-to-3’ orientation.

[0147] As described herein, a “percent (%) amino acid sequence identity” or “homology” with respect to the polypeptide or antibody sequences identified herein refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide or antibody being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, % amino acid sequence identity values are generated using the sequence comparison computer program BLAST.

[0148] “Binding” or “coupling” as used herein generally refers to a covalent or non-covalent interaction between two molecules (referred to herein as “binding partners”, e.g., a substrate and an enzyme or an antibody and an epitope). Binding between binding partners may be specific or non-specific.

[0149] As used herein, “specifically binds” or “binds specifically” generally refers to an interaction between binding partners (e.g., a binding partner and a cognate molecule) such that the binding partners bind to one another, but do not bind to other molecules that may be present in the environment (e.g., in a biological sample, in tissue, in an in vitro assay) under a set of conditions. A specific binding interaction may entail a binding partner that binds to a cognate molecule. The specific binding interaction may entail the binding of the binding partner to its cognate molecule at a significantly or substantially higher level or with greater affinity as compared to the binding of the binding partner to a non-cognate molecule. A specific binding interaction may entail a first binding partner that has greater selectivity of binding to the cognate molecule as compared to a non-cognate molecule.

[0150] The binding agents described herein, e.g., antibodies, antibody fragments, nucleic acid binding agents, etc. can include derivatized binding agents. Derivatized binding agents can comprise a chemical or enzymatic modification. The modification can be covalent or noncovalent. Non-limiting examples modifications include, but are not limited to, acylation, acetylation,alkylation (including methylation), biotinylation, butyrylation, carb amyl ati on, carbonylation, amidation, deamidation, deiminiation, diphthamide formation, disulfide bridge formation, eliminylation, flavin attachment, formylation, gamma-carboxylation, glutamyl ati on, glycylation, glycosylation, glypiation, heme C attachment, hydroxylation, hypusine formation, iodination, isoprenylation, lipidation, lipoylation, malonylation, methylation, myristolylation, oxidation, transglutamination, palmitoylation, pegylation, phosphopantetheinylation, phosphorylation, prenylation, propionyl ati on, retinylidene Schiff base formation, S-glutathionylation, S- nitrosylation, S-sulfenylation, selenation, succinylation, sulfination, ubiquitination, sumoylation, disulfide bond formation, and C-terminal amidation. A modification can occur on the amino terminus and / or the carboxyl terminus of a peptide. Modifications of the terminal amino group include, but are not limited to, des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include, but are not limited to, amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications (e.g., wherein lower alkyl is C1-C4 alkyl). A modification can include chemical modifications such as a protecting or blocking group. The binding agents can contain one or more non-natural amino acids.

[0151] The terms “nucleic acid”, “nucleic acid molecule”, “oligonucleotide” and “polynucleotide” may be used interchangeably herein and generally refer to a polymeric form of naturally occurring or synthetic nucleotides, or analogs thereof, of any length. A nucleic acid molecule may comprise one or more deoxyribonucleotides, deoxynucleotide triphosphates, dideoxynucleotide triphosphates, ribonucleotides, hexitol nucleotides, cyclohexane nucleotides, or analogs or combinations thereof. A nucleic acid molecule may comprise, e.g., DNA, RNA, HNA, and modified forms thereof. A nucleic acid molecule may comprise nucleotides that are linked by phosphodiester bonds. A nucleic acid molecule may have any two- or three-dimensional structure, and may perform any function, known or unknown. A nucleic acid molecule may be single stranded, double stranded, or partially double stranded. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, noncoding RNA, small interfering RNA, short hairpin RNA, micro RNA, scaRNA, ribozymes, riboswitches, viral RNA, complementary DNA (cDNA), cosmid DNA, mitochondrial DNA, chromosomal or genomic DNA, viral DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, nucleic acid adapters, and primers. The nucleic acid molecule may be linear, circular, or any other geometry. Examples of polynucleotide analogs include but are not limited to xeno nucleic acid (XNA), bridged nucleic acid (BNA), glycol nucleic acid (GNA), hexitol nucleic acid (HNA), cyclohexane nucleic acid(CeNA), 2 ’-F- Arabinonucleic acids (2’-F-ANA), peptide nucleic acids (PNAs), yPNAs, morpholino polynucleotides, locked nucleic acids (LNAs), threose nucleic acid (TNA), 2'-O- Methyl polynucleotides, 2'-O-alkyl ribosyl substituted polynucleotides, phosphorothioate polynucleotides, and boronophosphate polynucleotides. A polynucleotide analog may possess purine or pyrimidine analogs, including for example, 7-deaza purine analogs, 8-halopurine analogs, 5-halopyrimidine analogs, or universal base analogs that can pair with any base, including hypoxanthine, nitroazoles, isocarbostyril analogues, azole carboxamides, and aromatic triazole analogues, or base analogs with additional functionality, such as a biotin moiety for affinity binding.

[0152] As used herein, the terms “amino acid” generally refers to an organic compound that combines to form a protein or peptide. An amino acid generally comprises an amine group, a carboxylic acid group, and a side-chain specific to each amino acid, which serve as a monomeric subunit of a peptide. An amino acid may include the 20 standard, naturally occurring or canonical amino acids as well as non-standard amino acids. The standard, naturally-occurring or canonical amino acids include Alanine (A or Ala), Cysteine (C or Cys), Aspartic Acid (D or Asp), Glutamic Acid (E or Glu), Phenylalanine (F or Phe), Glycine (G or Gly), Histidine (H or His), Isoleucine (I or He), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q or Gin), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Vai), Tryptophan (W or Trp), and Tyrosine (Y or Tyr). An amino acid may be an L-amino acid or a D-amino acid. Non-standard amino acids may be modified amino acids, amino acid analogs, amino acid mimetics, non-standard proteinogenic amino acids, or non- proteinogenic amino acids that occur naturally or are chemically synthesized. Examples of nonstandard amino acids include, but are not limited to, selenocysteine, pyrrolysine, and N- formylmethionine, (3 -amino acids, Homo-amino acids, Proline and Pyruvic acid derivatives, 3- substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, and N-methyl amino acids.

[0153] As used herein, the term “amino acid type” generally refers to one of the standard, naturally-occurring or canonical amino acids, e.g., one member of the group consisting of Alanine (A or Ala), Cysteine (C or Cys), Aspartic Acid (D or Asp), Glutamic Acid (E or Glu), Phenylalanine (F or Phe), Glycine (G or Gly), Histidine (H or His), Isoleucine (I or He), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q or Gin), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Vai), Tryptophan (W or Trp), Tyrosine (Y or Tyr), derivatives thereof, and modified forms of any of the aforementioned amino acids. The term “amino acid type” may be used herein todistinguish a plurality of amino acids that comprise different side chain groups, rather than a plurality of amino acids that are identical (e.g., different positional amino acids of a single peptide that have the same side chain).

[0154] As used herein, the term “post-translational modification” refers to modifications that occur on a peptide or amino acid subsequent to translation. A post-translational modification may be a covalent modification or enzymatic modification. Examples of post-translation modifications include, but are not limited to, acylation, acetylation, alkylation (including methylation), biotinylation, butyrylation, carb amyl ati on, carbonylation, deamidation, deiminiation, diphthamide formation, disulfide bridge formation, eliminylation, flavin attachment, formylation, gamma-carboxylation, glutamyl ati on, glycylation, glycosylation, glypiation, heme C attachment, hydroxylation, hypusine formation, iodination, isoprenylation, lipidation, lipoylation, malonylation, methylation, myristolylation, oxidation, transglutamination, palmitoylation, pegylation, phosphopantetheinylation, phosphorylation, prenylation, propionyl ati on, retinylidene Schiff base formation, S-glutathionylation, S-nitrosylation, S-sulfenylation, S-adenosylation, selenation, succinylation, sulfination, ubiquitination, sumoylation, disulfide bond formation, and C-terminal amidation. A post-translational modification includes modifications of the amino terminus and / or the carboxyl terminus of a peptide. Modifications of the terminal amino group include, but are not limited to, des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include, but are not limited to, amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications (e.g., wherein lower alkyl is C1-C4 alkyl). A post-translational modification also includes modifications, such as but not limited to those described above, of amino acids falling between the amino and carboxy termini. The term post-translational modification can also include peptide modifications that include one or more detectable labels. A post-translational modification may be naturally occurring or synthetic.

[0155] As used herein, the term “binding agent” refers to a molecule, e.g., a nucleic acid molecule, a peptide, a polypeptide, a protein, carbohydrate, a synthetic molecule, or a small molecule that binds to, associates with, unites with, recognizes, or combines with another molecule. The binding agent may bind to a macromolecule or a component or feature of a macromolecule. A binding agent may form a covalent association or non-covalent association with a molecule, a macromolecule, or a component or feature of a macromolecule. A binding agent may also be a chimeric binding agent, composed of two or more types of molecules, such as a nucleic acid molecule-peptide chimeric binding agent, a carbohydrate-peptide chimeric binding agent, or a lipid-peptide chimeric binding agent. A binding agent may be a naturallyoccurring, synthetically produced, or recombinantly expressed molecule. A binding agent may bind to a single monomer or subunit of a polymeric analyte, such as a macromolecule (e.g., a single amino acid of a peptide) or bind to a plurality of linked subunits of a macromolecule (e.g., a di-peptide, tri-peptide, or higher order peptide of a longer peptide, polypeptide, or protein molecule). A binding agent may bind to a linear molecule or a molecule having a three- dimensional structure (also referred to as conformation). For example, an antibody binding agent may bind to linear peptide, polypeptide, or protein, or bind to a conformational peptide, polypeptide, or protein. A binding agent may bind to an N-terminal peptide, a C-terminal peptide, or an intervening peptide of a peptide, polypeptide, or protein molecule. A binding agent may bind to an N-terminal amino acid, C-terminal amino acid, or an intervening amino acid of a peptide molecule. A binding agent may preferably bind to a chemically modified or labeled amino acid over a non-modified or unlabeled amino acid. For example, a binding agent may preferably bind to an amino acid that has been modified with an acetyl moiety, guanyl moiety, dansyl moiety, PTC moiety, DNP moiety, SNP moiety, etc., over an amino acid that does not possess such a moiety. A binding agent may bind to a post-translational modification, either naturally occurring or synthetic, of a peptide molecule. A binding agent may exhibit selective binding to a component or feature of a macromolecule (e.g., a binding agent may selectively bind to one of the 20 possible natural amino acid residues and with bind with very low affinity or not at all to the other 19 natural amino acid residues). A binding agent may exhibit less selective binding, where the binding agent is capable of binding a plurality of components or features of a macromolecule (e.g., a binding agent may bind with similar affinity to two or more different amino acid residues). A binding agent may comprise a tag, which may be coupled to the binding agent via a linker.

[0156] As used herein, the term “linker” generally refers to a molecule or moiety that is involved in joining two or more molecules. A linker may facilitate a covalent or noncovalent interaction of two or more molecules. A linker may be a crosslinker. The linker can be unifunctional, bifunctional, trifunctional, quadrifunctional, or polyfunctional. A linker can be or comprise a nucleotide, a nucleotide analog, an amino acid, a peptide, a polypeptide, or a nonnucleotide chemical moiety, such as an organic or inorganic compound. A linker may comprise a polymer, such as a polyethylene glycol (PEG), poly-L-lysine (PLL), poly (DL-lactic acid) (PLA), poly (DL-lactide-co-glycoside) (PLGA), polyornithine, polyarginine, etc. A linker may comprise one or more reactive ends, e.g., an amine-reactive group, a carboxyl-reactive group, a sulfhydrylreactive group, a hydroxyl-reactive group, etc. In some examples, a linker may be used to join different molecule types, e.g., different biomolecule types such as a peptide with a nucleic acid molecule, a lipid with a peptide, a carbohydrate with a peptide, etc.; non-biomolecule types; or abiomolecule to a non-biomolecule. For example, a linker may be used to join a binding agent with a tag, a tag with a macromolecule (e.g., peptide, nucleic acid molecule), a macromolecule with a solid support, a tag with a solid support, etc. A linker may join two molecules via enzymatic reaction or chemistry reaction (e.g., click chemistry). A linker may join more than two molecules, e.g., via enzymatic or chemical reactions.

[0157] The term “conjugated” as used herein generally refers to a covalent or ionic interaction between two entities, e.g., molecules, compounds, or combinations thereof.

[0158] As used herein, the term “tag” generally refers to a molecule or moiety that is conjugated to a molecule. A tag may comprise a detectable label, e.g., a fluorophore or fluorescent protein, a radioactive isotope, an enzyme (e.g., a chromogenic or fluorescent protein, proteins that can catalyze chromogenic substrates), a mass tag, a hapten (e.g., biotin, digoxigenin, urushiol, fluorescein), a vibrational or FTIR tag (e.g., alkyne group). A tag may comprise a biomolecule, such as a nucleic acid molecule, a protein, a lipid, a carbohydrate, or a combination thereof. A tag may comprise one or more nucleic acid molecules, which may optionally encode information regarding the tag or the molecule onto which a tag is conjugated (e.g., a binding agent, such as an antibody). For example, a tag may comprise a nucleic acid barcode molecule. A tag may comprise an organic compound or an inorganic compound.

[0159] As used herein, the term “barcode” generally refers to an identifying feature that may be used to distinguish similar items. A barcode may comprise a nucleic acid molecule of about 2 to about 30 bases (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 bases), which may provide a unique identifier tag or origin information for a molecule (e.g., protein, polypeptide, peptide), a binding agent, a set of binding agents from a binding cycle, a sample molecule, a set of samples, molecules within a compartment (e.g., droplet, bead, partition or separated location), macromolecules within a set of compartments, a fraction of macromolecules, a set of macromolecule fractions, a spatial region or set of spatial regions, a library of macromolecules, or a library of binding agents. A barcode can be an artificial sequence or a naturally occurring sequence including peptides, proteins, protein complexes, carbohydrates, and synthetic polymeric materials. In certain embodiments, each barcode within a population of barcodes is different. In other embodiments, a portion of barcodes in a population of barcodes is different, e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the barcodes in apopulation of barcodes is different. A population of barcodes may be randomly generated or non- randomly generated. A population of barcodes may comprise error correcting barcodes. Barcodes can be used to computationally deconvolute sequence reads derived from an individual molecule, sample, library, etc. Barcodes may comprise multiplexed information, e.g., arising from different samples, compartments, individual molecules, etc. A barcode can also be used for deconvolution of a collection of molecules that have been distributed into small compartments for enhanced mapping. For example, rather than mapping a peptide back to the proteome, the peptide can be mapped back to its originating protein molecule or protein complex. A barcode may comprise any useful structure moiety or motif, e.g., hairpins, loop sequences, or spacers. Barcodes can comprise artificial or modified nucleic acids, e.g., locked nucleic acids (LNA), protein nucleic acids (PNA), hexitol nucleic acids (HNA), cyclohexane nucleic acids (CeNA), or a combination thereof. Barcodes may comprise or be generated using a protein, e.g., Tai effector, Cas protein (e.g., Cas9), Argonaut, or coiled coils.

[0160] As used herein, a “sample barcode”, also referred to as “sample tag” generally refers to a barcode molecule comprising identifying information of a sample from which a barcoded molecule derives.

[0161] As used herein, a “spatial barcode” generally refers to a barcode molecule comprising identifying information of a region of a 2-D or 3-D sample (e.g., a tissue section) from which a molecule originates or is derived. Spatial barcodes may be used for molecular pathology on tissue sections. A spatial barcode may allow for multiplex sequencing of a plurality of samples or libraries from tissue section(s).

[0162] As used herein, a “temporal barcode” generally refers to a barcode molecule comprising time-based information relating to the barcoded molecule. The types of time-based data encoded in a temporal barcode can include information such as a life-time of a barcoded molecule, a time of collection of a sample, a time or duration since the beginning of an experiment or induction with a stimulus, information on the age of a cell or tissue, a sequence of interactions between molecules, among others. It is possible for different types of barcodes (e.g., spatial, temporal, cell-specific) to be combined in one multiplexed barcode.

[0163] As used herein, the term “nucleic acid sequence” or “oligonucleotide sequence” generally refers to a contiguous string of nucleotide bases and may refer to the particular placement of nucleotide bases in relation to each other as they appear in an oligonucleotide. Similarly, the term “polypeptide sequence” or “amino acid sequence” refers to a contiguous string of amino acids and may refer to the particular placement of amino acids in relation to each other as they appear in a polypeptide.

[0164] A “nucleic acid molecule” according to the present invention may include any polymer or oligomer of nucleotides such as pyrimidine and purine bases, such as cytosine, thymine, and uracil, and adenine and guanine, respectively and combinations thereof. The nucleotide sequence may comprise any deoxyribonucleotide, ribonucleotide, hexitol -nucleotide, cyclohexanenucleotide, peptide nucleic acid component, and any chemical variants thereof, such as methylated, 7-deaza purine analogs, 8-halopurine analogs, hydroxymethyl ated or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogeneous or homogenous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced. A nucleic acid molecule may comprise DNA, RNA, HNA, CeNA or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.

[0165] The terms “complementary” or “complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by Watson-Crick base-pairing rules. For example, the sequence “5 -AGT-3',” is complementary to the sequence “5'- ACT-3'”. Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules, or there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands can have significant effects on the efficiency and strength of hybridization between nucleic acid strands under defined conditions.

[0166] As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (e.g., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, and the melting temperature of the formed hybrid. Hybridization methods involve the annealing of one nucleic acid to another, complementary nucleic acid, e.g., based on Watson-Crick base pairing.

[0167] As used herein, the term “proteomics” generally refers to quantitative and / or qualitative analysis of the proteome within a sample, such as biological sample, e.g., from cells, tissues, or bodily fluids. Proteomics may include the analysis of spatial distributions of proteins within a sample (e.g., cell and / or tissues). Proteomics may include studies of the dynamic state of the proteome, e.g., how one or more proteins change in time. A proteome may comprise multiple “-omes”, e.g., a kinome; a secretome; a receptome (e.g., GPCRome); an immunoproteome; a nutriproteome; a proteome subset defined by a post-translational modification (e.g., phosphorylation, ubiquitination, methylation, acetylation, glycosylation, oxidation, lipidation, and / or nitrosylation), such as a phosphoproteome (e.g., phosphotyrosine-proteome, tyrosine- kinome, and tyrosine-phosphatome), a glycoproteome, etc.; a proteome subset associated with atissue or organ, a developmental stage, or a physiological or pathological condition (e.g., cancer or disease); a proteome subset associated a cellular process, such as cell cycle, differentiation (or de-differentiation), cell death, senescence, cell migration, transformation, or metastasis; or any combination thereof.

[0168] The terminal amino acid at one end of the peptide chain that has a free amino group may be referred to herein as the “N-terminal amino acid” (NTAA). The terminal amino acid at the other end of the chain that has a free carboxyl group may be referred to herein as the “C-terminal amino acid” (CTAA). The amino acids making up a peptide may be numbered in order, with the peptide being “n” amino acids in length. As used herein, in some instances, NTAA may be considered the nth amino acid (also referred to herein as the “n NTAA”). In such cases, the next amino acid is the n-1 amino acid, then the n-2 amino acid, and so on down the length of the peptide from the N-terminal end to C-terminal end. Alternatively, CTAA may be considered the nth amino acid (also referred to herein as the “n CTAA”). In such cases, the next amino acid is the n-1, then the n-2 amino acid, and so on down the length of the peptide from the C-terminal end to N-terminal end. An NTAA, CTAA, or both may be modified or labeled with a chemical moiety.

[0169] As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.

[0170] As used herein, the term “unique molecular identifier” or “UMI” generally refers to a molecule barcode comprising indexing information. A UMI may comprise a nucleic acid molecule of about 3 to about 150 bases (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 bases) in length. A UMI may provide a unique identifier tag for each molecule (e.g., peptide, binding agent, a nucleic acid molecule) that comprises or is coupled to a UMI. A UMI may comprise a random sequence (e.g. a random N- mer).

[0171] As used herein, a “derivative” of a nucleic acid molecule generally refers to a nucleic acid molecule that is derived from an originating nucleic acid molecule. The derivative may have the same or substantially the same nucleotide sequence as the originating nucleic acid molecule, or the derivative may comprise a complement or partial complement as the originating nucleic acid molecule. A derivative may be the same type of nucleic acid (e.g., DNA or RNA) as the originating nucleic acid molecule, or the derivative may be a different type of nucleic acid (e.g., cDNA generated from an RNA molecule). A nucleic acid molecule derivative may displaysequence identity as the originating nucleic acid molecule. The derivative nucleic acid molecule may also be subjected to additional processing from the originating nucleic acid molecule, e.g., chemical or enzymatic modification, splicing, ligation, polymerization, fragmentation, tagmentation (e.g., using a transposase), digestion, etc.

[0172] A derivative polypeptide or peptide may be derived from an originating polypeptide (or peptide). A derivative may comprise the same amino acid sequence as the originating polypeptide, or the sequence may be different. The derivative polypeptide may result from or be subjected to additional processing from the originating polypeptide, e.g., chemical or enzymatic modification. The derivative polypeptide may comprise one or more tags, nucleic acid molecules, barcode molecules, labels (e.g., detectable labels), fluorophores, probes, linkers, post-translational modifications, chemical protecting groups, or other chemical moieties.

[0173] A derivative of an amino acid may generally refer to a modified amino acid that is derived from an originating amino acid. The derivative may have the same or substantially the same chemical composition as the originating amino acid, or the derivative may comprise a different chemical composition. The derivative may be a chemically modified or enzymatically modified amino acid. The derivative may be naturally occurring or synthetic.

[0174] As used herein, the term “compartment” or “partition” generally refers to a physical area or volume that separates or isolates a subset of molecules from a sample of molecules. For example, a compartment may separate an individual cell from other cells, or a subset of a sample’s proteome from the rest of the sample's proteome. A compartment may be an aqueous compartment (e.g., microfluidic droplet), a solid compartment (e.g., picotiter well or microtiter well on a plate, tube, vial, gel bead), or a separated region on a surface. A compartment may comprise one or more beads to which macromolecules may be immobilized.

[0175] As used herein, the term “solid support”, “solid surface”, or “solid substrate” or “substrate” refers to any solid material, including porous and non-porous materials, to which a molecule can be associated directly or indirectly. The molecule may be associated with the substrate by covalent or non-covalent interactions, or a combination thereof. A substrate may be two-dimensional (e.g., planar surface) or three-dimensional (e.g., gel matrix or bead). A solid support may comprise, in non-limiting examples, a bead, a microbead, an array, a glass surface, a silicon surface, a plastic surface, a filter, a membrane, nylon or other polymer, a silicon wafer chip, a flow through chip, a flow cell (e.g., custom designed or commercially available flow cells such as Illumina® HiSeq, iSeq, MiniSeq, NextSeq, NovaSeq, MiSeq flow cells), a microfluidic device or chip or a surface thereof, a biochip including signal transducing electronics, a channel, a microtiter well, an ELISA plate, a spinning interferometry disc, a nitrocellulose membrane, anitrocellulose-based polymer surface, a polymer matrix, a nanoparticle, or a microsphere. Materials for a solid support include but are not limited to acrylamide, agarose, cellulose, nitrocellulose, glass, gold, quartz, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, poly silicates, polycarbonates, Teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, functionalized silane, polypropylfumerate, collagen, glycosaminoglycans, polyamino acids, dextran, or any combination thereof. Solid supports further include thin film, membrane, bottles, dishes, fibers, woven fibers, shaped polymers such as tubes, particles, beads, microspheres, microparticles, or any combination thereof. For example, when solid surface is a bead, the bead can include, but is not limited to, a ceramic bead, polystyrene bead, a polymer bead, a methylstyrene bead, an agarose bead, an acrylamide bead, a solid core bead, a porous bead, a magnetic or paramagnetic bead, a glass bead, a carboxyl bead, or a controlled pore bead. A bead may be spherical or an irregularly shaped. A bead's size may range from nanometers, e.g., 100 nm, to millimeters, e.g., 1 mm. In certain embodiments, beads range in size from about 0.2 micron to about 200 microns, or from about 0.5 micron to about 5 microns. In some embodiments, beads can be about 1, 1.5, 2, 2.5, 2.8, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100 pm in diameter. In certain embodiments, “a bead” solid support may refer to an individual bead or a plurality of beads. A solid support may assume any useful geometry, e.g., pyramid, cube, cylinder, helix, sphere, spheroid, rod, disc, arrow, spring, teardrop, prism, tetrapod, or any other useful geometry.

[0176] As used herein, “sequencing” generally refers to determining the order of: (A) nucleotides (base sequences) in a nucleic acid sample, e.g., DNA or RNA; or determining the order of (B) amino acids in all or part of a polymer, such as a protein, peptide, or other multimeric molecule. Many techniques are available, such as Sanger sequencing or High Throughput Sequencing technologies (HTS). Sanger sequencing may involve sequencing via detection through (capillary) electrophoresis, in which up to 384 capillaries may be sequence analyzed in one run. High throughput sequencing involves the parallel sequencing of thousands or millions or more sequences at once. HTS can be defined as Next Generation sequencing (NGS), i.e. techniques based on solid phase pyrosequencing or as Next-Next Generation sequencing based on single nucleotide real time sequencing (SMRT). HTS technologies are available such as offered by Roche, Illumina and Applied Biosystems (Life Technologies). Further high throughputsequencing technologies are described by and / or available from Helicos, Pacific Biosciences, Complete Genomics, Ion Torrent Systems, Oxford Nanopore Technologies, Nabsys, ZS Genetics, GnuBio.

[0177] As used herein, “next generation sequencing” refers to high-throughput sequencing methods that allow the sequencing of millions to billions of molecules in parallel. Examples of next generation sequencing methods include sequencing by synthesis, sequencing by ligation, sequencing by hybridization, polony sequencing, ion semiconductor sequencing, nanopore sequencing, and pyrosequencing. By attaching primers to a solid substrate and a complementary sequence to a nucleic acid molecule, a nucleic acid molecule can be hybridized to the solid substrate via the primer and then multiple copies can be generated in a discrete area on the solid substrate by using polymerase to amplify (these groupings are sometimes referred to as polymerase colonies or polonies). Consequently, during the sequencing process, a nucleotide at a particular position can be sequenced multiple times (e.g., hundreds or thousands of times) — this depth of coverage is referred to as “deep sequencing.” Examples of high throughput nucleic acid sequencing technology include platforms provided by Illumina, BGI, Qiagen, ThermoFisher, and Roche, including formats such as parallel bead arrays, sequencing by synthesis, sequencing by ligation, capillary electrophoresis, electronic microchips, “biochips,” microarrays, parallel microchips, and single-molecule arrays, zero mode waveguide based sequencing, some of which are reviewed by Service (Science 311 : 1544-1546, 2006).

[0178] As used herein, “analyzing” the macromolecule means to quantify, characterize, distinguish, or a combination thereof, all or a portion of the components of a molecule (e.g., a macromolecule, a biological molecule such as a protein, amino acid, nucleic acid molecule, etc.). For example, analyzing a peptide, polypeptide, or protein may comprise determining all or a portion of the amino acid sequence (contiguous or non-continuous) of the peptide. Analyzing a macromolecule may include partial identification of a component of the macromolecule. For example, partial identification of amino acids in a protein sequence can identify an amino acid in the protein as belonging to a subset of possible amino acids. Analysis may be performed sequentially, e.g., beginning with analysis of the n NTAA, and then proceeding to the next amino acid of the peptide (i.e., n-1, n-2, n-3, and so forth). In such instances, sequencing may be performed by cleavage of the n NTAA, thereby converting the n-1 amino acid of the peptide to an N-terminal amino acid (referred to herein as the “n-1 NTAA”). Similarly, analysis of a peptide may begin from C-terminus towards the N-terminus with each round of cleavage from the C- terminus creating a new CTAA. Cleavage of the n CTAA converts the n-1 amino acid of the peptide to a C-terminal amino acid, referred to herein as an “n-1 CTAA”. Analyzing the peptidemay also include determining a presence and frequency of post-translational modifications on the peptide, which may or may not include information regarding the sequential order of the post- translational modifications on the peptide. Analyzing the peptide may also include determining the presence and frequency of epitopes in the peptide, which may or may not include information regarding the sequential order or location of the epitopes within the peptide. Analyzing the peptide may include combining different types of analysis, for example obtaining epitope information, amino acid sequence information, post-translational modification information, or any combination thereof.

[0179] As used herein, the term “analyte” generally refers to a substance that is of interest to be further identified, characterized, or measured. An analyte can be, in non-limiting examples, an ion, chemical, compound, small molecule, element, particle, metal, biomolecule, macromolecule, metabolite, lipid, carbohydrate, peptide or protein, nucleic acid molecule, organelle, or cell. An analyte may be naturally occurring or synthetic. The analyte may be a solid, semi-solid, liquid, semi-liquid, gas, or plasma. The analyte may be characterized qualitatively or quantitatively. A portion of an analyte may be analyzed. For example, an analyte may be a peptide and the constituent amino acids may be analyzed. The analyte may comprise a polymer, also referred to herein as “polymeric analyte”, which generally refers to an analyte of interest that comprises one or more monomers. A polymeric analyte can be, in non-limiting examples, a group of ions, chemicals, compounds, small molecules, elements, particles, metals, or a biomolecule, macromolecule, metabolite, lipid, carbohydrate, peptide or protein, nucleic acid molecule, organelle, or cell.

[0180] As used herein, the term “array” generally refers to a population of molecules that is attached to one or more solid supports such that the molecules at one address can be distinguished from molecules at other addresses. An array can include different molecules that are each located at different addresses on a solid support. Alternatively, an array can include separate solid supports each functioning as an address that bears a different molecule, wherein the different molecules can be identified according to the locations of the solid supports on a surface to which the solid supports are attached, or according to the locations of the solid supports in a liquid such as a fluid stream. The molecules of the array can be, for example, nucleic acids such as SNAPs, polypeptides, proteins, peptides, oligopeptides, enzymes, ligands, or receptors such as antibodies, functional fragments of antibodies or aptamers. The addresses of an array can optionally be optically observable and, in some configurations, adjacent addresses can be optically distinguishable when detected using a method or apparatus set forth herein.

[0181] As used herein, the term “functionalized” refers to any material or substance that has been modified to include a functional group. A functionalized material or substance may be naturally or synthetically functionalized. For example, a polypeptide can be naturally functionalized with a phosphate group, oligosaccharide (e.g., glycosyl, glycosylphosphatidylinositol or phosphoglycosyl), nitrosyl, methyl, acetyl, lipid (e.g., glycosyl phosphatidylinositol, myristoyl or prenyl), ubiquitin or other naturally occurring post-translational modification. A functionalized material or substance may be functionalized for any given purpose, including altering chemical properties (e.g., altering hydrophobicity or changing surface charge density) or altering reactivity (e.g., capable of reacting with a moiety or reagent to form a covalent bond to the moiety or reagent).

[0182] As used herein, the term “click reaction,” “click chemistry,” or “bioorthogonal reaction” refers to single-step, thermodynamically favorable conjugation reaction utilizing biocompatible reagents. A click reaction may utilize no toxic or biologically incompatible reagents (e.g., acids, bases, heavy metals) or generate no toxic or biologically incompatible byproducts. A click reaction may utilize an aqueous solvent or buffer (e.g., phosphate buffer solution, Tris buffer, saline buffer, MOPS, etc.). A click reaction may be thermodynamically favorable if it has a negative Gibbs free energy of reaction, for example a Gibbs free energy of reaction of less than about -5 kiloJoules / mole (kJ / mol), -10 kJ / mol, -25 kJ / mol, -50 kJ / mol, -100 kJ / mol, -200 kJ / mol, -300 kJ / mol, -400 kJ / mol, or less than -500 kJ / mol. Exemplary bioorthogonal and click reactions are described in detail in WO 2019 / 195633A1, which is herein incorporated by reference in its entirety. Exemplary click reactions may include metal-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, strain-promoted azide- nitrone cycloaddition, strained alkene reactions, thiolene reaction, Diels-Alder reaction, inverse electron demand Diels-Alder reaction, [3+2] cycloaddition, [4+1] cycloaddition, nucleophilic substitution, dihydroxylation, thiolyne reaction, photoclick, nitrone dipole cycloaddition, norbomene cycloaddition, oxanob ornadiene cycloaddition, tetrazine ligation, and tetrazole photoclick reactions. Exemplary functional groups or reactive handles utilized to perform click reactions may include alkenes (e.g., linear alkenes or cyclic alkenes such as trans-cyclooctene (TCO)), alkynes (e.g., linear alkynes or cycloalkynes (e.g., cyclooctynes or derivatives thereof, e.g., aza-dimethoxycyclooctyne (DIMAC), symmetrical pyrrolocyclooctyne (SYPCO), pyrrolocyclooctyne (PYRROC), difluorocyclooctyne (DIFO), a,a- bis(trifluoromethyl)pyrrolocyclooctyne (TRIPCO), bicyclo[6.1.0]nonyne(BCN), dibenzocyclooctyne (DIBO), difluorinated cyclooctyne (DIFO), difluorobenzocyclooctyne (DIFBO), dibenzoazacyclo-octyne (DBCO), difluoro-aza-dibenzocyclooctyne (F2-DIBAC), biaryl-azacyclooctynone (BARAC), difluorodimethoxy dibenzocyclooctynol (FMDIBO), difluorodimethoxy dibenzocyclooctynone (keto-FMDIBO), and 3,3,6,6-tetramethylthiacycloheptyne (TMTH)), TMTH-sulfoximine (TMTHSI), azides, epoxides, amines, thiols, nitrones, isonitriles, isocyanides, aziridines, activated esters, and tetrazines, triazoles, and combinations, variations, or derivatives thereof. The click chemistry moi eties may be subjected to conditions sufficient to react the first click chemistry moiety to the second click chemistry moiety, e.g., provision of metal catalysts, appropriate solvents, pH, temperature, ionic concentration, or light / energy, for any useful duration of time.

[0183] As used herein, the terms “group” and “moiety” are intended to be synonymous when used in reference to the structure of a molecule. The terms refer to a component or part of the molecule. The terms do not necessarily denote the relative size of the component or part compared to the molecule, unless indicated otherwise. The terms do not necessarily denote the relative size of the component or part compared to any other component or part of the molecule, unless indicated otherwise. A group or moiety can contain one or more atoms.

[0184] As used herein, “primers” generally refer to nucleic acid molecules which can prime the synthesis of a nucleic acid molecule (e.g., DNA or RNA). A primer may be single stranded. A primer may comprise one or more recognition sites for a protein (e.g., a polymerizing enzyme, a restriction enzyme, a cleaving enzyme, a nuclease, etc.) to bind to the primer or a primer hybridized to a template strand. A primer may comprise DNA, RNA, or other nucleic acid analogs or noncanonical bases (e.g., spacer moieties, uracils, abasic sites). A primer may optionally comprise any number of functional sequences such as sequencing primer sequences (e.g., P5 or P7 sequences), sequencing primer-binding sequences, read sequences (e.g., R1 or R2 sequences), enzyme-recognition sites, e.g., restriction sites or nuclease-recognition sites, abasic sites, cleavage sites, transposition sites, spacer sequences, a barcode sequence, a unique molecular identifier (UMI), etc.

[0185] “Amplification” or “amplifying” generally refers to a polynucleotide amplification reaction, namely, a population of polynucleotides that are replicated from one or more starting sequences. Amplifying may refer to a variety of amplification reactions, including but not limited to polymerase chain reaction (PCR), linear polymerase reactions, nucleic acid sequence- based amplification, rolling circle amplification and similar reactions. An amplification reaction may generate an amplicon.

[0186] An “adapter” as referred to herein, generally refers to a short nucleic acid molecule (e.g., about 10 to about 100 base pairs in length). An adapter may comprise a short double-stranded DNA molecule. An adapter may be attached, e.g., via polymerization or ligation, to an end of aDNA fragments or amplicons. Adapters may comprise synthetic oligonucleotides, e.g., oligonucleotides that have nucleotide sequences which are at least partially complementary to each other. An adapter may have blunt ends, may have staggered ends (also referred to herein as a 3’ or 5’ “overhang sequence” or “sticky end”, or a blunt end and a staggered end. Adapters may be attached (e.g., via ligation) to fragments to provide an adapter-ligated fragment; the adapter- ligated fragment may serve as a starting point for subsequent manipulation e.g., for amplification or sequencing. An adapter may be functionalized, e.g., conjugated with a tag, probe, detectable label, affinity capture reagent (e.g., biotin or streptavidin).

[0187] The term “capture moiety” as used herein generally refers to a molecule that is configured to be coupled to another moiety or molecule. A capture moiety can be a biomolecule, e.g., a lipid, carbohydrate, sugar, amino acid, peptide or protein, nucleotide, nucleic acid molecule, metabolite, or a combination thereof (e.g., glycoproteins, lipoproteins, glycosaminoglycans, etc.). A capture moiety can be a small molecule, organic compound, inorganic compound, metal, polymer, ion, or other molecule or molecular compound. A capture moiety may comprise a macromolecule. A capture moiety may comprise an enzyme, antibody, antibody fragment, nanobody, aptamer, biotin, streptavidin, avidin, neutravidin, or analogs or derivatives thereof. A capture moiety may comprise more than one molecule, e.g., a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, etc. A capture moiety can be a solid substrate or part of a solid substrate, or the capture moiety can be separate from a substrate, e.g., in a fluidic medium (e.g., air, in a liquid solution). A capture moiety may have specificity to a binding partner or a plurality of binding partners. A capture moiety may be able to bind to one molecule or moiety (univalent), or a plurality of molecules or moieties (multivalent).

[0188] As used herein, the abbreviations for the natural 1 -enantiomeric amino acids are conventional and can be as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine (C, Cys); glutamic acid (E, Glu); glutamine (Q, Gin); glycine (G, Gly); histidine (H, His); isoleucine (I, He); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Vai). Unless otherwise specified, X can indicate any amino acid. In some aspects, X can be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R). References to these amino acids are also in the form of “[amino acid] [residues / residues]” (e.g., lysine residue, lysine residues, leucine residue, leucine residues, etc.).

[0189] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can beused in the practice or testing of the present disclosure, suitable methods and materials are described below.Amino Acid Binding Agents

[0190] Provided herein are methods, compositions, and kits for processing and analyzing polymeric analytes e.g., peptides, polymers, nucleic acid molecules, etc. in a highly-parallelized and accurate manner. The present disclosure provides for approaches for sequencing polymeric analytes that comprise monomers via the use of binding agents that specifically or semi- specifically recognize and bind to individual monomer types of the polymeric analytes. The binding agents may be labeled with a detectable label, e.g., fluorophore or fluorescent protein, mass tag, radioisotope, nucleic acid molecule (e.g., nucleic acid barcode molecule), that can identify or be used to identify the binding agent or its cognate molecule. In some aspects, the systems and methods of the present disclosure may comprise providing a polymeric analyte comprising a plurality of monomers, cleaving a monomer from the polymeric analyte to thereby generate a free monomer, coupling (e.g., via local tethering) the monomer to a capture moiety, and providing a monomer-specific binding agent that recognizes the cleaved (or free) monomer. Additional example methods and systems of identifying monomers of polymeric analytes are described elsewhere herein and also provided in PCT / US2023 / 017954, filed on April 7, 2023, which is incorporated by reference herein in its entirety.

[0191] In some aspects of the present disclosure, the binding agents provided herein are used to sequence or characterize proteins or peptides comprising amino acids. A binding agent of the present disclosure may bind to one type of proteinogenic amino acid or derivative thereof, e.g., a chemically or enzymatically derivatized amino acid, which can include post-translationally modified amino acids, with greater specificity or with higher affinity than the binding agent binds to all other (e.g., all 19 other) types of proteinogenic amino acids or derivatives thereof. Accordingly, the binding agents may be used to identify individual amino acids or derivative thereof with high accuracy. In some aspects, the binding agents provided herein are configured to bind free amino acids or derivatives thereof. The free amino acids (or derivatives thereof) may be generated, for example, by cleaving an amino acid from a peptide or protein, thereby generating a free amino acid. The free amino acid can be an internal amino acid or a terminal amino acid, e.g., an N-terminal amino acid or a C-terminal amino acid.

[0192] In some embodiments, the binding agent specifically binds to alanine (A, Ala) or derivative thereof; in some embodiments, the binding agent specifically binds to arginine (R, Arg) or derivative thereof; in some embodiments, the binding agent specifically binds toasparagine (N, Asn) or derivative thereof; in some embodiments, the binding agent specifically binds to aspartic acid (D, Asp) or derivative thereof; in some embodiments, the binding agent specifically binds to cysteine (C, Cys) or derivative thereof; in some embodiments, the binding agent specifically binds to glutamic acid (E, Glu) or derivative thereof; in some embodiments, the binding agent specifically binds to glutamine (Q, Gin) or derivative thereof; in some embodiments, the binding agent specifically binds to glycine (G, Gly) or derivative thereof; in some embodiments, the binding agent specifically binds to histidine (H, His) or derivative thereof; in some embodiments, the binding agent specifically binds to isoleucine (I, He) or derivative thereof; in some embodiments, the binding agent specifically binds to leucine (L, Leu) or derivative thereof; in some embodiments, the binding agent specifically binds to lysine (K, Lys) or derivative thereof; in some embodiments, the binding agent specifically binds to methionine (M, Met) or derivative thereof; in some embodiments, the binding agent specifically binds to phenylalanine (F, Phe) or derivative thereof; in some embodiments, the binding agent specifically binds to proline (P, Pro) or derivative thereof; in some embodiments, the binding agent specifically binds to serine (S, Ser) or derivative thereof; in some embodiments, the binding agent specifically binds to threonine (T, Thr) or derivative thereof; in some embodiments, the binding agent specifically binds to tryptophan (W, Trp) or derivative thereof; in some embodiments, the binding agent specifically binds to tyrosine (Y, Tyr) or derivative thereof; in some embodiments, the binding agent specifically binds to valine (V, Vai) or derivative thereof. In some embodiments, the binding agent specifically binds to a post- translationally modified amino acid or derivative thereof.

[0193] In some embodiments, the binding agent binds specifically to an amino acid derivative. An amino acid derivative may be a chemically or enzymatically modified amino acid and may or may not comprise a post-translational modification. The amino acid derivative may be derived from a cleaved or free amino acid, or the amino acid derivative may arise from the cleaving process. For instance, in the methods and systems described herein, an amino acid of a peptide may be contacted with a linker comprising an amino acid reactive group and cleaved from the peptide to generate a free amino acid or amino acid-linker complex; accordingly, the binding agents provided herein may bind specifically and recognize the free amino acid, the amino acid-linker complex, or a portion of the free amino acid or amino acid-linker complex. In one such example, the peptide may be reacted with a linker comprising a phenylisothiocyanate (PITC) moiety (an amino acid reactive group). The PITC moiety can react with the N-terminal amino acid of the peptide to generate a phenylthiocarbamyl (PTC)-derivatized amino acid, which may be recognized by the binding agent. Alternatively, the binding agent may recognizeother derivatives of the PITC reaction with the amino acid, including an anilinothiazolinone (ATZ) or phenylthiohydantoin (PTH) derivatized amino acid. In some instances, the ATZ derivative is generated from cleaving the amino acid from the peptide. In some instances, a derivatized amino acid (e.g., ATZ derivative) is further derivatized (e.g., to a PTH or PTC derivative), which further derivatized amino acid may be recognized by the binding agent.

[0194] The amino acid derivative can comprise any number of chemical modifications, which may be naturally occurring or synthetic. Non-limiting examples of chemical modifications of an amino acid include alkylation, e.g., of cysteine residues (e.g., using 4- vinylpyridine, iodoacetamide, iodoacetate, chloroacetate); acetylation, e.g., to react serine or threonine residues to form an ester (e.g., using acetyl chloride) or using acetic anhydride; oxidation, e.g., to convert cysteine residues to cysteic acid; reduction (e.g., using a reducing agent such as dithiothreitol, P-mercaptoethanol, or TCEP); addition of a protecting group, e.g., phosphorylated residues may be protected (e.g., using a p-elimination of a phosphate group, with an optional Michael addition of a thiol group, e.g., as described in Knight, et al. Nat. Biotechnology. 21, 1047-1054 (2003), which is incorporated by reference herein in its entirety), etc. The amino acid may be modified or derivatized with a protecting group or moiety, such as a methyl, formyl, ethyl, acetyl, t-butyl, anisyl, benzyl, tifluoroacetyl, N-hydroxysuccinimide, t- butyloxycarbonyl (Boc), benzoyl, 4-methyl benzyl, thioanizyl, thiocresyl, benzyloxymethyl, 4- nitrophenyl, benzyloxycarbonyl, 2-nitrobenzoyl, 2-nitrophenylsulphenyl, 4-toluenesulphonyl, pentafluorophenyl, diphenylmethyl, 2-chlorobenzyloxycarbonyl, 2,4,5-trichlorophenyl, 2- bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC), triphenylmethyl, or 2,2,5,7,8-pentamethyl-chroman-6-sulphonyl group. The amino acid may be modified or derivatized with a protecting agent, e.g., carboxy ethyl methanethiosulfonate (CEMTS), thiazolidine, mercaptophenyl acetic acid, cyanobenzothiazole (e.g., for lipidation ofN-terminal cysteines), acetamidomethyl, 2-methylsulfonylethyl-oxycarbonyl, etc. In some instances, the lysine residues are modified or derivatized (e.g., the primary amines of lysine residues may be reacted) using an isothiocyanate (e.g., PITC) to generate a modified lysine residue.

[0195] In some instances, the binding agent binds specifically to an amino acid derivative and not to the native amino acid, e.g., when the amino acid is present in the peptide. In some instances, the binding agent specifically binds to or recognizes a free amino acid (e.g., a cleaved amino acid) or a free amino acid derivative (e.g., an amino acid-linker complex) but not the amino acid whilst the amino acid is connected to the peptide. For example, a linker comprising an amino acid reactive agent may be reacted with a terminal amino acid of a peptide, thereby generating an amino acid-linker complex. The amino acid-linker complex may be cleaved fromthe peptide, thereby generating a cleaved amino-acid linker complex. In some instances, the binding agent may bind to and recognize the cleaved amino acid-linker complex but not the amino acid-linker complex that is attached to the peptide. In some instances, the binding agent may bind to and recognize the cleaved amino acid-linker complex but not the amino acid when the amino acid is attached to the peptide.

[0196] A derivative of an amino acid may refer to the free or cleaved amino acid, an amino acid-linker complex, or a portion of the amino acid, the free (e.g., cleaved) amino acid, or the amino acid-linker complex. The derivative of the amino acid can also include a further derivatized amino acid (e.g., an amino acid that has undergone more than one derivatization). For example, a derivatized amino acid generated from using a linker comprising PITC include PTC, PTH, or ATZ derivatized amino acids. Accordingly, the binding agents described herein may be able to recognize and bind to a specific amino acid type or a derivatized amino acid type, the amino acid-linker complex, the cleaved amino acid, or the amino acid-linker complex. Additional examples of linkers are contemplated and described elsewhere herein.

[0197] In some instances, the amino acid derivative may be an enzymatically modified amino acid. The enzyme used to generate the enzymatically modified amino acid may comprise, for example, a modified cleaving enzyme, e.g., a protease, such as an Edmanase, cruzain, a cleaving protein (e.g., ClpS, ClpX), Proteinase K, exopeptidase, aminopeptidase, diaminopeptidase, serine protease, cysteine protease, threonine protease, aspartic protease, aspartic protease, glutamic protease, metalloprotease, asparagine peptide lyase, pepsin, trypsin, pancreatin, Lys-C, Glu-C, Asp-N, chymotrypsin, carboxypeptidase (e.g., carboxypeptidase A, carboxypeptidase B, carboxypeptidase Y), SUMO protease, elastase, papain, endoproteinase, proteinase, TrypZean®, bromelain, collagenase, hyaluronase, thermolysin, ficin, keratinase, tryptase, fibroblast activation, enterokinase, chymotrypsinogen, chymase, clostripain, calpain, alpha-lytic protease, proline specific endopeptidase, furin, thrombin, subtilisin, genenase, PCSK9, cathepsin, prolidase, methionine aminopeptidase, cathepsin C, 1-cyclohexen-l-yl- boronic acid pinacol ester, pyroglutamate aminopeptidase, renin, kininogen, kallikrein, DPPIV / CD26, thimet oligopeptidase, prolyl oligopeptidase, leucine aminopeptidase, dipeptidylpeptidase, or other enzyme or protease, or a combination or variation (e.g., engineered mutant or variant) thereof. The enzyme may chemically modify and / or cleave the amino acid, thereby generating the derivative of an amino acid.

[0198] In some embodiments, the binding agent may bind to a C-terminal amino acid or derivative thereof. The C-terminal amino acid or derivative thereof may be connected to a peptide, or the C-terminal amino acid or derivative may be free or cleaved from the peptide. Insome instances, the C-terminal amino acid comprises a free or cleaved amino acid using a C- terminal degradation approach. C-terminal degradation may comprise Edman-like degradation approaches. C-terminal degradation may employ the use of activating reagents that react with the C-terminal carboxyl group of a peptide, and a derivatizing agent (e.g., a thiocyanate to generate a peptide-thiocyanate or peptide-thiohydantoin). Non-limiting examples of activating reagents include acetyl chloride and acetic anhydride. Alternatively, or in addition to, single-step C-terminal derivatization of a peptide to a peptidyl-thiohydantoin may be performed, e.g., using Schlack-Kumpf approach, in which a peptide is reacted with thiocyanic acid (e.g., in acetone) to generate a peptidyl-thiohydantoin. The peptide-thiohydantoin may be cleaved, e.g., using basic conditions, to generate an amino acid thiohydantoin and remaining peptide. In such an example, the binding agent may recognize the amino acid thiohydantoin or a derivative thereof.

[0199] In some embodiments, the binding agent does not bind to a particular amino acid or derivative thereof. For instance, the binding agent may bind a first amino acid type but not to a second amino acid type under a given set of conditions. In some instances, the binding agent binds to one or more amino acid types that have similar side chain groups; for example, the binding agent may bind to one or more nonpolar aliphatic side chain amino acids such as alanine, glycine, valine, leucine, methionine, and / or isoleucine. A binding agent may bind to one or more amino acids comprising a positively charged side chain such as lysine, arginine, and / or histidine. A binding agent may bind to one or more amino acids comprising a negatively charged side chain, e.g., aspartate and glutamate. A binding agent may bind to one or more amino acids comprising a nonpolar, aromatic side chain such as phenylalanine, tyrosine, and / or tryptophan. A binding agent may bind to one or more amino acids comprising a polar uncharged side chain such as serine, threonine, cysteine, proline, asparagine, and / or glutamine. In some embodiments, the binding agent may bind to two or more amino acids with different side chain characteristics (e.g., an amino acid with an aliphatic side chain and an amino acid with a charged side chain).

[0200] The binding agent may bind to one or more amino acid types with differential specificity or affinity. In an example, under a given set of conditions, a binding agent (e.g., antibody, antibody fragment) may bind to isoleucine and valine, or derivatives thereof, but the binding agent may bind to isoleucine with greater affinity than the affinity of the binding agent to valine, when measured using an ELISA. In another example, under a given set of conditions, a binding agent may bind to phenylalanine, tyrosine, and tryptophan, or derivatives thereof, but the binding agent may bind to phenylalanine preferentially over tyrosine and tryptophan under a given set of conditions, e.g., when measured using an ELISA using a particular concentration ofthe binding agent, the amino acid target, or both. It will be appreciated that the above-listed examples are merely for demonstration purposes, and that other binding agents that can differentially bind different combinations of amino acids or derivatives thereof are contemplated.

[0201] The binding properties of the binding agent to a target (e.g., a particular amino acid type) can be measured using any useful technique, including, but not limited to immunoassays, e.g., Western blot, a direct or indirect enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), biolayer interferometry (BLI), fluorescence activated cell sorting (FACS), imaging, or radioimmunoprecipitation. Non-limiting examples of useful binding properties that are measurable include affinity (e.g., the equilibrium dissociation constant (Kd) or association constant (Ka)), kinetic rate constants (e.g., konor koff), specificity, avidity, and selectivity. In some examples, binding can be measured, for example, by determining binding of a binding agent to a particular amino acid type or derivative thereof as compared to the binding of the binding agent to a control molecule, which may be a structurally similar molecule. In some examples, specific binding may be determined using a competition assay, e.g., competition ELISA.

[0202] The binding agents provided herein may bind to an amino acid or derivative thereof at a relatively high affinity. A binding agent may bind to the amino acid or derivative thereof at a dissociation constant, e.g., as determined by BLI or SPR, of about 500 nanomolar (nM), about 100 nM, about 10 nM, about 1 nM, about 500 picomolar (pM), about 100 pM, about 10 pM, about 1 pM, or less. In some instances, the binding agent binds to the amino acid or derivative thereof at a dissociation constant of at most about 500 nanomolar (nM), at most about 100 nM, at most about 10 nM, at most about 1 nM, at most about 500 picomolar (pM), at most about 100 pM, at most about 10 pM, at most about 1 pM, or less. The dissociation constant may fall within a range of values, e.g., from about 100 pM to about 1 nM, from about 500 pM to about 900 pM, etc.

[0203] As described elsewhere herein, the binding agent may comprise a protein or peptide. In some instances, the binding agent comprises an antibody, antibody fragment, e.g., Fab, single-chain variable fragment (scFv), or nanobody. In some embodiments, the binding agent comprises an antibody, such as an immunoglobulin G (IgG). The antibody may be a monoclonal antibody or a polyclonal antibody (e.g., of a population of antibodies). A monoclonal antibody may be obtained from a polyclonal mixture of antibodies. The binding agent may be a purified binding agent (e.g., a purified antibody from sera of an animal or from a monoclonalhybridoma), or the binding agent may be recombinant. In some instances, the binding agent comprises an scFv.

[0204] In some instances, the binding agent is engineered using any suitable approach such as directed evolution approaches including, but not limited to phage display, yeast display, yeast surface display, ribosome display, mammalian surface display, bacterial display, mRNA display, droplet-based directed evolution, continuous evolution approaches (e.g., OrthoRep). Display technologies may include or incorporate genetic diversification strategies (e.g., error-prone mutagenesis), as described elsewhere herein. In some instances, the binding agent is engineered using rational design or computational modeling, e.g., using RosettaDesign, PyMol, Schrodinger, MOE, and WAM, e.g., as described in Sevy and Meiler, 2018. Microbiology Spectrum.

[0205] Alternatively, or in addition to, amino acid substitutions, which may be conservative or non-conservative, may be performed to identify important residues of the binding agents that bind to the amino acids or derivatives thereof, thereby allowing for rational design to change a binding property (e.g., binding affinity, specificity, selectivity, dissociation rate, etc.) of the antibodies. In one example of conservative amino acid substitution, a native amino acid residue can be substituted with another amino acid residue such that there is little or no effect on the polarity, charge, or other property (e.g., hydrophobicity, hydrophilicity, isoelectric point) of the amino acid residue at that position. In another example, alanine scanning mutagenesis may be performed to determine the important residues for binding of the binding agent (e.g., antibody or antibody fragment). Conservative substitution may encompass non-naturally occurring amino acid residues, which can be incorporated by chemical peptide synthesis or synthesis in biological systems (e.g., orthogonal translation, expansion of the genetic code).

[0206] The binding agent may comprise or be coupled to a detectable label, e.g., fluorophore, fluorescent protein, radioisotope, mass tag, or an identifying polymerizable molecule (e.g., nucleic acid barcode molecule). The detectable label may be coupled the binding agent using any suitable approach, e.g., protein engineering to generate a fusion protein comprising a detectable protein (e.g., fluorescent or chromogenic protein such as a green fluorescent protein (GFP), peroxidase), SNAP -tag, CLIP -tag, a cysteine tag, SpyCatcher, SpyTag, chemical conjugation strategies, e.g., using NHS esters, click chemistry, affinity-based interactions, e.g., biotin-avidin or similar molecules such as desthiobiotin, streptavidin, neutravidin, etc. Additional conjugation strategies and example linkers are provided elsewhere herein.

[0207] In some embodiments, the binding agent comprises a noncanonical amino acid (ncAA), which may be used for coupling a molecule such as a detectable label to the binding agent. The binding agent may be engineered to comprise the ncAA using any useful technique, e.g., site-specific incorporation of noncanonical amino acids using an expansion of the genetic code of organisms, e.g., using archaeal tRNAs and aminoacyl-tRNA synthetases into bacterial or eukaryotic systems; genome engineering or recoding (e.g., adding or removing codons from the genetic code); engineered ribosomes; molecular evolution approaches, e.g., as described in Arranz-Gibert et al, 2018. Curr Opinion Chem. Bio., 46; 203-211, and as reviewed in De la Torre and Chin, 2021. Nature Reviews Genetics, 22, 169-184, each of which is incorporated by reference herein in its entirety. In some instances, the noncanonical amino acid comprises a click chemistry moiety (e.g., azide, DBCO, BCN, TCO, tetrazine, or other click chemistry moiety), which can facilitate conjugation of the detectable label via a complementary click chemistry (e.g., azide-DBCO click reaction).

[0208] Libraries of Binding Agents: The binding agent may be provided as part of a library of binding agents, which library can collectively recognize and specifically or semi-specifically bind to any number of proteinogenic amino acid types or derivatives thereof, under a given set of conditions. In some embodiments, the library of binding agents comprises a plurality of binding agents that can recognize and specifically bind to at least 2, at least 3, at least 4, 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or all 20 different types of proteinogenic amino acids. The library of binding agents can comprise any number of binding agents to recognize any number of amino acid types or derivatives thereof. The library of binding agents may comprise binding agents with single-specificity or specificity to a set of amino acid types. In an example, the library may comprise a first binding agent with specificity to a single amino acid type, whereas a second binding agent may have specificity to more than a single amino acid type (e.g., may bind to 2, 3, 4, or 5 amino acids). In some instances, fewer than 20 binding agents are required to recognize all 20 proteinogenic amino acids or derivatives thereof. In some instances, more than 20 binding agents may be provided to recognize all 20 proteinogenic amino acids or derivatives thereof. In one such example, multiple interrogations of a single amino acid or derivative thereof using one or more binding agents that recognize at least one type of amino acid or derivative thereof may be performed, which can improve the accuracy of identification of the amino acid.

[0209] The library of binding agents may comprise different types of binding agents. For example, a first binding agent may comprise an antibody, a second binding agent may comprisean antibody fragment, and a third binding agent may comprise a tRNA synthetase. Alternatively, all of the binding agents in the library may be of the same type (e.g., all antibodies, all antibody fragments, all engineered tRNA synthetases, all engineered ClpS or ClpX proteins, etc.).

[0210] Advantageously, the use of a library of binding agents may increase efficiency and specificity of some binding agents within the library of binding agents. For instance, two or more binding agents within the library of binding agents may bind differentially to a target amino acid or derivative thereof and may thus competitively bind for the target amino acid, thereby reducing non-specific binding. Similarly, binding agents with varying levels of specificity may be used in a particular sequence or order, which may help render a less-specific binding agent to be more specific, simply based on the sequence in which it is provided. For example, a first binding agent may be capable of specifically binding to a first amino acid or derivative thereof, and a second binding agent may be capable of binding to both the first amino acid or derivative thereof and a second amino acid or derivative thereof. The first binding agent may be provided and contacted with the first amino acid or derivative thereof and second amino acid or derivative thereof. Since the first binding agent is specific to the first amino acid or derivative thereof, the first binding agent will bind exclusively to the first amino acid or derivative thereof. Subsequently, the second binding agent may be provided; however, since the first amino acid or derivative thereof is bound to the first binding agent, the first amino acid or derivative thereof may be inaccessible (e.g., sterically blocked) to the second binding agent. As such, the second binding agent may bind only to the second amino acid or derivative thereof. Accordingly, identification of the first binding agent and second binding agent (e.g., through detection of a label / tag or through sequencing of polymerizable molecules coupled to the binding agents and optionally transferred to a substrate), may allow for identification of the first amino acid or derivative thereof and the second amino acid or derivative thereof.

[0211] The binding agents may be provided at any useful concentration. The binding agents may be provided at a concentration of about 1 pg / mL, about 10 pg / mL, about 100 pg / mL, about 1 pg / mL, about 10 pg / mL, about 100 pg / mL, about 1 mg / mL, or greater. In some instances, the binding agent may be provided at a concentration of at most about 1 mg / mL, at most about 100 pg / mL, at most about 10 pg / mL, at most about 1 pg / mL, at most about 0.1 pg / mL, or lesser. The binding agents in a library of binding agents may be provided at the same concentration or at different concentrations. For example, each binding agent may be titrated to its cognate molecule (e.g., amino acid type or derivative thereof) to achieve optimal specificity.

[0212] The binding agents may comprise or be coupled to any useful number of functional moieties, as described elsewhere herein. For example, the binding agent may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more functional moieties attached thereto.

[0213] Also provided herein are kits and compositions, which may comprise a plurality of binding agents. A kit or composition of the present disclosure may comprise a plurality of binding agents, in which the plurality of binding agents specifically or semi-specifically bind to a plurality of different types of proteinogenic amino acids or derivatives thereof. In some embodiments, the plurality of binding agents may bind to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or all 20 types of proteinogenic amino acids or derivatives thereof. In some embodiments, the plurality of binding agents may bind to at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or all 20 different types of proteinogenic amino acids or derivatives thereof. In some embodiments, the binding agents comprise antibodies or antibody fragments.

[0214] Also provided herein are antibody or antibody fragments that comprise a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs) and a light chain variable region (VL) comprising three CDRs, wherein the VH CDR #1 sequence has at least 80% homology to a sequence selected from Table 1, wherein the VH CDR #2 sequence has at least 80% homology to a sequence selected from Table 2, wherein the VH CDR #3 sequence has at least 80% homology to a sequence selected from Table 3, wherein the VL CDR #1 sequence has at least 80% homology to a sequence selected from Table 4, wherein the VL CDR #2 sequence has at least 80% homology to a sequence selected from Table 5, and wherein the VL CDR #3 sequence has at least 80% homology to a sequence selected from Table 6.

[0215] Table 1

[0216] Table 2

[0217] Table 3

[0218] Table 4

[0220] Table 6

[0221] In some embodiments, the heavy chain sequence has at least 80% homology to a sequence selected from Table 7.

[0222] Table 7

[0223] In some embodiments, the light chain sequence has at least 80% homology to a sequence selected from Table 8.

[0224] Table 8

[0225] In some embodiments, the VH sequence has at least 80% homology to a sequence selected from Table 9.

[0226] Table 9

[0227] In some embodiments, the VL sequence has at least 80% homology to a sequence selected from Table 10.

[0228] Table 10

[0229] In some aspects of the present disclosure, provided herein in is a binding agent that selectively binds to tryptophan or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof.

[0230] In some aspects of the present disclosure, provided herein in is a binding agent that selectively binds to phenylalanine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof.

[0231] In some aspects of the present disclosure, provided herein in is a binding agent that selectively binds to leucine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof.

[0232] In some aspects of the present disclosure, provided herein in is a binding agent that selectively binds to isoleucine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof.

[0233] In some aspects of the present disclosure, provided herein in is a binding agent that selectively binds to valine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof.

[0234] In some aspects of the present disclosure, provided herein in is a binding agent that selectively binds to tyrosine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof.

[0235] In some aspects of the present disclosure, provided herein in is a binding agent that selectively binds to proline or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof.Methods and Compositions for Producing Amino Acid Binding Agents

[0236] Also provided herein, in some aspects, are methods for producing the binding agents described herein that recognize individual monomer (e.g., amino acid) types. In one aspect, the binding agents described herein are produced using an immunization method. In some embodiments, the binding agent comprises an antibody or an antibody fragment. In some embodiments, the binding agent is produced by a method, the method comprising: immunizing an animal with an immunogenically effective amount of a composition comprising an amino acid or derivative thereof and a polymeric linker molecule, and obtaining the binding agent; optionally, the obtaining of the binding agent may comprise additional processing or engineering of the binding agent. The composition used to immunize the animal may further comprise a carrier molecule coupled to the polymeric linker molecule and the amino acid or derivative thereof.

[0237] In one example, a binding agent may be produced using a method that comprises immunizing an animal with an immunogenically effective amount of a composition comprising a carrier molecule coupled to (i) a polymeric linker molecule and (ii) an amino acid or derivative thereof, and obtaining a binding agent from the animal. The binding agent may comprise an antibody, such as an immunoglobulin G (IgG). In some instances, the binding agent may be further processed or engineered to generate an amino acid binding agent that may recognize or bind to a single or several amino acid types. Examples of processing or engineering antibodies or antibody fragments (e.g., single chain variable fragments (scFvs)) include hybridoma technology to generate monoclonal antibodies, or cloning and directed evolution approachessuch as phage display and phage panning, yeast display, yeast surface display, mammalian display, bacterial display, ribosome display, mRNA display, droplet-based directed evolution, and continuous evolution approaches, e.g., OrthoRep, phage-assisted continuous evolution (PACE) or periplasmic PACE (pPACE). In some instances, the directed evolution approaches may comprise cloning one or more sequences of the antibodies or antibody fragments (e.g., a VH and VL sequence) into an expression vector, expressing in a host cell (e.g., bacterial, yeast, mammalian cell), purifying, and assaying for binding.

[0238] Polymeric linker'. The composition used for immunizing the animal may comprise a carrier molecule that is coupled to at least one polymeric linker. The polymeric linker can comprise any useful polymer. The polymer may be naturally occurring or synthetic, such as acrylics, nylons, silicones, viscose, rayon, polyesters, polycarboxylic acids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol (PEG), polyurethane, polylactic acid, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, poly(chlorotrifluoroethylene), poly(ethylene oxide), polyethylene terephthalate), polyethylene, polyisobutylene, poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde, polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene dichloride), poly(vinylidene difluoride), poly(vinyl fluoride), poly-L-lysine (PLL), poly (DL-lactic acid) (PLA), poly (DL-lactide-co-glycoside) (PLGA), polycaprolactone (PCL), polyvinyl acid (PVA), polyethylene terephthalate (PET), polystyrene (PSt), poly ornithine, polyarginine, or other useful polymer. The polymer may comprise a biopolymer, e.g., a polypeptide or a nucleic acid molecule e.g., DNA molecule, RNA molecule, or DNA:RNA hybrid.

[0239] The polymeric linker may comprise one or more functional groups, which may allow for conjugation of the polymeric linker to the amino acid or derivative thereof and / or the carrier molecule. Examples of useful functional moieties include, but are not limited to, click chemistry moieties such as azide, alkyne and cycloalkynes such as BCN and DBCO, nitrone, alkene (e.g., a strained alkene), tetrazine, methyltetrazine, triazole, tetrazole, phosphite, and phosphine; photoreactive moieties such as phenyl azides, benzophenone, psoralen, diazirines; carboxy-reactive crosslinker groups; amino-reactive crosslinker groups such as NHS esters; an EDC group, a maleimide, a thiol, a cystamine, an aldehyde, a succinimidyl group, an epoxide, an acrylate, or other crosslinking or reactive group. Additional examples of linkers and functional moieties are provided elsewhere herein. The polymeric linker may comprise any useful number of functional groups, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional groups, which may be the same or different functional groups.

[0240] The polymeric linker may comprise any useful number of monomers or have any desired length or molecular weight, e.g., to control the molecular distance between the carrier protein and the amino acid or derivative thereof. The polymeric linker may comprise about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more monomers. The polymeric linker may comprise at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, or greater monomers. Alternatively, the polymeric linker may comprise at most about 100, at most about 50, at most about 10, at most about 5, or fewer monomers.

[0241] The polymeric linker may comprise any useful length under a given set of conditions. For example, the polymeric linker in an aqueous solution may be about 0.01 nm, about 0.1 nm, about 0.2 nm, about 0.3 nm, about 0.4 nm, about 0.5 nm, about 0.6 nm, about 0.7 nm, about 0.8 nm, about 0.9 nm, about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm or greater in length. The polymeric linker may be at least about 0.01 nm, at least about 0.1 nm, at least about 0.2 nm, at least about 0.3 nm, at least about 0.4 nm, at least about 0.5 nm, at least about 0.6 nm, at least about 0.7 nm, at least about 0.8 nm, at least about 0.9 nm, at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 20 nm, at least about 30 nm, at least about 40 nm, at least about 50 nm, at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm or greater in length. Alternatively, the polymeric linker may be at most about 100 nm, at most about 90 nm, at most about 80 nm, at most about 70 nm, at most about 60 nm, at most about 50 nm, at most about 40 nm, at most about 30 nm, at most about 20 nm, at most about 10 nm, at most about 5 nm, at most about 1 nm or smaller in length.

[0242] Carrier molecule: The carrier molecule can comprise any useful molecule that facilitates an immune response of the animal to the composition. The carrier molecule may comprise a nanoparticle (e.g., gold nanoparticles), a liposome, a vesicle, or a biomolecule, such as a protein. Examples of carrier molecules include, in non-limiting examples, keyhole limpet hemocyanin (KLH), concholepas concholepas hemocyanin (CCH), Q virus-like particle, outer membrane protein complex (OMPC), genetically modified cross-reacting material (CRM) of diphtheria toxin, tetanus toxoid (TT), diphtheria toxoid (DT), H. influenzae protein D (HiD),glycoengineered outer membrane vesicles (geOMVs), recombinant Hla, rEPA, and recombinant pneumolysine, among others, e.g., as reviewed by Micolia et al. 2018. Molecules. 23(6): 1451, which is incorporated by reference herein. Additional examples of carrier proteins include albumin (e.g., bovine serum albumin, cationic bovine serum albumin, ovalbumin, cationic ovalbumin), biotin, streptavidin, hemocyanin carrier protein, and Blue Carrier™ protein.

[0243] As described elsewhere herein, the binding agent may be configured to recognize and bind to an amino acid derivative, including chemically modified amino acids. In some instances, the amino acid derivative comprises an amino acid that has been modified via a reaction with a linker. In one example, the linker comprises an amino acid reactive group, e.g., an isothiocyanate such as PITC, which may react with the N-terminal amino acid of a peptide, e.g., under basic conditions. The reaction of the PITC with the N-terminal amino acid generates a PTC-derivatized amino acid, which may further undergo cleavage, e.g., using acidic and / or heat conditions, thereby generating a cleaved ATZ-derivatized amino acid that is free from the peptide. The ATZ-derivatized amino acid may, in some instances, be subjected to further derivatization conditions, e.g., to generate a PTH or PTC-derivatized amino acid.

[0244] Accordingly, the methods provided herein for generating a binding agent include providing the compositions comprising the amino acid derivatives. The composition may comprise a carrier molecule, such as a carrier protein, that is coupled to a polymeric linker (e.g., a bifunctional linker comprising a polymer region) and a derivatized amino acid (e.g., an amino acid-linker complex). In one particular example, the derivatized amino acid may be generated by reacting a first bifunctional linker comprising a PITC moiety and a click chemistry moiety (e.g., azide), as described elsewhere herein, to generate an amino acid-linker complex that comprises a PTC-derivatized amino acid and the click chemistry moiety. The click chemistry moiety (e.g., azide) of amino acid-linker complex may be used to couple the amino acid-linker complex to a carrier molecule (e.g., carrier protein), e.g., a carrier molecule that comprises a click chemistry moiety, or via a polymeric linker that comprises a click chemistry moiety (e.g., DBCO). In some examples, the carrier molecule comprises a bovine serum albumin (BSA) protein that is conjugated to a PEG polymeric linker that comprises a click chemistry moiety (e.g., DBCO). The PEG linker may be coupled to the BSA using, for example, an NHS ester reaction (e.g., a NHS-PEG-DBCO) with amine groups (e.g., of the lysine residues) of the BSA. The click chemistry moiety of the PEG polymeric linker may join with the click chemistry moiety of the amino acid-linker complex (e.g., DBCO-azide click reaction), thereby generating a BSA-PEG- derivatized amino acid complex. In some examples, the amino acid need not be derivatized prior to conjugation to the carrier molecule or the polymeric linker.

[0245] The carrier molecule may be coupled to any useful number of molecules of the amino acids or derivatives thereof, e.g., to generate an immune response in the animal. The carrier molecule may be coupled to about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or a greater number of molecules of the amino acids or derivatives thereof. The carrier molecule may be coupled to at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or greater number of molecules of the amino acids or derivatives.

[0246] Similarly, any number of polymeric linkers may be used to couple the amino acids or derivatives thereof to the carrier molecule. The carrier molecule may be coupled to about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or a greater number of polymeric linkers. The polymeric linkers may be provided at any useful ratio to the amino acids or derivatives thereof. For instance, the polymeric linkers may be provided at a ratio of approximately 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 1 :50, 1 : 100, 1 :1000, 1 : 10,000 or less to the number of amino acids or derivatives thereof.

[0247] The compositions described herein contemplate all 20 proteinogenic amino acid types as well as modified amino acids (e.g., amino acid derivatives, post-translationally modified amino acids). Individual compositions for each or a subset of the amino acid types may be provided. The methods described herein for generating binding agents using an immunization approach may include using one or more animals per composition per amino acid type. For example, a first composition may be generated that comprises a carrier molecule, a polymeric linker, and one amino acid type of the 20 amino acid types. A second composition may be generated that comprises a carrier molecule, a polymeric linker, and another amino acid type of the 20 amino acid types that is different than that of the first composition. The first composition and the second composition may be separately injected into animals for generation of unique binding agents (e.g., antibodies) toward the target antigen (the particular amino acid type).

[0248] The compositions described herein may additionally comprise or be provided with an adjuvant, including but not limited to aluminum hydroxide, complete Freund’s adjuvant (CFA or FC A), incomplete Freund’s adjuvant (IFA or FIA), Ribi adjuvants, Titermax, Specol, or others.

[0249] In some embodiments, the method for generating the binding agent comprises obtaining a polyclonal mixture of antibodies from the animal, which polyclonal mixture of antibodies comprises a plurality of antibodies that can recognize and bind preferentially to one type of proteinogenic amino acid or derivative thereof (e.g., the amino acid type for which the animal was immunized).

[0250] In some instances, the binding agent is a monoclonal antibody. The monoclonal antibody may be generated using any useful technique, e.g., hybridoma technology. In some examples, the monoclonal antibody is generated by immunizing an animal with a composition comprising the antigen of interest (e.g., an amino acid or amino acid derivative), recovering lymphatic cells (e.g., B cells) from the animal, and fusing the recovered cells with an immortal cell line (e.g., myeloid-type cell lines such as myeloma cells), thereby generating a hybridoma. The hybridoma cell lines can then be screened and selected to identify individual hybridoma cells that produce antibodies specific to the antigen of interest. The antibodies may be harvested directly from the cells (e.g., from culture medium). In some instances, the hybridomas that produce the antibodies may be sequenced, and recombinant antibodies or antibody fragments (e.g., scFvs) can be generated using the DNA or the RNA sequences (e.g., RNA sequences from the hybridoma cell may be obtained, reverse transcribed, and sequenced).

[0251] Alternatively, or in addition to, the binding agent may comprise a recombinant antibody or antibody fragment that is generated using genetic recombination. For example, a monoclonal cell (e.g., a B cell harvested from an animal) or hybridoma cell that produces a particular antibody may be genomically or transcriptomically sequenced to obtain the DNA or RNA sequence or sequences that correspond to the antibody or portions of the antibody, e.g., the variable heavy chain region, the variable light chain region, etc. The DNA or RNA sequence can then be used to generate recombinant antibodies (e.g., via translation of the DNA or RNA sequence, transformation or transduction of the gene in a host cell, or protein synthesis of the corresponding amino acid sequence). In some instances, recombinant antibody fragments, e.g., scFvs are generated, e.g., by isolation of RNA from a B cell from an immunized animal, reverse transcription of the RNA to obtain the VH and VL sequences, and recombining the VH and VL sequences in a vector. The VH and VL sequences may be input into a phagemid vector, a yeast vector, a mammalian cell vector, a plasmid, etc., and transformed into a carrier or host cell (e.g., yeast, mammalian, bacteria, phage) in order to generate the recombinant antibody fragment. Bioconjugation of the binding agent may be performed to attach useful functional moieties to the binding agent. For example, bioconjugation approaches may allow for attachment of detectable labels such as nucleic acid barcodes, fluorophores, proteins (e.g., fluorescent proteins,antibodies or antibody fragments), mass tags, etc. to the binding agent. Non-limiting examples of bioconjugation strategies include chemical strategies such as use of silane, e.g., aminosilane (e.g., APTES), amino-PEG-silane, oxidation of sugars and reaction with amines, PLP transamination and reaction with alkoxyamine, aminooxy or hydroxylamine or hydrazine, aldehyde and organoboronate, o-aminophenols and catechols, activated esters (e.g., NHS), sulfonyl chloride, isocyanates, isothiocyanates, aldehydes, sulfonyl acrylate, maleimide, haloacetamide, alkynyl carboxylic acid derivatives, 5-methylene pyrrolone, 5,5'-dithiobis-(2- nitrobenzoic acid), cyclooctyne, diazonium salt, in situ generated imine, 4-phenyl-3H- 1,2,4- triazole-3,5(4H)-dione, phenothiazine, 9-azabicyclo [3.3.1]nonane-3-one-N-oxyl, N-substituted pyridinium salts, oxaziridine, hypervalent iodine, lumiflavin, phosphorus(V)reagent and DBU, tetrazole derivatives, aldehyde and isocyanide, H-azirine-based reagents, dicarbonyl compounds, p-azidophenylglyoxalhydrate, 2-cyclohexenone, thiophosphoro alkyne dichloridate, sulfinate salts, light promoted reaction with C4-alkyl-l,4-dihydropyridine reagents, or other reactive moieties. Examples of enzymatic approaches to bioconjugation include sortase A, microbial transglutaminase, tyrosinase (e.g., tyrosinase mediated oxidative coupling), famesyltransferase, N-myristoytransferase, phosphopantetheinyl transferases, tubulin tyrosine ligase, lipoic acid ligase, biotin ligase, formylglycine generating enzyme, SnoopTag / SnoopCatcher, SnoopLigase, SpyTag / SpyCatcher, etc.

[0252] In some instances, bioconjugation may be achieved using expansion of the genetic code, as described elsewhere herein. Non-limiting examples of noncanonical amino acids include the incorporation of click chemistry moieties, which can mediate conjugation of additional molecules via click chemistry reactions, e.g., via CuAAC, SPAAC, IEDDA, and other click reactions. Additional examples of conjugation approaches (e.g., for peptides to substrates) are provided elsewhere herein and are applicable for bioconjugation of useful molecules to the binding agents.

[0253] Any useful number of functional moieties may be attached to the binding agent. The binding agent may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more functional moieties attached thereto. The binding agents may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or greater functional moieties attached thereto. If a plurality of binding agents is used, each binding agent may comprise the same or different number of functional moieties attached thereto.

[0254] In some instances, the method for generating the binding agent further comprises engineering a precursor binding agent to arrive at a binding agent that has improved bindingproperties (e.g., affinity, specificity) to its target antigen (e.g., amino acid type). Examples of engineering antibodies include directed evolution approaches, which can include derivatizing the antibodies into antibody fragments, such as scFvs, and / or using any number of useful evolution approaches, e.g., phage display and phage panning, yeast display, yeast surface display, mammalian display, mammalian surface display, bacterial display, ribosome display, mRNA display, etc. Additional genetic diversity, e.g., for generating additional mutations of the binding agent sequences, may be implemented, e.g., using error-prone mutagenesis, e.g., error prone PCR, error prone rolling circle amplification (epRCA), random insertional-deletional strand exchange (RAISE), tandem repeat insertion (TRINS), mini-mu transposon based techniques, DNA shuffling, SELEX, staggered expansion process (StEP), random chimeragenesis on transient templates (RACHITT), Incremental Truncation for the Creation of Hybrid enzymes (ITCHY) and SCRATCHY, Sequence Homology-Independent Protein Recombination (SHIPREC), Sequence Independent Site Directed Chimeragenesis (SISDC), Kunkel mutagenesis, Mutagenic Organized Recombination Process by Homologous in vivo Grouping (MORPHING), saturation mutagenesis, site- saturation mutagenesis, Stepwise Loop Insertion Strategy (StLois), FACS-enabled high throughput screening, plate-based automated enzymatic assays, MS-based methods, QUEST, cofactor regeneration coupling, in vitro compartmentalized self-replication, Multiplex Automated Genome Engineering (MAGE), CRISPR-based mutagenesis, phage-assisted continuous evolution (PACE), periplasmic PACE, and others, e.g., as reviewed by Vidal et al. 2023. RSC Chem. Biol. 4, 271-291. In some instances, increased genetic diversity may be generated using cloning or amplification with degenerate codons, DNA shuffling, CDR recombination, or other approach.Analysis of Polymeric Analytes via Local Tethering

[0255] The present disclosure also provides for approaches for processing and analyzing polymeric analytes, e.g., peptides, polymers, nucleic acid molecules, etc. in a highly-parallelized and accurate manner. Systems and methods of the present disclosure may comprise cleaving a monomer from the polymeric analyte, coupling (e.g., via local tethering) of the monomer to a capture moiety, and detecting the monomer. In some embodiments, detecting comprises using monomer-specific binding agents to recognize and bind the cleaved monomers. In some embodiments, the monomer-specific binding agents are used for direct or indirect detection; for example, the binding agents may comprise a detectable label (e.g., fluorophore, mass tag, radioisotope, fluorescent protein) that can be detected directly, or the binding agent may comprise a polymerizable molecule with encoded information, which may be transferred via coupling to orcopying of the encoded information onto the capture moiety or an additional polymerizable molecule. In some instances, the additional polymerizable molecule or the capture moiety is located adjacent to the polymeric analyte. Alternatively, or in addition to, the binding agents may be used to sort the cleaved monomers, e.g., into separate partitions or compartments for downstream labeling, e.g., with identifying barcode molecules. The operations may be iterated or repeated any number of times to obtain information on all or a subset of the monomers of the polymeric analyte and optionally, the sequence of the monomers relative to the polymeric analyte. The information may be read out from the polymerizable molecules using, for example, conventional next-generation sequencing or nanopore sequencing approaches. Beneficially, by cleaving the monomer from the polymeric analyte, the monomers are removed from the adjacent monomers, and binding agents can specifically bind to individual monomers without the influence of the adjacent surrounding monomers. As such, the methods disclosed herein enable more accurate molecular identification and polymeric sequencing, which has applications in diagnosing disease, monitoring protein dynamics or protein interactions, single-cell proteomics, developing or characterizing therapeutics, and more.

[0256] In some embodiments, the methods provided herein comprise performing an intramolecular expansion of a polymeric analyte, e.g., a peptide, to generate a modified monomer (e.g., modified amino acid). Such an intramolecular expansion process may comprise providing a linker, coupling the linker to a monomer of the polymeric analyte to generate a monomer-linker complex, coupling the linker or the monomer-linker complex to a capture moiety, and cleaving the monomer from the polymeric analyte, thereby yielding a modified monomer. In some instances, the method may further comprise repeating the intramolecular expansion or one or more operations of the intramolecular expansion on the next monomer of the polymeric analyte to generate another modified monomer. In some instances, the monomer-linker complex or the modified monomer resulting from a round or cycle of intramolecular expansion may be coupled to that of the previous round or cycle of intramolecular expansion to generate a stacked plurality of modified monomers, e.g., linked by a polymerizable molecule backbone. In some instances, the modified monomer or stacked plurality of modified monomers is detected using a nanopore sequencer, thereby outputting the identity of the individual monomers of the polymeric analyte.

[0257] One or more methods of the present disclosure may employ the use of a linker that is capable of coupling to (i) a monomer of the polymeric analyte and (ii) a capture moiety, which may be used to locally tether the monomer, once cleaved, adjacent to the polymeric analyte or directly attached to the polymeric analyte. The methods and systems disclosed herein may, in some instances, additionally comprise a substrate for the local tethering; the substrate may becoupled, for example, to the polymeric analyte, the capture moiety, and an additional polymerizable molecule. In some instances, the additional polymerizable molecule is encoded with information from the polymerizable molecules of the binding agents. In other instances, the binding agent comprises a detectable label that can indicate a binding event between the binding agent and the monomer. Alternatively, or in addition to, one or more processes described herein may be performed in solution or in the absence of a substrate.

[0258] Methods of the present disclosure for processing a polymeric analyte comprising a plurality of monomers may comprise cleaving of a monomer of the polymeric analyte and coupling the monomer to a capture moiety (e.g., coupled to a substrate, the polymeric analyte, or provided in solution) for subsequent processing or analysis. In one example, a method of the present disclosure may comprise: providing (i) a polymeric analyte comprising a plurality of monomers and (ii) a capture moiety; coupling a monomer of the plurality of monomers to the capture moiety to generate a monomer-capture moiety complex; cleaving the monomer; contacting the cleaved monomer-capture moiety complex with the binding agent; and coupling a first polymerizable molecule to a second polymerizable molecule or to the capture moiety. In some instances, the first polymerizable molecule is coupled to the binding agent and comprises information about the binding agent, such as the identity of the binding agent or its cognate molecule, which information may be transferred to the second polymerizable molecule or to the capture moiety. In some instances, the capture moiety may be a copy or identical molecule as the second polymerizable molecule. Alternatively, or in addition to, the binding agent may be used to sort a mixture of cleaved monomers by identity or type, and subsequent to sorting, an identifying label or barcode that identifies the monomer type may be coupled to the capture moiety. Example methods and systems of such processing approaches and systems are described in U.S. Pat. No. 11,499,979, International Pub. No. WO / 2023 / 114732, International Pat. App. Pub. No. WO / 2023 / 196642, and U.S. Pat. App. No. 18 / 740,088, filed June 11, 2024, and L. Zheng et al. 2024. Peptide sequencing via reverse translation of peptides into DNA. BioRxiv., each of which is incorporated by reference herein in its entirety.

[0259] In some embodiments, the coupling of the barcode molecule to the polymeric analyte, the capture moiety, or the polymerizable molecule is mediated using a nuclease. The nuclease may be an editing endonuclease, such as a CRISPR-associated (Cas) enzyme or a prime editor enzyme. The Cas enzyme or prime editor enzyme may comprise any useful Cas enzyme, e.g., a DNA-targeting Cas enzyme such as Cas9, Cas 3, Casl2a, Casl4, or an RNA- targeting Cas enzyme such as Casl3 or Cas 7-11, or a modified variant thereof. The prime editor enzyme may comprise polymerizing enzyme, e.g., a polymerase such as a DNA polymerase,RNA polymerase, reverse transcriptase, or modified variant thereof. In some instances, the prime editor enzyme comprises a modified Cas nickase (e.g., a modified Cas9 nickase) and a modified reverse transcriptase. Beneficially, use of a prime editor enzyme may allow for accurate polymeric sequencing by allowing for efficient approaches to barcoding of individual monomers that do not involve generating double-stranded DNA breaks and which allow for efficient stacking of barcode molecules.

[0260] In some embodiments, the polymerizable molecule comprises a tandem array of nuclease target sites, e.g., CRISPR-Cas9 target sites, restriction sites, transposition sites, etc. In one such example, the tandem array comprises a plurality of CRISPR-Cas9 target sites, and the barcode molecule comprises a prime-editing guide RNA (pegRNA). Coupling or copying of the barcode molecule to the polymerizable molecule may be mediated using a DNA typewriter approach, e.g., as described in J. Choi et al. 2022. Nature. 608, pages 98-107, which is incorporated by reference herein in its entirety. In such an approach, the polymerizable molecule may comprise a tandem array of nuclease recognition sites, e.g., CRISPR-Cas target sites (such as CRISPR-Cas9 target sites). The tandem array of nuclease recognition sites may, for example, comprise a plurality of CRISPR-Cas9 target sites (for simplicity, “target sites”) located adjacent to one another. A first target site of the plurality of target sites may be active and the other target sites of the plurality of target sites except for the first target site may be inactive. For example, the plurality of target sites, except for the first target site, may be truncated such that the target site is inactive or not recognized by the requisite nuclease (e.g., Cas9 or prime editor enzyme comprising a modified Cas9). For instance, the plurality of target sites, except for the first target site, may be truncated at the 5’ ends of the target sites. The barcode molecule may comprise a pegRNA molecule that comprises a key sequence or complement thereof that completes or activates one of the target sites. For instance, the pegRNA molecule may comprise a key sequence or complement thereof that activates a second target site of the plurality of target sites, which second target site is located adjacent to the first target site. The pegRNA molecule may additionally comprise a barcode sequence (or complement thereof). In such an example, a Cas or prime editor enzyme may be used to couple the pegRNA or a complement thereof to the first target site, thereby inserting a barcode sequence and the key sequence adjacent to the first target site and thereby inactivating or deactivating the first target site whilst activating the second target site. Such an approach can be mediated using a prime editor enzyme comprising a Cas protein (e.g., a Cas enzyme or Cas nickase) and a polymerizing enzyme (e.g., reverse transcriptase). For example, in prime editing, the Cas protein may cleave a first strand of the polymerizable molecule, and a portion of the pegRNA (e.g., a primer-binding site) mayhybridize to a portion of the cleaved strand. An extension reaction, e.g., reverse transcription, may then generate a complementary DNA segment that is complementary to at least a portion of the remaining pegRNA. The generated complementary DNA segment may then be incorporated into the polymerizable molecule by hybridizing to the second strand of the polymerizable molecule. Additional operations may be performed, such as flap cleavage and ligation. In some instances, the prime editor comprises a Cas nickase, which may increase the efficiency of incorporation of the cDNA segment into the polymerizable molecule. Additional barcoding operations, e.g., in iterating the barcoding process or the entire workflow for processing of additional monomers of the polymeric analyte, may subsequently deactivate the second target site whilst activating a third target site adjacent to the second target site, and so forth.

[0261] In some instances, the barcode molecule comprises a pegRNA molecule that is used for editing the polymerizable molecule, e.g., without insertion of additional nucleotides. In one such example, the barcode molecule may comprise a key sequence and a barcode sequence that replaces a sequence of the first target site, which results in inactivation of the first target site and activation of the second target site. In such an example, the polymerizable molecule is edited to include a barcode sequence, rather than elongated via insertion of additional nucleotides or sequences.

[0262] Alternatively, or in addition to, the polymerizable molecule may be coupled to the barcode molecule using a peCHYRON approach, e.g., as described by T. Loveless et al. 2021. BioRxiv. 11.05.467507, which is incorporated by reference herein in its entirety. In such an approach, the polymerizable molecule may comprise a protospacer adjacent motif (PAM) and a first nuclease target site, e.g., a CRISPR-Cas9 target site (“target site”). In such an example, the barcode molecule may comprise a pegRNA molecule that comprises a barcode sequence (or complement thereof) and a propagator sequence (or complement thereof) that comprises a second target site. A Cas or prime editor enzyme may be used to couple the pegRNA to the first target site, e.g., between the target site and the PAM, thereby inserting the barcode sequence and the propagator sequence adjacent (or complements thereof) to the first target site. Such insertion may result in the deactivation of the first target site and activation of the second target site and elongate the polymerizable molecule. Additional barcoding operations, e.g., in iterating the barcoding process or the entire workflow for processing of additional monomers of the polymeric analyte, may subsequently add additional target sites to the first target site and the third target site, e.g., a third target site, a fourth target site, and so forth.

[0263] FIG. 1A shows an example workflow for processing a polymeric analyte for sequencing. In workflow 100, a polymeric analyte 103 is provided, sequentially disassembledinto individual monomers via contacting with capture moieties and cleavage, and the individual cleaved monomers complexed with the capture moieties are contacted with binding agents comprising polymerizable molecules (e.g., barcode molecules) that identify or encode for the binding agents. The polymerizable molecule (e.g., a barcode molecule) of a binding agent is coupled to an additional polymerizable molecule, and the monomer is cleaved from the capture moiety or blocked to prevent downstream recognition from additional binding agents. In FIG. 1A Panel A, a substrate 101 is coupled to a polymeric analyte 103 (e.g., a peptide to be sequenced), a capture moiety 105 (e.g., a nucleic acid primer), and an additional polymerizable molecule 107 (e.g., an additional nucleic acid primer). In some instances, the capture moiety 105 and the additional polymerizable molecule 107 are identical molecule types (e.g., both nucleic acid molecules that comprise the same sequence). The polymeric analyte may be contacted with a bifunctional linker 109, which comprises a terminal monomer-coupling group (e.g., an amino acid-reactive group such as PITC) and a click chemistry moiety (e.g., azide). In some instances, the bifunctional linker 109 couples to a terminal monomer (e.g., terminal amino acid such as the N-terminal amino acid (NTAA)). In FIG. 1A Panel B, a linking nucleic acid molecule 111 comprising a click chemistry moiety (e.g., alkyne) is reacted with the bifunctional linker 109 and covalently linked. In some instances, the linking nucleic acid molecule 111 and the bifunctional linker 109 are provided pre-coupled (see, e.g., FIG. 2). In FIG. 1A Panel C, the linking nucleic acid molecule 111 is coupled to the capture moiety 105, thereby generating a monomer-capture moiety complex. The coupling may be mediated by hybridization of the linking nucleic acid molecule 111 to the capture moiety 105 (hybridization not shown), or by using a splint oligonucleotide 113 comprising sequences complementary to a sequence of the linking nucleic acid molecule 111 and the capture moiety 105. In some instances (not shown), the linking nucleic acid molecule 111 comprises a self-splinting sequence, such that the linking nucleic acid molecule 111 may couple to the capture moiety 105 in the absence of a separate splint molecule. A ligase may be used to covalently link the linking nucleic acid molecule 111 to the capture moiety 105. Alternatively, the linking nucleic acid molecule 111 may comprise a first reactive moiety (e.g., click chemistry moiety not shown) that can react with a second reactive moiety (not shown) of the capture moiety 105. In FIG. 1A Panel D, the system is subjected to conditions sufficient to cleave the terminal monomer (e.g., amino acid) from the polymeric analyte 103 (e.g., peptide), thereby generating a cleaved monomer-capture moiety complex. The conditions may include performing an Edman degradation reaction. The cleavage of the monomer from the polymeric analyte results in a cleaved monomer-capture moiety complex comprising the cleaved monomer, bifunctional linker 109, linking nucleic acid molecule 111,and capture moiety 105. In FIG. 1A Panel E, a binding agent 115 (e.g., antibody) comprising another polymerizable molecule 117 (e.g., nucleic acid barcode molecule) is provided. The binding agent 115 may be specific to the monomer (e.g., to an amino acid type) or to the monomer-linker complex (e.g., PITC-amino acid complex, which may be derivatized to a phenylthiocarbamoyl, phenylthiohydantoin, or anilinothiazolinone moiety). The polymerizable molecule 117 of the binding agent may comprise information on the identity of the binding agent or the specific monomer (e.g., single amino acid) to which the binding agent binds. The polymerizable molecule 117 of the binding agent may comprise additional sequences, such as a barcode sequence, UMI, restriction site, transposition site, a sequence to represent a cycle or iteration number, or other functional sequence. The polymerizable molecule 117 of the binding agent may couple to the additional polymerizable molecule 107 that is coupled to the substrate 101. In some instances, an extension reaction may be performed (e.g., using a polymerase), to copy the sequence of the polymerizable molecule 117 of the binding agent to the additional polymerizable molecule 107 that is coupled to the substrate 101. Alternatively, the polymerizable molecule 117 of the binding agent may be ligated to the additional polymerizable molecule 107, either chemically (e.g., via complementary click chemistry) or enzymatically (e.g., using a ligating enzyme, ribozyme or DNAzyme, or a nuclease such as a prime editor enzyme (see also, FIG. ID). Optionally, the polymerizable molecule 117 may be cleaved from the binding agent 115 (not shown). In FIG. 1A Panel F, the monomer may be decoupled (e.g., removed or cleaved) from the capture moiety 105. For example, the monomer, bifunctional linker 109, and all or a portion of the linking nucleic acid molecule 111 may be cleaved (depicted as a star). The cleavage may be performed chemically, mechanically, or enzymatically. In an example of enzymatic cleavage, the linking nucleic acid molecule 111 may comprise a restriction site or other cleavage site (e.g., a uracil), and cleavage occurs by introduction of a restriction enzyme or cleaving enzyme (e.g., uracil DNA glycosylase) to cleave the restriction / cleavage site. Alternatively, the cleaved monomer-capture moiety complex may be blocked with a blocking agent (not shown). The workflow 100 may then be iterated or repeated to process and sequence all or a portion of the polymeric analyte 103.

[0264] FIG. IB schematically shows another example of processing and characterizing a polymeric analyte 103, e.g., a peptide. A polymeric analyte 103 may be tagged (or provided pretagged) with a capture moiety 105, e.g., a polymerizable molecule such as a nucleic acid molecule. The capture moiety 105 may, for example, be attached at a terminus of a peptide (e.g., as shown at the C-terminus) or at an internal residue. The capture moiety 105 may, in some instances, comprise a barcode sequence. In some instances, the capture moiety 105 may not beattached to the peptide but may be associated to the peptide (e.g., via an indirect interaction). In other examples (not shown), the peptide is coupled (directly or indirectly) to a non-nucleic acid molecule (e.g., a polymerizable molecule such as an additional peptide, or other detectable label such as a mass tag, fluorophore, radioisotope, etc.). A linker 109 comprising a linking nucleic acid molecule I l l is provided. The linking nucleic acid molecule 111 may comprise any useful sequences, such as a primer sequence, a barcode sequence, a UMI, restriction site, etc. In some instances, the linking nucleic acid molecule 111 comprises a cleavable moiety, e.g., a restriction site, an abasic site, a uracil, a transposition site, etc. In process 110, the linker 109 couples to a monomer (e.g., terminal amino acid) of the polymeric analyte to generate a monomer-linker complex. Prior to, during, or subsequent to the coupling of the linker to the monomer, the linking nucleic acid molecule 111 may couple to the capture moiety 105, e.g., via hybridization (not shown), ligation, or splinted ligation (not shown), thereby generating a monomer-capture moiety complex. In process 113, the monomer is cleaved (e.g., chemically or enzymatically) from the rest of the polymeric analyte 103, thereby providing a cleaved monomer-capture moiety complex comprising the cleaved monomer coupled to the linker 109, linking nucleic acid molecule 111, and capture moiety 105 or complements thereof. In some instances, the cleaved monomer-capture moiety complex remains coupled to the polymeric analyte 103 via the linking nucleic acid molecule 111 and the additional polymerizable molecule 107. The cleaved monomer-capture moiety complex can be further processed for downstream analysis.

[0265] The downstream processing and analysis may comprise sorting, barcoding, detection, or a combination thereof. In some instances, a plurality of binding agents 115 is provided; the binding agents 115 may recognize and bind different monomer types (e.g., different amino acid types). The binding agents 115 may be contacted with a plurality of cleaved monomer-capture moiety complexes comprising different (cleaved) monomer types (e.g., different amino acids) and bind to their respective targets. In some instances (not shown), the binding agents 115 comprise polymerizable molecules, e.g., nucleic acid molecules (e.g., nucleic acid barcode molecules), that identify the binding agent or its cognate molecule; the polymerizable molecules may be coupled or transferred to the capture moiety 105 (not shown), or to the linking nucleic acid molecule 111 (not shown), e.g., via nucleic acid extension, ligation, transposition, prime editing, etc. Alternatively or in addition to, in process 119, the binding agents 115 may be separated or sorted, e.g., using complementary nucleic acid sequences to the nucleic acid molecules of the binding agents, into separate compartments (not shown). Alternatively, or in addition to, the binding agent may comprise a sorting tag, e.g., a reporter molecule, mass tag, fluorophore or fluorescent protein, which can enable sorting of the different binding agent types.In one such example, a first binding agent against a first monomer type (e.g., an amino acid residue) may comprise a GFP tag and a second binding agent against a second monomer type may comprise an RFP tag that is sortable by fluorescence (e.g., using FACS) or affinity sorting (e.g., using beads with anti-GFP and anti-RFP antibodies).

[0266] Optionally, subsequent to process 119, the linker 109 or the linking nucleic acid molecule 111 or portion thereof may be removed from the monomer-linker complex or cleaved monomer-linker complex, e.g., via restriction digest or cleavage of a uracil (e.g. using UDG or USER enzymes) of the linking nucleic acid molecule 111.

[0267] In some instances, barcoding of the cleaved monomer-capture moiety complexes may be performed. For example, as described above, the binding agents 115 may comprise polymerizable barcode molecules, e.g., nucleic acid barcode molecules, that identify the binding agent or its cognate molecule; the polymerizable barcode molecules may be coupled or transferred to the capture moiety 105 (not shown), thereby barcoding the capture moiety. Alternatively, or in addition to, subsequent to sorting in process 119, the sorted cleaved monomer-capture moiety complexes may be barcoded. For instance, as each compartment comprises a known monomer (e.g., amino acid) type according to the binding profile (e.g., specificity to a particular monomer type) of the binding agent, the cleaved monomer-capture moiety complex may be labeled with an identifying polymerizable molecule 117 (e.g., nucleic acid barcode molecule) that comprises the identity of the particular monomer type of the cleaved monomer-capture moiety complex, thereby generating a barcoded capture moiety.

[0268] Subsequent to barcoding, the contents of the separate compartments may then be pooled, and the process repeated to iteratively cleave and attach an identifying barcode for each monomer in the polymeric analyte. The subsequent barcode molecules may attach to the barcoded capture moiety to generate a multi-barcoded additional polymerizable molecule (e.g., a concatenated or stacked barcoded nucleic acid molecule). Alternatively, or in addition to, the barcodes may be added onto separate polymerizable molecules, e.g., amplicons (not shown) of the capture moiety 105 or the linking nucleic acid molecule 111. In some instances, the polymerizable molecules 117 or the linking nucleic acid molecule 111 may comprise temporal information (e.g., a round or cycle number). After any useful number of rounds or iterations, the barcoded additional polymerizable molecule (or multi -barcoded additional polymerizable molecule) may be removed and sequenced, e.g., using NGS approaches, thereby outputting the identity of each monomer type that has been processed and the order or position in which they occur, based on the temporal information, in the polymeric analyte.

[0269] FIG. 1C schematically shows another example workflow of processing or sequencing a polymeric analyte. A plurality of polymeric analytes (e.g., peptides) may be coupled to a substrate 101, along with a plurality of capture moi eties. For illustration purposes, further sequencing workflow operations are shown for a single polymeric analyte 103; however, it will be appreciated that the workflow operations of FIGs. 1A-1C may be performed on all or a subset of the polymeric analytes in parallel. In process 110, a linker 109 is provided. The linker 109 is capable of coupling to (i) a monomer of the polymeric unit (e.g., a terminal amino acid, such as the N-terminal amino acid) and (ii) a capture moiety 105, which may be used to locally tether the monomer adjacent to the polymeric analyte. In an example, the linker 109 may comprise an amino acid-reactive group such as phenyl isothiocyanate (PITC), which enables the linker to couple to an N-terminal amino acid. The linker may additionally comprise a substratetethering moiety that can couple to the capture moiety. For example, the sub state-tethering moiety may comprise a click chemistry moiety that can couple to a click chemistry capture moiety, e.g., through an azide-alkyne or azide-cycloalkyne reaction. In other examples, the substrate-tethering moiety may comprise a nucleic acid molecule that can couple, e.g., via hybridization, ligation, or both, to a nucleic acid capture moiety (e.g., as shown in FIG. 1A). In process 112, the substrate-tethering moiety of the linker may couple to the capture moiety. In process 113, the monomer may be cleaved from the polymeric analyte. In some embodiments, cleavage of the monomer may be mediated by a stimulus, e.g., chemical reaction or pH change (such as addition of acid). Subsequent to cleavage, a binding agent 115 may be provided. The binding agent, e.g., an antibody or antibody fragment, may be specific to a particular monomer of a plurality of monomers, e.g., specific to a particular amino acid type or derivative thereof. The binding agent may comprise a detectable label (e.g., fluorophore, radioisotope, mass tag, etc.). The detectable label may be detected (e.g., using microscopy or imaging). To determine the identity of the cleaved terminal monomer (e.g., amino acid). Subsequently, the linker may be removed or cleaved, and the process may be repeated or reiterated to sequence the remaining monomers of the polymeric analyte.

[0270] FIG. ID shows another example workflow for processing a polymeric analyte for sequencing using a DNA typewriter approach in which a nuclease is used to facilitate barcoding of a polymerizable molecule associated with a polymeric analyte. In such an example, a polymeric analyte 103 is provided, sequentially disassembled into individual monomers via contacting with capture moieties and cleavage, and the individual cleaved monomers complexed with the capture moieties are contacted with binding agents comprising barcode molecules that identify or encode for the binding agents. The barcode molecule of a binding agent is coupled toa polymerizable molecule using a nuclease or used as an editing agent to edit the polymerizable molecule to comprise a barcode. The polymerizable molecule may comprise one or more nuclease target sites, e.g., CRISPR-Cas target sites. In FIG. ID Panel A, a substrate 101 is coupled to a polymeric analyte 103 (e.g., a peptide to be sequenced), a capture moiety 105 (e.g., a nucleic acid primer), and a polymerizable molecule 107 (e.g., an additional nucleic acid primer). The polymerizable molecule 107 may comprise a tandem array of CRISPR-Cas9 target sites (“target sites”), as described above. In some instances, the tandem array comprises a plurality of target sites, including, for example, a first target site 107a and a second target site 107b. In some instances, the first target site 107a is active whilst the second target site 107b and all other target sites of the tandem array are inactive (e.g., truncated at a 5’ region or end of each target site). The polymerizable molecule 107 may be associated with the polymeric analyte 103; for instance, the polymerizable molecule may comprise a barcode sequence which identifies the polymeric analyte (e.g., a barcode that identifies a peptide), a spatial location, or other useful information. Alternatively, or in addition to, the polymerizable molecule 107 may be coupled (directly or indirectly) to the polymeric analyte, e.g., using linkers (e.g., a trifunctional linker), noncovalent interactions, attachment to a substrate, etc.

[0271] In FIG. ID Panel B, the polymeric analyte may be contacted with a bifunctional linker 109, which comprises a terminal monomer-coupling group (e.g., an amino acid-reactive group such as PITC) and a click chemistry moiety (e.g., azide). In some instances, the bifunctional linker 109 couples to a terminal monomer (e.g., terminal amino acid such as the N- terminal amino acid (NTAA)). In FIG. ID Panel B, a linking nucleic acid molecule 111 comprising a click chemistry moiety (e.g., alkyne) is reacted with the bifunctional linker 109 and covalently linked. In some instances, the linking nucleic acid molecule 111 and the bifunctional linker 109 are provided pre-coupled (see, e.g., FIG. 2). In FIG. ID Panel C, the linking nucleic acid molecule 111 is coupled to the capture moiety 105, thereby generating a monomer-capture moiety complex. The coupling may be mediated by hybridization of the linking nucleic acid molecule 111 to the capture moiety 105 (hybridization not shown), or by using a splint oligonucleotide 113 comprising sequences complementary to a sequence of the linking nucleic acid molecule 111 and the capture moiety 105. In some instances (not shown), the linking nucleic acid molecule 111 comprises a self-splinting sequence, such that the linking nucleic acid molecule 111 may couple to the capture moiety 105 in the absence of a separate splint molecule. A ligase may be used to covalently link the linking nucleic acid molecule 111 to the capture moiety 105. Alternatively, the linking nucleic acid molecule 111 may comprise a first reactive moiety (e.g., click chemistry moiety not shown) that can react with a second reactive moiety(not shown) of the capture moiety 105. In FIG. ID Panel D, the system is subjected to conditions sufficient to cleave the terminal monomer (e.g., amino acid) from the polymeric analyte 103 (e.g., peptide), thereby generating a cleaved monomer-capture moiety complex. The conditions may include using a chemical stimulus, e.g., performing an Edman degradation reaction, or a biological stimulus, e.g., an enzyme. The cleavage of the monomer from the polymeric analyte results in a cleaved monomer-capture moiety complex comprising the cleaved monomer, bifunctional linker 109, linking nucleic acid molecule 111, and capture moiety 105. In FIG. ID Panel E, a binding agent 115 (e.g., antibody) comprising a barcode molecule 117 (e.g., nucleic acid barcode molecule) is provided. The binding agent 115 may be specific to the monomer (e.g., to an amino acid type) or to the monomer-linker complex (e.g., PITC-amino acid complex, which may be derivatized to a phenylthiocarbamoyl, phenylthiohydantoin, or anilinothiazolinone moiety, amino acid-guanidinylating agent complexes, or amino acid- dithioester or amino acid-thiobenzoyl complexes). The barcode molecule 117 of the binding agent may comprise information on the identity of the binding agent or the specific monomer (e.g., single amino acid) to which the binding agent binds. The barcode molecule 117 of the binding agent may comprise additional sequences, such as a barcode sequence, UMI, restriction site, transposition site, a sequence to represent a cycle or iteration number, or other functional sequence. In some instances, the barcode molecule comprises a pegRNA that comprises a barcode sequence. In some instances, the pegRNA further comprises a key sequence, which key sequence is configured to activate the next (adjacent) inactive target site that is immediately adjacent the active target site. For example, if the first target site 107a is active, and the second target site 107b and all other target sites are inactive, the key sequence may be configured to be inserted between the first target site 107a and the second target site 107b, in which case the insertion results in inactivation of the first target site 107a and activation of the second target site 107b. In some instances, the inactive sites of the tandem array are truncated, such that provision or coupling of the key sequence allows for a complete target site that is active.

[0272] In FIG. ID Panel E, the barcode molecule 117 of the binding agent may couple to or be inserted into the polymerizable molecule 107. The coupling may occur via use of a nuclease, such as a Cas protein or a prime editor comprising a modified Cas protein. For instance, a prime editor enzyme may be used to insert the pegRNA or a complement thereof adjacent to the active target site (e.g., the first target site 107a), thereby inactivating the active target site and activating the adjacent target site (e.g., the second target site 107b). In one such example, the prime editor may comprise a Cas protein and a reverse transcriptase; the Cas protein may be used to cleave a first strand of the polymerizable molecule, and a portion of the pegRNA (e.g., aprimer-binding site) may hybridize to a portion of the cleaved strand. An extension reaction, e.g., reverse transcription, may then generate a complementary DNA segment that is complementary to at least a portion of the remaining pegRNA. The generated complementary DNA segment may then be incorporated into the polymerizable molecule by hybridizing to the second strand of the polymerizable molecule, and optionally, flap cleavage and ligation. Additional nucleic acid reactions may be performed, e.g., extension, amplification, ligation, restriction digestion, etc. Optionally, the barcode molecule 117 may be cleaved from the binding agent 115 (not shown). In FIG. 1A Panel F, the monomer may be decoupled (e.g., removed or cleaved) from the capture moiety 105. For example, the monomer, bifunctional linker 109, and all or a portion of the linking nucleic acid molecule 111 may be cleaved (depicted as a star). The cleavage may be performed chemically, mechanically, or enzymatically. In an example of enzymatic cleavage, the linking nucleic acid molecule 111 may comprise a restriction site or other cleavage site (e.g., a uracil), and cleavage occurs by introduction of a restriction enzyme or cleaving enzyme (e.g., uracil DNA glycosylase) to cleave the restriction / cleavage site. Alternatively, the cleaved monomer-capture moiety complex may be blocked with a blocking agent (not shown). The workflow may then be iterated or repeated to process and sequence all or a portion of the polymeric analyte 103 and resulting in polymerizable molecule 107 comprising multiple barcode sequences arising from binding of the binding agents to their targets for all or a subset of the monomers of the polymeric analyte.

[0273] The polymerizable molecule may comprise any useful number of target sites. The polymerizable molecule may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more target sites. In some instances, the number of target sites is predetermined, e.g., based on a number of iterations or cycles of the workflow that are to be conducted. For instance, in some instances, it may be sufficient to only have around 5-6 rounds of sequencing in order to determine the identity of the polymeric analyte (e.g., peptide). As such, the polymerizable molecule may comprise 5 or 6 target sites.

[0274] In some instances, the polymerizable molecule comprises a single target site, and additional target sites may be added via the pegRNA, e.g., using a peCHYRON approach. In such an approach, the polymerizable molecule may comprise a protospacer adjacent motif (PAM) and a first target site. The pegRNA molecule of the barcode molecule coupled to the binding agent may comprise a barcode sequence and a propagator sequence comprising a second target site. As such, coupling of the barcode molecule to the polymerizable molecule results in insertion of the propagator sequence adjacent to the PAM and the first target site. Such an insertion may result in deactivation of the first target site and activation of the second target site.

[0275] The peCHYRON and DNA typewriter approaches may similarly be used to barcode the capture moiety, e.g., as shown in FIG. IB. In one such example, and referring again to FIG. IB, the capture moiety 105 may comprise one or more target sites. For instance, a peCHYRON approach may be used in which the capture moiety 105 comprises a first target site and a PAM sequence. The barcode molecule 117 may comprise a pegRNA molecule, which may comprise a barcode sequence (e.g., encoding the identity of the binding agent or monomer), a propagator sequence, and a second target site. Coupling of the barcode molecule 117 to the first target site may be performed using a prime editor enzyme, as described above, thereby inactivating the first target site and inserting and activating the second site. Alternatively, a DNA typewriter approach may be used in which the capture moiety 105 comprises a tandem array of target sites, all of which except the first site is inactive. The barcode molecule 117 may comprise a pegRNA molecule, which may comprise a barcode sequence (e.g., encoding the identity of the binding agent or monomer), and a key sequence. Insertion of the barcode molecule 117 or complement thereof adjacent to the first target site may be performed using a prime editor enzyme, thereby inactivating the first target site, inserting the barcode sequence (or complement thereof), and activating the second site. In either instance, iteration of the workflow may be performed to sequentially disassemble and barcode the individual monomers of the polymeric analyte.

[0276] Additional processing may be performed at any useful or convenient step, e.g., subsequent to barcoding. For instance, a nucleic acid reaction may be performed, e.g., nucleic acid ligation, nucleic acid extension reaction, amplification, DNA repair, etc.

[0277] FIG. IE shows another workflow of sequencing a polymeric analyte. A polymeric analyte 103 and a capture moiety 105 are provided, which optionally are coupled to a substrate 101. The capture moiety 105 may comprise a first nucleic acid molecule (e.g., DNA). In process 106, a linker 207 and a polymerizable molecule, e.g., a linking nucleic acid molecule 111 are provided. In some instances, the linker 109 is pre-tethered to the polymerizable molecule (depicted as a linking nucleic acid molecule 111); alternatively, the linker 109 and the polymerizable molecule may be provided separately. In process 106, the linker 109 may couple to a monomer, e.g., an amino acid (e.g., NTAA) of the polymeric analyte 103 (e.g., a peptide) to generate a monomer-linker complex. In process 112, the monomer-linker complex may couple to the capture moiety 105, thereby generating a monomer-capture moiety complex. Coupling of the monomer-linker complex to the capture moiety 105 may be mediated by the polymerizable molecule, e.g., the linking nucleic acid molecule 111. Optionally, the monomer-linker complex and the capture moiety 105 may be covalently linked together (e.g., the linking nucleic acid molecule 111 may be covalently linked to the capture moiety 105), using chemical (e.g., clickchemistry) or enzymatic (e.g., a ligase) approaches. Alternatively, or in addition to, the polymerizable molecule may comprise a first sequence that is complementary and may hybridize to a second sequence of the capture moiety 105 (not shown), or the polymerizable molecule may be linked to the capture moiety 105 via a splint or bridge molecule, which may comprise sequences that are complementary to the first sequence of the polymerizable molecule and the second sequence of the capture moiety 105 (not shown). In process 113, the monomer may be cleaved from the polymeric analyte 103, thereby providing a cleaved monomer-capture moiety complex comprising the cleaved monomer coupled to the linker 109, polymerizable molecule (shown as a linking nucleic acid molecule 111), and capture moiety 105. In process 114, a binding agent 115 (e.g., an antibody, binding protein, etc.) may be contacted with the monomer-capture moiety complex. The binding agent may be configured to recognize all or a portion of the monomer-capture moiety complex. For example, the binding agent may recognize the monomer, the monomer-linker complex, or the entirety of the monomer-capture moiety complex. In one example, the linker may comprise a PITC moiety, and the binding agent may recognize the PITC-amino acid, or a derivative thereof, e.g., a phenylthiocarbamyl, a thiazolone, or a phenylthiohydantoin. In some instances, the binding agent 115 may comprise a detectable moiety (not shown) or may be contacted with an additional binding agent (e.g., a secondary antibody) which may optionally comprise a detectable moiety (not shown).

[0278] Any of the processes, e.g., 106, 112, 113, or 114 may be iterated and repeated any number of times (“rounds”) using additional linkers 109 and polymerizable molecules (optionally comprising cycle / round information), and tethering the additional polymerizable molecules together (e.g., tethering an additional polymerizable molecule to the polymerizable molecule of the monomer-capture moiety complex). Multiple rounds may be performed until all or a subset of the monomers in the polymeric analyte 103 are cleaved and tethered together. In some instances, processes 106, 112, and 113 may be iterated to generate a stacked polymerizable molecule 123 comprising a set of cleaved monomers, e.g., a concatenated set of monomer- linker-polymerizable complexes. The stacked set of polymerizable molecules may then be contacted with a library of binding agents, which can bind to their respective monomer targets (e.g., an amino acid type).

[0279] Further downstream analysis may be performed, e.g., using a nanopore or nanogap system. In one such example, the stacked polymerizable molecule 123, which optionally may be coupled to binding agents, may be prepared and translocated through a nanopore sequencing system, which may output the identity of the polymerizable molecules (e.g., a nucleic acid sequence), the monomer type, and, if prevalent, the individual binding agents.

[0280] Alternatively, in some instances, no binding agents may be required in order to sequence the polymerizable molecule. FIG. IF schematically illustrates an example workflow of sequencing a polymeric analyte. In such an example workflow, the same operations of the workflow shown in FIG. IE are performed, omitting process 114. The workflow may be iterated numerous times to generate a stacked polymerizable molecule 123, which may be further processed, e.g., analyzed using a nanopore or nanogap sequencing system, to output the identity of the polymerizable molecules and the individual monomers.

[0281] Similarly, FIG. 1G schematically illustrates another example workflow of sequencing a polymeric analyte in the absence of binding agents and that can be prepared in the presence or absence of a substrate. In such an example, a polymeric analyte 103 and a capture moiety 105 are provided. The capture moiety 105 may comprise a first nucleic acid molecule (e.g., DNA molecule) and may comprise identifying information of the polymeric analyte 103, e.g., an identifying barcode sequence. The capture moiety 105 may additionally comprise a releasable or cleavable moiety. The polymeric analyte and the capture moiety 105 may, in some instances, be coupled to a substrate (FIG. 1G inset). In one such example, the substrate (e.g., bead, a surface of a flow cell) may comprise an anchor molecule to which the capture moiety 105 may couple. For instance, the capture moiety 105 may comprise a nucleic acid sequence that is complementary to an anchor sequence on a substrate (e.g., bead, flat surface). In process 106, a linker 109 and polymerizable molecule, such as a linking nucleic acid molecule 111, are provided. In some instances, the linker 109 is pre-tethered to the polymerizable molecule (linking nucleic acid molecule 111); alternatively, the linker 109 and the polymerizable molecule (linking nucleic acid molecule 111) may be provided separately. The polymerizable molecule may comprise identifying temporal information, e.g., the cycle or round in which it is provided. In process 106, the linker 109 may couple to a monomer, e.g., an amino acid (e.g., NTAA) of the polymeric analyte 103 to generate a monomer-linker complex. In process 112, the monomer-linker complex may couple to the capture moiety 105. Coupling of the monomer- linker complex to the capture moiety 105 may be mediated by the polymerizable molecule and optionally an additional polymerizable molecule 116. Optionally, the monomer-linker complex and the capture moiety may be covalently linked together (e.g., via ligation). Alternatively, or in addition to, the polymerizable molecule may comprise a first sequence that is complementary and may hybridize to a second sequence of the capture moiety 105 (not shown), or the polymerizable molecule may be linked to the capture moiety 105 via a splint or bridge molecule, which may comprise sequences that are complementary to the first sequence of the polymerizable molecule and the second sequence of the capture moiety 105 (not shown). Inprocess 113, the monomer may be cleaved from the polymeric analyte 103 to generate monomer-capture moiety complex, which comprises the cleaved monomer, the linker 109, the polymerizable molecule (e.g., linking nucleic acid molecule 111), and the capture moiety 105. Processes 106, 112, and 113 may be iterated and repeated any number of times (“rounds”) using additional linkers 109 and polymerizable molecules (e.g., linking nucleic acid molecule 111, which may comprise the same or different sequences), and tethering the additional polymerizable molecules together (e.g., tethering an additional polymerizable molecule to the monomer-capture moiety complex). Multiple rounds may continue until all or a subset of the monomers of the polymeric analyte 103 are tethered together. For example, the process may be iterated to generate a stacked polymerizable molecule 123 comprising a set of cleaved monomers, e.g., a concatenated set of monomer-linker-polymerizable complexes. After any useful number of rounds, the stacked polymerizable molecule 123 may be cleaved from the or at the capture moiety 105, e.g., using the cleavable moiety. The cleaved product may then be sequenced using a nanopore or nanogap sequencing instrument.

[0282] In some instances, the polymerizable molecule, e.g., linking nucleic acid molecule 111 comprises temporal information on the cycle in which it is provided; as such, the temporal information may be used for conducting quality control. For example, if a missing cycle number is missing, then it can be inferred that an amino acid is missing or was not present in the peptide, that cleavage of the amino acid did not occur, or other error.

[0283] Iteration: In some instances, one or more of the operations described herein may be iterated or repeated. Iteration of the operations may allow for sequential processing, analysis, or identification of the individual monomers of the polymeric analyte, which can allow for reconstruction of the entire polymeric analyte. For example, referring to FIG. 1A, the operations of the workflow 100 may be conducted to encode the identity (e.g., via the polymerizable molecule 117) of a terminal amino acid (e.g., NTAA) onto the additional polymerizable molecule 107. The operations of workflow 100 may then be repeated to encode the identities of the n-1 terminal amino acid, the n-2 terminal amino acid, the n-3 terminal amino acid, etc. until the entire or portion of the peptide is processed. The encoding may occur on the same (additional) polymerizable molecule 107, e.g., to generate a stacked polymerizable molecule comprising multiple polymerizable molecules from multiple binding agents, or the encoding may occur on additional polymerizable molecules (not shown) present on the substrate. In the former situation, in some instances, the polymerizable molecule of the second (or third, fourth, fifth, . . . nth) cycle may be configured to only couple to the first (or second, third, fourth, . . .n- 1th) polymerizable molecule. For example, the first cycle binding agent polymerizable moleculemay comprise a unique binding sequence that is absent on the additional polymerizable (or capture) molecules of the substrate, and to which the second cycle binding agent polymerizable molecule can bind. Accordingly, the second cycle binding agent polymerizable molecule can only bind to the first cycle binding agent polymerizable molecule and not to any of the additional polymerizable (or capture) molecules of the substrate. In the event that no binding occurs (a “null” event), a bridging polymerizable molecule may be provided that encodes for a null binding event but comprises the unique binding sequence, such that subsequent rounds may continue, even if a binding agent does not bind the cleaved monomer.

[0284] Similarly, any of the operations depicted in FIGS. 1B-1G may be iterated to sequentially analyze all or a subset of the monomers of the polymeric analyte. For instance, referring to FIG. IB, the operations of the workflow may be conducted to encode the identity (e.g., via the polymerizable molecule 117) of a terminal monomer (e.g., NTAA) onto an additional polymerizable molecule (not shown) or onto the barcoded capture moiety. The operations may be repeated to encode the identities of the n-1 terminal amino acid, the n-2 terminal amino acid, the n-3 terminal amino acid, etc. until the entire or portion of the peptide is processed. The encoding may occur on the same capture moiety 105, e.g., to generate a stacked polymerizable molecule comprising multiple barcode polymerizable molecules 117, or the encoding may occur on additional polymerizable molecules (not shown). In the former situation, in some instances, the polymerizable molecule of the second (or third, fourth, fifth, . . . nth) cycle may be configured to only couple to the first (or second, third, fourth, . . .n-lth) polymerizable molecule, as described above.

[0285] In instances where one or multiple additional polymerizable molecules are used, the polymerizable molecules 117 (e.g., coupled to the binding agents or provided separately after sorting) may additionally encode temporal information, e.g., for the cycle or iteration number, such that the order of the individual monomers may be determined. For example, for a given peptide, the terminal amino acid may be coupled to a capture moiety and cleaved, then contacted with a binding agent comprising a barcode molecule comprising a barcode sequence that identifies (i) the identity of the amino acid (e.g., any one of twenty proteinogenic amino acids, a post-translationally modified amino acid, etc.) and (ii) the cycle number (e.g., cycle 1) (not shown). The information encoded by the barcode sequence may be coupled to an adjacent (additional) polymerizable molecule (not shown) or to the capture moiety 105 or barcoded capture moiety to generate a stacked barcoded capture moiety. Following cleavage of the monomer from the capture moiety (e.g., as shown in process 121 of FIG. IB), the workflow may be repeated for the n-1 terminal amino acid, which may again be coupled to a capturemoiety, cleaved, and contacted with a binding agent, which may comprise an additional barcode sequence that identifies (i) the identity of the amino acid (e.g., any one of twenty proteinogenic amino acids) and (ii) the cycle number (e.g., cycle 2). The information encoded by the additional barcode sequence may be transferred to the same capture moiety 105 or barcoded capture moiety, or to an additional polymerizable molecule (not shown). In the former situation, the polymerizable molecule may then comprise information on the (i) the identity of the terminal amino acid, (ii) the cycle number of the terminal amino acid (cycle 1), (iii) the identity of the n-1 terminal amino acid, and (iv) the cycle number of the n-1 terminal amino acid (cycle 2), and so forth. Alternatively, or in addition to, the temporal information may be provided on another molecule, such as the capture moiety 105, the linking nucleic acid molecule 111, additional polymerizable molecule 116, etc.

[0286] Alternatively, or in addition to, the binding agent may comprise a sorting tag, and a barcode sequence may be provided subsequent to sorting. For example, the binding agents may be used to sort different cleaved amino acid-linker complexes (as shown in process 119), and a polymerizable molecule 117 comprising the identity of the amino acid type may be provided for each sorted, cleaved amino acid-linker complex. The polymerizable molecule 117 may also comprise temporal information, e.g., the round or cycle in which it is provided.

[0287] In some instances, temporal information may be provided separately. For example, prior to, during, or subsequent to coupling of a polymerizable molecule 117 comprising barcode information to either an additional polymerizable molecule 107 (FIG. 1A) or the capture moiety 105 or linking nucleic acid molecule 111 (FIG. IB), a temporal barcode may be provided that can couple to the polymerizable molecule 117 (FIGs. 1A-1B), the additional polymerizable molecule 107 (FIG. 1A), the capture moiety 105 or linking nucleic acid molecule 111 (FIGS. 1A, IB, 1D-1G), or a combination thereof. The temporal barcode may comprise any useful agent, including a nucleic acid molecule, a peptide, a lipid, a carbohydrate, an enzyme (e.g., a chromogenic or fluorogenic enzyme) or a ribozyme or DNAzyme, a fluorophore, a dye, an intercalating agent, a dideoxynucleotide, a fluorescent nucleic acid molecule or nucleotide, a radioisotope, a mass tag, or other detectable label that can indicate the time or cycle (or iteration) number in which it is provided. In some instances, the temporal barcode comprises a cycle-specific nucleic acid barcode molecule, which can couple to the polymerizable molecule 117 (comprising the identity of the monomer) or to a terminal polymerizable molecule of a stacked polymerizable molecule comprising polymerizable molecules from multiple rounds or iterations. The temporal barcode may comprise any additional useful functional sequences, e.g., primer sites, sequencing sites, restriction sites, abasic or cleavable sites, etc. In some instances,the temporal barcode may comprise an amplification site that allows for bridge amplification of the temporal barcode and optionally, the coupled polymerizable molecules, to other capture or polymerizable molecules.

[0288] In some instances, the barcoding occurs only on a single polymerizable molecule. For instance, referring to FIG. ID, using a peCHYRON approach or a DNA typewriter approach for barcoding may allow for increased stacking efficiency of barcode molecules, e.g., from specific binding events of the binding agents comprising the barcode molecules to their monomer target. The stacked barcoded molecules may then be efficiently read out using NGS approaches, as described elsewhere herein, to identify and sequence the polymeric analyte.

[0289] Polymeric Analytes: The polymeric analyte may be a biomolecule, macromolecule, or synthetic molecule. The polymeric analyte may be a biomolecule or other biological molecule that comprises one or more monomers. Non-limiting examples of polymeric biomolecules include nucleic acid molecules (e.g., DNA molecule, RNA molecule, DNA:RNA hybrids, aptamers), peptides and proteins, polysaccharides, lipid polymers (e.g., diglycerides, triglycerides and other fatty acids). The polymeric analyte may be a synthetic molecule, e.g., a peptoid or synthetic polymer, or a peptidomimetic (e.g., a peptoid, a beta-peptide, a D-peptide peptidomimetic). Non-limiting examples of synthetic polymers include acrylics, nylons, silicones, viscose, rayon, polyesters, polycarboxylic acids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol, polyurethane, polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(ethylene terephthalate), polyethylene, polyisobutylene, poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde, polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene dichloride), poly(vinylidene difluoride), poly(vinyl fluoride), or a combination thereof. The polymeric analytes may comprise a single polymer type (e.g., a homopolymer) or more than one polymer type (e.g., a copolymer) and may comprise random or arranged monomers. The polymeric analytes may be a block polymer, alternating copolymer, periodic copolymer, statistical copolymer, stereoblock copolymer, gradient copolymer, branched copolymer, graft copolymer, etc. In some embodiments, the polymeric analytes described herein are proteins or peptides that comprise monomeric amino acids.

[0290] The polymeric analytes may be any size or comprise a range of sizes. The polymeric analyte may be about 1 nanometer (nm), about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about800 nm, about 900 nm, about 1 micrometer (pm), about 10 m, about 100 pm, about 1 millimeter mm in size or greater. A plurality of polymeric analytes may comprise polymeric analytes of similar size or within a range of sizes, e.g., between about 10 nm to about 100 nm, between about 50 nm to about 1 p.m. Similarly, the polymeric analytes may have any molecular weight or range of molecular weights. The polymeric analytes may be about 10 daltons (Da), 100 Da, 500 Da, 1 kilodalton (kDa), 10 kDa, 100 kDa, 1,000 kDa, 10,000 kDa, 100,000 kDa, or greater. The polymeric analytes may comprise polymeric analytes of similar molecular weight or within a range of molecular weights.

[0291] The monomers of the polymeric analytes may comprise any size or range of sizes that is less than that of the entire polymeric analyte. A monomer may be about 0.1 nanometer (nm), about 0.5 nm, 1 about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1 micrometer (pm), about 10 pm, about 100 pm, about 1 millimeter mm in size or greater. The monomers may have any molecular weight or range of molecular weights. The monomers may be about 1 dalton (Da), 10 Da, 100 Da, 500 Da, 1 kilodalton (kDa), 10 kDa, 100 kDa, 1,000 kDa, 10,000 kDa, 100,000 kDa, or greater. The monomers or polymeric analytes may range in size of molecular weight; for example, a polymeric analyte may comprise a peptide comprising amino acid monomers, which may vary in molecular weight from 75 Da (glycine) to 204 Da (tryptophan).

[0292] The polymeric analytes may comprise any number of monomers. The polymeric analytes may comprise about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 50,000, 100,000 or more monomers. The polymeric analytes may comprise at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 500, at least about 1,000, at least about 5,000, at least about 10,000, at least about 50,000 at least about 100,000 or greater monomers. Alternatively, the polymeric analytes may comprise at most about 100,000, at most about 50,000, at most about 10,000, at most about 5,000, at most about 1,000, at most about 500, at most about 100, at most about 50, at most about 10, at most about 5, or fewer monomers. The polymeric analytes may comprise a range of monomers; for example, a polymeric analyte may comprise about 5 monomers whereas another polymeric analyte may comprise about 500 monomers.

[0293] In some instances, the polymeric analyte comprises a peptide comprising amino acid monomeric units. The peptide may be naturally occurring or synthetic. The peptide may comprise any number of amino acids. The amino acids may be one of 20 proteinogenic amino acids and may comprise any number of post-translational modifications. The peptides or any of the constituent amino acids may be processed, e.g., contacted with protecting groups, alkylated, beta-elimination of phosphate groups, etc., as is described elsewhere herein. In some instances, the peptides are derived from larger peptides or proteins and are fragmented.

[0294] Substrates'. One or more operations described herein may be performed using a substrate. For example, one or more molecules described herein (e.g., polymeric analyte, capture moiety, polymerizable molecule) may be coupled to a substrate. In some instances, the polymeric analyte, capture moiety, and one or more polymerizable molecules (e.g., the first or second polymerizable molecule), or a combination thereof may be provided coupled to one or more substrates. In one example, the polymeric analyte, capture moiety, and the second polymerizable molecule are coupled to a substrate. In some instances, more than one substrate may be used. In such cases, the substrates may comprise the same material or different material.

[0295] The substrate may be made from any suitable material, e.g., glass, silicon, gel, polymer, etc., as is described elsewhere herein. In some instances, the substrate may be a bead or a gel bead (e.g., polyacrylamide, agarose, or TentaGel® bead). The substrate may be functionalized. One or more molecules, e.g., a capture moiety and the polymeric analyte (e.g., a peptide) may be coupled to the substrate via a covalent or non-covalent interaction. The capture moiety and polymeric analyte (e.g., peptide) can be coupled to the substrate using any suitable chemistry, e.g., click chemistry moieties (e.g., alkyne-azide coupling), photoreactive groups (e.g., benzophenone), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (e.g., to couple amino-oligos or peptides), N-hydroxysulfosuccinimide (NHS), Sulfo-NHS, or NHS-esters (e.g., to couple sulfhydryl oligos), maleimides, hydrazines, hydroxyl amines, thiols, biotin-streptavidin interactions, cystamine, glutaraldehyde, formaldehyde, succinimidyl 4-(N- maleimidomethyl)cyclohexame-l -carboxylate (SMCC), Sulfo-SMCC, 4-(4,6-Dimethoxy-l,3,5- triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), silane (e.g., amino silanes), combinations thereof, etc. In some instances, the substrate may be functionalized to comprise a coupling chemistry to couple the polymeric analyte or the capture moiety. In one non-limiting example, a substrate (e.g., bead or surface) may comprise an alkyne such as dibenzocyclooctyne (DBCO), which may be configured to react to an amine (e.g., DBCO-alcohol, DBCO-Boc, DBCO-NHS), a carboyxl or carbonyl (e.g., DBCO, DBCO-silane), a sulfhydryl, etc. An azide- functionalized nucleic acid or protein may react with DBCO to link the nucleic acid or protein tothe DBCO substrate. In other examples, linkers such as bifunctional linkers may be used to attach a molecule to a substrate; such bifunctional linkers may comprise the same reactive moiety on both ends or a different moiety at each end (e.g., heterobifunctional linker).

[0296] In some instances, a molecule (e.g., polymeric analytes, capture moi eties, polymerizable molecules) may be coupled to the substrate using an enzymatic approach, e.g., as described elsewhere herein. For example, a chemical linker or moiety such as a click chemistry moiety may be attached to a polymeric analyte (e.g., peptide) using an enzyme. The chemical linker or moiety may be able to react with another chemical linker or moiety (e.g., click chemistry moiety) of a substrate, capture moiety, or polymerizable molecule.

[0297] The substrates may be coupled to any useful number of molecules (e.g., polymeric analytes, capture moieties, polymerizable molecules). In some instances, a substrate may comprise a plurality of polymeric analytes, a plurality of capture moieties, and / or a plurality of polymerizable molecules, which may be provided at any useful ratio or density. For example, the ratio of polymeric analytes to capture moieties or polymerizable molecules may be about 1 : 1, 1 :5, 1 : 10, 1 :20, 1 : 100, 1 : 1000, 1 : 10,000, 1 : 100,000, 1: 1,000,000 or lower. In some instances, the ratio of polymeric analytes to capture moieties or polymerizable molecules may be at most about 1 : 1, at most about 1 :5, at most about 1 : 10, at most about 1 :20, at most about 1 : 100, at most about 1 : 1000, at most about 1 : 10,000, at most about 1 : 100,000, at most about 1 : 1,000,000 or lower.

[0298] Similarly, the molecules (e.g., polymeric analytes, capture moieties, or polymerizable molecules) may be coupled to the substrate at any useful density, for example about 1 molecule / square micron (pm2), about 10 molecules / pm2, about 100 molecules / pm2, about 1,000 molecules / pm2, about 10,000 molecules / pm2, about 100,000 molecules / pm2, about 1,000,000 molecules / pm2, about 10,000,000 molecules / pm2, about 100,000,000 molecules / pm2, about 1,000,000,000 molecules / pm2, about 10,000,000,000 molecules / pm2, about 100,000,000,000 molecules / pm2, or greater. The polymeric analytes, capture moieties, and polymerizable molecules may be coupled to the substrate at a range of densities, e.g., from about 100 to about 10,000 molecules / pm2, or from about 10 to about 1,000 molecules / pm2. The density of the polymeric analytes, capture moieties, and polymerizable molecules may be the same or different. For example, the density of the polymerizable molecules may be 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, 100,000- fold, 1,000,000-fold or greater-fold lower than that of the polymeric analyte.

[0299] In some instances, the molecules coupled to the substrate may be spaced apart at a designated or controlled distance. For example, the average spacing or distance between thepolymerizable molecules coupled to the substrate may be about 1 nanometer (nm), about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 8 nm, about 9 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 500 nm, about 1pm or greater. In some instances, the spacing between the polymerizable molecules coupled to the substrate may be at most about 1pm, at most about 500 nm, at most about 100 nm, at most about 90 nm, at most about 80 nm, at most about 70 nm, at most about 60 nm, at most about 50 nm, at most about 40 nm, at most about 30 nm, at most about 20 nm, at most about 10 nm, at most about 5 nm, or less. Similarly, the spacing or distance between a polymeric analyte and a polymerizable molecule or capture moiety may be about 1 nanometer (nm), about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 8 nm, about 9 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 500 nm, about 1pm or greater. In some instances, the average spacing between the polymerizable molecule and the polymeric analyte coupled to the substrate may be at most about 1 m, at most about 500 nm, at most about 100 nm, at most about 90 nm, at most about 80 nm, at most about 70 nm, at most about 60 nm, at most about 50 nm, at most about 40 nm, at most about 30 nm, at most about 20 nm, at most about 10 nm, at most about 5 nm, or less. A range of average distances between the polymerizable molecules from one another or from the polymeric analytes may be used, e.g., from about 1 nm to about 40 nm, from about 2 nm to about 10 nm, etc.

[0300] The concentration or density of the molecules attached to the substrate may be modulated using one or more suitable approaches, including patterning or random deposition approaches. Examples of methods to control the concentration or density of the molecules attached to the substrate include limited dilution, addition of chaotropes (e.g., guanidine, formamide, urea), using metal organic compounds, etc. The molecules may be attached to the substrate in a patterned fashion, e.g., using self-assembling monolayers, photopatteming, lithography, etching, or a combination thereof, or the molecules may be randomly arranged.

[0301] The substrate may comprise any useful size or dimension (e.g., length, width, height, diameter, radius), surface area, volume, or ratio or combination thereof. The substrate may comprise a bead or particle that may comprise a diameter of about 1 nanometer (nm), about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 8 nm, about 9 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 500 nm, about 1 m, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 20 pm, about 30 pm, about 40 pm, about 50pm, about 60pm, about 70 pm, about 80 pm, about 90 pm, about100 pm, about 200 pm, about 300 pm, about 400 pm, about 500 pm, about 600 pin, about 700 pm, about 800 pm, about 900 pm, about 1 millimeter (mm) or greater. The substrate may comprise a surface area of about 1 square nanometer (nm2), about 10 nm2, about 100 nm2, about 1,000 nm2, about 10,000 nm2, about 100,000 nm2, about 1 pm2, about 10 pm2, about 100pm2, about 1,000 pm2, about 10,000 pm2, about 100,000 pm2, about 1 mm2, about 10 mm2, about 100 mm2, about 1,000 mm2, about 10,000 mm2, about 100,000 mm2, about 1,000,000 mm2 or greater.

[0302] The molecules may be coupled to the substrate in an ordered or random arrangement. In ordered arrangements, the molecules may be patterned using any conventional approach such as lithography (e.g., soft lithography, photolithography), etching (e.g., ion etching, photo etching), or other patterning approach. In some instances, a linker (e.g., bifunctional linker) may be used to facilitate the coupling of the molecules (e.g., polymeric analytes, polymerizable molecules, capture moieties) to the substrate; such linkers may be patterned using any useful technique such as self-assembling monolayers, photopatteming, lithography, etching. In some instances, the molecules may be coupled to the substrate in a random arrangement. For example, the molecules may be provided at a stoichiometric ratio or controlled concentration to couple the molecules at any useful ratio or density.

[0303] Polymerizable Molecules'. The polymerizable molecules, including the barcode molecules described herein, may comprise any useful type of polymerizable molecule. The polymerizable molecules may by naturally occurring, such as biological polymers (e.g., nucleic acid molecules, peptides, polysaccharides, fatty acids), or other naturally occurring polymers, e.g., rubber, cellulose, starches, polyhydroxyalkanoates, chitosan, dextran, structural proteins (e.g., collagen, hyaluronic acid, glycosaminoglycans), agarose, carrageenan, isphagula, acacia, agar, gelatin, shellac, xanthan gum, guar gum, alginate, etc. The polymerizable molecules may be synthetic, e.g., acrylics, nylons, silicones, viscose, rayon, polyesters, polycarboxylic acids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol, polyurethane, polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene terephthalate, poly(chlorotrifluoroethylene), polyethylene oxide), poly(ethylene terephthalate), polyethylene, polyisobutylene, poly(methyl methacrylate), poly(oxymethylene), poly formaldehyde, polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene dichloride), poly(vinylidene difluoride), poly(vinyl fluoride) and combinations thereof. The polymerizable molecules may comprise one or more reactive moieties (e.g., radical groups) to initiate polymerization or may be polymerized via contacting of an initiating agent (e.g., ammoniumpersulfate, peroxide, or other radicalizing agent). The polymerizable molecules may be polymerizable via contacting of an enzyme (e.g., polymerizing enzyme such as polymerases), ribozyme or DNAzyme. Alternatively or in addition to, the polymerizable molecules may be polymerizable via self-assembly. The polymerizable molecules may comprise a single polymer type (e.g., a homopolymer) or more than one polymer type (e.g., a copolymer) and may comprise random or arranged monomers. The polymerizable molecules may be a block polymer, alternating copolymer, periodic copolymer, statistical copolymer, stereoblock copolymer, gradient copolymer, branched copolymer, graft copolymer, etc.

[0304] The same or different types of polymerizable molecules may be used in the methods described herein. For example, the first polymerizable molecule comprised by or coupled to the binding agent may be a nucleic acid molecule, and the second polymerizable molecule may be a peptide. In another example, both the first polymerizable molecule and the second polymerizable molecule are nucleic acid molecules. In such an example, the first polymerizable molecule may be coupled to the second polymerizable molecule via ligation or hybridization. For instance, the first polymerizable molecule may comprise a first nucleic acid sequence and the second polymerizable molecule may comprise a second nucleic acid sequence. The first nucleic acid sequence may be complementary or partially complementary to the second nucleic acid sequence, and the coupling may comprise hybridizing the first nucleic acid sequence or portion thereof to the second nucleic acid sequence or portion thereof. Alternatively, the first nucleic acid sequence and the nucleic acid sequence may be complementary to two sequences of a splint or bridge oligonucleotide, and coupling may be mediated via hybridization to the splint oligo. The first nucleic acid sequence may be ligated to the second nucleic acid sequence, either chemically (e.g., via click chemistry approaches in which the first polymerizable molecule and the second polymerizable molecule comprise one member of a click chemistry pair) or enzymatically (e.g., using a ligase).

[0305] The polymerizable molecules may comprise functional portions. For example, the polymerizable molecules may comprise a nucleic acid molecule comprising a functional sequence, such as a primer sequence (e.g., universal priming site), a sequencing sequence, a read sequence, a unique molecular identifier (UMI), a barcode sequence, a cleavage sequence (e.g., a restriction site, a Cas-binding sequence), a transposition sequence (e.g., a mosaic end sequence), or a combination thereof.

[0306] The polymerizable molecules may be any useful size. The polymerizable molecules may be about 1 angstrom, about 2 angstrom, about 3 angstrom, about 4 angstrom, about 5 angstrom, about 6 angstrom, about 7 angstrom, about 8 angstrom, about 9 angstrom, about 10angstrom, about 20 angstrom, about 30 angstrom, about 40 angstrom, about 50 angstrom, about 60 angstrom, about 70 angstrom, about 80 angstrom, about 90 angstrom, about 100 angstrom, about 200 angstrom, about 300 angstrom, about 400 angstrom, bout 500 angstrom, about 600 angstrom, about 700 angstrom, about 800 angstrom, about 900 angstrom, about 1000 angstrom, about 10,000 angstrom, about 100,000 angstrom or greater in size, length, or another dimension. In some instances, the polymerizable molecule (e.g., the first polymerizable molecule or the second polymerizable molecule) comprises a nucleic acid molecule comprising one or more nucleotide bases. The polymerizable molecule may comprise any useful number of nucleotide bases, e.g., about 1 base, about 2 bases, about 3 bases, about 4 bases, about 5 bases, about 6 bases, about 7 bases, about 8 bases, about 9 bases, about 10 bases, about 20 bases, about 30 bases, about 40 bases, about 50 bases, about 60 bases, about 70 bases, about 80 bases, about 90 bases, about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, about 1000 bases, or a greater number of bases.

[0307] The polymerizable molecules may comprise a nucleic acid molecule. The nucleic acid molecule can be single stranded, double stranded, or partially double-stranded. The nucleic acid molecule may comprise a modified nucleotide or non-canonical base. For instance, the polymerizable molecules may comprise a pseudo-complementary base, a bridged nucleic acid (BNA), a xenonucleic acid (XNA), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a gamma-PNA molecule, a morpholino, or a combination thereof. In some instances, a polymerizable molecule may comprise a hexitol nucleic acid (HNA) or a cyclohexyl nucleic acid (CeNA), which may be useful in rendering the polymerizable molecule more resistant to acid degradation (e.g., as used in conventional Edman degradation). Alternatively or in addition to, a polymerizable molecule may comprise naturally occurring bases that are more resistant to acid degradation, e.g., be composed of primarily thymine or cytosine. For example, a nucleic acid molecule may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% thymines or cytosines, which can render the nucleic acid molecule more acid resistant as compared to a nucleic acid molecule comprising adenines or guanines.

[0308] Linkers: One or more operations of the method may be mediated using a linker. In some instances, the coupling of the monomer (e.g., amino acid) to the capture moiety to generate a monomer-capture moiety complex is mediated using a linker. The coupling of the linker to the monomer or capture moiety may be covalent or noncovalent. In an example, a linker may comprise a first reactive group that is able to couple to a monomer of the polymeric analyte (e.g., an amino acid of a peptide) and optionally, cleave the amino acid from a peptide. Forexample, the first reactive group may be an amino-acid reactive group, e.g., an isothiocyanate (ITC) such as phenyl isothiocyanate (PITC), 3-pyridyl isothiocyanate (PYITC), 2- piperidinoethyl isothiocyanate (PEITC), 3-(4-morpholino) propyl isothiocyanate (MP ITC), 3- (diethylamino)propyl isothiocyanate (DEPTIC) or naphthylisothiocyanate (NITC), fluorescein isothiocyanate (FITC), ammonium thiocyanate, potassium thiocyanate, trimethyl silyl isothiocyanate (TMS-ITC), phenyl phosphoroisothiocyanatidate, acetyl isothiocyanate (AITC), or an aldehyde group, e.g., ortho-phthalaldehyde (OP A), 2,3-naphthalenedicarboxyaldehyde (ND A), 2-pyridinecarboxyaldehyde, which can react with an N-terminal amino acid (NTAA). The linker may additionally comprise a second reactive group that is capable of coupling, either directly or indirectly, to the capture moiety. In an example of direct coupling, the capture moiety may comprise a click chemistry moiety (e.g., alkyne), and the second reactive group of the linker may comprise an additional click chemistry moiety (e.g., azide) that can react with the click chemistry moiety of the capture moiety. Alternatively, the linker may be coupled indirectly to the capture moiety, e.g., via noncovalent interaction or via an intermediate linking molecule. In some instances, the intermediate linking molecule may comprise a third polymerizable molecule (e.g., a polymer or nucleic acid molecule) that can couple the linker to the capture moiety. In one such example, the third polymerizable molecule may comprise (i) a third reactive group that is capable of coupling to the second reactive group (e.g., via alkyne-azide click chemistry) of the linker and (ii) a moiety that can couple to the capture moiety (e.g., another orthogonal click chemistry reaction, avidin-biotin interaction, nucleic acid coupling or hybridization). In some instances, the third polymerizable molecule comprises a nucleic acid molecule that comprises (i) a click chemistry moiety (e.g., alkyne) that can conjugate to the first reactive group (e.g., azide) of the linker and (ii) a nucleic acid sequence that can couple to the capture moiety, e.g., via ligation, splint ligation, or hybridization. In some instances, the linker comprises a linking nucleic acid molecule that comprises a self-splinting moiety.

[0309] When applicable, the click chemistry moieties of the linker and capture moiety or intermediate linking molecule may comprise any suitable bioorthogonal moieties, as described elsewhere herein, e.g., alkenes, alkynes, azides, epoxides, amines, thiols, nitrones, isonitriles, isocyanides, aziridines, activated esters, and tetrazines, and combinations, variations, or derivatives thereof. The linker may be subjected to conditions sufficient to react the first click chemistry moiety to the second click chemistry moiety, e.g., provision of metal catalysts, appropriate solvents, pH, temperature, ionic concentration, or light / energy for any useful duration of time.

[0310] The first reactive group of the linker may be an amino acid-reactive moiety. The amino acid- reactive moiety of the linker may be any useful moiety that enables the reactive moiety to conjugate to and optionally cleave an amino acid. In some examples, the first reactive moiety can react with a terminal amino acid (e.g., NTAA or CTAA). In such examples, the first reactive moiety may comprise any primary amine or carboxylic group reactive group, including but not limited to isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, phenyl esters, isothiocyanates (e.g., phenyl isothiocyanate, sodium isothiocyanate, ammonium isothiocyanates (e.g., tetrabutylammonium isothiocyanate, tetrabutylammonium isothiocyanate), diphenylphosphoryl isothiocyanate), acetyl chloride, cyanogen bromide, carboxypeptidases, azide, alkyne, DBCO, maleimide, succinimide, thiol-thiol disulfide bonds, tetrazine, TCO, vinyl, methylcyclopropene, acryloyl, allyl, among others. Additional examples of amino acid reactive groups are provided in U.S. Pat. No. 11,499,979, U.S. Pat. Pub. No. 2020 / 0217853, International Pat. Pub. No. WO / 2024 / 107755, and International Pat. App. No. PCT / US2024 / 013211, each of which is incorporated by reference herein in its entirety.

[0311] The linker may comprise any additional useful moieties. For example, the linker may comprise a releasable or cleavable moiety, which may facilitate removal of the monomer from the polymeric analyte, or portion thereof, or from the substrate. Such a releasable or cleavable moiety may comprise, for example, a disulfide bond, which may be releasable by contacting with a reducing agent (e.g., DTT, TCEP). In some examples, the linker may couple to the third polymerizable molecule via the releasable or cleavable moiety, alternatively or in addition to the coupling via click chemistry moieties. As such, the coupling between the polymerizable molecule and the linker may be reversible. The linker may additionally or alternatively comprise any number of spacing moieties, e.g., polymers (e.g., PEG, PVA, polyacrylamide), aminohexanoic acid, nucleic acids, alkyl chains, etc. Such spacing moieties may increase the distance between any other moieties of the linker, e.g., the amino acid-reactive group and the polymerizable molecule-reactive group. The linker may comprise or be coupled to a detectable moiety, e.g., a fluorophore, radioisotope, mass tag, nucleic acid molecule (which can also act as a releasable or cleavable moiety), or other detectable moiety. In some examples, the linker comprises a fluorophore, which can enable localization visualization of the linker using singlemolecule imaging or fluorescence lifetime imaging. In another example, the monomer may be labeled with a first fluorophore and the linker may comprise a second fluorophore to enable localization visualization of the linker and the monomer (e.g., using two-channel imaging or FRET).

[0312] Use of a linker comprising two reactive groups may allow for coupling of the linker to (i) the monomer of the polymeric analyte and (ii) the intermediate linking molecule (e.g., third polymerizable molecule) or (iii) the capture moiety. In some instances, when an intermediate linking molecule is used, the linker may be pre-coupled to the intermediate linking molecule. For example, a precursor linker may comprise a monomer-binding group (e.g., PITC) and a click chemistry moiety (e.g., azide), which may be reacted with a polymerizable molecule (e.g., oligonucleotide) comprising complementary click chemistry moiety (e.g., alkyne) to generate a linker that is capable of coupling to the monomer and the capture moiety (e.g., another oligonucleotide). In some instances, the linker may be provided pre-coupled to the intermediate linking molecule.

[0313] FIG. 2 schematically shows an example linker that may be used in sequencing polymeric analytes such as peptides. FIG. 2 Panel A shows a bifunctional linker 203 (e.g., 1- (but-3-yn-l-yl)-4-isothiocyanatobenzene) comprising an amino acid reactive moiety (e.g., PITC) and an alkyne click chemistry moiety, which may be reacted with a polymerizable molecule 201 (e.g., a linking nucleic acid molecule) comprising a complementary azide click chemistry moiety. The bifunctional linker may also comprise a spacer moiety, e.g., an alkyl chain (an ethyl group is depicted) of any length, a polymer (e.g., PEG) of any length, etc. The spacer moiety may be located between the amino acid reactive moiety and the click chemistry moiety. FIG. 2 Panel B shows the product of a click chemistry cycloaddition reaction between the azide and alkyne groups to generate a linker molecule comprising the polymerizable molecule and the amino acid reactive moiety. The conjugation of the polymerizable molecule 201 to the bifunctional linker 203 may occur at any useful or convenient step. In alternative examples (not shown), the bifunctional linker 203 may comprise an azide group, e.g., l-(2-azidoethyl)-4- isothiocyanatobenzene, which can be reacted to a polymerizable molecule 201 comprising an alkyne moiety (e.g., linear alkyne or cycloalkyne such as DBCO).

[0314] In some instances, e.g., where the polymeric analyte comprises a peptide that comprises amino acid monomers, the coupling of the linker to an amino acid (e.g., NTAA or CTAA) modifies the chemical structure of the amino acid. For example, if using a linker comprising an isothiocyanate moiety, the amino acid may be derivatized to a thiocarbamyl group (e.g., under mildly alkaline conditions) during or subsequent to contact with the isothiocyanate moiety. One or more further derivatizations may be performed. For instance, the amino acid or amino acid derivative (e.g., thiocarbamyl-derivatized amino acid) may be further derivatized to a thiazolone group (e.g., under acid conditions, which may cleave the amino acidfrom the peptide), a thiohydantoin group, or other chemical moiety. Similarly, a thiazolone group or thiohydantoin group may be further derivatized to a thiocarbamyl group.

[0315] Capture Moieties: The capture moieties may couple to a monomer of the polymeric analyte via any suitable mechanism. The coupling of the monomer to the capture moiety may comprise a covalent interaction or a noncovalent interaction. The coupling may occur by interaction of binding pairs, e.g., biotin and avidin (or streptavidin), cyclodextrins and small hydrophobic molecules (e.g., alkanes, benzene, polycyclics), cucurbiturils and adamantaneammonium or trimethylammoniomethyl ferrocene, cyclophane (e.g., calixarenes, cavitands, pillararenes, tetralactams), etc.

[0316] In some instances, the capture moiety comprises an additional polymerizable molecule (e.g., a nucleic acid molecule). In such instances, the monomer may be first coupled with a complementary polymerizable molecule (e.g., to generate a peptide-oligonucleotide conjugate) and tethered to the capture moiety, e.g., via complementary base pairing directly or via a splint molecule. Alternatively, the monomer may be coupled to the capture moiety via a linker, as is described above. For example, the linker may comprise a monomer-coupling group (e.g., PITC, which can couple to or react with an amino acid of a peptide) and a nucleic acid molecule. The capture moiety may comprise an additional nucleic acid molecule, which may be coupled to the nucleic acid molecule of the linker via hybridization, ligation, or both.

[0317] The capture moiety may comprise a nucleic acid molecule, which can comprise any naturally occurring, non-naturally occurring or engineered nucleotide base. For example, the nucleic acid molecule may comprise a pseudo-complementary base, a bridged nucleic acid, a xenonucleic acid, a locked nucleic acid, a peptide nucleic acid (PNA), a gamma-PNA, a morpholino, etc., as is described elsewhere herein.

[0318] The capture moiety may comprise one or more functional sequences, including, but not limited to a priming sequence, sequencing sequence, sequencing read sequence, a mosaic end sequence, a transposase recognition sequence, a cleavage site (e.g., restriction site), a UMI, a blocking group, a spacer sequence, a barcode sequence, or other functional sequence. In some instances, the capture moiety comprises a cleavable or releasable moiety (e.g., a restriction enzyme recognition site, an abasic site, a uracil which can be cleaved using USER® or uracil DNA glycosylase, a disulfide bond that can be releasable upon addition of a reducing agent).

[0319] In some instances, the capture moiety and a polymerizable molecule are provided coupled to a substrate. In one example, the substrate comprises, coupled thereto, a first nucleic acid molecule, a second nucleic acid molecule, and a capture moiety, which may be a third nucleic acid molecule. In some instances, a substrate may comprise identical nucleic acidmolecules across the substrate; these identical nucleic acid molecules may act as both a capture moiety and a polymerizable molecule to which additional polymerizable molecules (e.g., coupled to binding agents) may couple.

[0320] The capture moiety may comprise any useful moiety or functional group. The capture moiety may have a monomer-capture group, a substrate-tethering group or linker, or any additional functional groups or moieties, e.g., for coupling or tethering to other molecules or for detection. In some examples, the capture moiety comprises a nucleic acid molecule that comprises a substrate-tethering group (e.g., biotin, a click chemistry moiety such as an azide) that can couple to a substrate (e.g., comprising streptavidin or a complementary click chemistry). The capture moiety may additionally comprise a binding sequence, to which another nucleic acid molecule (e.g., a linking nucleic acid molecule, a linker-monomer complex, or a binding agent nucleic acid barcode molecule) can couple, e.g., via hybridization, ligation, or both. In some instances, the capture moiety comprises a single-stranded oligonucleotide or a singlestranded region in which a complementary oligonucleotide can hybridize. The complementary oligonucleotide may comprise a detectable label (e.g., fluorophore) that allows for detection of the capture moiety.

[0321] Cleaving: The cleaving of the monomer from the polymeric analyte may be achieved using any suitable mechanism, such as via application of a stimulus. The stimulus can be, for example, a chemical stimulus, a biological stimulus, a thermal stimulus (e.g., application of heat), a photo-stimulus, a physical or mechanical stimulus, or other type of stimulus or a combination of stimuli. In some instances, the stimulus may be a chemical stimulus, e.g., a change in pH, addition of a lytic agent, initiating agent, radical -generating agent, reducing agent, etc. In some instances, the stimulus may be a biological stimulus, e.g., enzyme (e.g., Edmanase, protease, endonuclease, artificial protease such as artificial peptidase) that can cleave or catalyze cleavage of the monomer from the polymeric analyte.

[0322] In some examples, the polymeric analyte comprises a peptide and the monomer comprises an amino acid (e.g., an NTAA, CTAA, or internal amino acid). The method may comprise using a linker comprising an amino acid reactive group (e.g., PITC) by coupling the amino acid reactive group of the linker with the amino acid, and cleaving the amino acid from the peptide using a stimulus (e.g., change in pH, temperature). In an example, PITC may couple to an NTAA under mildly alkaline conditions to generate a phenylthiocarbamoyl (PTC) derivative of the NTAA, and cleavage of the NTAA from the peptide may be achieved using an Edman degradation reaction (e.g., application of an acid such as trifluoroacetic acid with heat), to generate a thiazolinone (ATZ) derivative or a phenylthiohydantoin (PTH) derivative. Asdescribed elsewhere herein, the linker may comprise a moiety or molecule (e.g., nucleic acid molecule or polymerizable molecule) that can also couple to the capture moiety such that, subsequent to cleavage, the cleaved amino acid may be coupled to the capture moiety.

[0323] In some instances, more than one monomer may be cleaved from the polymeric analyte at a time. The cleaving may comprise cleaving 2 monomers, 3 monomers, 4 monomers, 5 monomers, 6 monomers, 7 monomers, 8 monomers, 9 monomers, 10 monomers, or more. For example, the polymeric analyte may comprise a peptide comprising a plurality of amino acid monomers, and single amino acids, di-peptides, tri-peptides, quadri-peptides, or larger may be cleaved in the methods described herein. In some instances, at most about 10 monomers, at most about 9 monomers, at most about 8 monomers, at most about 7 monomers, at most about 6 monomers, at most about 5 monomers, at most about 4 monomers, at most about 3 monomers, or fewer monomers may be cleaved in a given cleavage event. In some instances, cleavage of greater than one monomer (e.g., amino acid) may be mediated using an enzyme (e.g., Edmanase, protease) that is capable of recognizing or cleaving more than a single amino acid.

[0324] Cleavage of the monomer (or plurality of monomers) may be conducted using a biological stimulus, such as an enzyme. The enzyme can be any useful cleaving enzyme, e.g., a protease, such as an Edmanase, cruzain, x protein (e.g., ClpS, ClpX), Proteinase K, exopeptidase, aminopeptidase, diaminopeptidase, serine protease, cysteine protease, threonine protease, aspartic protease, aspartic protease, glutamic protease, metalloprotease, asparagine peptide lyase, pepsin, trypsin, pancreatin, Lys-C, Glu-C, Asp-N, chymotrypsin, carboxypeptidase (e.g., carboxypeptidase A, carboxypeptidase B, carboxypeptidase Y), SUMO protease, elastase, papain, endoproteinase, proteinase, TrypZean®, bromelain, collagenase, hyaluronase, thermolysin, ficin, keratinase, tryptase, fibroblast activation, enterokinase, chymotrypsinogen, chymase, clostripain, calpain, alpha-lytic protease, proline specific endopeptidase, furin, thrombin, subtilisin, genenase, PCSK9, cathepsin, prolidase, methionine aminopeptidase, cathepsin C, 1-cyclohexen-l-yl-boronic acid pinacol ester, pyroglutamate aminopeptidase, renin, kininogen, kallikrein, DPPIV / CD26, thimet oligopeptidase, prolyl oligopeptidase, leucine aminopeptidase, dipeptidylpeptidase, or other enzyme or protease, or a combination or variation (e.g., engineered mutant or variant) thereof. In some instances, the cleaving enzyme or ribozyme or DNAzyme may be configured or engineered to cleave a terminal monomer or plurality of monomers; alternatively, the cleaving enzyme or ribozyme or DNAzyme may be configured or engineered to cleave off-site at a non-terminal location of the polymeric analyte, e.g., at an internal monomer within the polymeric analyte, at an n-1, n-2, n-3,n-4, n-5, n-6, n-7, n-8, n-9, n-10, etc. position (where n is the number of monomers in the polymeric analyte).

[0325] In the instances of enzymatic cleavage, additional reagents may be provided to catalyze or induce the cleavage. For instance, metalloproteases, aminopeptidases, or exopeptidases may facilitate cleavage of an amino acid or plurality of amino acids in the presence of a catalyst, e.g., metal or metal ion (e.g., cobalt). Accordingly, a catalyst may be provided in order to facilitate the binding of the enzyme to an amino acid or the subsequent cleavage of the amino acid from the peptide. In some examples, cleavage may be mediated by an apo-enzyme, which is inactive in the absence of a metal catalyst of cofactor, and cleavage may be controlled by addition of metal or metal ions.

[0326] Other examples of cleaving stimuli may include: a photo stimulus (e.g., application of UV, X-rays, gamma rays, or other wavelength of light), mechanical stimulus (e.g., sonication, high pressure), thermal stimulus (e.g., application of heat), or chemical stimulus. In some instances, the polymeric analyte may comprise or be altered to comprise a cleavable or labile bond that can be cleaved upon application of the appropriate stimulus, e.g., disulfide bonds (e.g., cleavable upon application of a chemical stimulus such as a reducing agent), ester linkages (e.g., cleavable with a change of pH), a vicinal-diol linkage (e.g., cleavable with sodium periodate), a Diels- Alder linkage (e.g., cleavable upon application of heat), a sulfone linkage (e.g., cleavable via a base), a silyl ether linkage (e.g., cleavable via an acid), a glycosidic linkage (e.g., cleavable via an amylase), a peptide linkage (e.g., cleavable via a protease), or a phosphodiester linkage (e.g., cleavable via a nuclease (e.g., DNAse)).

[0327] Modifications of Monomers: In some instances, one or more monomers of the polymeric analyte may be modified. Modifications may be naturally-occurring (e.g., post translational modifications) or non-naturally occurring, such as by labeling or tagging, e.g., with an amino acid- or amine-reactive agent such as an isothiocyanate (e.g., PITC, NITC), 1-fluoro- 2,-4-dinitrobenzene (DNFB), dansyl chloride, 4-sulfonyl-2-nitrobfluorobenzene (SNFB), an acetylating agent, an acylating agent, an alkylating agent, a guanidination agent, a thioacetylation agent, a thioacylation agent, a thiobenzoylation agent, or a derivative or combination thereof. Alternatively, or in addition to, the one or more monomers may be modified to comprise any useful moiety such as an adduct (e.g., a polymer such as PEG, a polymerizable molecule such as a nucleic acid molecule, a nanoparticle or nanotube, a peptide or protein), a lipid, a carbohydrate, a metabolite, a fluorophore, a hapten, a quencher, a tag (e.g., a fluorescent tag, a magnetic tag, a radioactive tag), a barcode, or other moiety. In some instances, a monomer of the polymeric analyte may be modified to facilitate recruitment of anenzyme to recognize or cleave a terminal monomer (e.g., a NTAA or CTAA of a peptide, the 5’ or 3’ nucleotide of a nucleic acid molecule, or the first or last monomer of a polymer) or set of monomers. For example, a terminal amino acid of a peptide analyte may be modified with a saccharide in order to recruit a lectin or lectin-bound protease. In another example, one or more monomers of a polymeric analyte may comprise or be coupled to a nucleic acid molecule having a first sequence that is complementary to a second sequence comprised by an oligo-bound protease. Hybridization of the first sequence to the second sequence may facilitate local recruitment of the protease to the monomer to be cleaved. In yet another example, a peptide analyte may be modified with PITC, which may allow for recruitment and cleavage by an Edmanase. In some examples, modifications to monomers of a polymeric analyte may include epitope tags, which can facilitate binding of a binding agent (e.g., subsequent to cleavage of the monomer from the polymeric analyte). Examples of such epitope tags include fluorophores, nucleic acid molecules, peptides, haptens, polymers, chemical moieties, or other adduct molecule. Additional examples of modifications to polymeric analytes are described elsewhere herein.

[0328] The polymeric analyte may comprise one or more modified monomers. The modification of the monomers may be naturally occurring, or synthetic. Synthetic modifications may be performed prior to, during, or subsequent to cleavage of a monomer from the polymeric analyte and may be advantageous in preserving the identity of the monomer. For instance, during standard Edman degradation reactions to cleave a terminal amino acid (monomer) from the peptide, some amino acid residues may be altered or rendered undetectable by the reaction conditions. In an example, the conditions of Edman degradation may cause oxidation of cysteine residues, dehydration or destruction of the phenylthiohydantoin (PTH) forms of serine or threonine, react with and modify lysine residues, or render some post-translational modifications undetectable. As such, it modifying a peptide prior to analysis, e.g., to protect some of the amino acid residues or post-translational modifications may be useful in more accurately identifying each of the amino acid residues. In an example of a modification that can be performed prior to cleavage, a peptide or portion thereof may be alkylated, e.g., to alkylate the cysteine residues (e.g., using 4-vinylpyridine, iodoacetamide, iodoacetate, chloroacetate or aminoethylated, e.g., using 2-bromoethylamine); acetylated, e.g., to react serine or threonine residues form an ester (e.g., using acetyl chloride) or using acetic anhydride; oxidized, e.g., to convert cysteine residues to cysteic acid; reduced (e.g., using a reducing agent such as dithiothreitol, [3-mercaptoethanol, or TCEP); subjected to native chemical ligation; contacted with a protecting group, e.g., phosphorylated residues may be protected (e.g., using a P-elimination of a phosphate group,with an optional Michael addition of a thiol group, e.g., as described in Knight, et al. Nat. Biotechnology. 21, 1047-1054 (2003), which is incorporated by reference herein in its entirety), etc. The polymeric analyte or monomer may be modified with a protecting group or moiety, such as a methyl, formyl, ethyl, acetyl, t-butyl, anisyl, benzyl, tifhioroacetyl, N- hydroxysuccinimide, t-butyloxycarbonyl (Boc), benzoyl, 4-methyl benzyl, thioanizyl, thiocresyl, benzyloxymethyl, 4-nitrophenyl, benzyloxycarbonyl, 2-nitrobenzoyl, 2-nitrophenylsulphenyl, 4- toluenesulphonyl, pentafluorophenyl, diphenylmethyl, 2-chlorobenzyloxycarbonyl, 2,4,5- tri chlorophenyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC), triphenylmethyl, or 2,2,5,7,8-pentamethyl-chroman-6-sulphonyl group. The polymeric analyte or monomer may be treated with a protecting agent, e.g., carboxyethyl methanethiosulfonate (CEMTS), thiazolidine, mercaptophenyl acetic acid, cyanobenzothiazole (e.g., for lipidation of N-terminal cysteines), acetamidomethyl, 2-methylsulfonylethyl-oxycarbonyl, etc. In some instances, the lysine residues may be blocked (e.g., the primary amines of lysine residues may be reacted) using an isothiocyanate (e.g., PITC), and optionally carrying out a single round of Edman degradation to generate a new N-terminal exposed end.

[0329] In some instances, a monomer of the polymeric analyte may be modified to facilitate cleavage of the monomer from the polymeric analyte. For example, an amino acid monomer of a peptide polymeric analytic may be modified such that it is recognized by an enzyme, e.g., acetylation of an amino acid, which can facilitate acyl peptide hydrolase cleavage of the acetylated amino acid. Additional or alternative modifications to the monomers, such as those described herein, may also facilitate recognition by or interaction with an engineered cleaving enzyme.

[0330] In some instances, a monomer comprising a naturally-occurring modification may be treated to remove or alter the naturally-occurring modification to render the polymeric analyte or monomer more amenable to the processing operations disclosed herein. For example, acetylation, formylation, methylation, and pyrrolidone carboxylic acid post-translational modifications may be removed prior to sequencing. Acetylation modifications may be removed with acyl peptide hydrolase or acid treatment (e.g., using IN HC1). Methylation may be removed using aminopeptidases. Formylation modifications may be removed, for example, using acid treatment (e.g., 0.6M HC1 treatment). Pyrrolidone carboxylic acid (PCA) may be removed with pyroglutamate aminopeptidase. Exemplary C-terminal modifications may include amidation and methylation, both of which may be removed using carb oxy peptidases.

[0331] Binding agents: The binding agent may be contacted with the monomer (e.g., subsequent to cleavage and monomer-capture moiety coupling). The binding agent may be anyuseful molecule that can couple to the monomer or monomer-capture moiety complex. For example, as described herein, a binding agent may be or comprise a protein or peptide (e.g., an antibody, antibody fragment, single chain variant fragment (scFv), nanobody, anticalin, tRNA synthetase or tRNA-acyl synthetase, a fibronectin domain), a peptide mimetic, a peptidomimetic (e.g., a peptoid, a beta-peptide, a D-peptide peptidomimetic), a polysaccharide, a nucleic acid molecule (e.g., aptamer), a somamer, a polymer, an inorganic compound, an organic compound, a small molecule, or derivatives (e.g., engineered variants) or combinations thereof. In instances where the polymeric analyte comprises a peptide, the binding agent may be able to bind to a modified or derivatized amino acid (e.g., an amino acid coupled to a linker) or portion thereof. The binding agent may comprise a recognition site that specifically recognizes an amino acid, modified amino acid (e.g., an amino acid bound to a linker comprising a PITC moiety), or a derivatized (and optionally modified) amino acid. For example, the binding agent may be configured to recognize or have binding specificity to a moiety of a modified amino acid, such as a specific amino acid residue, the residue-linker complex, or derivatized amino acid (e.g., a thiocarbomyl-derivatized residue, a thiazolone-derivatized residue, a thiohydantoin-derivatized residue, etc.), or a portion of a modified amino acid. In some instances, the binding agent may be derived or engineered from a naturally-occurring enzyme or protein, e.g., an aminopeptidase, exopeptidase, metalloprotease, antibody, anticalin, N-recognin protein, Clp protease, endoprotease (e.g. trypsin), or tRNA synthetase. In some examples, a binding agent may be a cleaving enzyme (e.g., trypsin, endoprotease) that has been modified to remove the peptidase activity. The binding agent may also recognize a terminal amino acid that is attached to a substrate; for example, after all but the final monomer of a polymeric analyte has been coupled to the capture moiety or capture moieties and cleaved, the final monomer may remain coupled to a substrate. Accordingly, the binding agent may recognize and bind the substrate-coupled monomer.

[0332] The binding agent may be monospecific, bispecific, trispecific, or have specificity to a plurality of different molecules. In some instances, the binding agent may comprise a plurality of binding domains or moieties that can recognize and bind to different molecules. Alternatively, or in addition to, the binding agent may have multiple binding domains or moieties that bind to the same target, e.g., a bivalent, trivalent, or multivalent binding agent. In some embodiments, the binding agent is a bispecific antibody.

[0333] The binding agents may be contacted with and specifically bind to cleaved monomers, monomer-linker complex, monomer-linker-capture moiety, or monomer-capture moiety complexes (altogether referred to herein as “monomeric analytes”). For example, a-Homonomeric analyte may fall in any size or range of sizes that is less than that of the entire polymeric analyte. A monomeric analyte complex may be about 0.1 nanometer (nm), about 0.5 nm, 1 about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1 micrometer (pm), about 10 pm, about 100 pm, about 1 millimeter mm in size or greater. The monomeric analyte may have any molecular weight or range of molecular weights. The monomeric analyte may be about 1 dalton (Da), 10 Da, 100 Da, 500 Da, 1 kilodalton (kDa), 10 kDa, 100 kDa, 1,000 kDa, 10,000 kDa, 100,000 kDa, or greater. The monomeric analyte may vary in molecular weight or length, e.g., depending on the amino acid residue.

[0334] The binding agent may comprise or be coupled, directly or indirectly, to a polymerizable molecule. The polymerizable molecule may be the same type of molecule as the binding agent (e.g., both peptides, both nucleic acid molecules, etc.), or they may be different. In some instances, the binding agent comprises a peptide (e.g., antibody or antibody fragment) and the polymerizable molecule comprises a nucleic acid molecule. In some instances, the binding agent and the polymerizable molecule comprise proteins (e.g., an antibody and a fluorescent protein, two joined antibodies, e.g., a bispecific antibody).

[0335] The polymerizable molecule may be conjugated to the binding agent via a chemical conjugation approach, e.g., using linkers such as SMCC, (N-e- maleimidocaproyloxy)succinimide ester (EMCS), succinimidyl-4-(p-maleimidophenyl)butryate (SMPB), succinimidyl-(N-maleimidopropionamido-ethyleneglycol) ester (SMPEG), Succinimidyl (NHS) esters, succinimidyl-4-formylbenzamide (S-4FB), succinidmidyl-6- hydrazino-nicotinamide (S-HyNic), 4-Phenyl-3H-l,2,4-triazoline-3.5(4H)diones (PT AD) or other diazonium, l-ethyl-3-3-dimethylaminoproyl carbodiimide hydrochloride (EDC), etc. Synthesis of the peptide-nucleic acid molecule conjugate may also be carried out using solidphase synthesis, fragment conjugation (e.g., using heterobifunctional crosslinkers such as those comprising an aliphatic chain and a maleimide group on one end and NHS on the other), click chemistry (e.g., strain-promoted azide alkyne cycloaddition, inverse-electron-demand Diels- Alder reactions), or combinations of approaches or chemistries. In some instances, the polymerizable molecule may be conjugated to the binding agent using an enzymatic approach. For example, a DNA-protein conjugate may be generated using a truncated a nuclease (e.g., Cas protein such as Cas9), a relaxase (e.g., VirD2), or other enzyme, ribozyme, or DNAzyme. In some instances, the polymerizable molecule may be conjugated to the binding agent using a SpyTag and SpyCatcher interaction (e.g., generating a binding agent comprising a fusion proteincomprising SpyCatcher and conjugating to a Spy Tag-conjugated polymerizable molecule), a biotin-avidin interaction, a SNAP -tag, or other interaction. Optional purification may be performed, e.g., using ion-exchange chromatography, affinity chromatography, HPLC, or other purification technique.

[0336] The binding agent may be coupled to the polymerizable molecule via a noncovalent interaction. For instance, the binding agent may comprise an avidin or streptavidin tag, to which biotin-conjugated polymerizable molecules can bind. Alternatively, the binding agent may comprise a biotin tag to which an avidin or streptavidin-conjugated polymerizable molecule can bind.

[0337] The polymerizable molecule may comprise identifying information of the binding agent. For example, the polymerizable molecule may comprise a nucleic acid barcode molecule comprising a barcode sequence. The barcode sequence may encode for the identity of the binding agent or the binding partner. For example, a monomer (e.g., amino acid) of a polymeric analyte (e.g. peptide comprising a plurality of amino acids) may be cleaved and coupled to the capture moiety (e.g., on a substrate) and may be contacted with a binding agent (e.g., antibody, antibody fragment, nanobody). The binding agent may specifically recognize the amino acid residue or derivative thereof (e.g., a PTH, PTC, ATZ derivatized form) over other amino acid residues or derivatives thereof. The nucleic acid barcode molecule may comprise information that identifies the binding agent, which, due to the specificity of the binding agent to its target, may also identify the particular amino acid residue (or derivative).

[0338] In some instances, the binding agent comprises or is coupled to a guide RNA (gRNA), such as a prime editing guide RNA (pegRNA). For example, the binding agent may comprise a barcode molecule that comprises a pegRNA. The pegRNA may comprise a barcode sequence which identifies the binding agent or the cognate molecule of the binding agent (e.g., the monomer identity or type). The gRNA may comprise useful functional sequences, e.g., a key sequence (e.g., a key sequence that is configured to insert into a tandem array of CRISPR-Cas9 target sites), a UMI, barcode sequence, primer site, restriction site, cleavable moiety, abasic site, transposition site, spacer moiety, etc. In some instances, the gRNA comprises a propagator sequence that comprises a CRISPR-Cas9 target site. The propagator sequence may be configured to insert into a polymerizable molecule that comprises an additional target site and a protospacer-adjacent motif. In some instances, the propagator sequence is configured to be inserted into the polymerizable molecule between the protospacer-adjacent motif and the additional target site.

[0339] The polymerizable molecule of the binding agent may comprise additional multiplexed information. For example, the polymerizable molecule, e.g., a nucleic acid molecule, may comprise sequences that encode cycle or other temporal information or spatial information. In one such example, an array of peptides and capture moieties may be provided on a substrate. The array may comprise a plurality of individually addressable units, in which each (or a subset of) individually addressable units of the array comprises a peptide to be analyzed and a capture moiety. The binding agents, and the polymerizable molecules comprised therein or coupled thereto, may comprise spatial information (e.g., spatial barcode sequences) which uniquely identify the individually addressable units and thus the location of the array. The polymerizable molecules may additionally comprise temporal information (e.g., a cycle barcode that indicates the round or iteration in which the binding agent or polymerizable molecule is provided). Subsequent sequencing of the polymerizable molecule may be used to reveal the spatial information (e.g., the originating location in the array of a peptide or amino acid). In some instances, the polymerizable molecule may comprise a unique molecular identifier (UMI), which may be used to determine the quantity of a given binding agent or monomer (e.g., amino acid) for a given peptide, substrate, array, or sample. In some instances, the polymerizable molecule may comprise one or more detectable labels, e.g., fluorophores, radioisotopes, mass tags, etc. For instance, the polymerizable molecule may comprise a nucleic acid molecule or peptide comprising a plurality of fluorophores. The unique combination of fluorophores may be used as a barcode or to encode additional information to the polymerizable molecule.

[0340] Alternatively, the binding agent that recognizes the monomer-capture moiety complex may not comprise or be coupled to a polymerizable molecule. In such cases, subsequent to the binding of the binding agent to the monomer-capture moiety complex, an additional molecule (e.g., a secondary binding agent) comprising a detectable label, e.g., fluorophore, radioisotope, mass tag, or an identifying polymerizable molecule (e.g., nucleic acid barcode molecule) may be contacted with and bind to the binding agent that is bound to the monomer-capture moiety complex. In some examples, the additional molecule comprises an identifying polymerizable molecule, and the identifying polymerizable molecule may be coupled to or transferred to the additional polymerizable molecule. In one non-limiting example, the binding agent comprises a primary antibody or antibody fragment that recognizes the monomer- capture moiety complex (e.g., terminal amino acid- linker-capture moiety complex) or portion thereof (e.g., the terminal amino acid, or the terminal amino acid-linker complex); subsequent to binding of the primary antibody or antibody fragment to the monomer-capture moiety complex or portion thereof, a secondary antibody or antibody fragment comprising or coupled to apolymerizable molecule (e.g., nucleic acid barcode molecule) is coupled to the primary antibody. The polymerizable molecule of the secondary antibody or antibody fragment may comprise information on the secondary antibody or antibody fragment, the primary antibody or antibody fragment, or other information. Transfer or coupling of the polymerizable molecule of the secondary antibody or antibody fragment to the additional polymerizable molecule can be mediated by any suitable technique, e.g., hybridization of nucleic acid molecules optionally mediated by a splint molecule, click chemistry, or association of high affinity molecules (e.g., streptavidin and biotin).

[0341] In some instances, the method may comprise contacting the monomer-capture moiety complex with a library of binding agents. The library of binding agents may comprise a plurality of binding agents that have specificity to different analytes. For example, the library of binding agents may comprise a plurality of binding agents that recognize different amino acids or derivatives thereof (e.g., derivatized amino acids such as the PTH, PTC, or ATZ forms), clusters of amino acids (e.g., dipeptides, tripeptides, etc.), or combinations of amino acids (e.g., amino acids with similar side chain groups). In one such example, a given binding agent may recognize and bind to more than one amino acid, optionally with different affinities or binding kinetics. A given binding agent may recognize and bind to a single amino acid, two different amino acids, three different amino acids, four different amino acids, etc. For instance, a given binding agent may bind to amino acids with similar residues, e.g., amino acids with positively- charged side chains (e.g., arginine, histidine, lysine), negatively-charged side chains (aspartic acid, glutamic acid), amino acids with polar uncharged side chains (e.g., serine, threonine, asparagine, glutamine), amino acids with hydrophobic side chains (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, trytophan), or a combination thereof. Altogether, the library of binding agents may specifically recogni...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A binding agent that binds to one type of proteinogenic amino acid or derivative thereof of a set of two or more proteinogenic amino acids or derivatives thereof with greater specificity or with higher affinity than to all other types of proteinogenic amino acids or derivatives thereof in said set, wherein said one type of amino acid is not tryptophan.

2. The binding agent of claim 1, wherein said set of two or more proteinogenic amino acids or derivatives thereof comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different proteinogenic amino acids or derivatives thereof.

3. The binding agent of claim 1 or 2, wherein said set of two or more proteinogenic amino acids or derivatives thereof comprises naturally occurring proteinogenic amino acids.

4. The binding agent of any one of claims 1 to 3, wherein said set of two or more proteinogenic amino acids or derivatives thereof comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 standard proteinogenic amino acids or derivatives thereof.

5. The binding agent of any one of claims 1 to 3, wherein said set of two or more proteinogenic amino acids or derivatives thereof consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 standard proteinogenic amino acids or derivatives thereof.

6. The binding agent of any one of claims 1-5, wherein said binding agent is part of a library of binding agents, wherein said library of binding agents comprises a plurality of binding agents that collectively can recognize and specifically bind to at least 5 different types of proteinogenic amino acids.

7. The binding agent of claim 6, wherein said binding agent is part of a library of binding agents, wherein said library of binding agents comprises a plurality of binding agents that collectively can recognize and specifically bind to all 20 types of standard proteinogenic amino acids.

8. The binding agent of claim 7, wherein a binding agent of said library of binding agents can recognize and specifically bind to at least one post-translationally modified amino acid.

9. The binding agent of any one of claims 6-8, wherein said library of binding agents comprises a plurality of polyclonal antibodies.

10. The binding agent of any one of claims 6-9, wherein said library of binding agents comprises a plurality of monoclonal antibodies.

11. The binding agent of any one of claims 1-10, wherein said binding agent comprises an antibody, an antibody fragment, a single-chain variable fragment (scFv), or a nanobody.

12. The binding agent of claim 11, wherein said binding agent comprises an antibody.

13. The binding agent of claim 12, wherein said antibody is an immunoglobulin G (IgG).

14. The binding agent of claim 12 or 13, wherein said antibody is generated by immunizing an animal with an immunogenically effective amount of a composition comprising a carrier protein coupled to an amino acid or amino acid derivative.

15. The binding agent of claim 14, wherein said composition comprises said carrier protein comprising a polymeric linker that couples to said amino acid or amino acid derivative.

16. The binding agent of claim 15, wherein said polymeric linker comprises polyethylene glycol (PEG).

17. The binding agent of any one of claims 12-16, wherein said antibody is a monoclonal antibody.

18. The binding agent of claim 1-17, wherein said binding agent comprises an engineered scFv.

19. The binding agent of claim 18, wherein said engineered scFv is derived from an IgG.

20. The binding agent of claim 18 or 19, wherein said engineered scFv is engineered using directed evolution.

21. The binding agent of claim 20, wherein said engineered scFv is affinity matured using yeast surface display, phage display, ribosome display, or continuous evolution.

22. The binding agent of claim 20 or 21, wherein said engineered scFv has improved specificity to said one type of proteinogenic amino acid or derivative thereof through use of yeast surface display.

23. The binding agent of any one of claims 1-22, wherein said derivative or said derivatives thereof comprise a chemically modified amino acid.

24. The binding agent of claim 23, wherein said chemically modified amino acid comprises a phenylthiohydantoin, phenylthiocarbamyl, or anilinothiazolinone derivative of said amino acid.

25. The binding agent of claim 23 or 24, wherein said chemically modified amino acid comprises an amino acid coupled to a linker.

26. The binding agent of claim 25, wherein said linker is coupled to said amino acid at an N- terminal end.

27. The binding agent of claim 26, wherein said linker coupled to said chemically modified amino acid comprises a phenylthiohydantoin, phenylthiocarbamyl, or anilinothiazolinone moiety.

28. The binding agent of claim 25, wherein said linker is coupled to said amino acid at a C- terminal end.

29. The binding agent of any one of claims 25-28, wherein said linker comprises a linking nucleic acid molecule.

30. The binding agent of any one of claims 25-28, wherein said linker comprises an amino acid reactive group and a linking nucleic acid molecule.

31. The binding agent of any one of claims 1-30, wherein binding of said binding agent specifically to said one type of proteinogenic amino acid or derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in said set is measured using an indirect enzyme-linked immunosorbent assay (ELISA).

32. The binding agent of one of claim 1-31, wherein binding of said binding agent specifically to said one type of proteinogenic amino acid or derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in said set is measured using surface plasmon resonance.

33. The binding agent of any one of claims 1-32, wherein binding of said binding agent specifically to said one type of proteinogenic amino acid or derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in said set is measured using bio layer interferometry.

34. The binding agent of any one of claims 1-33, wherein said binding agent comprises a detectable label.

35. The binding agent of claim 34, wherein said detectable label comprises a nucleic acid molecule, a fluorophore, a mass tag, or a protein.

36. The binding agent of claim 35, wherein said detectable label comprises a protein, wherein said protein comprises an additional antibody or additional antibody fragment.

37. The binding agent of claim 35, wherein said detectable label comprises a nucleic acid molecule, wherein said nucleic acid molecule comprises a barcode sequence.

38. The binding agent of any one of claims 34-37, wherein said detectable label is coupled to said binding agent using a moiety selected from the group consisting of biotin, desthiobiotin, avidin, streptavidin, neutravidin, SpyCatcher, SpyTag, SNAP tag, a click chemistry moiety, and a cysteine tag.

39. The binding agent of any one of claims 34-38, wherein said binding agent comprises a noncanonical amino acid, wherein said detectable label is coupled to said binding agent via said noncanonical amino acid.

40. The binding agent of claim 39, wherein said noncanonical amino acid comprises a click chemistry moiety.

41. The binding agent of any one of claims 1-40, wherein said binding agent specifically binds to phenylalanine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in said set.

42. The binding agent any one of claims 1-40, wherein said binding agent specifically binds to leucine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in said set.

43. The binding agent of any one of claims 1-40, wherein said binding agent specifically binds to valine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in said set.

44. The binding agent of any one of claims 1-40, wherein said binding agent specifically binds to tyrosine or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in said set.

45. The binding agent of any one of claims 1-40, wherein said binding agent specifically binds to proline or a derivative thereof over all other types of proteinogenic amino acids or derivatives thereof in said set.

46. The binding agent of any one of claims 1-45, wherein said binding agent is derived from a mouse.

47. An antibody fragment that binds to one type of proteinogenic amino acid or derivative thereof of a set of two or more proteinogenic amino acids or derivatives thereof with greater specificity or with higher affinity than to all other types of proteinogenic amino acids or derivatives thereof in said set.

48. The antibody fragment of claim 47, wherein said set of two or more proteinogenic amino acids or derivatives thereof comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different proteinogenic amino acids or derivatives thereof.

49. The antibody fragment of claim 47 or 48, wherein said set of two or more proteinogenic amino acids or derivatives thereof comprises naturally occurring proteinogenic amino acids.

50. The antibody fragment of any one of claims 47-49, wherein said set of two or more proteinogenic amino acids or derivatives thereof comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 standard proteinogenic amino acids or derivatives thereof.

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