Methods and systems for processing polymeric analytes

Abstract

Provided herein are novel methods for treating polymeric analytes, such as biopolymers. A method for treating a polymeric analyte can include coupling a monomer of the polymeric analyte to a capture moiety, cleaving the monomer from the polymeric analyte, and analyzing the cleaved monomer, for example, using a binding agent. The methods described herein may employ the use of an activator.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 423,602, filed on November 8, 2022, and U.S. Provisional Patent Application No. 63 / 328,981, filed on April 8, 2022, each of which is incorporated herein by reference in its entirety.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with U.S. government support under Grant No. HG012563 awarded by the National Institutes of Health. The government has certain rights in this invention. Background Art

[0005] The analysis of naturally occurring and synthetic polymer analytes is important for characterizing systems, understanding biology and pathology, and developing therapeutics. Biopolymers, such as proteins, drive cellular processes, including differentiation, migration, and disease. Therefore, direct measurement and characterization of biopolymers is essential for disease modeling and medicine.

[0006] While technological advances have made significant progress in characterizing some types of biopolymers (e.g., nucleic acid molecules), challenges remain in selectively measuring and analyzing proteins in a high-throughput manner. Current methods for studying proteins can be limited in selectivity, sensitivity, throughput, or require prior knowledge. Therefore, new methods for characterizing and analyzing proteins are needed. Summary of the Invention

[0007] It is recognized herein that there is a need for novel methods for characterizing and analyzing polymer analytes such as proteins in an accurate and high-throughput manner. Provided herein are methods, systems, and compositions for analyzing (e.g., via sequencing) polymer analytes for addressing the above needs. The method disclosed herein may include providing a polymer analyte comprising a plurality of monomers, cutting a monomer from the plurality of monomers, and analyzing the cut monomer. The analysis of the cut monomer may include contacting the cut monomer with a binding agent or portion thereof that recognizes the cut monomer. The method disclosed herein may also employ local tethering of polymerizable molecules. In some cases, the cut monomer may be coupled to a capture portion, and the binding agent may be coupled to the cut monomer. A first polymerizable molecule comprising the identification information of the monomer may be coupled to a second polymerizable molecule adjacent to the capture portion. Subsequently, the process may be repeated to analyze all or a subset of the monomers of the polymer analyte. Further characterization of the polymerizable molecule may be performed, which may help identify a single monomer of the polymer analyte.

[0008] In one aspect, provided herein is a method for sequencing peptides with single-molecule sensitivity, the method comprising: (a) providing a peptide, wherein the peptide comprises a plurality of amino acids; and (b) sequencing the peptide to determine the identity and order of at least a subset of the plurality of amino acids, wherein the individual read accuracy of the amino acids is greater than 50% for at least 5 different amino acids.

[0009] In some embodiments, at least one of the plurality of amino acids comprises a post-translational modification. In some embodiments, (a) comprises providing a plurality of peptides comprising a peptide. In some embodiments, the individual read accuracy of an amino acid is greater than 50%, wherein the individual read accuracy is not affected by the side chains of adjacent amino acids. In some embodiments, the amino acid comprises one or more amino acids. In some embodiments, the one or more amino acids are less than 6 amino acids. In some embodiments, the amino acid comprises only one amino acid.

[0010] In some embodiments, (b) comprises: (i) cleaving an amino acid from the peptide; (ii) coupling the amino acid to a capture moiety to generate an amino acid-capture moiety complex; (iii) contacting the amino acid-capture moiety complex with a binding agent; (iv) providing a first polymerizable molecule; and (v) coupling the first polymerizable molecule to a second polymerizable molecule. In some embodiments, (ii) occurs before (i). In some embodiments, (ii) occurs during (i). In some embodiments, (ii) occurs after (i). In some embodiments, (iv) occurs before (iii). In some embodiments, (iv) occurs during (iii). In some embodiments, (iv) occurs after (iii). In some embodiments, during (iv), the binding agent is not coupled to the first polymerizable molecule. In some embodiments, the binding agent comprises an activator, wherein the activator promotes coupling of the first polymerizable molecule to the second polymerizable molecule. In some embodiments, the activator comprises an activated polymerizable molecule, and further comprises, after (iv), coupling the first polymerizable molecule to the activated polymerizable molecule. In some embodiments, the coupling is mediated using a splint molecule, which comprises a sequence complementary to at least a portion of (i) the first polymerizable molecule, (ii) the second polymerizable molecule, and (iii) the activation polymerizable molecule. In some embodiments, the first polymerizable molecule and the activation polymerizable molecule are nucleic acid molecules, and wherein the coupling of the first polymerizable molecule and the activation polymerizable molecule occurs via hybridization chain reaction (HCR). In some embodiments, the activator includes an enzyme. In some embodiments, the enzyme includes peroxidase, Cas protein, ligase, kinase, or restriction enzyme. In some embodiments, the enzyme includes kinase, wherein the kinase is configured to phosphorylate the second polymerizable molecule, thereby allowing the first polymerizable molecule to be attached to the second polymerizable molecule. In some embodiments, the binding agent is indirectly coupled to the first polymerizable molecule. In some embodiments, the binding agent includes activation polymerizable molecule, and wherein the first polymerizable molecule is coupled to the activation polymerizable molecule. In some embodiments, the binding agent includes an activator, wherein the activator is configured to promote the coupling of the first polymerizable molecule and the second polymerizable molecule. In some embodiments, the activator includes an enzyme or a nucleic acid molecule. In some embodiments, the activator comprises an anchoring nucleic acid molecule, wherein the anchoring nucleic acid molecule is configured to couple to an additional second polymerizable molecule adjacent to the second polymerizable molecule. In some embodiments, the activator comprises an enzyme selected from the group consisting of a peroxidase, a Cas protein, a ligase, a kinase, and a restriction enzyme. In some embodiments, the enzyme is a kinase, and further comprising using the kinase to phosphorylate the second polymerizable molecule, thereby allowing the first polymerizable molecule to couple to the second polymerizable molecule.In some embodiments, the enzyme is horseradish peroxidase (HRP) or ascorbate peroxidase (APEX), wherein HRP or APEX activates the first polymerizable molecule or the second polymerizable molecule, thereby coupling the first polymerizable molecule to the second polymerizable molecule. In some embodiments, the enzyme is a restriction enzyme, and wherein the second polymerizable molecule comprises a restriction recognition site, and further includes cutting the second polymerizable molecule with a restriction enzyme before the first polymerizable molecule is coupled to the second polymerizable molecule. In some embodiments, the binding agent includes an activated nucleic acid molecule, and wherein the first polymerizable molecule is a nucleic acid molecule, and further includes, before (iii), coupling the first polymerizable molecule to the activated nucleic acid sequence. In some embodiments, the method further includes generating the first polymerizable molecule via hybridization chain reaction (HCR). In some embodiments, generation includes (A) providing a first hairpin molecule and a second hairpin molecule; (B) coupling the first hairpin molecule to the activated nucleic acid sequence; and (C) coupling the second hairpin molecule to the first hairpin molecule, thereby generating the first polymerizable molecule coupled to the activated nucleic acid molecule. In some embodiments, after (C), the first polymerizable molecule includes a flap sequence and further includes using the flap sequence to couple the first polymerizable molecule to the second polymerizable molecule. In some embodiments, the method further includes repeating (i)-(v) at least once. In some embodiments, the capture portion comprises a third polymerizable molecule. In some embodiments, the first polymerizable molecule, the second polymerizable molecule, or the third polymerizable molecule comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule includes a DNA molecule with pseudo-complementary bases, a bridged nucleic acid (BNA), a heterologous nucleic acid (XNA), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a γ-PNA molecule, a morpholino, or a combination thereof. In some embodiments, the nucleic acid molecule includes a hexose nucleic acid (HNA). In some embodiments, greater than 70% of the nucleotides in the nucleic acid molecule are thymine or cytosine. In some embodiments, the third polymerizable molecule includes a DNA molecule that includes a priming site, a sequencing site, a unique molecular identifier, a barcode sequence, a cleavage site, or a combination thereof. In some embodiments, the first polymerizable molecule includes a first nucleic acid molecule, and wherein the third polymerizable molecule includes a second nucleic acid molecule. In some embodiments, the coupling in (iv) includes hybridization of at least a portion of the first nucleic acid molecule with at least a portion of the second nucleic acid molecule. In some embodiments, the method further includes performing a nucleic acid extension reaction. In some embodiments, the cutting in (i) includes cutting one or more amino acids from the peptide. In some embodiments, the first polymerizable molecule includes a first nucleic acid molecule and the second polymerizable molecule includes a second nucleic acid molecule. In some embodiments, the coupling in (ii) includes connection or hybridization of the first nucleic acid molecule with the second nucleic acid molecule.In some embodiments, the coupling in (ii) is mediated by a splint molecule. In some embodiments, the first polymerizable molecule comprises a first reactive portion, and the second polymerizable molecule comprises a second reactive portion capable of reacting with the first reactive portion. In some embodiments, the coupling in (ii) includes reacting the first portion and the second portion. In some embodiments, the first reactive portion or the second reactive portion comprises a click chemistry portion. In some embodiments, the method further includes decoupling the amino acid from the capture portion. In some embodiments, the method further includes repeating (b). In some embodiments, the method further includes identifying at least a portion of the first polymerizable molecule or its derivative. In some embodiments, identification includes sequencing the first polymerizable molecule or its derivative. In some embodiments, sequencing is performed using next-generation DNA sequencing. In some embodiments, identification includes contacting the first polymerizable molecule or its derivative with a probe. In some embodiments, the coupling in (ii) is mediated using a linker. In some embodiments, the linker is a bifunctional linker. In some embodiments, the linker comprises a first reactive group and a second reactive group, wherein the first reactive group is capable of coupling to the amino acid and wherein the second reactive group is capable of coupling to the capture portion. In some embodiments, the amino acid is a terminal amino acid. In some embodiments, the first reactive group comprises a thiocyanate, an aldehyde, dansyl chloride, or fluorobenzene. In some embodiments, the fluorobenzene is dinitrofluorobenzene. In some embodiments, the thiocyanate comprises a phenyl isothiocyanate (PITC) moiety, and wherein the coupling in (ii) generates a phenylthiocarbamoyl (PTC) derivative of the amino acid. In some embodiments, the method further comprises, after (e), derivatizing the phenylthiocarbamoyl (PTC) derivative to generate a thiazolinone (ATZ) derivative or a phenylthiohydantoin (PTH) derivative. In some embodiments, the method further comprises derivatizing the ATZ derivative or PTH derivative to another PTC derivative. In some embodiments, the cleavage in (i) is performed by applying a stimulus. In some embodiments, the stimulus comprises a change in pH. In some embodiments, the change in pH comprises the use of an acid. In some embodiments, the change in pH comprises the use of a base. In some embodiments, the stimulus comprises the use of a Lewis acid. In some embodiments, the Lewis acid comprises boron trifluoromethanesulfonate (BF3), boron trifluoride ethyl etherate, boron trichloride, boron tribromide, boron triiodide or scandium trifluoromethanesulfonate. In some embodiments, the stimulation comprises electromagnetic radiation. In some embodiments, the electromagnetic radiation is applied using microwaves. In some embodiments, the capture moiety comprises a third reactive group capable of reacting with the second reactive group. In some embodiments, the first reactive group, the second reactive group or the third reactive group comprises a click chemistry moiety.In some embodiments, the second reactive group and the third reactive group comprise a click chemistry pair. In some embodiments, the linker comprises an amino acid reactive group and an additional polymerizable molecule, wherein the additional polymerizable molecule is configured to be coupled to the capture portion. In some embodiments, the amino acid reactive group is capable of cleaving amino acids. In some embodiments, the additional polymerizable molecule comprises an enzyme recognition site. In some embodiments, the enzyme recognition site is recognized by a nuclease. In some embodiments, the method further comprises, after (iv), cleaving the additional polymerizable molecule, thereby releasing the amino acid from the capture portion. In some embodiments, the nuclease is a restriction enzyme, a Cas protein, or an Argonaut nuclease. In some embodiments, the cleavage in (i) comprises applying a stimulus. In some embodiments, the stimulus comprises a change in pH. In some embodiments, the stimulus comprises using a Lewis acid. In some embodiments, the Lewis acid comprises boron trifluoromethanesulfonate (BF3), boron trifluoride etherate, boron trichloride, boron tribromide, boron triiodide, or scandium trifluoromethanesulfonate. In some embodiments, the stimulus comprises electromagnetic radiation. In some embodiments, the electromagnetic radiation is applied using microwaves. In some embodiments, the stimulus comprises biostimulation. In some embodiments, biostimulation comprises the use of an enzyme. In some embodiments, the enzyme is a metalloprotease, an aminopeptidase, or an exopeptidase. In some embodiments, the method further comprises providing a metal catalyst. In some embodiments, the amino acid comprises a modification, and wherein the enzyme recognizes the modification. In some embodiments, the first polymerizable molecule comprises a moiety that identifies the binding agent or amino acid. In some embodiments, the first polymerizable molecule comprises a nucleic acid molecule comprising a barcode sequence that identifies the binding agent or amino acid. In some embodiments, the method further comprises subjecting the amino acid-capture moiety complex to conditions sufficient to inhibit binding of the additional binding agent to the amino acid-capture moiety complex. In some embodiments, the conditions comprise chemical or enzymatic treatment. In some embodiments, the method further comprises blocking a carboxyl group or an amine group of the peptide. In some embodiments, the binding agent comprises an antibody, an antibody fragment, an aptamer, a scFv, a nanobody, an anticalin, a tRNA-acyl synthetase, a peptide, a peptide mimetic, a polysaccharide, or a derivative thereof. In some embodiments, the binding agent comprises an enzyme. In some embodiments, the enzyme comprises a metalloprotease, an aminopeptidase, or an exopeptidase. In some embodiments, the peptide, capture moiety, or second polymerizable molecule is coupled to a substrate. In some embodiments, the substrate is a solid support. In some embodiments, the substrate is substantially planar. In some embodiments, the substrate is a bead or particle. In some embodiments, the peptide, capture moiety or second polymerizable molecule is coupled to the substrate via a click chemistry moiety. In some embodiments, the peptide, capture moiety or second polymerizable molecule is coupled to the support via a functional group. In some embodiments, the functional group is added to the peptide, capture moiety or second polymerizable molecule using an enzyme.In some embodiments, the enzyme comprises an amidase. In some embodiments, the peptide, capture moiety, or second polymerizable molecule is coupled to a substrate using a linker molecule. In some embodiments, the substrate is functionalized with a functional group and further comprises attaching the peptide, capture moiety, or second polymerizable molecule to the functional group. In some embodiments, the peptide, capture moiety, and second polymerizable molecule are coupled to a substrate. In some embodiments, the method further comprises passivating the substrate. In some embodiments, passivation reduces non-specific binding of the binding agent. In some embodiments, at least one amino acid in a plurality of amino acids comprises a non-naturally occurring modification. In some embodiments, the method further comprises generating a peptide comprising a non-naturally occurring modification. In some embodiments, generating comprises alkylating or acetylating at least one amino acid, β-eliminating a phosphate group, or using PITC or acetic anhydride. In some embodiments, generating comprises converting a cysteine ​​residue into cysteic acid. In some embodiments, generating comprises using an oxidizing agent or a reducing agent.

[0011] In some embodiments, the peptide is derived from a biological sample.

[0012] In some embodiments, (b) comprises: (i) cleaving an amino acid from the peptide; (ii) coupling the amino acid to a capture moiety to generate an amino acid-capture moiety complex; (iii) contacting the amino acid-capture moiety complex with a binding agent, wherein the binding agent comprises an activator; and (iv) detecting a product from the activator. In some embodiments, the activator comprises an enzyme that generates the product. In some embodiments, the enzyme is horseradish peroxidase or ascorbate peroxidase.

[0013] In another aspect, provided herein is a method for processing a polymer analyte, the method comprising: (a) providing a polymer analyte and a capture portion, wherein the polymer analyte comprises a plurality of monomers; (b) cleaving a monomer of the plurality of monomers from the polymer analyte; (c) coupling the monomer to the capture portion to generate a monomer-capture portion complex; (d) after (b) and (c), contacting the monomer-capture portion complex with a binding agent comprising an activator; (e) providing a first polymerizable molecule; and (f) using the activator to couple the first polymerizable molecule to a second polymerizable molecule.

[0014] In some embodiments, (e) occurs before (d). In some embodiments, (e) occurs during (d). In some embodiments, (e) occurs after (d). In some embodiments, during (e), the binding agent is not coupled to the first polymerizable molecule. In some embodiments, the binding agent is indirectly coupled to the first polymerizable molecule. In some embodiments, the activator includes an activated polymerizable molecule, and further includes coupling the first polymerizable molecule to the activated polymerizable molecule. In some embodiments, the first polymerizable molecule, the second polymerizable molecule and the activated polymerizable molecule are nucleic acid molecules, and wherein the coupling is mediated using a splint molecule, which comprises a sequence complementary to at least a portion of (i) the first polymerizable molecule, (ii) the second polymerizable molecule and (iii) the activated polymerizable molecule. In some embodiments, the first polymerizable molecule includes a nucleic acid molecule, and further includes generating the first polymerizable molecule via a hybridization chain reaction (HCR). In some embodiments, generating includes (A) providing a first hairpin molecule and a second hairpin molecule; (B) coupling the first hairpin molecule to an activated polymerizable molecule; and (C) coupling the second hairpin molecule to the first hairpin molecule, thereby generating a first polymerizable molecule coupled to the activated polymerizable molecule. In some embodiments, after (C), the first polymerizable molecule includes a flap sequence, and further includes using the flap sequence to couple the first polymerizable molecule to the second polymerizable molecule. In some embodiments, the method further includes coupling an additional first hairpin molecule to the first polymerizable molecule and coupling an additional second hairpin molecule to the additional first hairpin molecule, thereby generating an additional first polymerizable molecule coupled to the first polymerizable molecule. In some embodiments, the method further includes coupling an additional first polymerizable molecule to an additional second polymerizable molecule. In some embodiments, the method further includes repeating (b)-(f), wherein the repeating includes generating an additional first polymerizable molecule by repeating (A)-(C). In some embodiments, the repetition of (A)-(C) includes providing additional hairpin molecules, wherein at least a subset of the additional hairpin molecules comprises a sequence different from the first hairpin molecule or the second hairpin molecule. In some embodiments, the subset of additional hairpin molecules comprises a sequence configured to be coupled to at least a portion of the first polymerizable molecule. In some embodiments, the method further comprises cutting a portion of the first polymerizable molecule. In some embodiments, the activator comprises an anchoring nucleic acid molecule, wherein the anchoring nucleic acid molecule is configured to be coupled to an additional second polymerizable molecule adjacent to the second polymerizable molecule. In some embodiments, the first polymerizable molecule is coupled to a binding agent. In some embodiments, the activator comprises an enzyme selected from the group consisting of a peroxidase, a Cas protein, a ligase, a kinase, and a restriction enzyme. In some embodiments, the enzyme is a kinase, and further comprises using a kinase to phosphorylate the second polymerizable molecule, thereby allowing the first polymerizable molecule to be coupled to the second polymerizable molecule.In some embodiments, the enzyme is horseradish peroxidase (HRP) or ascorbate peroxidase (APEX), wherein HRP or APEX activates the first polymerizable molecule or the second polymerizable molecule, thereby coupling the first polymerizable molecule to the second polymerizable molecule. In some embodiments, the enzyme is a restriction enzyme, and wherein the second polymerizable molecule includes a restriction recognition site, and further includes cutting the second polymerizable molecule with a restriction enzyme before the first polymerizable molecule is coupled to the second polymerizable molecule. In some embodiments, (c) is performed before (b). In some embodiments, (c) is performed during (b). In some embodiments, (c) is performed after (b). In some embodiments, the cutting in (b) includes cutting one or more monomers in a plurality of monomers. In some embodiments, the capture portion comprises a third polymerizable molecule. In some embodiments, the first polymerizable molecule, the second polymerizable molecule, or the third polymerizable molecule comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises a DNA molecule with pseudo-complementary bases, a bridged nucleic acid (BNA), a heterologous nucleic acid (XNA), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a γ-PNA molecule, a morpholino, or a combination thereof. In some embodiments, the nucleic acid molecule comprises a hexose nucleic acid (HNA). In some embodiments, greater than 70% of the nucleotides in the nucleic acid molecule are thymine or cytosine. In some embodiments, the third polymerizable molecule comprises a DNA molecule comprising a priming site, a sequencing site, a unique molecular identifier, a barcode sequence, a cleavage site, or a combination thereof. In some embodiments, the first polymerizable molecule comprises a first nucleic acid molecule, and wherein the third polymerizable molecule comprises a second nucleic acid molecule. In some embodiments, the coupling in (e) comprises hybridization of at least a portion of the first nucleic acid molecule with at least a portion of the second nucleic acid molecule. In some embodiments, the method further comprises performing a nucleic acid extension reaction. In some embodiments, the first polymerizable molecule comprises a first nucleic acid molecule and the second polymerizable molecule comprises a second nucleic acid molecule. In some embodiments, the coupling in (c) comprises connection or hybridization of the first nucleic acid molecule with the second nucleic acid molecule. In some embodiments, the coupling in (c) is mediated by a splint molecule. In some embodiments, the first polymerizable molecule comprises a first reactive portion, and the second polymerizable molecule comprises a reactive portion capable of reacting with the first reactive portion. In some embodiments, the coupling in (c) comprises reacting the first portion and the second portion. In some embodiments, the first reactive portion or the second reactive portion comprises a click chemistry portion. In some embodiments, the method further comprises decoupling the monomer from the capture portion. In some embodiments, the method further comprises repeating (b)-(e). In some embodiments, the method further comprises identifying at least a portion of the first polymerizable molecule or its derivative. In some embodiments, identification comprises sequencing the first polymerizable molecule or its derivative.In some embodiments, sequencing is carried out using next generation DNA sequencing. In some embodiments, identification includes hybridization using a probe. In some embodiments, the coupling in (c) is mediated by a joint. In some embodiments, the joint is a bifunctional joint. In some embodiments, the joint comprises a first reactive group and a second reactive group, wherein the first reactive group can be coupled to a monomer and wherein the second reactive group can be coupled to a capture portion. In some embodiments, the polymer analyte is a peptide comprising multiple amino acids, and the first reactive group can be coupled to an amino acid. In some embodiments, the amino acid is a terminal amino acid. In some embodiments, at least one amino acid in a plurality of amino acids comprises a post-translational modification. In some embodiments, the first reactive group comprises thiocyanate, an aldehyde group, dansyl chloride or fluorobenzene. In some embodiments, thiocyanate comprises phenyl isothiocyanate (PITC) moiety, and wherein the coupling in (c) generates an amino acid whose phenylthiocarbamoyl derivative. In some embodiments, the method further comprises, after (e), derivatizing the phenylthiocarbamoyl derivative to generate a thiazolinone (ATZ) derivative or a phenylthiohydantoin (PTH)-amino acid derivative. In some embodiments, the cleavage in (b) is performed by applying a stimulus. In some embodiments, the stimulus comprises a change in pH. In some embodiments, the change in pH comprises the use of an acid. In some embodiments, the change in pH comprises the use of a base. In some embodiments, the stimulus comprises the use of a Lewis acid. In some embodiments, the Lewis acid comprises boron trifluoromethanesulfonate (BF3), boron trifluoride ethyl etherate, boron trichloride, boron tribromide, boron triiodide, or scandium trifluoromethanesulfonate. In some embodiments, the stimulus comprises electromagnetic radiation. In some embodiments, electromagnetic radiation is applied using microwaves. In some embodiments, the capture portion comprises a third reactive group capable of reacting with the second reactive group. In some embodiments, the first reactive group, the second reactive group, or the third reactive group comprises a click chemistry moiety. In some embodiments, the second reactive group and the third reactive group comprise a click chemistry pair. In some embodiments, the linker comprises an amino acid reactive group and an additional polymerizable molecule, wherein the additional polymerizable molecule is configured to be coupled to the capture portion. In some embodiments, the additional polymerizable molecule comprises a nucleic acid molecule comprising an enzyme recognition site. In some embodiments, the enzyme recognition site is recognized by a nuclease. In some embodiments, the method further comprises, after (e), cutting the additional polymerizable molecule, thereby releasing one or more monomers from the capture portion. In some embodiments, the nuclease is a restriction enzyme, a Cas protein, or an Argonaut nuclease. In some embodiments, the polymer analyte comprises a macromolecule. In some embodiments, the macromolecule comprises a peptide comprising a plurality of amino acids, wherein the monomer is an amino acid in the plurality of amino acids.In some embodiments, the amino acid is a terminal amino acid. In some embodiments, at least one amino acid in the plurality of amino acids comprises a post-translational modification. In some embodiments, at least one amino acid in the plurality of amino acids comprises a non-naturally occurring modification. In some embodiments, the method further comprises generating a peptide comprising a non-naturally occurring modification. In some embodiments, generating comprises alkylating or acetylating at least one amino acid, β-eliminating a phosphate group, or using PITC or acetic anhydride. In some embodiments, generating comprises converting a cysteine ​​residue to cysteic acid. In some embodiments, generating comprises using an oxidizing or reducing agent. In some embodiments, the non-naturally occurring modification is located on an amino acid. In some embodiments, the macromolecule is derived from a biological sample. In some embodiments, the polymer analyte comprises a peptidomimetic. In some embodiments, the binding agent comprises an antibody, an antibody fragment, an aptamer, a scFv, a nanobody, an anticalin, a tRNA-acyl synthetase, a peptide, a peptide mimetic, a polysaccharide, or a derivative thereof. In some embodiments, the binding agent comprises an enzyme. In some embodiments, the enzyme comprises a metalloprotease, an aminopeptidase, or an exopeptidase. In some embodiments, the polymer analyte, the capture moiety, or the second polymerizable molecule is coupled to a substrate. In some embodiments, the substrate is a solid support. In some embodiments, the substrate is substantially planar. In some embodiments, the substrate is a bead or particle. In some embodiments, the polymer analyte, capture moiety or second polymerizable molecule is coupled to the substrate via a click chemistry portion. In some embodiments, the polymer analyte, capture moiety or second polymerizable molecule is coupled to the substrate via a functional group. In some embodiments, the functional group is added to the polymer analyte, capture moiety or second polymerizable molecule using an enzyme. In some embodiments, the enzyme includes an amidase. In some embodiments, the polymer analyte, capture moiety or second polymerizable molecule is coupled to the substrate using a linker molecule. In some embodiments, the substrate is functionalized with a functional group and further includes attaching the polymer analyte, capture moiety or second polymerizable molecule to the functional group. In some embodiments, the polymer analyte, capture moiety and second polymerizable molecule are coupled to the substrate. In some embodiments, the method further includes passivating the substrate. In some embodiments, passivation reduces the non-specific binding of the binding agent. In some embodiments, the cutting in (b) includes applying a stimulus. In some embodiments, the stimulus includes a change in pH. In some embodiments, the stimulation comprises the use of a Lewis acid. In some embodiments, the Lewis acid comprises boron trifluoromethanesulfonate (BF3), boron trifluoride ethyl etherate, boron trichloride, boron tribromide, boron triiodide, or scandium trifluoromethanesulfonate. In some embodiments, the stimulation comprises biostimulation. In some embodiments, the biostimulation comprises the use of an enzyme. In some embodiments, the enzyme is a metalloproteinase, an aminopeptidase, or an exopeptidase. In some embodiments, the method further comprises providing a metal catalyst.In some embodiments, the monomer comprises a modification, and wherein the enzyme recognizes the modification. In some embodiments, the first polymerizable molecule comprises a portion that recognizes a binding agent or a monomer. In some embodiments, the first polymerizable molecule comprises a nucleic acid molecule comprising a sequence that identifies a binding agent or a monomer. In some embodiments, the method further comprises subjecting the monomer-capture moiety complex to conditions sufficient to inhibit the binding of the additional binding agent to the monomer-capture moiety complex. In some embodiments, the conditions comprise chemical or enzymatic treatment. In some embodiments, the size of the monomer is <10 nm. In some embodiments, the monomer has a molecular weight of less than 210 Daltons.

[0015] In yet another aspect, disclosed herein is a method for processing an analyte, the method comprising: (a) providing (i) an analyte, (ii) a binding agent comprising an activator, (iii) a first polymerizable molecule, and (iv) a second polymerizable molecule, wherein the analyte has a size of <10 nm; (b) contacting the analyte with the binding agent; and (c) using the activator to couple the first polymerizable molecule to the second polymerizable molecule.

[0016] In some embodiments, the binding agent is not coupled to the first polymerizable molecule. In some embodiments, the binding agent is indirectly coupled to the first polymerizable molecule. In some embodiments, the activator includes activating the polymerizable molecule, and further includes coupling the first polymerizable molecule to the activated polymerizable molecule. In some embodiments, the first polymerizable molecule, the second polymerizable molecule and the activated polymerizable molecule are nucleic acid molecules, and wherein the coupling is mediated using a splint molecule, which comprises a sequence complementary to at least a portion of (i) the first polymerizable molecule, (ii) the second polymerizable molecule and (iii) the activated polymerizable molecule. In some embodiments, the first polymerizable molecule includes a nucleic acid molecule, and further includes generating the first polymerizable molecule via a hybridization chain reaction (HCR). In some embodiments, generation includes (A) providing a first hairpin molecule and a second hairpin molecule; (B) coupling the first hairpin molecule to the activated polymerizable molecule; and (C) coupling the second hairpin molecule to the first hairpin molecule, thereby generating the first polymerizable molecule coupled to the activated polymerizable molecule. In some embodiments, after (C), the first polymerizable molecule comprises a flap sequence, and further comprises using a flap sequence to couple the first polymerizable molecule to the second polymerizable molecule. In some embodiments, the activator comprises an anchoring nucleic acid molecule, wherein the anchoring nucleic acid molecule is configured to couple to an additional second polymerizable molecule adjacent to the second polymerizable molecule. In some embodiments, the first polymerizable molecule is coupled to a binding agent. In some embodiments, the activator comprises an enzyme selected from the group consisting of peroxidase, Cas protein, ligase, kinase, and restriction enzyme. In some embodiments, the enzyme is a kinase, and further comprises using a kinase to phosphorylate the second polymerizable molecule, thereby allowing the first polymerizable molecule to couple to the second polymerizable molecule. In some embodiments, the enzyme is horseradish peroxidase (HRP) or ascorbate peroxidase (APEX), wherein HRP or APEX activates the first polymerizable molecule or the second polymerizable molecule, thereby coupling the first polymerizable molecule to the second polymerizable molecule. In some embodiments, the enzyme is a restriction enzyme, and wherein the second polymerizable molecule comprises a restriction recognition site, and further comprises cutting the second polymerizable molecule with a restriction enzyme before the first polymerizable molecule is coupled to the second polymerizable molecule. In some embodiments, the analyte comprises a monomeric unit of a polymer. In some embodiments, the polymer is a peptide and the monomeric unit comprises amino acid. In some embodiments, at least one amino acid of the peptide comprises a post-translational modification. In some embodiments, the method further comprises, before (a), providing a polymer comprising a plurality of monomers, coupling the monomer in the plurality of monomers to a substrate, and cutting the monomer from the polymer, thereby producing an analyte coupled to the substrate. In some embodiments, the polymer comprises a peptide, and the monomer comprises amino acid or modified amino acid. In some embodiments, the coupling is mediated by a joint.In some embodiments, the linker comprises a first reactive moiety capable of coupling to a monomer and a second reactive moiety capable of coupling to a substrate.

[0017] In yet another aspect, provided herein is a method for processing an analyte, the method comprising: (a) providing (i) an analyte, (ii) a binding agent, and (iii) a first polymerizable molecule, wherein the first polymerizable molecule is not coupled to the binding agent; and (iv) a second polymerizable molecule, wherein the analyte has a size of <10 nm; (b) contacting the analyte with the binding agent; and (c) coupling the first polymerizable molecule to the second polymerizable molecule.

[0018] In some embodiments, the binding agent includes an activated polymerizable molecule, and further includes coupling the first polymerizable molecule to the activated polymerizable molecule. In some embodiments, the first polymerizable molecule, the second polymerizable molecule and the activated polymerizable molecule are nucleic acid molecules, and wherein the coupling is mediated using a splint molecule, which includes a sequence complementary to at least a portion of (i) the first polymerizable molecule, (ii) the second polymerizable molecule and (iii) the activated polymerizable molecule. In some embodiments, the first polymerizable molecule includes a nucleic acid molecule, and further includes generating the first polymerizable molecule via a hybridization chain reaction (HCR). In some embodiments, generation includes (A) providing a first hairpin molecule and a second hairpin molecule; (B) coupling the first hairpin molecule to the activated polymerizable molecule; and (C) coupling the second hairpin molecule to the first hairpin molecule, thereby generating the first polymerizable molecule coupled to the activated polymerizable molecule. In some embodiments, after (C), the first polymerizable molecule includes a flap sequence, and further includes using a flap sequence to couple the first polymerizable molecule to the second polymerizable molecule. In some embodiments, the binding agent includes an anchoring nucleic acid molecule, wherein the anchoring nucleic acid molecule is configured to be coupled to an additional second polymerizable molecule adjacent to the second polymerizable molecule. In some embodiments, the binding agent includes an enzyme coupled thereto, wherein the enzyme is selected from: peroxidase, Cas protein, ligase, kinase and restriction enzyme. In some embodiments, the enzyme is a kinase, and further includes using a kinase to phosphorylate the second polymerizable molecule, thereby allowing the first polymerizable molecule to be coupled to the second polymerizable molecule. In some embodiments, the enzyme is horseradish peroxidase (HRP) or ascorbic acid peroxidase (APEX), wherein HRP or APEX activates the first polymerizable molecule or the second polymerizable molecule, thereby coupling the first polymerizable molecule to the second polymerizable molecule. In some embodiments, the enzyme is a restriction enzyme, and wherein the second polymerizable molecule includes a restriction recognition site, and further includes cutting the second polymerizable molecule with a restriction enzyme before the first polymerizable molecule is coupled to the second polymerizable molecule. In some embodiments, the analyte includes a monomeric unit of a polymer. In some embodiments, the polymer is a peptide and the monomeric unit includes amino acids. In some embodiments, at least one amino acid of the peptide includes post-translational modification. In some embodiments, the method further comprises, before (a), providing a polymer comprising a plurality of monomers, coupling the monomers in the plurality of monomers to a substrate, and cutting the monomers from the polymer to produce an analyte coupled to the substrate. In some embodiments, the polymer comprises a peptide, and the monomer comprises an amino acid or a modified amino acid. In some embodiments, the coupling is mediated by a joint. In some embodiments, the joint comprises a first reactive portion that can be coupled to a monomer and a second reactive portion that can be coupled to a substrate.

[0019] In another aspect of the present disclosure, a method is provided herein comprising: (a) providing (i) a peptide comprising a plurality of amino acids, wherein an amino acid in the plurality of amino acids comprises a non-naturally occurring modification of a plurality of amino acids, (ii) a substrate coupled to the peptide, and (iii) a linker; (b) using a linker to couple an amino acid in the plurality of amino acids or an additional amino acid to the substrate to produce a substrate-coupled amino acid; and (c) after (b), cleaving the substrate-coupled amino acid from the peptide, thereby producing (i) the removed substrate-coupled amino acid and (ii) the remaining portion of the peptide. In some embodiments, the method further comprises, after (c), contacting the removed substrate-coupled amino acid with a binding agent. In some embodiments, the binding agent comprises a first polymerizable molecule, and wherein the substrate comprises a second polymerizable molecule. In some embodiments, the first polymerizable molecule comprises a portion that identifies the binding agent or amino acid. In some embodiments, the first polymerizable molecule comprises a nucleic acid molecule comprising a barcode sequence that identifies the binding agent or amino acid. In some embodiments, the method further comprises coupling the first polymerizable molecule to the second polymerizable molecule. In some embodiments, the method further includes sequencing the first polymerizable molecule. In some embodiments, sequencing is performed using next-generation DNA sequencing. In some embodiments, the first polymerizable molecule or the second polymerizable molecule comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises a DNA molecule with pseudo-complementary bases, a bridged nucleic acid (BNA), a heterologous nucleic acid (XNA), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a γ-PNA molecule, a morpholino, or a combination thereof. In some embodiments, the nucleic acid molecule comprises a hexose nucleic acid (HNA). In some embodiments, greater than 70% of the nucleotides in the nucleic acid molecule are thymine or cytosine. In some embodiments, the first polymerizable molecule comprises a first nucleic acid molecule and the second polymerizable molecule comprises a second nucleic acid molecule. In some embodiments, the coupling of the first polymerizable molecule and the second polymerizable molecule comprises a connection or hybridization of the first nucleic acid molecule with the second nucleic acid molecule. In some embodiments, the coupling of the first polymerizable molecule and the second polymerizable molecule is mediated by a splint molecule. In some embodiments, the coupling comprises hybridization of at least a portion of the first polymerizable molecule with at least a portion of the second polymerizable molecule. In some embodiments, the method further includes performing a nucleic acid extension reaction. In some embodiments, the first polymerizable molecule comprises a first reactive portion, and the second polymerizable molecule comprises a second reactive portion capable of reacting with the first reactive portion. In some embodiments, the coupling in (ii) comprises reacting the first portion and the second portion. In some embodiments, the first reactive portion or the second reactive portion comprises a click chemistry portion. In some embodiments, the binding agent comprises an antibody, an antibody fragment, an aptamer, a nanobody, a peptide, a peptide mimetic, a polysaccharide, or a derivative thereof. In some embodiments, the binding agent comprises an enzyme.In some embodiments, the enzyme comprises a metalloproteinase, an aminopeptidase, or an exopeptidase. In some embodiments, the cleavage in (c) comprises applying a stimulus. In some embodiments, the stimulus comprises a change in pH. In some embodiments, the stimulus comprises using a Lewis acid. In some embodiments, the Lewis acid comprises boron trifluoromethanesulfonate (BF3), boron trifluoride ethyl etherate, boron trichloride, boron tribromide, boron triiodide, or scandium trifluoromethanesulfonate. In some embodiments, the stimulus comprises electromagnetic radiation. In some embodiments, the electromagnetic radiation is applied using microwaves. In some embodiments, the stimulus is a biostimulation. In some embodiments, the biostimulation comprises using an enzyme. In some embodiments, the enzyme is a metalloproteinase, an aminopeptidase, or an exopeptidase. In some embodiments, the method further comprises providing a metal catalyst. In some embodiments, the amino acid comprises a modification, and wherein the enzyme recognizes the modification. In some embodiments, the method further comprises subjecting the removed substrate-coupled amino acid to conditions sufficient to inhibit the binding of an additional binding agent to the removed substrate-coupled amino acid. In some embodiments, the conditions comprise chemical or enzymatic treatment. In some embodiments, the method further comprises blocking a carboxyl group or an amine group of the peptide. In some embodiments, the cleavage in (c) includes cleaving one or more amino acids from the peptide. In some embodiments, the method further includes removing the amino acid of the substrate-coupled amino acid from the substrate. In some embodiments, the substrate comprises a capture portion, wherein the capture portion is coupled to the amino acid or another amino acid via a linker. In some embodiments, the capture portion comprises a polymerizable molecule. In some embodiments, the polymerizable molecule is a nucleic acid molecule. In some embodiments, the nucleic acid molecule includes a DNA molecule with pseudo-complementary bases, a bridged nucleic acid (BNA), a heterologous nucleic acid (XNA), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a γ-PNA molecule, a morpholino or a combination thereof. In some embodiments, the nucleic acid molecule includes a hexose nucleic acid (HNA). In some embodiments, greater than 70% of the nucleotides in the nucleic acid molecule are thymine or cytosine. In some embodiments, the nucleic acid molecule includes a priming site, a sequencing site, a unique molecular identifier, a barcode sequence, a cleavage site or a combination thereof. In some embodiments, the method further includes, before (a), generating a peptide comprising a non-naturally occurring modification. In some embodiments, generating includes alkylating or acetylating the amino acid residues of the peptide. In some embodiments, the generation includes beta-elimination of a phosphate group or addition of a sulfhydryl group. In some embodiments, the generation includes the use of phenyl isothiocyanate or acetic anhydride. In some embodiments, the non-natural modification is at the N-terminal or C-terminal amino acid of the peptide. In some embodiments, (c) is performed using a protease that recognizes the non-natural modification. In some embodiments, the coupling in (b) includes coupling the C-terminal amino acid to a substrate. In some embodiments, the method further includes generating a peptide comprising a non-natural modification.In some embodiments, generation includes subjecting the peptide to conditions sufficient to block all carboxyl groups. In some embodiments, the coupling of the C-terminal amino acid is carried out using a photoredox reaction or an enzymatic reaction. In some embodiments, the enzymatic reaction includes carboxypeptidase or amidase. In some embodiments, the coupling in (b) includes coupling the N-terminal amino acid to a substrate. In some embodiments, the joint is a bifunctional joint. In some embodiments, the joint includes a first reactive group and a second reactive group, wherein the first reactive group can be coupled to the amino acid and wherein the second reactive group can be coupled to the substrate. In some embodiments, the amino acid is a terminal amino acid. In some embodiments, the first reactive group includes thiocyanate, aldehyde, dansyl chloride or fluorobenzene. In some embodiments, thiocyanate includes phenyl isothiocyanate (PITC) moiety, and wherein the coupling in (b) includes coupling the PITC moiety to the amino acid to generate a phenylthiocarbamoyl (PTC) derivative of amino acid. In some embodiments, the method further includes, after (c), derivatizing the PTC derivative to generate a thiazolinone (ATZ) derivative or a phenylthiohydantoin (PTH)-amino acid derivative. In some embodiments, the cleavage in (c) is performed by applying a stimulus. In some embodiments, the stimulus includes a change in pH. In some embodiments, the change in pH includes the use of an acid. In some embodiments, the change in pH includes the use of a base. In some embodiments, the stimulus includes the use of a Lewis acid. In some embodiments, the Lewis acid includes boron trifluoromethanesulfonate (BF3), boron trifluoride ethyl etherate, boron trichloride, boron tribromide, boron triiodide, or scandium trifluoromethanesulfonate. In some embodiments, the stimulus includes electromagnetic radiation. In some embodiments, electromagnetic radiation is applied using microwaves. In some embodiments, the substrate comprises a capture portion comprising a third reactive group capable of reacting with a second reactive group. In some embodiments, the first reactive group, the second reactive group, or the third reactive group comprises a click chemistry portion. In some embodiments, the second reactive group and the third reactive group comprise a click chemistry pair. In some embodiments, the linker comprises an amino acid reactive group and a polymerizable molecule, wherein the polymerizable molecule is configured to be coupled to a substrate. In some embodiments, the polymerizable molecule comprises an enzyme recognition site. In some embodiments, the enzyme recognition site is recognized by a nuclease. In some embodiments, the nuclease is a restriction enzyme, a Cas protein, or an Argonaut nuclease. In some embodiments, the method further comprises contacting the removed substrate-coupled amino acid with a binding agent. In some embodiments, the binding agent comprises an antibody, an antibody fragment, an aptamer, a scFv, a nanobody, anticalin, tRNA-acyl synthetase, a peptide, a peptide mimetic, a polysaccharide, or a derivative thereof. In some embodiments, the binding agent comprises an enzyme.In some embodiments, the enzyme includes a metalloproteinase, an aminopeptidase, or an exopeptidase. In some embodiments, the substrate is a solid support. In some embodiments, the substrate is substantially planar. In some embodiments, the substrate is a bead or particle. In some embodiments, the peptide is coupled to the support via a click chemistry moiety. In some embodiments, the peptide is coupled to the support via a functional group. In some embodiments, the functional group is added to the peptide, capture moiety, or a second polymerizable molecule using an enzyme. In some embodiments, the enzyme includes an amidase. In some embodiments, the peptide is coupled to the support via a linker molecule. In some embodiments, the substrate is functionalized with a functional group, and further includes attaching the peptide to the functional group. In some embodiments, the method further includes passivating the substrate. In some embodiments, the cutting in (c) includes applying a stimulus. In some embodiments, the stimulus includes a change in pH. In some embodiments, the stimulus includes using a Lewis acid. In some embodiments, the Lewis acid includes boron trifluoromethanesulfonate (BF3), boron trifluoride etherate, boron trichloride, boron tribromide, boron triiodide, or scandium trifluoromethanesulfonate. In some embodiments, the stimulation comprises electromagnetic radiation. In some embodiments, the electromagnetic radiation is applied using microwaves. In some embodiments, the stimulation is a biological stimulation. In some embodiments, the biological stimulation comprises the use of an enzyme. In some embodiments, the enzyme is a metalloproteinase, an aminopeptidase, or an exopeptidase. In some embodiments, the method further comprises providing a metal catalyst. In some embodiments, the amino acid comprises a modification, and wherein the enzyme recognizes the modification. In some embodiments, the method further comprises blocking a carboxyl group or an amine group of the peptide. In some embodiments, the method further comprises generating a peptide comprising a non-naturally occurring modification. In some embodiments, generating comprises alkylating or acetylating at least one amino acid, beta-eliminating a phosphate group, or using PITC or acetic anhydride. In some embodiments, generating comprises converting a cysteine ​​residue into cysteic acid. In some embodiments, generating comprises using an oxidizing agent or a reducing agent. In some embodiments, the peptide is derived from a biological sample.

[0020] In another aspect, provided herein is a method for sequencing a peptide, the method comprising: (a) providing a substrate comprising a peptide and a nucleic acid molecule; (b) providing a linker, wherein the linker comprises an amino acid reactive group and a substrate tethering portion; (c) coupling the linker to the terminal amino acid of the peptide and the substrate; (d) cleaving the terminal amino acid, thereby providing a cleaved amino acid-linker complex coupled to the substrate; (e) contacting the cleaved amino acid-linker complex with a binding agent comprising an activator; (f) providing a nucleic acid barcode molecule; and (g) using the activator to couple the nucleic acid barcode molecule to the nucleic acid molecule.

[0021] In some embodiments, the substrate comprises a capture portion, wherein the substrate tethered portion is configured to be coupled to the capture portion. In some embodiments, the capture portion comprises an additional nucleic acid molecule, wherein the joint comprises a connecting nucleic acid molecule, and wherein in (c), the connecting nucleic acid molecule is coupled to the additional nucleic acid molecule. In some embodiments, the capture portion comprises a first click chemistry portion and wherein the joint comprises a second click chemistry portion, and wherein the coupling in (c) comprises reacting the first click chemistry portion with the second click chemistry portion. In some embodiments, the activator comprises an activation nucleic acid molecule, and further comprises coupling the nucleic acid barcode molecule to the activation polymerizable molecule. In some embodiments, the coupling is mediated using a splint molecule, which comprises a sequence complementary to at least a portion of (i) nucleic acid barcode molecules, (ii) nucleic acid molecules, and (iii) activation nucleic acid molecules. In some embodiments, the method further comprises generating nucleic acid barcode molecules via hybridization chain reaction (HCR). In some embodiments, generation includes (A) providing a first hairpin molecule and a second hairpin molecule; (B) coupling the first hairpin molecule to an activator; and (C) coupling the second hairpin molecule to the first hairpin molecule, thereby generating a nucleic acid barcode molecule. In some embodiments, the activator includes an activation nucleic acid molecule. In some embodiments, after (C), the nucleic acid barcode molecule comprises a flap sequence, and wherein (g) uses a flap sequence to mediate to couple the nucleic acid barcode molecule to the nucleic acid molecule. In some embodiments, the nucleic acid barcode molecule comprises the first hairpin molecule or the second hairpin molecule. In some embodiments, the activator includes an anchoring nucleic acid molecule, wherein the anchoring nucleic acid molecule is configured to couple to an additional nucleic acid molecule of an adjacent nucleic acid molecule. In some embodiments, the nucleic acid barcode molecule is coupled to a binding agent. In some embodiments, the activator includes an enzyme selected from the following items: peroxidase, Cas protein, ligase, kinase, and restriction enzyme. In some embodiments, the enzyme is a kinase, and further includes using a kinase to phosphorylate the nucleic acid molecule, thereby allowing the nucleic acid barcode molecule to couple to the nucleic acid molecule. In some embodiments, the enzyme is horseradish peroxidase (HRP) or ascorbate peroxidase (APEX), wherein HRP or APEX activates the nucleic acid barcode molecule or the nucleic acid molecule, thereby coupling the nucleic acid barcode molecule to the nucleic acid molecule. In some embodiments, the enzyme is a restriction enzyme, and wherein the nucleic acid molecule comprises a restriction recognition site, and further comprising cleaving the nucleic acid molecule with the restriction enzyme prior to coupling the nucleic acid barcode molecule to the nucleic acid molecule. In some embodiments, at least one amino acid of the peptide comprises a post-translational modification. In some embodiments, the coupling in (c) is mediated by a linker.

[0022] In another aspect, provided herein is a method for characterizing multiple analytes, the method comprising: (a) providing a first analyte, a second analyte, a first binding agent capable of coupling to the first analyte and not coupling to the second analyte, and a second binding agent capable of coupling to both the first analyte and the second analyte; (b) contacting the first analyte and the second analyte with the first binding agent; (c) after (b), contacting the first analyte and the second analyte with the second binding agent; (d) identifying the first binding agent and the second binding agent; and (e) using the first binding agent and the second binding agent identified in (d) to identify the first analyte and the second analyte.

[0023] Another aspect of the present disclosure provides a non-transitory computer-readable medium comprising machine-executable code that, when executed by one or more computer processors, implements any of the methods described above or elsewhere herein.

[0024] Another aspect of the present disclosure provides a system comprising one or more computer processors and a computer memory coupled thereto, wherein the computer memory comprises machine executable code that, when executed by the one or more computer processors, implements any method described above or elsewhere herein.

[0025] Additional aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0026] Incorporation by reference

[0027] 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 that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The novel features of the present 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 and accompanying drawings (also referred to herein as "Figures" and "FIG.") which set forth illustrative embodiments in which the principles of the invention are utilized, wherein:

[0029] Figure 1A An example workflow for processing and analyzing polymer analytes in a highly accurate manner is schematically illustrated. Figure 1B Another example workflow for analyzing polymeric analytes using activated polymerizable molecules is schematically illustrated. Figure 1C Another example workflow for analyzing polymeric analytes using activated polymerizable molecules is schematically illustrated. Figure 1D Another example workflow for analyzing multiple monomers of a polymeric analyte is schematically shown. Figure 1E Example activators coupled to polymerizable molecules for analysis of polymeric analytes are schematically shown. Figure 1F Another example of an activator coupled to a polymerizable molecule for analyzing a polymeric analyte is schematically shown.

[0030] Figure 2 Shown are examples of linkers that can be used to couple to polymerizable molecules and monomers of polymer analytes, which can be used in a workflow for sequencing polymer analytes, as described herein.

[0031] Figure 3 An example workflow using a library of binders with different monomer specificities is shown.

[0032] Figure 4 Examples of monomers that modify polymeric analytes are schematically shown.

[0033] Figure 5 An example of modification of a cleaved monomer to prevent binding agent conjugation is schematically shown.

[0034] Figure 6 An example workflow for modifying a cleaved monomer to prevent binding agent conjugation is schematically shown.

[0035] Figure 7 Another example for modifying a cleaved monomer to prevent binding agent conjugation is schematically shown.

[0036] Figure 8 Example methods for controlling the distance between polymerizable molecules as described herein are schematically illustrated.

[0037] Figure 9 Methods for coupling amino acid residues to polymerizable molecules are schematically shown.

[0038] Figure 10 The method for barcoding peptides using the bridge amplification method is schematically shown.

[0039] Figure 11An example workflow for processing a sample containing cells by partitioning the cells into individual partitions, releasing proteins contained in the cells, and barcoding the proteins is schematically shown.

[0040] Figure 12 An example workflow for partitioning cells and barcoding their proteins or peptides is schematically shown.

[0041] Figure 13 An example workflow for spatially barcoding proteins in tissue samples is schematically shown.

[0042] Figure 14 Another example workflow for spatially barcoding proteins in tissue samples is schematically shown.

[0043] Figure 15 Another example workflow for spatially barcoding proteins in tissue samples is schematically shown.

[0044] Figure 16A An example model system for demonstrating coupling of monomers to capture moieties using nucleic acid molecules is schematically shown. Figure 16B Example data for the coupling of a first nucleic acid molecule to a second nucleic acid molecule is shown, representing the coupling of a monomer to a capture moiety across different substrate types. Figure 16C Schematic representation of a substrate comprising a linker for coupling nucleic acid molecules and example data for coupling a first nucleic acid molecule to a second nucleic acid molecule, representing coupling of a monomer to a capture moiety across the substrate. Figure 16D Example data is shown for a cleavage event of a first nucleic acid molecule from a second nucleic acid molecule, representing cleavage of a monomer from a capture moiety.

[0045] Figure 17A An example experimental workflow for studying the effect of Edman degradation treatment on the cleavage event of a first nucleic acid molecule from a second nucleic acid molecule is shown. Figure 17B Shown Figure 17A Example data for the experimental workflow in .

[0046] Figure 18A An exemplary method for coupling a linker to an amino acid of a peptide to generate a linker-peptide complex is shown. Figure 18B Shown are exemplary alternative Edman degradation methods for cleaving terminal amino acids from peptides and the efficiency of such methods. Figure 18C Another exemplary alternative Edman degradation method for cleaving terminal amino acids from peptides and the efficiency of such a method are shown. Figure 18D Shown are exemplary methods for cleaving terminal amino acids from peptides, as well as different quantifications of the efficiency of such methods. Figure 18EThe cleavage efficiency of the peptides using the Edman degradation method is shown. Figure 18F Another exemplary method for cleaving terminal amino acids from a peptide is shown. Figure 18G Shown are the cleavage efficiencies of peptides cleaved using the Edman degradation method and two non-standard Edman degradation methods.

[0047] Figure 19 Exemplary data on the stability of nucleic acid molecules in an alternative Edman degradation method are shown.

[0048] Figure 20 An example of a sequencing method for analyzing or characterizing a polymer analyte is schematically shown.

[0049] Figure 21 A computer system is shown that is programmed or otherwise configured to implement the methods provided herein.

[0050] Figure 22A A schematic diagram of a substrate-polymer analyte-linker complex (e.g., bead-peptide-linked nucleic acid molecule) is shown, which is used as model input for validation studies of local tethering, cleavage, and detection of monomers (e.g., N-terminal amino acid) via coupling of the monomer to a capture portion. Figure 22B Example data of local tethering efficiency are shown. Figure 22C Example data for cutting efficiency is shown. Figure 22D Shown is a schematic diagram of the detection of monomers using binding agents across two rounds of local tethering, cleavage, and detection. Figure 22E Example data for the first round of detection is shown. Figure 22F Example data for the second round of detection is shown.

[0051] Figure 23 Example data showing the stacking efficiency of a second nucleic acid barcode molecule on a first nucleic acid barcode molecule.

[0052] Figure 24 Schematically depicted is a linker comprising a linking nucleic acid molecule comprising a self-splitting region.

[0053] Figure 25A Example sequences for generating polymerizable molecules using the hybridization chain reaction are shown. Figure 25B Another example of using a hybridization chain reaction to generate a polymerizable molecule containing a flap sequence is shown. Figure 25C Example data for a hybridization chain reaction are shown. Figure 25D Another example of using a hybridization chain reaction to generate a polymerizable molecule containing a flap sequence is shown. Figure 25E Example data for a hybridization chain reaction are shown. Figure 25FAn example of coupling of a polymerizable molecule generated from a hybridization chain reaction to a substrate-binding capture moiety is shown. Figure 25G Example data are shown for coupling of a polymerizable molecule to a substrate-binding capture moiety.

[0054] Figure 26A The workflow for detecting monomeric analytes using a binding agent comprising an enzyme activator is schematically shown. Figure 26B Example monomeric analytes are shown schematically. Figure 26C Example data showing the coupling efficiency of enzyme substrates using enzyme activators. Figure 26D Example data for absorbance are shown as a measure of coupling efficiency. Figure 26E Example data for fluorescence as a measure of coupling efficiency are shown. Figure 26F Additional example data are shown for fluorescence as a measure of coupling efficiency. DETAILED DESCRIPTION

[0055] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions may occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0056] definition

[0057] When the term "at least," "greater than," or "greater than or equal to" precedes the first value in a series of two or more values, the term "at least," "greater than," or "greater than or equal to" applies to every value in the series. 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.

[0058] When the term "not more than," "less than," or "less than or equal to" precedes the first value in a series of two or more values, the term "not more than," "less than," or "less than or equal to" applies to every value in the series. 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.

[0059] References to "one embodiment," "an embodiment," "example embodiment," "some embodiments," "certain embodiments," "various embodiments," etc., indicate that embodiments of the disclosed technology so described may include a particular feature, structure, or characteristic, but not every embodiment is required to include that particular feature, structure, or characteristic. Furthermore, repeated use of the phrase "in one embodiment" does not necessarily refer to the same embodiment, although it may.

[0060] In this article, ranges can be expressed as from "about" or "approximately" or "substantially" a specific value and / or to "about" or "approximately" or "substantially" another specific value. When such a range is expressed, other exemplary embodiments include from one specific value and / or to another specific value. Further, the term "about" means within an acceptable error range of a specific value as determined by those skilled 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, according to the practice of this art, "about" can mean within an acceptable standard deviation. Alternatively, "about" can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and still more preferably 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 times of a value. When describing a specific value in the application and claims, unless otherwise indicated, the term "about" is implicit and means within an acceptable error range of the specific value in this context.

[0061] “Comprising” or “containing” or “including” means that at least the specified compound, element, particle or method step is present in the composition, article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if other such compounds, materials, particles, method steps have the same function as that specified.

[0062] Throughout this specification, various components may be identified with specific values ​​or parameters, however, these items are provided as exemplary embodiments. In fact, the exemplary embodiments do not limit the various aspects and concepts of the present disclosure, as many comparable parameters, sizes, ranges and / or values ​​can be implemented. The terms "first," "second," etc., "primary," "secondary," etc. do not indicate any order, quantity, or importance, but are used to distinguish one element from another.

[0063] As used herein, the term "protein" generally refers to a molecule comprising two or more amino acids connected by a peptide bond. Proteins may also be referred to as "polypeptides," "oligopeptides," or "peptides." Proteins may be naturally occurring molecules or synthetic molecules. Proteins may comprise one or more non-natural amino acids, modified amino acids, or non-amino acid linkers. Proteins may contain D-amino acid enantiomers, L-amino acid enantiomers, or both. The amino acids of a protein may be modified naturally or synthetically, such as by post-translational modification or by chemical modification. In some cases, different proteins may be distinguished from each other based on the different genes, different primary sequence lengths, or different primary sequence compositions expressed in an organism. However, proteins expressed from the same gene may be different protein variants, such as those distinguished based on non-identical lengths, non-identical amino acid sequences, or non-identical post-translational modifications. Different proteins may be distinguished based on one or both of the gene of origin and the protein variant state.

[0064] As used herein, the term "peptide" can refer to any short single peptide chain. The length of the peptide can 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. The peptide can have a known or unknown biological function or activity. The peptide can include natural, synthetic, modified or degraded proteins or peptides, or a combination thereof.

[0065] As used herein, "polypeptide" refers to two or more amino acids linked together by peptide bonds. The term "polypeptide" includes proteins having a C-terminus and an N-terminus as generally known in the art, and can be synthetic or naturally occurring. As used herein, "at least a portion of a polypeptide" refers to two or more amino acids in a polypeptide. A polypeptide may comprise one or more peptides. Optionally, a portion of a polypeptide includes at least: 1, 5, 10, 20, 30, or 50 amino acids, continuous or with gaps, of the complete amino acid sequence of the polypeptide, or the full amino acid sequence of the polypeptide.

[0066] As used herein, the term "sample" refers to a collected substance or material that contains or is suspected to contain one or more analytes of interest (e.g., biomolecules, such as polypeptides). The sample can be modified for purposes such as storage or stability. The sample can be naturally occurring or synthetic. The sample can be processed to separate or remove unwanted fractions or impurities from the analyte of interest. The sample can be enriched or purified. For example, the sample can include fractions of a separation process (e.g., chromatography, fractionation, electrophoresis, etc.). Alternatively, the sample can not be processed to separate or remove any unwanted fractions or impurities from the analyte of interest. The sample can be obtained from any suitable source or position, including from an organism, a cell, a tissue, a cell preparation, a cell-free composition, an environment (e.g., air, water, dirt, soil, agriculture, soil, dust, sewage). The sample can be obtained from an organism or a part of an organism, such as from a fluid, a tissue, or a cell. The sample can include biological and / or non-biological components. As used herein, the term "biological sample" or "biological source" refers to a sample derived from a primarily biological system or organism, such as one or more viral particles, cells (e.g., personalized cells), organelles (e.g., personalized organelles), tissues, body fluids, bones, cartilage, and exoskeletons. A biological sample can comprise most biological materials based on mass, excluding the weight of the fluid within the sample. A biological sample can comprise one or more proteins, referred to herein as a protein sample. A biological sample can be obtained from various sources, for example, from clinical patient samples, such as blood, serum, plasma, cerebrospinal fluid (CSF), saliva, mucosal secretions, sputum, urine, lymph, sweat, vaginal fluid, semen, feces, amniotic fluid, sweat, synovial fluid, etc. A biological sample can be processed to purify and retain one or more biomolecules (e.g., proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, metabolites, etc.) from a biological sample. A biological sample (e.g., a protein sample) can be derived from cultured cells, which can be treated or untreated. Biological samples (e.g., protein samples) can also be generated from tissue samples, such as biopsy samples, which can optionally be treated to release the biomolecules (e.g., proteins) they contain. Tissue samples can also be derived from in vivo samples, including fresh, frozen, emergency, and fixed tissues.

[0067] As used herein, the terms "antibody" and "immunoglobulin" may generally refer to proteins that can recognize and bind to specific antigens. Antibodies or immunoglobulins may refer to fragments of antibody isotypes, antibodies, including but not limited to Fab, Fv, scFv and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and non-antibody proteins. Antibodies may be detectably labeled, for example, using fluorophores, radioisotopes, enzymes (such as peroxidases) or ribozymes or deoxyribozymes, fluorescent proteins, nucleic acid barcode sequences, etc. that generate detectable products. Antibodies may also be conjugated to other moieties, such as members of specific binding pairs, for example, biotin (members of biotin-avidin specific binding pairs), etc. The term also includes nanobodies, Fab', Fv, F(ab')2, scFv, and other antibody fragments that retain specific binding to an antigen. Antibodies can exist in a variety of other forms, including, for example, Fv, Fab, and (Fab)2, as well as bifunctional (e.g., bispecific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single-chain (e.g., Huston et al., Proc. Natl. Acad. Sci. USA, 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, NY, 2nd ed. (1984) and Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporated herein by reference). Naturally occurring immunoglobulin or antibody classes include immunoglobulin A, immunoglobulin G, immunoglobulin D, immunoglobulin E, immunoglobulin M, or other immunoreactive components.

[0068] As used herein, "binding" or "coupling" 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). The binding between the binding partners can be specific or non-specific. The binding or coupling of a molecule to another molecule can occur through hydrogen bonding, electrostatic interactions, ionic bonding, van der Waals forces, metallic bonding, molecular bonding, or any useful interaction.

[0069] 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 partner binds to another partner but does not bind to other molecules that may be present in the environment (e.g., in a biological sample, in a tissue, in an in vitro assay) under a set of conditions. Specific binding interactions can involve a binding partner that binds to a cognate molecule. Specific binding interactions can involve a binding partner that binds to its cognate molecule at a significantly or substantially higher level or with greater affinity than the binding of the binding partner to a non-cognate molecule. Specific binding interactions can involve a first binding partner that has greater selectivity for binding to a cognate molecule than to a non-cognate molecule.

[0070] The terms "nucleic acid," "nucleic acid molecule," "oligonucleotide," and "polynucleotide" are used interchangeably herein and generally refer to polymeric forms of naturally occurring or synthetic nucleotides or their derivatives of any length. A nucleic acid molecule may comprise one or more deoxynucleotides, deoxynucleotide triphosphates, dideoxynucleotide triphosphates, ribonucleotides, hexitol nucleotides, cyclohexane nucleotides, or analogs or combinations thereof. Nucleic acid molecules may include, for example, DNA, RNA, HNA, CeNA, and modified forms thereof. Nucleic acid molecules may comprise nucleotides linked by phosphodiester bonds. Nucleic acid molecules may have any two-dimensional or three-dimensional structure and may perform any known or unknown function. Nucleic acid molecules may be single-stranded, double-stranded, or partially double-stranded. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, non-coding DNA, small interfering RNA, short hairpin RNA, microRNA, 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. Nucleic acid molecules can be linear, circular, or any other geometric shape. Examples of polynucleotide analogs include, but are not limited to, xenonucleic acid (XNA), bridged nucleic acid (BNA), glycol nucleic acid (GNA), hexitol nucleic acid (HNA), cyclohexane nucleic acid (CeNA), peptide nucleic acid (PNA), γPNA, morpholino polynucleotides, locked nucleic acid (LNA), threose nucleic acid (TNA), 2′-O-methyl polynucleotides, 2′-O-alkylribosyl substituted polynucleotides, phosphorothioate polynucleotides, and boronate polynucleotides. Polynucleotide analogs can have purine or pyrimidine analogs, including, for example, 7-deazapurine analogs, 8-halogenated purine analogs, 5-halogenated pyrimidine analogs, or universal base analogs that can pair with any base, including hypoxanthine, nitroazoles, isoquinolone analogs, azole carboxamides, and aromatic triazole analogs, or base analogs with additional functionality, such as a biotin moiety for affinity binding. Nucleic acid molecules according to the present invention can include any polymer or oligomer of nucleotide, such as pyrimidine and purine bases, such as cytosine, thymine and uracil and adenine and guanine, and combinations thereof.Nucleic acid molecules can comprise any deoxyribonucleotide, ribonucleotide, hexitol nucleotide, cyclohexane nucleotide, peptide nucleic acid component and any chemical variant thereof, such as methylation, 7-deazapurine analogs, 8-halogenated purine analogs, the hydroxymethylation of these bases or glycosylation form etc.These polymers or oligomers can be heterogeneous or homogeneous in composition, and can be separated from naturally occurring sources or can be produced artificially or synthetically.Nucleic acid molecules may include DNA, RNA, HNA, CeNA, or mixtures thereof, and may exist permanently or transiently in single-stranded or double-stranded form (including homoduplex, heteroduplex, and hybrid states).

[0071] As used herein, the term "amino acid" generally refers to an organic compound that is combined to form a protein or peptide. Amino acid generally comprises an amine group, a carboxylic acid group, and a side chain specific to each amino acid, which serves as a monomeric subunit of a peptide. Amino acid can include 20 standards, naturally occurring or typical amino acids and non-standard amino acids. Standard, naturally occurring or typical 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 Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr). The amino acid can be an L-amino acid or a D-amino acid. Non-standard amino acids can be naturally occurring or chemically synthesized modified amino acids, amino acid analogs, amino acid mimetics, non-standard protein amino acids, or non-protein amino acids. Examples of non-standard amino acids include, but are not limited to, selenocysteine, pyrrolysine, and N-formylmethionine, 3-amino acids, homoamino 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.

[0072] As used herein, the term "post-translational modification" refers to a modification that occurs on a peptide after translation. A post-translational modification can be a covalent modification or an enzymatic modification. Examples of post-translational modifications include, but are not limited to, acylation, acetylation, alkylation (including methylation), biotinylation, butyrylation, carbamylation, carbonylation, deamidation, deimination, diphtheria amide formation, disulfide bridge formation, elimination, flavin attachment, formylation, γ-carboxylation, glutamylation, glycination, glycosylation, glycosylphosphatidylinositolization, heme C attachment, hydroxylation, hydroxybutyrylation, iodination, isoprene The post-translational modifications include modifications of the amino and / or carboxyl termini of the peptide. Modifications of the terminal amino group include, but are not limited to, deamination, N-low alkyl, N-di-low alkyl, and N-acyl modifications. Modifications of the terminal carboxyl group include, but are not limited to, amides, lower alkyl amides, dialkyl amides, and lower alkyl ester modifications (e.g., wherein the lower alkyl group is a C1-C4 alkyl). Post-translational modifications also include modifications to the amino acids falling between the amino and carboxyl termini, such as, but not limited to, those described above. The term post-translational modifications can also include modifications of peptides that include one or more detectable labels. Post-translational modifications can be naturally occurring or synthetic.

[0073] As used herein, the term "binding agent" refers to a molecule that binds, associates, unites, recognizes or combines another molecule, such as a nucleic acid molecule, peptide, polypeptide, protein, carbohydrate, synthetic molecule or small molecule. A binding agent can be bound to a macromolecule or a component or feature of a macromolecule. A binding agent can form a covalent association or non-covalent association with a molecule, macromolecule or a component or feature of a macromolecule. A binding agent can 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 can be a naturally occurring, synthetically produced or recombinantly expressed molecule. A binding agent can be bound to a polymer analyte such as a single monomer or subunit of a macromolecule (e.g., a single amino acid of a peptide) or to multiple connected subunits of a macromolecule (e.g., a dipeptide, tripeptide or higher order peptide of a longer peptide, polypeptide or protein molecule). A binding agent can be bound to a linear molecule or a molecule with a three-dimensional structure (also referred to as a conformation). For example, an antibody binding agent can bind to a linear peptide, polypeptide or protein, or to a conformational peptide, polypeptide or protein. The binding agent can bind to the N-terminal peptide, C-terminal peptide or intermediate peptide of a peptide, polypeptide or protein molecule. The binding agent can bind to the N-terminal amino acid, C-terminal amino acid or intermediate amino acid of a peptide molecule. The binding agent can preferably bind to chemically modified or labeled amino acids, compared to unmodified or unlabeled amino acids. For example, the binding agent can preferably bind to amino acids modified with acetyl moieties, amidino moieties, dansyl moieties, isothiocyanate moieties, 2,4-dinitrophenol (DNP) moieties, SNP moieties, etc., compared to amino acids without such moieties. The binding agent can bind to naturally occurring or synthetic post-translational modifications of peptide molecules. The binding agent can exhibit selective binding to components or features of macromolecules (for example, the binding agent can selectively bind to one of 20 possible natural amino acid residues and bind to the other 19 natural amino acid residues with very low affinity or not at all). A binding agent may exhibit less selective binding, where the binding agent is able to bind to multiple 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.

[0074] As used herein, the term "joint" generally refers to a molecule or part that is involved in joining two or more molecules. A joint can promote the covalent or non-covalent interaction of two or more molecules. A joint can be a cross-linking agent. A joint can be monofunctional, bifunctional, trifunctional, tetrafunctional or multifunctional. A joint can be or comprise a nucleotide, a nucleotide analog, an amino acid, a peptide, a polypeptide or a non-nucleotide chemical moiety, such as an organic or inorganic compound. A joint can comprise a polymer, such as polyethylene glycol (PEG), poly-L-lysine (PLL), poly (DL-lactic acid) (PLA), poly (DL-lactic-co-glycolic acid) copolymer (PLGA), polyornithine, polyarginine, etc. A joint can comprise one or more reactive ends, such as an amine reactive group, a carboxyl reactive group, a sulfhydryl reactive group, a hydroxyl reactive group, etc. In some examples, a joint can be used to join different molecular types, for example, different biomolecule types (such as peptides and nucleic acid molecules, lipids and peptides, carbohydrates and peptides, etc.); non-biological molecule types; or biomolecules to non-biological molecules. For example, a linker can be used to join a binding agent to a tag, a tag to a macromolecule (e.g., a peptide, a nucleic acid molecule), a macromolecule to a solid support, a tag to a solid support, etc. The linker can join two molecules via an enzymatic reaction or a chemical reaction. The linker can comprise one or more click chemistry moieties. The linker can join more than two molecules, for example, via an enzymatic or chemical reaction.

[0075] As used herein, the term "conjugate" generally refers to a covalent or ionic interaction between two entities (eg, molecules, compounds, or a combination thereof).

[0076] As used herein, the term "label" generally refers to a molecule or part that is conjugated to a molecule. Labels can include detectable markers, for example, fluorophores or fluorescent proteins, radioisotopes, enzymes (for example, color production or fluorescent proteins, proteins that can catalyze color production substrates) or ribozymes or deoxyribozymes, mass labels, metals, haptens (for example, biotin, digoxin, urushiol, fluorescein), vibration or FTIR labels (for example, alkynyl). Labels can include biomolecules, such as nucleic acid molecules, proteins, lipids, carbohydrates or combinations thereof. Labels can include one or more nucleic acid molecules that can optionally encode information about a label or a label conjugated to a molecule thereon (for example, a binding agent, such as an antibody). For example, labels can include nucleic acid barcode molecules. Labels can include organic compounds or inorganic compounds.

[0077] As used herein, the term "barcode" generally refers to an identifying feature that can be used to distinguish between similar items. 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, 101, 102, 103, 104, 105, 106, 107, 108, 109 Barcodes can be artificial or naturally occurring sequences, including peptides, polypeptides, peptides, binding agents, binding agent collections from binding cycles, sample molecules, sample collections, molecules within compartments (e.g., droplets, beads, partitions, or separate locations), macromolecules within a compartment collection, fractions of macromolecules, collections of macromolecule fractions, spatial regions or collections of spatial regions, libraries of macromolecules, or libraries of binding agents. In certain embodiments, each barcode within a barcode population is different. In other embodiments, a portion of the barcodes in a barcode population are different, for example, 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 the barcode population are different. The barcode population can be generated randomly or non-randomly. The barcode population can include error correction barcodes. The barcodes can be used to computationally deconvolute sequence reads derived from a single molecule, sample, library, etc. The barcodes can contain multiplexed information, for example, generated by different samples, compartments, individual molecules, etc. The barcodes can also be used to deconvolve a collection of molecules that have been distributed into a small compartment for enhanced mapping. For example, instead of mapping peptides back to the proteome, peptides can be mapped back to the protein molecule or protein complex from which they originated.Barcodes can contain any useful structural part or motif, such as a hairpin, loop sequence, or spacer.The barcode can comprise an artificial or modified nucleic acid, e.g., locked nucleic acid (LNA), protein nucleic acid (PNA), hexitol nucleic acid (HNA), cyclohexane nucleic acid (CeNA), or a combination thereof. The barcode can comprise a protein (e.g., a Tal effector, a Cas protein (e.g., Cas9), Argonaut, or coiled-coil) or be generated using a protein.

[0078] As used herein, a "sample barcode," also referred to as a "sample tag," generally refers to a barcode molecule that contains identification information of a sample from which the barcoded molecule is derived.

[0079] As used herein, "spatial barcode" generally refers to a barcode molecule that contains identification information for a 2D or 3D sample (e.g., a tissue section) region from which the molecule originates or is derived. Spatial barcodes can be used for molecular pathology on tissue sections. Spatial barcodes can allow multiplexed sequencing of multiple samples or libraries from tissue sections.

[0080] As used herein, "temporal barcodes" generally refer to barcode molecules that contain time-based information associated with the barcoded molecule. Time-based data types encoded in a temporal barcode can include information such as the lifetime of the barcoded molecule, sample collection time, time or duration since the start of the experiment or induction with a stimulus, information about the age of the cell or tissue, the sequence of interactions between molecules, the number of cycles in an iterative process, etc. It is possible to combine different types of barcodes (e.g., spatial, temporal, cell-specific) in a single multiplexed barcode.

[0081] As used herein, the term "nucleic acid sequence" or "oligonucleotide sequence" generally refers to a continuous string of nucleotide bases, and may refer to the specific placement of the nucleotide bases relative to each other when they occur in an oligonucleotide. Similarly, the term "polypeptide sequence" or "amino acid sequence" refers to a continuous string of amino acids, and may refer to the specific placement of the amino acids relative to each other when they occur in a polypeptide.

[0082] The terms "complementary" or "complementarity" refer to polynucleotides (i.e., sequences of nucleotides) that are related by the base pairing rules. For example, the sequence "5'-AGT-3'" is complementary to the sequence "5'-ACT-3'." Complementarity can be "partial," where only some of the bases of the nucleic acids match according to the base pairing rules, or there can be "complete" or "perfect" complementarity between nucleic acids. The degree of complementarity between nucleic acid strands can have a significant effect on the efficiency and strength of hybridization between nucleic acid strands under defined conditions.

[0083] As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids. Hybridization and hybridization strength (e.g., the strength of association between nucleic acids) are affected by factors such as the degree of complementarity between the nucleic acids, the GC content, the stringency of the conditions involved, and the melting temperature of the hybrid formed. Hybridization methods involve annealing one nucleic acid to another complementary nucleic acid, for example, based on Watson-Crick base pairing.

[0084] As used herein, the term "proteomics" generally refers to the quantitative and / or qualitative analysis of a proteome within a sample (such as a biological sample, e.g., from a cell, tissue, or body fluid). Proteomics can include analysis of the spatial distribution of proteins within a sample (e.g., a cell and / or tissue). Proteomics can include studying the dynamic state of a proteome, e.g., how one or more proteins change over time. A proteome can include multiple "-groups," such as a kinase group, a secretome, a receptor group (e.g., a GPCome), an immune proteome, a nutritional proteome, a subset of proteomes defined by post-translational modifications (e.g., phosphorylation, ubiquitination, methylation, acetylation, glycosylation, oxidation, lipidation, and / or nitrosylation), such as a phosphorylated proteome (e.g., a phosphotyrosine proteome, a tyrosine kinase group, and a tyrosine phosphate group), a glycoprotein group, etc., a subset of proteomes associated with a tissue or organ, a developmental stage, or a physiological or pathological condition, a subset of proteomes associated with a cellular process such as a cell cycle, differentiation (or dedifferentiation), cell death, aging, cell migration, transformation, or metastasis, or any combination thereof.

[0085] The terminal amino acid with a free amino group at one end of the peptide chain may be referred to as "N-terminal amino acid" (NTAA) in this article. The terminal amino acid with a free carboxyl group at the other end of the chain may be referred to as "C-terminal amino acid" (CTAA) in this article. The amino acids that make up the peptide can be numbered in sequence, where the length of the peptide is "n" amino acids. As used herein, in some cases, NTAA may be considered to be the nth amino acid (also referred to as "nNTAA" in this article). In such cases, the next amino acid is the n-1th amino acid, followed by the n-2th amino acid, so reducing the length of the peptide from the N-terminal to the C-terminal. Alternatively, CTAA may be considered to be the nth amino acid (also referred to as "n CTAA" in this article). In such cases, the next amino acid is the n-1th amino acid, followed by the n-2th amino acid, so reducing the length of the peptide from the C-terminal to the N-terminal. NTAA, CTAA or both may be modified or labeled with chemical moieties.

[0086] As used herein, the terms "determine," "measure," "evaluate," and "assay" are used interchangeably and include both quantitative and qualitative determinations.

[0087] As used herein, the term "unique molecular identifier" or "UMI" generally refers to a molecular barcode that contains indexing information. A UMI can comprise a length 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 , 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). A UMI can provide a unique identifier tag for each molecule (e.g., a peptide, a binding agent, a nucleic acid molecule) that comprises or is conjugated to a UMI. A UMI can include a random sequence (e.g., a random N-mer). UMI can be used to count the number of molecules in a given cell, partition, sample, etc., for example, where the given cell, partition, etc. contains a common barcode sequence. For example, a partition can contain multiple barcode molecules that include a common barcode sequence, where each barcode molecule has a unique molecule identifier sequence. The common barcode sequence can be used to attribute all barcoded molecules to the same given cell, partition, sample, etc., while the unique molecule identifier can be used to analyze the number of each original molecule (barcoded) present in the cell, partition, etc.

[0088] As used herein, " derivative " of nucleic acid molecules refers to the nucleic acid molecules derived from origin nucleic acid molecules in general.Derivative can have the nucleotide sequence identical or substantially identical with origin nucleic acid molecules, or derivative can comprise complement, partial complement, reverse complement or part reverse complement as origin nucleic acid molecules.Derivative can be the nucleic acid of the same type as origin nucleic acid molecules (for example, DNA or RNA), or derivative can be different types of nucleic acid (for example, cDNA generated from RNA molecules).Nucleic acid molecule derivative can show sequence identity or the complementarity as origin nucleic acid molecules.Derivative nucleic acid molecules can also be carried out additional processing from origin nucleic acid molecules, for example, chemical or enzymatic modification, montage, connection, polymerization, amplification, fragmentation, label cutting (tagmentation) (for example, using transposase), digestion etc.

[0089] Derivative polypeptide or peptide can be derived from origin polypeptide (or peptide).Derivative can comprise the amino acid sequence identical with origin polypeptide, or sequence can be different.Derivative polypeptide can be produced by origin polypeptide or can carry out additional processing to derivative polypeptide from origin polypeptide, for example chemical or enzymatic modification.Derivative polypeptide can comprise one or more labels, nucleic acid molecules, bar code molecules, mark (for example, detectable label), fluorophore, probe, joint, post-translational modification, chemical protecting group or other chemical moiety.

[0090] 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 can separate individual cells from other cells, or separate a subset of the proteome of a sample from the rest of the proteome of the sample. A compartment can be an aqueous compartment (e.g., a microfluidic droplet), a solid compartment (e.g., a picotiter well or microtiter well on a plate, tube, vial, gel bead), or an isolated area on a surface. A compartment can contain one or more beads to which a macromolecule can be affixed.

[0091] In some embodiments, the partitions as described herein are droplets. The terms "drop," "droplet," and "microdroplet" are used interchangeably herein to refer to small, generally spherical structures containing at least a first fluid phase (e.g., an aqueous phase (e.g., water)) bounded by a second fluid phase (e.g., oil) that is immiscible with the first fluid phase. In some embodiments, droplets according to the present disclosure may contain a first fluid phase (e.g., oil) bounded by a second immiscible fluid phase (e.g., an aqueous phase fluid (e.g., water)). In some embodiments, the second fluid phase will be an immiscible phase carrier fluid. Thus, droplets according to the present disclosure can be provided as water-in-oil emulsions or oil-in-water emulsions. As described herein for discrete entities, droplets can be sized and / or shaped. For example, the diameter range of droplets according to the present disclosure is generally 1 μm to 1000 μm, inclusive. Droplets according to the present disclosure can be used to encapsulate cells, nucleic acids (e.g., DNA), enzymes, ribozymes, deoxyribozymes, reagents, and various other components. The term droplet may be used to refer to droplets generated in, on, by, and / or flowing from, or applied by, a microfluidic device.

[0092] As used herein, the term "carrier fluid" refers to a fluid configured or selected to contain one or more discrete entities (e.g., droplets), as described herein. The carrier fluid can include one or more substances and can have one or more properties, such as viscosity, that allow it to flow through a microfluidic device or a portion thereof, such as a delivery orifice. In some embodiments, the carrier fluid includes, for example, oil or water, and can be in a liquid or gas phase.

[0093] As used herein, the terms "solid support," "solid surface," or "solid substrate" or "substrate" refer to any solid material to which molecules can associate directly or indirectly, including porous and non-porous materials. Molecules can associate with a substrate through covalent or non-covalent interactions, or a combination thereof. The substrate can be two-dimensional (e.g., a planar surface) or three-dimensional (e.g., a gel matrix or beads). In non-limiting examples, the solid support can include beads, microbeads, arrays, glass surfaces, silicon surfaces, plastic surfaces, filters, membranes, nylon or other polymers, silicon wafer chips, flow-through chips, flow cells, biochips including signal transduction electronic devices, channels, microtiter wells, ELISA plates, rotating interferometry disks, nitrocellulose membranes, nitrocellulose-based polymer surfaces, polymer matrices, nanoparticles, or microspheres. The material for solid support includes but is not limited to acrylamide, agarose, cellulose, nitrocellulose, glass, silicon dioxide, gold, quartz, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicate, polycarbonate, Teflon, fluorocarbon, nylon, silicone rubber, polyanhydride, polyglycolic acid, polylactic acid, polyorthoester, functionalized silane, polypropyl fumarate, collagen, glycosaminoglycan, polyamino acid, dextran or its any combination.Solid support further includes film, membrane, bottle, dish, fiber, braided fiber, shaped polymer such as pipe, particle, bead, microsphere, particulate or its any combination.For example, when solid surface is bead, bead can include but is not limited to ceramic beads, polystyrene beads, polymer beads, methyl styrene beads, agarose beads, acrylamide beads, solid beads, porous beads, magnetic or paramagnetic beads, glass beads or controlled pore beads.Bead can be spherical or irregularly shaped. The beads can range in size from nanometers (e.g., 100 nm) to millimeters (e.g., 1 mm). In certain embodiments, the beads range in size from about 0.2 microns to about 200 microns or from about 0.5 microns to about 5 microns.In some embodiments, the diameter of the 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 94, 95, 96, 97, 98, 99, or 100 μm. In certain embodiments, a "bead" solid support can refer to a single bead or a plurality of beads.

[0094] As used herein, " sequencing " generally refers to determining: the nucleotide (base sequence) order in (A) nucleic acid sample (for example, DNA or RNA); Or determining the amino acid sequence in all or part of (B) polymer (such as protein, peptide or other polymer molecules). Many technologies are available, such as Sanger sequencing or high-throughput sequencing technology (HTS). Sanger sequencing can involve sequencing via detection by (capillary) electrophoresis, in which a run can carry out sequence analysis to up to 384 capillaries. High-throughput sequencing relates to the parallel sequencing of thousands or millions or more sequences. HTS can be defined as next generation sequencing (NGS), that is, based on solid phase pyrophosphate sequencing technology or based on single nucleotide real-time sequencing (SMRT) - next generation sequencing technology. HTS technology is available, such as provided by Roche, Illumina and Applied Biosystems (Life Technologies). Additional high-throughput sequencing technologies are described by and / or available from: Helicos, Pacific Biosciences, Complete Genomics, Ion Torrent Systems, Oxford Nanopore Technologies, Nabsys, ZS Genetics, GnuBio.

[0095] As used herein, " next generation sequencing " refers to a high-throughput sequencing method that allows parallel sequencing of millions to billions of molecules. The example of next generation sequencing method includes synthetic sequencing, connection sequencing, hybridization sequencing, polymerase colony sequencing (polony sequencing), ion semiconductor sequencing, nanopore sequencing and pyrophosphate sequencing. By attaching primers to a solid substrate and attaching complementary sequences to nucleic acid molecules, nucleic acid molecules can hybridize with the solid substrate via primers, and then multiple copies can be generated in discrete areas on the solid substrate by using polymerase to amplify (these groupings are sometimes referred to as polymerase colonies or polymerase colonies). Therefore, in the sequencing process, the nucleotides at a specific position can be sequenced multiple times (for example, hundreds or thousands of times)--this coverage depth is referred to as "deep sequencing". Examples of high-throughput nucleic acid sequencing technologies include platforms offered 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, as reviewed by Service (Science 311: 1544-1546, 2006).

[0096] As used herein, "analysis" molecules generally refer to quantifying, characterizing, distinguishing or combining all or part of the components of molecules (e.g., macromolecules, biomolecules such as proteins, amino acids, nucleic acid molecules, etc.). For example, analyzing peptides, polypeptides or proteins can include determining all or part of the amino acid sequence (continuous or discontinuous) of the peptide. Analyzing macromolecules can include partial identification of the components of macromolecules. For example, the partial identification of amino acids in the protein sequence can identify the amino acids in the protein as belonging to a possible subset of amino acids. Analysis can be performed sequentially, for example, starting with the analysis of n NTAA, and then proceeding to the next amino acid of the peptide (i.e., n-1, n-2, n-3, etc.). In such cases, sequencing can be performed by cutting n NTAA, thereby converting the n-1 amino acid of the peptide into an N-terminal amino acid (referred to herein as "n-1 NTAA"). Similarly, analyzing peptides can start from the C-terminus to the N-terminus, and each round of cutting from the C-terminus produces a new CTAA. The cutting of nCTAA converts the n-1 amino acid of the peptide into a C-terminal amino acid, which is referred to herein as "n-1 CTAA". Analyzing the peptides can also include determining the presence and frequency of post-translational modifications on the peptides, which may or may not include information about the sequential order of the post-translational modifications on the peptides. Analyzing the peptides can also include determining the presence and frequency of epitopes in the peptides, which may or may not include information about the sequential order or position of the epitopes within the peptides. Analyzing the peptides can include combining different types of analyses, for example, to obtain epitope information, amino acid sequence information, post-translational modification information, or any combination thereof.

[0097] As used herein, the term "analyte" generally refers to a substance of interest to further identify, characterize or measure. In a non-limiting example, an analyte can be an ion, chemical, compound, small molecule, element, particle, metal, biomolecule (e.g., protein, lipid, carbohydrate, nucleic acid molecule present in nature), macromolecule, metabolite, lipid, carbohydrate, peptide or protein, nucleic acid molecule, organelle or cell. The analyte can be naturally occurring or synthetic. The analyte can be a solid, semi-solid, liquid, semi-liquid, gas or plasma. The analyte can be characterized qualitatively or quantitatively. A part of the analyte can be analyzed. For example, the analyte can be a peptide, and the constituent amino acids can be analyzed. The analyte can include a polymer, also referred to herein as a "polymer analyte", which generally refers to an analyte of interest comprising one or more monomers. In a non-limiting example, a polymer analyte can be a group of ions, chemicals, compounds, small molecules, elements, particles, metals or biomolecules, macromolecules, metabolites, lipids, carbohydrates, peptides or proteins, nucleic acid molecules, organelles or cells.

[0098] As used herein, the term "array" generally refers to a population of molecules attached to one or more solid supports so that molecules at one address can be distinguished from molecules at other addresses. An array can include different molecules at different addresses, each located on a solid support. Alternatively, an array can include a separate solid support, each solid support acting as an address with different molecules, wherein the different molecules can be identified according to the position of the solid support on the surface to which the solid support is attached or according to the position of the solid support 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, ribozymes, deoxyribozymes, ligands or receptors (such as antibodies, functional fragments of antibodies or aptamers). The addresses of the array can optionally be optically observable, and in some configurations, adjacent addresses can be optically distinguishable when detected using the methods or equipment described herein.

[0099] As used herein, the term "functionalized" refers to any material or substance that has been modified to include a functional group. Functionalized materials or substances can be natural or synthetic functionalized. For example, polypeptides can be naturally functionalized with phosphate groups, oligosaccharides (e.g., glycosyl, glycosylphosphatidylinositol, or phosphoglycosyl), nitrosyl, methyl, acetyl, lipids (e.g., glycosylphosphatidylinositol, myristoyl, or isoprenyl), ubiquitin, or other naturally occurring post-translational modifications. Functionalized materials or substances can be functionalized for any given purpose, including changing chemical properties (e.g., changing hydrophobicity or changing surface charge density) or changing reactivity (e.g., being able to react with a moiety or reagent to form a covalent bond with the moiety or reagent), adding new functions, features, capabilities, or properties to a surface or material. Functionalization of a surface or substrate can be achieved by using, adding, or placing a linker, as described elsewhere herein.

[0100] As used herein, the term "anchoring group" refers to a molecule or particle that serves as an intermediate for attaching a protein or peptide to a surface (e.g., a solid support or microbead). Anchoring groups can be covalently or non-covalently attached to a surface and / or polypeptide. Anchoring groups can be biomolecules, polymers, particles, nanoparticles, or any other entity that can attach to a surface or polypeptide. In some cases, anchoring groups can be structured nucleic acid particles.

[0101] As used herein, the term "click reaction", "click chemistry", "click chemical reaction" or "bioorthogonal reaction" refers to a single-step, thermodynamically favorable conjugation reaction using biocompatible reagents. The click reaction can not use toxic or bioincompatible reagents (e.g., acids, bases, heavy metals) or does not generate toxic or bioincompatible by-products. The click reaction can use an aqueous solvent or buffer (e.g., phosphate buffer solution, Tris buffer, saline buffer, MOPS, etc.). The click reaction can be thermodynamically favorable if it has a negative reaction Gibbs free energy, for example, 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 incorporated herein by reference in its entirety. Exemplary click reactions can include metal-catalyzed azide-alkyne cycloadditions, strain-promoted azide-alkyne cycloadditions, strain-promoted azide-nitrone cycloadditions, strained ene reactions, thiol-ene reactions, Diels-Alder reactions, inverse electron demand Diels-Alder reactions, [3+2] cycloadditions, [4+1] cycloadditions, nucleophilic substitutions, dihydroxylation reactions, thiol-alkyne reactions, photoclick reactions (photoclick), nitrone dipolar cycloadditions, norbornene cycloadditions, oxa-norbornadiene cycloadditions, tetrazine connections, and tetrazole photoclick reactions. Exemplary functional groups or reactive handles for performing click reactions can include alkenes, alkynes (including cycloalkynes), azides, epoxides, amines, thiols, nitrones, isonitriles, isocyanides, aziridines, activated esters, and tetrazines.

[0102] As used herein, the terms "group" and "moiety" are intended to be synonymous when used to refer to the structure of a molecule. The terms refer to a component or portion of a molecule. Unless otherwise indicated, the terms do not necessarily indicate the relative size of the component or portion compared to the molecule. Unless otherwise indicated, the terms do not necessarily indicate the relative size of the component or portion compared to any other component or portion of the molecule. A group or moiety may contain one or more atoms.

[0103] As used herein, "primer" generally refers to a nucleic acid molecule that can trigger the synthesis of a nucleic acid molecule (e.g., DNA or RNA). A primer can be single-stranded. A primer can include one or more recognition sites for binding of a protein (e.g., polymerase, restriction enzyme, cutting enzyme, nuclease, etc.) to a primer or to a primer hybridized with a template strand. A primer can include DNA, RNA, or other nucleic acid analogs or atypical bases (e.g., spacer portion, uracil, abasic site). A primer can optionally include 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), restriction sites, nuclease recognition sites, abasic sites, cutting sites, transposition sites, barcode sequences, unique molecular identifiers (UMIs), etc.

[0104] "Amplification" or "amplification" generally refers to a polynucleotide amplification reaction, i.e., a polynucleotide population replicated from one or more starting sequences. Amplification can refer to various amplification reactions, including but not limited to polymerase chain reaction (PCR), linear polymerase reaction, nucleic acid sequence-based amplification, rolling circle amplification, loop-mediated isothermal amplification, helicase-dependent amplification, multiple displacement amplification, strand invasion-based amplification, strand displacement amplification, recombinase polymerase amplification, nickase amplification reaction, nucleic acid sequence-based amplification, gp32-based amplification, and similar reactions. An amplification reaction can generate an amplicon. The amplification reaction can be performed isothermally or can require a temperature change.

[0105] As mentioned herein, "adapter" generally refers to a short nucleic acid molecule (e.g., a length of about 10 to about 100 base pairs). An adaptor can include a short double-stranded DNA molecule. An adaptor can be attached to the end of a DNA fragment or an amplicon, for example, via polymerization or connection. An adaptor can include a synthetic oligonucleotide, for example, an oligonucleotide having a nucleotide sequence that is at least partially complementary to each other. An adaptor can have a blunt end, can have a staggered end (also referred to herein as a 3' or 5' "overhang sequence" or "sticky end"), or a blunt end and a staggered end. An adaptor can be attached to a fragment (e.g., via connection) to provide a fragment to which the adaptor is connected; the fragment to which the adaptor is connected can serve as a starting point for subsequent manipulation, for example, for amplification or sequencing. An adaptor can be functionalized, for example, conjugated to a label, a probe, a detectable label, an affinity capture reagent (e.g., biotin or streptavidin).

[0106] As used herein, the term "capturing moiety" generally refers to a molecule that can be coupled to another part or molecule. The capturing moiety can be a biomolecule, such as a lipid, carbohydrate, sugar, amino acid, peptide or protein, nucleotide, nucleic acid molecule, metabolite or a combination thereof (e.g., glycoprotein, lipoprotein, glycosaminoglycan, etc.). The capturing moiety can be a small molecule, an organic compound, an inorganic compound, a metal, a polymer, an ion or other molecule or molecular compound. The capturing moiety can include a macromolecule. The capturing moiety can include an enzyme, a ribozyme, a deoxyribozyme, an antibody, an antibody fragment, a nanobody, an aptamer, biotin, streptavidin, avidin, neutravidin (neutravidin) or its analogs or derivatives. The capturing moiety can include more than one molecule, such as a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, etc. The capturing moiety can be a solid substrate or a part of a solid substrate, or the capturing moiety can be separated from the substrate, such as in a fluid medium (e.g., air, in a liquid solution). The capture moiety can be specific for a binding partner or multiple binding partners. The capture moiety can be capable of binding to one molecule or moiety (monovalent) or multiple molecules or moieties (multivalent). The capture moiety can be or include a reactive moiety or group, such as a click chemistry moiety.

[0107] As used herein, the abbreviations for the natural 1-enantiomer amino acids are conventional and may 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, Gln); glycine (G, Gly); histidine (H, His); isoleucine (I, Ile); 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, Val). Unless otherwise indicated, X may represent any amino acid. In some aspects, X can be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R). These amino acids are also referred to as "[amino acid] [residue / residue]" (e.g., lysine residue, lysine residues, leucine residue, leucine residues, etc.).

[0108] 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. Suitable methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.

[0109] Analysis of polymer analytes via local tethering

[0110] The present disclosure provides a novel method for processing and analyzing polymer analytes in a highly parallel and accurate manner. The system and method of the present disclosure can be related to the molecular analysis and identification (e.g., with single molecule sensitivity) of a single monomer of a polymer analyte. The system and method of the present disclosure can include cutting a monomer from a polymer analyte, coupling the monomer (e.g., via local tethering) to a capture portion (e.g., located near the polymer analyte), and contacting the monomer with a binding agent. The binding agent can be specifically or semi-specifically bound to a monomer or monomer type, and can include or be coupled to a polymerizable molecule with coded information (e.g., about the identity of a binding agent or its homologous molecule), which can be transferred via coupling and optionally copying the coded information to additional polymerizable molecules. Alternatively, the polymerizable molecule with coded information can be provided separately from the binding agent (e.g., not coupled with the binding agent), and in some cases, the binding agent can include an activator that helps the polymerizable molecule to couple with additional polymerizable molecules. The operation can be iterated or repeated any number of times to obtain information about all or a subset of the monomers of the polymer analyte, and optionally the sequence of the monomer relative to the polymer analyte. For example, information can be read out from polymerizable molecules using next generation sequencing methods. The systems and methods provided herein enable novel methods for sequencing polymer analytes (such as peptides), determining the identity and order of at least a subset of amino acids in a peptide with high accuracy for a given number of amino acids. In another aspect, provided herein are systems and methods for processing analytes less than 10 nanometers (nm) in size, comprising providing an analyte, a binding agent, a first polymerizable molecule and a second polymerizable molecule, contacting the analyte with the binding agent, and coupling the first polymerizable molecule and the second polymerizable molecule. The first polymerizable molecule can contain identification information of the analyte, and further analysis (e.g., sequencing) of the first polymerizable molecule can identify the analyte. In a further further aspect, provided herein are systems and methods for processing peptides comprising amino acids comprising non-naturally occurring modifications; such methods can include providing a peptide and a substrate, and coupling an amino acid or another amino acid to the substrate, and cleaving the substrate-coupled amino acid from the peptide to obtain the removed substrate-coupled amino acid and the remainder of the peptide. In a further further aspect, disclosed herein are methods for characterizing multiple analytes using an ordered binding method.

[0111] Advantageously, some methods disclosed herein include identifying cleaved monomers, thereby eliminating the "local environment" problem, in which the ability or specificity of a binder to bind to a particular monomer is affected by neighboring monomers. By first cleaving a monomer from a polymeric analyte, the monomer is removed from neighboring monomers, and the binder can bind to a single monomer without being affected by neighboring surrounding monomers. Thus, the methods disclosed herein enable more accurate molecular identification and sequencing of polymeric analytes, which have applications in diagnosing disease, monitoring protein dynamics or protein interactions, single-cell proteomics, and developing or characterizing therapeutic agents.

[0112] As further described herein, one or more methods of the present disclosure may employ a linker capable of coupling to (i) a monomer of a polymeric analyte and (ii) a capture moiety, which can be used to locally tether a monomer to a neighboring polymeric analyte. The methods and systems disclosed herein may further include a substrate for local tethering; the substrate can be coupled, for example, to a polymeric analyte, a capture moiety, and an additional polymerizable molecule that can encode information by copying or transferring the information of the encoded polymerizable molecule to the additional polymerizable molecule. Further analysis of the encoded additional polymerizable molecule can be performed after copying or transferring the information, for example, using an NGS sequencing method, which can identify individual monomers and their sequences (or positions) present in the polymeric analyte.

[0113] The methods and systems disclosed herein have applications in studying polymer analytes. Polymer analytes may include biomolecules, such as proteins or peptides, nucleic acid molecules, lipids, carbohydrates, or combinations thereof. The methods and systems may also be used to study non-biological or synthetic polymers, such as peptidomimetics, plastics, polyesters, and the like. Thus, the methods and systems disclosed herein may be used to interrogate and characterize analytes across a wide range of fields, including proteomics, genomics, transcriptomics, metabolomics, and polymer science. The methods and systems disclosed herein may provide for analyzing polymer analytes with single-molecule level sensitivity in a high-throughput format.

[0114] In one aspect of the present disclosure, methods are provided herein for processing a polymer analyte comprising a plurality of monomers. Such methods may include cleaving a monomer of the polymer analyte and coupling the monomer to a capture portion for subsequent analysis. The method may include providing (i) a polymer analyte comprising a plurality of monomers and (ii) a capture portion; cleaving a monomer from the plurality of monomers; coupling the monomer to the capture portion to generate a monomer-capture portion complex; contacting the monomer-capture portion complex with a binding agent and a first polymerizable molecule; and coupling the first polymerizable molecule to a second polymerizable molecule. The first polymerizable molecule may contain information about the binding agent, such as the identity of the binding agent or its cognate molecule, or the identity of the monomer. In some cases, the binding agent includes an activator, and the activator promotes the coupling of the first polymerizable molecule to the second polymerizable molecule. The information of the first polymerizable molecule can be transferred to the second polymerizable molecule. Subsequent reading or identification of the first polymerizable molecule can generate information about the identity of the binding agent or the cleaved monomer. One or more operations can be iterated for any number of cycles to identify the sequence and identity of all or a subset of the plurality of monomers.

[0115] Polymer analytes: Polymer analytes can be biomolecules, macromolecules, or synthetic molecules. Polymer analytes can be biomolecules or other biomolecules (e.g., derived from a sample) comprising one or more monomers. Non-limiting examples of polymer biomolecules include nucleic acid molecules (e.g., DNA molecules, RNA molecules, DNA:RNA hybrids, aptamers), peptides and proteins, polysaccharides, lipid polymers (e.g., diglycerides, triglycerides, and other fatty acids). Polymer analytes can be synthetic molecules, such as peptidomimetics or synthetic polymers, or peptide mimetics (e.g., peptidomimetics, β-peptides, D-peptide peptidomimetics). Non-limiting examples of synthetic polymers include acrylic acid, nylon, silicone, viscose, rayon, polyester, polycarboxylic acid, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol, polyurethane, polylactic acid, silicon dioxide, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(vinyl terephthalate), polyethylene, polyisobutylene, poly(methyl methacrylate), poly(oxymethylene), polyoxymethylene, polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene chloride), poly(vinylidene fluoride), poly(vinyl fluoride), or combinations thereof. Polymeric analytes can include a single polymer type (e.g., a homopolymer) or more than one polymer type (e.g., a copolymer) and can include random or arranged monomers. The polymer analyte can be a block polymer, an alternating copolymer, a periodic copolymer, a statistical copolymer, a stereoregular block copolymer, a gradient copolymer, a branched copolymer, a graft copolymer, and the like.

[0116] Polymer analytes can be in any size or include a series of sizes.The size of polymer analytes can be about 1 nanometer (nm), about 5nm, about 10nm, about 20nm, about 30nm, about 40nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, about 100nm, about 200nm, about 300nm, about 400nm, about 500nm, about 600nm, about 700nm, about 800nm, about 900nm, about 1 micron (μm), about 10 μm, about 100 μm, about 1 millimeter (mm) or larger.Multiple polymer analytes can include the polymer analytes in similar size or size range, for example, about 10nm to about 100nm, about 50nm to about 1 μm.Similarly, polymer analytes can have any molecular weight or molecular weight range. The polymeric analyte can 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 analyte can include polymeric analytes of similar molecular weight or within a range of molecular weights.

[0117] The monomer of polymer analyte can include any size or size range that is less than the size of whole polymer analyte.The size of monomer can be about 0.1 nanometer (nm), about 0.5nm, 1 about 1nm, about 5nm, about 10nm, about 20nm, about 30nm, about 40nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, about 100nm, about 200nm, about 300nm, about 400nm, about 500nm, about 600nm, about 700nm, about 800nm, about 900nm, about 1 micron (μm), about 10 μm, about 100 μm, about 1 millimeter (mm) or larger.Monomer can have any molecular weight or molecular weight range. Monomers can 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. Monomer or polymeric analytes can range in molecular weight; for example, polymeric analytes can include peptides comprising amino acid monomers, the molecular weight of which can vary from 75 Da (glycine) to 204 Da (tryptophan).

[0118] The polymer analyte can include any number of monomers. The polymer analyte can contain 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 analyte 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 more monomers. Alternatively, the polymeric analyte 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. A polymeric analyte may include a range of monomers; for example, a polymeric analyte may include about 5 monomers, while another polymeric analyte may include about 500 monomers.

[0119] In some cases, polymer analytes include peptides comprising amino acid monomers. The peptide can be naturally occurring or synthetic and can be a part of a sample or derived from a sample. The peptide can comprise any number of amino acids. The amino acid can be one of 20 proteinogenic amino acids and can comprise any number of post-translational modifications. The peptide or any of its constituent amino acids can be processed to comprise non-naturally occurring modifications, such as contacting with protecting groups, alkylation, β-elimination of phosphate groups, etc., as described elsewhere herein. In some cases, the peptide is derived from a larger peptide or protein and is fragmented.

[0120] Substrate: One or more operations described herein can be performed using a substrate. For example, one or more molecules described herein (e.g., polymer analytes, capture moieties, polymerizable molecules) can be coupled to a substrate. In some cases, a polymer analyte, a capture moiety, and one or more polymerizable molecules (e.g., a first or second polymerizable molecule) or a combination thereof can be provided to be coupled to one or more substrates. In one example, a polymer analyte, a capture moiety, and a polymerizable molecule (e.g., a second polymerizable molecule coupled to a first polymerizable molecule comprising coded information) are coupled to a substrate. In some cases, more than one substrate can be used. In such cases, the substrate can comprise the same material or different materials.

[0121] The substrate can be made of any suitable material, e.g., glass, silica, silicon, gel, polymer, etc., as described elsewhere herein. In some cases, the substrate can be particles, beads, or gel beads (e.g., polyacrylamide, agarose, polyethylene glycol, or Beads). The substrate can be functionalized. One or more molecules such as the capture moiety and the polymer analyte (e.g., a peptide) can be coupled to the substrate via covalent or non-covalent interactions. The coupling or attachment can be direct or indirect. The capture moiety and the polymer analyte (e.g., a peptide) can be coupled to the substrate using any suitable chemistry, such as click chemistry moieties (e.g., alkyne-azide coupling), photoreactive groups (e.g., benzophenone, phenyldiazirine, phenyl azide, 3-cyanovinylcarbazole phosphoramidite (CNVK)), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) N-hydroxysulfosuccinimide (NHS), sulfonamides.

[00145] The invention also includes but is not limited to the following: succinimidyl-NHS or NHS-ester, maleimide, thiol, biotin-streptavidin interaction, cystamine, glutaraldehyde, formaldehyde, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), sulfo-SMCC, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium hydrochloride (DMTMM), silane (e.g., aminosilane), hydrazine, hydroxylamine, and the like, or combinations thereof.

[0122] In some cases, the substrate can be functionalized to include a coupling chemistry to couple a polymer analyte or capture moiety. In one non-limiting example, the substrate (e.g., a bead or surface) can include an alkyne, such as dibenzocyclooctyne (DBCO, e.g., DBCO-alcohol, DBCO-Boc, DBCO-NHS, DBCO-silane), which can be configured to react with amines, carboxyl groups (carboyxl) or carbonyl groups, sulfhydryl groups, etc. The DBCO-functionalized substrate can be conjugated to a polymerizable molecule, such as an azide-functionalized polymerizable molecule (e.g., nucleic acids, proteins) or a polymer analyte via a click chemistry reaction. In other examples, linkers such as bifunctional linkers can be used to attach molecules to substrates; such bifunctional linkers can include the same reactive moiety at both ends or different moieties at each end (e.g., heterobifunctional linkers). Additional examples of linkers are described elsewhere herein.

[0123] In some cases, a molecule (e.g., a polymeric analyte, a capture moiety, a polymerizable molecule) can be coupled to a substrate using an enzymatic method, e.g., as described elsewhere herein. For example, an enzyme can be used to attach a chemical linker or moiety (such as a click chemistry moiety) to a polymeric analyte (e.g., a peptide). The chemical linker or moiety can be capable of reacting with another chemical linker or moiety (e.g., a click chemistry moiety) of a substrate, a capture moiety, or a polymerizable molecule.

[0124] The substrate can be coupled to any useful number of molecules (e.g., polymeric analytes, capturing moieties, polymerizable molecules). In some cases, the substrate can include multiple polymeric analytes, multiple capturing moieties, and / or multiple polymerizable molecules, which can be provided in any useful ratio or density. For example, the ratio of polymeric analytes to capturing moieties or polymerizable molecules can 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 less. In some cases, the ratio of polymer analyte to capture moiety or polymerizable molecule can 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 less.

[0125] Similarly, molecules (eg, polymeric analytes, capture moieties, or polymerizable molecules) can be coupled to the substrate at any useful density, such as about 1 molecule per square micrometer (μm). 2 ), about 10 molecules / μm 2 , about 100 molecules / μm 2 , about 1,000 molecules / μm 2, about 10,000 molecules / μm 2 , about 100,000 molecules / μm 2 , about 1,000,000 molecules / μm 2 , about 10,000,00 molecules / μm 2 , about 100,000,000 molecules / μm 2 , about 1,000,000,000 molecules / μm 2 , about 10,000,000,000 molecules / μm 2 , about 100,000,000,000 molecules / μm 2 or greater. The polymer analyte, capture moiety, and polymerizable molecule can be coupled to the substrate at a range of densities, for example, from about 100 to about 10,000 molecules / μm 2 or about 10 to about 1,000 molecules / μm 2 The densities of the polymeric analyte, capture moiety, and polymerizable molecule can be the same or different. For example, the density of the polymerizable molecule can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000, 10,000, 100,000, 1,000,000, or more times lower than the density of the polymeric analyte.

[0126] In some cases, the molecules (e.g., polymer analytes, capture moieties, or polymerizable molecules) coupled to the substrate can be spaced apart at a specified or controlled distance. For example, the average spacing or distance between the polymerizable molecules coupled to the substrate can 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 1 μm, or more. In some cases, the spacing between polymerizable molecules coupled to the substrate can be at most about 1 μm, 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 the polymerizable analyte and the polymerizable molecule or capture moiety can 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 1 μm, or more. In some cases, the average separation between the polymerizable molecules and the polymer analyte coupled to the substrate can be at most about 1 μm, 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 and each other or the polymer analyte can be used, for example, from about 1 nm to about 40 nm, from about 2 nm to about 10 nm, etc.

[0127] The concentration or density of molecules attached to the substrate can be adjusted using one or more suitable methods, including patterned or random deposition methods. Examples of methods for controlling the concentration or density of molecules attached to the substrate include limiting dilution, adding chaotropic agents (e.g., guanidine, formamide, urea), using metal organic compounds, etc. The molecules can be attached to the substrate in a patterned manner, such as using a self-assembled monolayer, photopatterning, lithography, etching, or a combination thereof, or the molecules can be randomly arranged.

[0128] The substrate can comprise any useful size or dimension (e.g., length, width, height, diameter, radius), surface area, volume, or ratios or combinations thereof. The substrate can comprise beads or particles having 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 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, or about 6 μm. The surface area of ​​the substrate can be about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 60 mm, about 70 mm, about 80 mm, about 90 mm, about 100 mm, about 200 mm, about 300 mm, about 400 mm, about 500 mm, about 600 mm, about 700 mm, about 800 mm, about 900 mm, about 1 mm or more. The surface area of ​​the substrate can include about 1 square nanometer (nm) or more. 2 ), about 10nm 2 , about 100nm 2 , about 1,000nm 2 , about 10,000nm 2 , about 100,000nm 2 , about 1μm 2 , about 10μm 2 , about 100μm 2 , about 1,000μm 2 , about 10,000μm 2 , about 100,000μm 2 , about 1mm 2 , about 10mm 2 , about 100mm 2 , about 1,000mm 2 , about 10,000mm 2 , about 100,000mm 2 , about 1,000,000mm 2 or larger.

[0129] The substrate may comprise a substantially planar substrate. Non-limiting examples of substantially planar substrates include microscope slides, microfluidic devices, flow cells, and multiwell plates. In some cases, the substrate may comprise a commercially available substrate such as a DNA bead, chip, or flow cell, e.g. HiSeq, iSeq, MiniSeq, NextSeq, NovaSeq flow cells. In such cases, the substrate-coupled molecule (e.g., polymer analyte, capture moiety, or polymerizable molecule) can be designed to couple to a pre-provided DNA molecule (e.g., a P5 or P7 sequence), or a pre-provided DNA molecule can be used as a capture moiety or polymerizable molecule, or used to couple to the polymer analyte.

[0130] In some cases, molecules (e.g., polymer analytes, capture moieties or polymerizable molecules) can be coupled to substrates in an ordered or random arrangement. In an ordered arrangement, any conventional method can be used for patterning molecules, such as lithography (e.g., soft lithography, photolithography), etching (e.g., ion etching, photoetching) or other patterning methods. In some cases, joints (e.g., bifunctional joints) can be used to promote the coupling of molecules (e.g., polymer analytes, polymerizable molecules, capture moieties) to substrates; such joints can be patterned using any useful technology (e.g., self-assembled monolayers, photopatterning, lithography, etching). In some cases, molecules can be coupled to substrates in a random arrangement. For example, molecules can be provided with any useful ratio or density to couple molecules with a stoichiometric ratio or a controlled concentration. Other examples of substrate types, substrate functionalization and conjugation and molecule attachment to substrates are provided elsewhere herein.

[0131] Polymerizable molecules: The polymerizable molecules described herein can be any useful type of polymerizable molecule. The polymerizable molecules can be naturally occurring, such as biopolymers (e.g., nucleic acid molecules, peptides, polysaccharides, fatty acids) or other naturally occurring polymers, for example, rubber, cellulose, starch, polyhydroxyalkanoates, chitosan, dextran, structural proteins (e.g., collagen, hyaluronic acid, glycosaminoglycans), agarose, carrageenan, isphagula, gum arabic, agar, gelatin, shellac, xanthan gum, guar gum, alginates, and the like. The polymerizable molecules can be synthetic, for example, acrylic, nylon, silicone, viscose, rayon, polyester, polycarboxylic acid, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol, polyurethane, polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(vinyl terephthalate), polyethylene, polyisobutylene, poly(methyl methacrylate), poly(oxymethylene), polyoxymethylene, polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene chloride), poly(vinylidene fluoride), poly(vinyl fluoride), and combinations thereof. The polymerizable molecules can contain one or more reactive moieties (e.g., free radicals) to initiate polymerization or can be polymerizable via contact with an initiator (e.g., ammonium persulfate, peroxide, or other radicalizing agent). The polymerizable molecule can be polymerizable via a catalase (e.g., a polymerizing enzyme such as a polymerase), a ribozyme, or a deoxyribozyme. Alternatively or additionally, the polymerizable molecule can be polymerizable via self-assembly. The polymerizable molecule can include a single polymer type (e.g., a homopolymer) or more than one polymer type (e.g., a copolymer) and can include random or arranged monomers. The polymerizable molecule can be a block polymer, an alternating copolymer, a periodic copolymer, a statistical copolymer, a stereoregular block copolymer, a gradient copolymer, a branched copolymer, a graft copolymer, etc.

[0132] The same or different types of polymerizable molecules can be used for methods described herein. For example, the first polymerizable molecule (for example, encoding polymerizable molecule) can be a nucleic acid molecule, and the second polymerizable molecule (for example, for attaching and transferring the information of the first polymerizable molecule) can be a peptide. In another example, both the first polymerizable molecule and the second polymerizable molecule are nucleic acid molecules. In such examples, the first polymerizable molecule can be coupled to the second polymerizable molecule via connection or hybridization. For example, the first polymerizable molecule can include a first nucleic acid sequence, and the second polymerizable molecule can include a second nucleic acid sequence. The first nucleic acid sequence can be complementary or partially complementary to the second nucleic acid sequence, and coupling can include hybridizing the first nucleic acid sequence or its portion with the second nucleic acid sequence or its portion. Alternatively, the first nucleic acid sequence and the second nucleic acid sequence can be complementary to two sequences of splints or bridging oligonucleotides, and coupling can be mediated via hybridization with splint oligonucleotides. The first nucleic acid sequence can be chemically (for example, via click chemistry, wherein the first polymerizable molecule and the second polymerizable molecule both include one or more members of click chemistry pairs) or enzymatically (for example, using a ligase or polymerase) connected to the second nucleic acid sequence.

[0133] The polymerizable molecule may comprise a functional moiety. For example, the polymerizable molecule may comprise a member of a binding pair (e.g., biotin-streptavidin), a reactive moiety, a functional site, etc. The polymerizable molecule may comprise a nucleic acid molecule comprising a functional sequence, such as a primer sequence (e.g., a universal priming site), a sequencing sequence (e.g., an Illumina P5 or P7 sequence), a reader sequence (e.g., an R1 or R2 sequence), a unique molecular identifier (UMI), a barcode sequence, a cleavage site or sequence (e.g., a uracil or abasic site, a restriction site, a Cas binding sequence), a transposition sequence (e.g., a chimeric end sequence), or a combination thereof.

[0134] In some cases, polymerizable molecule (for example, the first polymerizable molecule or the second polymerizable molecule) includes a nucleic acid molecule comprising one or more nucleotide bases. The polymerizable molecule can include any useful number of nucleotide bases, for example, 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.

[0135] The polymerizable molecule may include a nucleic acid molecule that may include modified nucleotides or atypical bases. For example, the polymerizable molecule may include pseudo-complementary bases, bridged nucleic acids (BNAs), heterologous nucleic acids (XNAs), locked nucleic acids (LNAs), peptide nucleic acids (PNAs), γ-PNA molecules, morpholinos, or combinations thereof. In some cases, the polymerizable molecule may include hexitol nucleic acids (HNAs) or cyclohexane nucleic acids (CeNAs), which may be used to make the polymerizable molecule more resistant to acid degradation (e.g., as used in traditional Edman degradation). Alternatively or additionally, the polymerizable molecule may include naturally occurring bases that are more resistant to acid degradation, for example, consisting primarily of pyrimidines, such as thymine or cytosine. For example, the nucleic acid molecule may include at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% thymine or cytosine, which may make the nucleic acid molecule more acid-resistant compared to nucleic acid molecules comprising purines (adenine or guanine). Other examples of modifying polymerizable molecules to make them more resistant to certain reaction conditions are described elsewhere herein.

[0136] In some cases, the polymerizable molecule is the same molecule as the capture moiety. For example, a substrate can include two identical nucleic acid molecules, one of which can serve as a capture moiety for a monomer coupled to a polymer analyte, and the other can serve as a polymerizable molecule for coupling to a polymerizable molecule of a binding agent. Thus, a substrate can include multiple polymerizable molecules (e.g., nucleic acid molecules) that can collectively serve as tetherable molecules for coupling to monomers and additional polymerizable molecules (e.g., encoded polymerizable molecules).

[0137] Linkers: One or more operations of the method can be mediated using a linker. In some cases, the coupling of a monomer to a 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 can be covalent or non-covalent. In an example, the linker can include a first reactive group that is capable of coupling to a monomer of a polymeric analyte (e.g., an amino acid of a peptide) and optionally cleaving an amino acid from the peptide. For example, the first reactive group can be a thiocyanate conjugate, for example, an isothiocyanate (ITC) such as phenyl isothiocyanate (PITC), 3-pyridyl isothiocyanate (PY1TC), 2-piperidyl ethyl isothiocyanate (PEITC), 3-(4-morpholino)propyl isothiocyanate (MPITC), 3-(diethylamino)propyl isothiocyanate (DEPTIC) or naphthyl isothiocyanate (NITC), fluorescein isothiocyanate (FITC), ammonium thiocyanate, potassium thiocyanate, trimethylsilyl isothiocyanate (TMS-ITC), phenyl phosphoroisothiocyanatidate, acetyl isothiocyanate (AITC) or an aldehyde group, for example, o-phthalaldehyde (OPA), 2,3-naphthalenedicarboxylic acid (NDA), 2-pyridinecarboxaldehyde, which can react with an N-terminal amino acid (NTAA). The linker can additionally comprise a second reactive group capable of being coupled directly or indirectly to the capture moiety. In the example of direct coupling, the capture portion may include a click chemistry moiety (e.g., alkyne), and the second reactive group of the joint may include another click chemistry moiety (e.g., azide) that can react with the click chemistry moiety of the capture portion. Alternatively, the joint can be indirectly coupled to the capture portion, for example, via non-covalent interactions or via an intermediate linking molecule. In some cases, the intermediate linking molecule may include a third polymerizable molecule (e.g., polymer or nucleic acid molecule) that can couple the joint to the capture portion. In one such example, the intermediate linking molecule includes connecting nucleic acid molecules, which may include (i) a third reactive group (e.g., via alkyne-azide click chemistry) that can be coupled to the second reactive group of the joint and (ii) a part (e.g., another orthogonal click chemistry reaction, avidin-biotin interaction, nucleic acid coupling or hybridization) that can be coupled to the capture portion. In some cases, the third intermediate linker molecule comprises a nucleic acid molecule comprising (i) a click chemistry moiety (e.g., alkyne) that can be conjugated to a first reactive group (e.g., azide) of a linker and (ii) a nucleic acid sequence that can be coupled to a capture moiety, e.g., via ligation, splinting, or hybridization. In some cases, the linker comprises a linker nucleic acid molecule comprising a self-splinting moiety (e.g., see Figure 24 ).

[0138] When applicable, the click chemistry portion of the linker, polymerizable molecule, capture moiety, or intermediate connecting molecule can include any suitable bioorthogonal moiety, as described elsewhere herein, such as an alkene, an alkyne (e.g., a cyclooctyne or a derivative thereof, such as aza-dimethoxycyclooctyne (DIMAC), symmetric pyrrolocyclooctyne (SYPCO), pyrrolocyclooctyne (PYRROC), difluorocyclooctyne (DIFO), α,α-bis(trifluoromethyl)pyrrolocyclooctyne (TRIPCO), bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctyne (DIBO), , difluorobenzocyclooctyne (DIFBO), dibenzoazepine octyne (DBCO), difluoro-aza-dibenzocyclooctyne (F2-DIBAC), biaryl-azacyclooctyne ketone (BARAC), difluorodimethoxydibenzocyclooctyne alcohol (FMDIBO), difluorodimethoxydibenzocyclooctyne ketone (keto-FMDIBO and 3,3,6,6-tetramethylthicycloheptyne (TMTH)), azides, epoxides, amines, thiols, nitrones, isonitriles, isocyanides, aziridines, activated esters and tetrazines, and combinations, variants or derivatives thereof. The click chemistry moieties can be subjected to conditions sufficient to react the first click chemistry moiety with the second click chemistry moiety, for example, providing a metal catalyst, a suitable solvent, pH, temperature, ion concentration or light / energy for any useful duration.

[0139] The first reactive group of the linker can be an amino acid reactive portion. The amino acid reactive portion of the linker can be any useful portion that enables the reactive portion to be conjugated to an amino acid and optionally cleave an amino acid. In some examples, the first reactive portion can react with a terminal amino acid (e.g., NTAA or CTAA). In such examples, the first reactive moiety can comprise any primary amine or carboxyl reactive group, including but not limited to isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, ethylene oxide, carbonate, aryl halide, imido ester, carbodiimide, anhydride, phenyl ester, isothiocyanate (e.g., phenyl isothiocyanate, sodium isothiocyanate, ammonium isothiocyanate (e.g., tetrabutylammonium isothiocyanate, tetrabutylammonium isothiocyanate), diphenylphosphoryl isothiocyanate), acetyl chloride, cyanogen bromide, carboxypeptidase, azide, alkyne, DBCO, maleimide, succinimide, thiol-thiol disulfide bond, tetrazine, TCO, vinyl, methylcyclopropene, allyloyl, allyl, etc. Additional examples of amino acid reactive groups are provided in U.S. Pat. No. 11,499,979, which is incorporated herein by reference in its entirety.

[0140] Joint can comprise any additional useful part.For example, joint can comprise releasable or cleavable part, and this can promote to remove monomer from polymer analyte or its part or from substrate.Such releasable or cleavable part can comprise, for example, disulfide bond, and it can be by contacting with reducing agent (for example, DTT, TCEP) or nucleic acid molecule comprising cutting or restriction site and releasable.In some examples, joint can be coupled to intermediate joint molecule via releasable or cleavable part, alternatively or additionally via click chemistry coupling.Therefore, the coupling between intermediate joint molecule and joint can be reversible.Additionally or alternatively, joint can comprise any number of spacer parts, for example, polymer (for example, PEG, PVA, polyacrylamide), aminocaproic acid, nucleic acid, alkyl chain etc.Such spacer parts can increase the distance between any other part (for example, amino acid reactive group and polymerizable molecule reactive group) of joint.

[0141] The joint can include or be coupled to a detectable portion, for example, a fluorophore, a radioisotope, a mass label, a nucleic acid molecule (also as a releasable or cleavable portion) or other detectable portion. In some examples, the joint includes a fluorophore, which can be used to visualize the position of the joint using single-molecule imaging. In another example, the monomer can be labeled with a first fluorophore and the joint can include a second fluorophore to enable visualization of the position of the joint and the monomer (e.g., using dual-channel imaging or FRET).

[0142] In some cases, when using an intermediate linking molecule, the joint can be coupled to the intermediate linking molecule in advance. For example, a precursor joint can include a monomer binding group (for example, PITC) and a click chemistry moiety (for example, an azide), which can react with an intermediate linking molecule (for example, connecting nucleic acid molecules) comprising a complementary click chemistry moiety (for example, alkynes such as DBCO) to generate a joint comprising connecting nucleic acid molecules and capable of being coupled to a monomer and capturing moiety (for example, another oligonucleotide). In some cases, a joint coupled to an intermediate linking molecule in advance or comprising an intermediate linking molecule can be provided.

[0143] In some cases, polymer analytes include peptides comprising amino acid monomers, and the coupling of joints and amino acids (for example, NTAA or CTAA) can change amino acid whose chemical structure. For example, if using a joint comprising an isothiocyanate moiety, during or after contact with the isothiocyanate moiety, amino acid can be derived into thiocarbamoyl (for example, under mild alkaline conditions). One or more further derivatizations can be carried out. For example, amino acid or amino acid derivatives (for example, the amino acid derived into thiocarbamoyl) can be further derived into thiazolone groups (for example, under acidic conditions such as Edman degradation or cutting), thiohydantoin groups or other chemical moieties. Similarly, thiazolone groups or thiohydantoin groups can be further derived into thiocarbamoyl.

[0144] Capture moiety: The capture moiety can be coupled to the monomer of the polymer analyte via any suitable mechanism. The coupling of the monomer to the capture moiety can include covalent interactions or non-covalent interactions. Coupling can occur through the interaction of binding pairs, for example, biotin and avidin (or streptavidin), an antigen or epitope and an antibody or antibody fragment, a cyclodextrin and a hydrophobic small molecule (e.g., an alkane, benzene, a polycyclic compound), a cucurbitane and adamantane or trimethylaminomethylferrocene, a diphenylene cycloalkanes (e.g., calixarenes, cavitands, pillararens, tetralactams), etc. In some embodiments, the coupling of the monomer to the capture moiety occurs through the coupling of nucleic acid molecules (e.g., hybridization to each other or to a splint molecule).

[0145] In some cases, the capture portion includes additional polymerizable molecules (e.g., nucleic acid molecules or peptides). Monomers can be coupled to complementary polymerizable molecules (e.g., to generate monomer-oligonucleotide conjugates) and, for example, directly or via a splint molecule via complementary base pairing, are tethered to the capture portion. Alternatively, as described above, monomers can be coupled to the capture portion via a joint. In some examples, the joint comprises a monomer coupling group (e.g., PITC, which can be coupled or reacted with the amino acids of the peptide) and a nucleic acid molecule. The capture portion can include additional nucleic acid molecules that can be coupled to the nucleic acid molecules of the joint via hybridization (directly or via a splint), connection, or both.

[0146] The nucleic acid molecule of the capture portion can comprise any naturally occurring, non-naturally occurring or engineered nucleotide base. For example, the nucleic acid molecule can comprise pseudo-complementary bases, bridged nucleic acids, heterologous nucleic acids, locked nucleic acids, peptide nucleic acids (PNA), γ-PNA, morpholinos, etc., as described elsewhere herein. The capture portion can comprise one or more functional sequences, including but not limited to a priming sequence, a sequencing sequence (e.g., a P5 or P7 sequence), a sequencing read sequence (e.g., an R1 or R2 sequence), a chimeric end sequence, a transposase recognition sequence, a cleavage site (e.g., a restriction site), a UMI, a blocking group, a spacer sequence, a barcode sequence, or other functional sequences. In some cases, the capture portion comprises a cleavable or releasable portion, e.g., a restriction enzyme recognition site, an abasic site, a removable portion ... Or uracil cleaved by uracil DNA glycosylase, disulfide bonds that can be released when a reducing agent is added, etc.

[0147] In some cases, a capture moiety and a polymerizable molecule coupled to a substrate are provided. In one example, the substrate includes a first nucleic acid molecule, a second nucleic acid molecule, and a capture moiety coupled thereto, which capture moiety can be a third nucleic acid molecule. In some cases, the substrate can include the same nucleic acid molecule or set of same nucleic acid molecules across the substrate; these same nucleic acid molecules can serve as both capture moieties and polymerizable molecules, to which additional polymerizable molecules (e.g., containing coding information) can be coupled. In some examples, a bead, flow cell, or chip comprising a plurality of nucleic acid molecules can be provided, and a subset of the plurality of nucleic acid molecules can serve as capture moieties, and another subset of the plurality of nucleic acid molecules can serve as polymerizable molecules, to which additional polymerizable molecules containing coding information can be coupled. In some cases, commercially available beads (e.g., DNA beads or barcoded beads), flow cells, or chips, such as HiSeq, iSeq, MiniSeq, NextSeq, NovaSeq, etc. can be used as substrates as described herein. In some cases, the substrate comprises multiple sequencing primer sequences (e.g., P5 or P7 sequences) or read sequences (e.g., R1 or R2), which can be used as capture moieties or polymerizable molecules to which additional polymerizable molecules containing encoded information can be coupled.

[0148] The capture portion can be coupled to a substrate. In such cases, the capture portion can include a substrate tethered group or a joint or an additional functional group. In some examples, the capture portion includes a nucleic acid molecule comprising a substrate tethered group (e.g., biotin), a click chemistry portion (such as an azide) that can be coupled to a substrate (e.g., a substrate comprising streptavidin) or a complementary click chemistry portion that can react with a substrate tethered group. The capture portion can additionally include a binding sequence, to which another nucleic acid molecule (e.g., a connecting nucleic acid molecule, a joint-monomer complex, or a nucleic acid barcode molecule) can be coupled via hybridization, connection, or both. In some cases, the capture portion includes a single-stranded region in which a single-stranded oligonucleotide or a complementary oligonucleotide can hybridize. The complementary oligonucleotide can include a detectable label (e.g., a fluorophore) that allows detection of the capture portion.

[0149] Cutting: Cutting monomers from polymer analytes can be achieved using any suitable mechanism (such as via applying a stimulus). The stimulus can be, for example, a chemical stimulus, a biological stimulus, a thermal stimulus (e.g., applying heat), a light stimulus, a physical or mechanical stimulus, or other types of stimuli or a combination of stimuli. In some cases, the stimulus includes a chemical stimulus, such as a change in pH, the application of an acid or base, the addition of a solubilizer, an initiator, a free radical generator, a reducing agent, etc. In some cases, the chemical stimulus includes applying a Lewis acid (e.g., boron trifluoromethanesulfonate, boron trifluoride etherate, boron trichloride, boron tribromide, boron triiodide, or scandium trifluoromethanesulfonate). In some cases, the stimulus includes a biological stimulus, such as an enzyme (e.g., Edmanase, protease, endonuclease) or a ribozyme or deoxyribozyme that can cut or catalyze the cutting of a monomer from a polymer analyte.

[0150] In some examples, the polymer analyte includes a peptide and the monomer includes an amino acid (e.g., NTAA, CTAA or internal amino acid). The method can include using a joint comprising an amino acid reactive group (e.g., PITC) with an amino acid by coupling the amino acid reactive group of the joint, and using stimulation (e.g., pH, temperature change) to cut amino acids from the peptide. In an example, PITC can be coupled to NTAA under mild alkaline conditions to generate phenylthiocarbamoyl (PTC) derivatives of NTAA, and Edman degradation reaction (e.g., applying acid such as trifluoroacetic acid or trifluoromethanesulfonic acid boron, optionally with heating) can be used to achieve cutting NTAA from the peptide to produce thiazolinone (ATZ) derivatives or phenylthiohydantoin (PTH) derivatives. As described elsewhere herein, the joint can include a part or molecule (e.g., a nucleic acid molecule or a polymerizable molecule), which can also be coupled to the capture portion so that amino acids can be coupled to the capture portion.

[0151] In some cases, each cutting event can be from polymer analyte cutting more than one monomer.Cutting can comprise cutting 2 monomers, 3 monomers, 4 monomers, 5 monomers, 6 monomers, 7 monomers, 8 monomers, 9 monomers, 10 monomers or more monomers.For example, polymer analyte can comprise the peptide comprising multiple amino acid monomers, and can cut single amino acid, dipeptides, tripeptides, tetrapeptides or larger peptide with method described herein.In some cases, can cut 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 monomer less in given cutting event.In some cases, can use can identify or cut more than single amino acid enzyme (for example, Edmanase, protease) or ribozyme or deoxyribozyme to mediate the cutting of more than one monomer (for example, amino acid).

[0152] Cleavage of the monomer (or monomers) can be performed using a biological stimulus such as an enzyme or a ribozyme or a deoxyribozyme. The enzyme can be any useful cleavage enzyme, for example, a protease such as Edmanase, cruzain, cleavage proteins (e.g., ClpS, ClpX), proteinase K, exopeptidase, aminopeptidase, diaminopeptidase, serine protease, cysteine ​​protease, threonine protease, aspartic protease, aspartic protease, glutamic protease, metalloprotease, asparagine peptide cleavage enzyme, pepsin, trypsin, pancreatin, Lys-C, Glu-C, Asp-N, chymotrypsin, carboxypeptidase (e.g., carboxypeptidase A, carboxypeptidase B, carboxypeptidase Y), SUMO protease, elastase, papain, endoprotease, protease, Bromelain, collagenase, hyaluronidase, thermolysin, ficin, keratinase, tryptase, fibroblast activation, enterokinase, chymotrypsinogen, chymase, clostripain, calpain, alpha-lytic protease, proline-specific endopeptidase, furin, thrombin, subtilisin, genenase, PCSK9, cathepsin, aminoacylproline dipeptidase, methionine aminopeptidase, cathepsin C, 1-cyclohexen-1-yl-boronic acid pinacol ester, pyroglutamate aminopeptidase, renin, kininogen, kallikrein, DPPIV / CD26, thimet oligopeptidase, prolyl oligopeptidase, leucine aminopeptidase, dipeptidyl peptidase, or other enzymes or proteases, or combinations or variants thereof (e.g., engineered mutants or variants). In some cases, the cleavage enzyme or ribozyme or deoxyribozyme can be configured or engineered to cleave a terminal monomer or multiple monomers; alternatively, the cleavage enzyme or ribozyme or deoxyribozyme can be configured or engineered to perform exo-site cleavage at a non-terminal position of the polymer analyte, for example, at an internal monomer within the polymer analyte, at positions n-1, n-2, n-3, n-4, n-5, n-6, n-7, n-8, n-9, n-10, etc., where n is the number of monomers in the polymer analyte.

[0153] In the case of enzymatic cleavage, additional reagents can be provided to catalyze or induce cleavage. For example, metalloproteinases, aminopeptidases, or exopeptidases can promote the cleavage of an amino acid or multiple amino acids in the presence of a catalyst (e.g., a metal or metal ion (e.g., cobalt)). Thus, a catalyst can be provided to promote the binding of the enzyme to the amino acid or the subsequent cleavage of the amino acid from the peptide. In some examples, cleavage can be mediated by an apoenzyme that is inactive in the absence of a metal catalyst of a cofactor, and cleavage can be controlled by the addition of a metal or metal ion.

[0154] Other examples of cleavage stimuli include: optical stimulation (e.g., application of UV, X-rays, gamma rays, or other wavelengths of light), mechanical stimulation (e.g., sonication, high voltage, electromagnetic energy), thermal stimulation (e.g., application of heat), or chemical stimulation. In some cases, the polymer analyte may contain or be altered to contain a cleavable or labile bond that can be cleaved upon application of an appropriate stimulus, such as a disulfide bond (e.g., cleavable upon application of a chemical stimulus such as a reducing agent), an ester bond (e.g., cleavable with a change in pH), a vicinal diol bond (e.g., cleavable with sodium periodate), a Diels-Alder bond (e.g., cleavable upon application of heat), a sulfone bond (e.g., cleavable via a base), a silyl ether bond (e.g., cleavable via an acid), a glycosidic bond (e.g., cleavable via an amylase), a peptide bond (e.g., cleavable via a protease), or a phosphodiester bond (e.g., cleavable via a nuclease (e.g., DNAse)).

[0155] Modification of Monomers: In some cases, one or more monomers of a polymeric analyte can be modified. The modification can be naturally occurring (e.g., post-translational modification) or non-naturally occurring, such as via labeling or tagging, for example, with an amino acid or amine reactant such as an isothiocyanate (e.g., PITC, NITC), 1-fluoro-2,-4-dinitrobenzene (DNFB), dansyl chloride, 4-sulfonyl-2-nitrofluorobenzene (SNFB), an acetylating agent, an acylating agent, an alkylating agent, a guanidating agent, a thioacetylating agent, a thioacylating agent, a thenoylating agent, or a derivative or combination thereof. Alternatively or additionally, one or more monomers can be modified to include 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 cases, the monomers of the polymer analyte can be modified to promote the recruitment of an enzyme (or ribozyme or deoxyribozyme) to recognize or cut the terminal monomer (e.g., 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 a set of monomers. For example, the terminal amino acid of a peptide analyte can be modified with a sugar to recruit a lectin or a lectin-binding protease. In another example, one or more monomers of a polymer analyte can comprise or be coupled to a nucleic acid molecule having a first sequence that is complementary to a second sequence comprised by an oligonucleotide-binding protease. Hybridization of the first sequence with the second sequence can promote local recruitment of the protease to the monomer to be cut. In another example, a peptide analyte can be modified with PITC, which can allow recruitment and cutting by Edmanase. In some examples, the modification of the monomers of the polymer analyte can include an epitope tag that can promote the binding of a binding agent to the monomer. Examples of such epitope tags include fluorophores, nucleic acid molecules, peptides, haptens, polymers, chemical moieties, or other adduct molecules. Other examples of polymer analyte modifications are described elsewhere herein.

[0156] Polymer analytes can include one or more modified monomers. The modification of the monomer can be naturally occurring or synthetic. Synthetic modification can be carried out before, during or after cutting the monomer from the polymer analyte, and can be beneficial to maintaining the identity of the monomer. For example, during the standard Edman degradation reaction of cutting the terminal amino acid (monomer) from the peptide, some amino acid residues may be changed or become undetectable by the reaction conditions. In one example, the conditions of Edman degradation can cause oxidation of cysteine ​​residues, dehydration or destruction of serine or threonine in the form of phenylthiohydantoin (PTH), react with lysine residues and modify lysine residues or make some post-translational modifications undetectable. Therefore, modifying the peptide before processing, for example protecting some amino acid residues or post-translational modifications, can be useful in more accurately identifying each amino acid residue. In some examples, the peptide or portion thereof can be alkylated, for example, alkylation of cysteine ​​residues (e.g., using 4-vinylpyridine, iodoacetamide, iodoacetyl esters, chloroacetate); acetylated, for example, reacting serine or threonine residues to form esters (e.g., using acetyl chloride) or using acetic anhydride; oxidized, for example, converting cysteine ​​residues to cysteic acid; reduced (e.g., using a reducing agent such as dithiothreitol, β-mercaptoethanol, or TCEP); contacted with a protecting group, for example, phosphorylated residues can be protected (e.g., using β-elimination of the phosphate group, Michael addition with an optional sulfhydryl group, e.g., as described in Knight, et al. Nat. Biotechnology. 21, 1047-1054 (2003), which is incorporated herein by reference in its entirety), etc. The polymeric analyte or monomer can be modified with a protecting group or moiety, such as methyl, formyl, ethyl, acetyl, tert-butyl, anisyl, benzyl, trifluoroacetyl, N-hydroxysuccinimide, tert-butyloxycarbonyl (Boc), benzoyl, 4-methylbenzyl, thioformyl, thiocresol, benzyloxymethyl, 4-nitrophenyl, benzyloxycarbonyl, 2-nitrobenzoyl, 2-nitrobenzenesulfinyl, 4-toluenesulfonyl, pentafluorophenyl, diphenylmethyl, 2-chlorobenzyloxycarbonyl, 2,4,5-trichlorophenyl, 2-bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC), trityl, or 2,2,5,7,8-pentamethylchroman-6-sulfonyl. Polymer analytes or monomers can be treated with protecting agents such as carboxyethyl methanethiosulfonate (CEMTS), thiazolidine, mercaptophenylacetic acid, cyanobenzothiazole (e.g., for lipidation of N-terminal cysteine), acetamidomethyl, 2-methylsulfonylethyloxycarbonyl, etc. In some cases, lysine residues can be blocked using isothiocyanates (e.g., PITC) (e.g., the primary amine of the lysine residue can react); optionally, additional rounds of Edman degradation can be performed to generate new N-terminal exposed ends.

[0157] In some cases, the monomer of polymer analyte can be modified to promote the cutting monomer from polymer analyte.For example, the amino acid monomer of peptide polymer analyte can be modified so that it is recognized by enzyme or ribozyme or deoxyribozyme.In some examples, such modification can include amino acid acetylation, which can promote the acyl peptide hydrolase cutting of acetylated amino acids.Additional or alternative modification to monomer, such as those described herein, can also promote the identification by engineered cutting enzyme, ribozyme or deoxyribozyme or interact with engineered cutting enzyme, ribozyme or deoxyribozyme.

[0158] In some cases, the monomer comprising a naturally occurring modification can be treated to remove or change the naturally occurring modification so that the polymer analyte or monomer is more suitable for processing operations disclosed herein. For example, acetylation, formylation, methylation and pyrrolidonecarboxylic acid post-translational modification can be removed before sequencing. Acetylation modification can be removed with acyl peptide hydrolase or acid treatment (for example, using 1N HCl). Methylation can be removed, for example, using aminopeptidase. Formylation modification can be removed, for example, using acid treatment (for example, 0.6M HCl treatment). Pyrrolidonecarboxylic acid (PCA) can be removed with pyroglutamate aminopeptidase. Examples of C-terminal modifications can include amidation and methylation, both of which can be removed using carboxypeptidase.

[0159] Binder: The binder can be contacted with the monomer, for example, after the monomer is coupled to the capture portion and cleaved. The binder can be any useful molecule that can be coupled to the monomer or the monomer-capture portion complex. For example, the binder can 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, fibronectin domain), a simulated peptide, a peptide mimetic (e.g., a peptidomimetic, β-peptide, D-peptide peptide mimetic), a polysaccharide, a nucleic acid molecule (e.g., an aptamer), a somamer, a polymer, an inorganic compound, an organic compound, a small molecule, or a derivative thereof (e.g., an engineered variant), or a combination. In the case where the polymer analyte includes a peptide, the binder can be capable of binding to a modified amino acid (e.g., an amino acid coupled to a linker) or a portion thereof. The binder can comprise a recognition site that specifically recognizes an amino acid, a modified amino acid (e.g., an amino acid bound to a linker comprising a PITC portion), or a derivatized and optionally modified amino acid. For example, the binding agent can be configured to recognize or have binding specificity for a portion of a modified amino acid, such as a specific amino acid residue, a residue-linker complex, or a derivatized amino acid (e.g., a thiocarbamoyl-derived residue, a thiazolone-derived residue, a thiohydantoin-derived residue, etc.) or a portion of a modified amino acid. In some cases, the binding agent can recognize and bind to a cleaved or modified amino acid, but not to an amino acid when present in a peptide. In some cases, the binding agent can be derived from or engineered from a naturally occurring enzyme or protein, such as an aminopeptidase, an exopeptidase, a metalloprotease, an antibody, an anticalin, an N-recognition protein, a Clp protease, an endoprotease (e.g., trypsin), or a tRNA synthetase. In some examples, the binding agent can be or comprise a cleavage enzyme (e.g., trypsin, endoprotease) that has been modified to remove peptidase activity. The binding agent can also recognize the terminal amino acid attached to the substrate; for example, after all monomers of the polymer analyte except the final monomer have been coupled to the capture moiety or multiple capture moieties and cleaved, the final monomer can remain coupled to the substrate. Thus, the binding agent can recognize and bind to the surface-coupled monomer.

[0160] Binding agent can be contacted with and specifically binds to the cleaved monomer, monomer-joint complex, monomer-joint-capture moiety or monomer-capture moiety complex (collectively referred to herein as "monomer analyte"). For example, the monomer analyte can fall into any size or size range that is less than the entire polymer analyte. The size of the monomer analyte complex can be about 0.1 nanometer (nm), about 0.5 nm, 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 micron (μm), about 10 μm, about 100 μm, about 1 millimeter mm or larger. The monomer analyte can have any molecular weight or molecular weight range. Monomeric analytes can 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 molecular weight or length of monomeric analytes can vary, for example, depending on the amino acid residues.

[0161] Activator: A binding agent can comprise an activator or be coupled (e.g., directly or indirectly) to an activator. An activator can facilitate the coupling of a polymerizable molecule (e.g., a first polymerizable molecule) to a second polymerizable molecule, the first polymerizable molecule comprising identification information for the binding agent or a monomer (e.g., a monomer type such as an amino acid type). Activators can include any useful chemical or biological moiety, such as a peptide or protein (e.g., an enzyme), a ribozyme, a deoxyribozyme, a nucleic acid molecule, a lipid, a carbohydrate, a free radical or free radical generator, a catalyst, an ion, a small molecule, a mineral, a metal, a salt, a nucleophile, an electrophile, an oxidizing agent, a reducing agent, an acid, a base, or other reagent.

[0162] In some cases, the activator comprises a polymerizable molecule. The activator can include any useful type of polymerizable molecule (also referred to herein as an "activating polymerizable molecule"), such as a nucleic acid molecule, a peptide, or a protein (e.g., an enzyme). The activating polymerizable molecule can be the same type of molecule as the binding agent (e.g., both are peptides, both are nucleic acid molecules, etc.), or they can be different. In some cases, the binding agent comprises a peptide (e.g., an antibody or antibody fragment) and the activating polymerizable molecule comprises a nucleic acid molecule. The activated polymerizable molecules can be conjugated to the binding agent via chemical conjugation methods, for example, using linkers such as SMCC, (Ne-maleimidocaproyloxy) succinimide ester (EMCS), succinimidyl-4-(p-maleimidophenyl) butyrate (SMPB), succinimidyl-(N-maleimidopropionamido-ethylene glycol) ester (SMPEG), succinimidyl (NHS) esters, succinimidyl-4-formylbenzamide (S-4FB), succinimidyl-6-hydrazino-nicotinamide (S-HyNic), 4-phenyl-3H-1,2,4-triazolidine-3,5(4H)dione (PTAD) or other diazonium salts, 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride (EDC), etc. The synthesis of binder-activated polymerizable molecule conjugates (e.g., peptide-nucleic acid molecule conjugates) can also be performed using solid phase synthesis, fragment conjugation (e.g., using a heterobifunctional cross-linker such as a cross-linker comprising an aliphatic chain and a maleimide group on one end and NHS on the other end), click chemistry (e.g., strain-promoted azide-alkyne cycloaddition, reverse electron demand Diels-Alder reaction), or a combination of methods or chemistries. In some cases, enzymatic methods can be used to conjugate the activated polymerizable molecule to the binder. For example, DNA-protein conjugates can be generated using truncated nucleases (e.g., Cas proteins such as Cas9), relaxases (e.g., VirD2), or other enzymes, ribozymes, or deoxyribozymes. In some cases, the activated polymerizable molecule can be conjugated to the binder using SpyTag and SpyCatcher interactions, biotin-avidin interactions, SNAP-tags, or other interactions. Optional purification can be performed, for example, using ion exchange chromatography, affinity chromatography, high performance liquid chromatography, electrophoresis, electrofocusing or other purification or separation techniques.

[0163] In some cases, the activated polymerizable molecule is a nucleic acid molecule, which can facilitate the coupling of one or more other nucleic acid molecules. In one example, the first polymerizable molecule, the second polymerizable molecule, and the activated polymerizable molecule can all include nucleic acid molecules. The first polymerizable molecule can include a nucleic acid barcode molecule that encodes or contains identification information about the binding agent or monomer. The activated polymerizable molecule can be a splint oligonucleotide that can hybridize with the first polymerizable molecule and the second polymerizable molecule. In such examples, the activated polymerizable molecule includes a sequence that is complementary to at least a portion of the first polymerizable molecule and the second polymerizable molecule. In another example, the first polymerizable molecule is not complementary to the activated polymerizable molecule or is otherwise coupled; in such cases, additional splint oligonucleotides may be required to enable the activated polymerizable molecule to be coupled to the first polymerizable molecule, for example, via providing a splint oligonucleotide that includes a sequence that is complementary to at least a portion of the activator, the first polymerizable molecule, and an optional second polymerizable molecule.

[0164] In some cases, the first polymerizable molecule comprises a nucleic acid barcode molecule, and the methods described herein can include generating the nucleic acid barcode molecule. The nucleic acid barcode molecule can be generated using an activator (e.g., an activating polymerizable molecule) as a starting nucleic acid molecule. In one such example, the activator comprises a nucleic acid molecule, and subsequent barcode sequence segments can be added to the activator, for example, using a hybridization chain reaction (HCR, e.g., Figure 1C In some cases, the first polymerizable molecule can include a barcode sequence segment and a first polymerizable molecule. In some cases, the first polymerizable molecule can include a barcode sequence segment and a first polymerizable molecule. In some cases, the first polymerizable molecule can include a barcode sequence segment and a first polymerizable molecule. In some cases, the first polymerizable molecule can include a barcode sequence segment of any useful number, and can include additional functional sequences, such as primer sites, sequencing sites, restriction or cleavage sites, spacers, atypical nucleic acids, HNA, PNA, etc. In some cases, the binding agent can include an HCR component (for example, a first hairpin molecule), which can be coupled to an activator provided separately. Therefore, the activator can be coupled to the binding agent via the HCR reaction. In some cases, the first polymerizable molecule can include a flap sequence, which can promote the coupling of the first polymerizable molecule with the second polymerizable molecule or other polymerizable molecules.

[0165] In some cases, the activator can include a nucleic acid molecule that indirectly promotes the coupling of the first polymerizable molecule to the second polymerizable molecule. In an example, the binding agent can be coupled to a first polymerizable molecule (e.g., a nucleic acid barcode molecule) and an activating nucleic acid molecule. The activating nucleic acid molecule can serve as an anchoring nucleic acid molecule and couple to an additional polymerizable molecule (e.g., on a substrate comprising a plurality of identical polymerizable molecules, including a second polymerizable molecule). The coupling of the activating nucleic acid molecule to the additional polymerizable molecule can help anchor the binding agent to the monomer, thereby promoting the transfer or coupling of the first polymerizable molecule to the second polymerizable molecule (see, e.g., Figure 1E In some cases, the anchoring nucleic acid molecule increases the residence time of the binding agent on the monomer, thereby increasing the probability of the first polymerizable molecule coupling to the second polymerizable molecule (or other polymerizable molecules).

[0166] Alternatively or additionally, the activator can include a polymerizable molecule comprising a peptide or protein, such as an enzyme. The enzyme can directly or indirectly promote the coupling of the first polymerizable molecule to the second polymerizable molecule. The enzyme can be a ligase (e.g., a ligase such as T4 or T7 DNA ligase, a cyclizing ligase (Circleigase), Taq ligase, SplintR ligase, etc.), a polymerase (e.g., DNA or RNA polymerase, reverse transcriptase), a kinase (e.g., a protein kinase, a lipid kinase, a nucleic acid kinase such as polynucleotide kinase (PNK)), a phosphatase (e.g., an alkaline phosphatase), a free radical generator or an oxidase such as horseradish peroxidase (HRP) or ascorbate peroxidase (APEX), a nuclease (e.g., an endonuclease, a restriction enzyme, a Cas enzyme), a phosphorylase, an ATPase or a combination of other enzymes or enzymes. In some cases, the enzyme is a cleavage enzyme, e.g., a glycosylase (e.g., uracil glycosylase), a restriction endonuclease, a micrococcal nuclease, a transposase, a Cas protein (e.g., Cas9), an Argonaut endonuclease. In one such example, the second polymerizable molecule can comprise a cleavage or restriction site, and coupling of the first polymerizable molecule can occur only when the cleavage or restriction site is cleaved or removed.

[0167] The binding agent can be coupled to the activator (e.g., a polymerizable molecule such as a nucleic acid molecule or an enzyme) via non-covalent interactions. For example, the binding agent can include an avidin or streptavidin tag to which a biotin-conjugated polymerizable molecule can be bound. Alternatively, the binding agent can include a biotin tag to which avidin or streptavidin-conjugated polymerizable molecule can be bound. Alternatively, the binding agent can be covalently coupled to the activator, for example, using a chemical conjugation method as described herein or via a protein engineering method, such as generating a fusion protein comprising a polymerizable molecule (e.g., an enzyme) and a binding agent (e.g., an antibody, an antibody fragment, a nanobody), a SNAP tag and a SNAP-tag or other attachment or coupling strategy.

[0168] In some cases, the binding agent includes a first polymerizable molecule that contains identification information of the binding agent. For example, the first polymerizable molecule may include a nucleic acid barcode molecule that contains a barcode sequence. The barcode sequence can encode the identity of the binding agent or a homologous molecule. For example, a monomer (e.g., amino acid) of a polymer analyte (e.g., a peptide containing multiple amino acids) can be cut and coupled to a capture portion (e.g., on a substrate) and can be contacted with a binding agent (e.g., an antibody, an antibody fragment, a nanobody). The binding agent can specifically recognize an amino acid residue or its derivative (e.g., a form derived from PTH, PTC, or ATZ) relative to other amino acid residues or their derivatives. The nucleic acid barcode molecule can contain information for identifying the binding agent, which can also identify specific amino acid residues (or derivatives) due to the specificity of the binding agent for its target. Alternatively, the binding agent may not include a first polymerizable molecule that contains identification information of a binding agent or a homologous molecule (e.g., a monomer).

[0169] For a single coupling event of the first and second polymerizable molecules, more than one binding agent can be used. In some cases, multiple binding agents (for example, multiple monoclonal antibodies or antibody fragments, multiple polyclonal antibodies or antibody fragments) can be bound to a monomer analyte. In some cases, a first binding agent and a second binding agent that is bound to the first binding agent are used. In some examples, after the binding agent is combined with the monomer-capture part complex, it is possible to make other molecules (for example, second binding agent) comprising a detectable marker such as a fluorophore, a radioisotope, a mass label, an activator or an identification polymerizable molecule (for example, a nucleic acid barcode molecule) contact and combine with the binding agent that is bound to the monomer-capture part complex. In some examples, other molecules include identifying polymerizable molecules, and identifying that polymerizable molecules can be coupled or transferred to the second polymerizable molecule. In one non-limiting example, the binding agent includes a first antibody or antibody fragment (e.g., scFv or nanobody) that recognizes a monomeric analyte, such as a monomer-capture moiety complex (e.g., a terminal amino acid-linker-capture moiety complex) or a portion thereof (e.g., a terminal amino acid or a terminal amino acid-linker complex); after the first antibody or antibody fragment is bound to the monomer-capture moiety complex or a portion thereof, a second antibody or antibody fragment comprising or coupled to a polymerizable molecule (e.g., a nucleic acid barcode molecule) is coupled to the first antibody. The polymerizable molecule of the second antibody or antibody fragment can contain information about the second antibody or antibody fragment or cognate molecule, the first antibody or antibody fragment or cognate molecule, or other information. The transfer or coupling of the polymerizable molecule of the second antibody or antibody fragment to the additional polymerizable molecule can be mediated by any suitable technique, for example, hybridization of nucleic acid molecules, optionally mediated by a splint molecule, click chemistry, or association of high affinity molecules (e.g., streptavidin and biotin). Alternatively or additionally, the first or second binding agent may include an activator, for example, to activate a polymerizable molecule such as a nucleic acid molecule or an enzyme, as described elsewhere herein.

[0170] The binding agent can contain or be coupled to any useful number of polymerizable molecules or activators. The binding agent can contain or be coupled to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more polymerizable molecules or activators. The binding agent population of each binding agent can contain or be coupled to an average of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more polymerizable molecules or activators. The activator can contain or be coupled to a range of polymerizable molecules or activators, for example, 1 to 15 polymerizable molecules, about 3 to 6 polymerizable molecules, about 15 to 50 polymerizable molecules, etc.

[0171] In some cases, the binding agent comprises an activated polymerizable molecule that comprises additional multiplexing information. For example, the activated polymerizable molecule, such as a nucleic acid molecule, can comprise a sequence encoding a cycle or other time information or spatial information. In one such example, an array of peptides and capture moieties can be provided on a substrate. The array can comprise a plurality of individually addressable units, wherein each (or subset) individually addressable unit of the array comprises a peptide to be analyzed and a capture moiety. The binding agent and the activated polymerizable molecule contained therein or coupled thereto can comprise spatial information (e.g., a spatial barcode sequence) that uniquely identifies the individually addressable unit, thereby identifying the position of the array. The activated polymerizable molecule of the binding agent can additionally comprise time information, for example, a cycle barcode indicating the round or iteration in which the binding agent or activated polymerizable molecule is provided. Subsequently, sequencing of the activated polymerizable molecule can be used to reveal spatial information (e.g., the origin position in a peptide or amino acid array). In some cases, the activated polymerizable molecule can comprise a unique molecular identifier (UMI), which can be used to determine the amount of a given binding agent or monomer (e.g., amino acid) of a given peptide, substrate, array or sample.

[0172] Alternatively or additionally, the first polymerizable molecule comprising coded information about the type of binding agent or monomer can comprise multiplexing information. For example, the first polymerizable molecule can comprise a sequence encoding a cycle or other temporal information or spatial information (e.g., a spatial barcode sequence) that uniquely identifies a position or individually addressable unit (e.g., of an array). The first polymerizable molecule can additionally comprise temporal information, for example, a cycle barcode indicating the round or iteration in which the binding agent is provided or the polymerizable molecule is activated. Subsequent sequencing of the first polymerizable molecule can be used to reveal spatial information (e.g., position of origin in a peptide or amino acid array) or temporal information. In some cases, the first polymerizable molecule can comprise a unique molecular identifier (UMI), which can be used to determine the amount of a given binding agent or monomer (e.g., amino acid) for a given peptide, substrate, array, or sample. The first polymerizable molecule can comprise any additional useful functional sequences, such as spacers, atypical nucleic acids, restriction sites, sequencing primers, read primers, primer sites or primer binding sites, transposase sites, etc., as described elsewhere herein.

[0173] In some cases, the method may include contacting the monomer-capture moiety complex with a binding agent library. The binding agent library may include a variety of binding agents that are specific for different analytes. For example, the binding agent library may include multiple binding agents that recognize different amino acids or derivatives thereof (e.g., derived amino acids such as PTH, PTC, or ATZ forms), amino acid clusters (e.g., dipeptides, tripeptides, etc.), or amino acid combinations (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 example, a given binding agent can bind to amino acids with similar residues, such as amino acids with positively charged side chains (e.g., arginine, histidine, lysine), amino acids with 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, tryptophan), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine), hydroxyl or sulfur- or selenium-containing side chains (e.g., serine, cysteine, selenocysteine, threonine, methionine), aromatic side chains (e.g., phenylalanine, tyrosine, tryptophan), basic side chains (e.g., histidine, lysine, arginine), acidic side chains (e.g., aspartic acid, glutamic acid, asparagine, glutamine), or combinations thereof. In general, a library of binders can specifically recognize or bind to any number of different amino acids; for example, a library of binders can be configured to specifically 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 at least 20 different proteinogenic amino acids or derivatives thereof.

[0174] The binding agent library can include any useful number of binding agents, each of which can have different binding specificities. For example, the first binding agent can recognize one amino acid, and the second binding agent can recognize two amino acids, and the third binding agent can recognize three amino acids. In another example, the first binding agent can recognize one amino acid, the second binding agent can recognize different amino acids, and the third binding agent can recognize multiple amino acids. It will be understood that any number of binding agents can be provided, and each binding agent can be specific for one or more amino acids. In short, the binding agent library can be bound to all 20 protein amino acids or derivatives thereof, or a subset of amino acids (e.g., 10 or more, 15 or more). In the case of using the first and second binding agents (e.g., a second binding agent that is bound to the first binding agent), the first binding agent can be derived from different species such as rabbit, mouse, goat, chicken, llama, etc., and the second binding agent can selectively bind to the first binding agent (e.g., anti-rabbit, anti-mouse, anti-goat, anti-chicken, anti-llama antibodies).

[0175] In some cases, the inactivation of the binder or substrate can be performed before or during contact with the cleaved monomer. Inactivation can be achieved using a blocking agent or solution, such as a milk protein (e.g., lactoglobulin, lactalbumin, lactoferrin, casein, whey, immunoglobulins, insulin, growth factors, osteopontin), albumin (e.g., bovine serum albumin), Tween 20, a commercially available blocking solution, or a combination thereof. Alternatively or additionally, inactivation can be performed using a polymer (e.g., polyethylene glycol), an organic compound (e.g., oil, lipid), a sugar, a nanoparticle, an inorganic compound, an ion, or the like.

[0176] Coupling of polymerizable molecules: polymerizable molecules (e.g., a first polymerizable molecule and a second polymerizable molecule) can be coupled to each other using any useful method. Such coupling can include covalent interactions or non-covalent interactions (e.g., ionic interactions, hydrophobic interactions, van der Waals forces, etc.). In some cases, the first polymerizable molecule and the second polymerizable molecule include nucleic acid molecules and can be coupled via hybridization, connection, or both. For example, the first polymerizable molecule can include a first sequence that is complementary to the second sequence of the second polymerizable molecule, and the coupling can occur via hybridization of the first sequence with the second sequence. Alternatively, the first sequence and the second sequence may not be complementary to each other, but may be complementary to the third sequence and the fourth sequence of the splint or bridging oligonucleotide, respectively. Therefore, the coupling of the first polymerizable molecule to the second polymerizable molecule can be mediated by hybridization of the first and second sequences with the third and fourth sequences of the splint or bridging oligonucleotide, respectively.

[0177] In some cases, nucleic acid reaction can be carried out as a part of the coupling of the first polymerizable molecule and the second polymerizable molecule or in addition thereto. For example, the first sequence of the first polymerizable molecule can be hybridized with the second sequence of the second polymerizable molecule, and nucleic acid extension reaction (for example, using polymerase) can be carried out. Such extension reaction can allow the coded information of one (for example, the first polymerizable molecule) in the polymerizable molecule to be transferred to another polymerizable molecule (for example, the second polymerizable molecule). In another example, the first sequence of the first polymerizable molecule can be connected to the second sequence of the second polymerizable molecule, to provide the first polymerizable molecule covalently coupled to the second polymerizable molecule. In one such example, the first sequence can be hybridized with the first splint sequence of the splint oligonucleotide, and the second sequence can be hybridized with the second splint sequence of the splint oligonucleotide. Connection can be carried out so that the first sequence is covalently coupled to the second sequence. In some cases, extension reaction and ligation reaction can be carried out, so that information is coupled or transferred to the second polymerizable molecule from the first polymerizable molecule. Optionally, after coupling, polymerizable molecule can be decoupled, for example, via denaturation or cutting at the cleavage site.

[0178] Polymerizable molecules can be chemically coupled covalently or non-covalently. In some cases, the first polymerizable molecule can be chemically coupled to the second polymerizable molecule. For example, the first polymerizable molecule can include a first reactive moiety, and the second polymerizable molecule can include a second reactive moiety that can react with the first reactive moiety. The first reactive moiety can be contacted with the second reactive moiety and subjected to conditions sufficient to couple the first reactive moiety to the second reactive moiety, for example, via click chemistry. In other cases, the first polymerizable molecule can be coupled to the second polymerizable molecule via non-covalent or indirect interactions (for example, biotin-streptavidin).

[0179] In some cases, for example, the first polymerizable molecule comprising the identification information of a monomer (e.g., monomer identity such as amino acid type) or a binding agent can be coupled to other polymerizable molecules. For example, a substrate can include a polymer analyte and a capture portion coupled thereto, together with a plurality of additional polymerizable molecules. After the monomer is coupled to the capture portion and the monomer is cut from the polymer analyte, the monomer can be contacted with the same or different binding agents any number of times. The first polymerizable molecule of the binding agent can be iteratively contacted and coupled to any number of additional polymerizable molecules for repeated inquiries; for example, the first polymerizable molecule can be coupled to a first additional polymerizable molecule, as described herein, and then subsequently cut or removed (e.g., via dehybridization, enzymatic cutting), and contacted and coupled with a second additional polymerizable molecule. Such methods can be advantageous for transferring several copies of the first polymerizable molecule to a substrate, for example, to increase sensitivity. Alternatively or additionally, the first polymerizable molecule can be iteratively coupled to the same first additional polymerizable molecule, for example, to generate a stacked polymerizable molecule of multiple copies of the polymerizable molecule comprising a binding agent.

[0180] Alternatively or additionally, a plurality of first polymerizable molecules can be provided and coupled to additional polymerizable molecules. The plurality of first polymerizable molecules can include, for example, a plurality of nucleic acid barcode molecules comprising the same sequence. The plurality of first polymerizable molecules can be coupled to additional polymerizable molecules, which can improve the sensitivity of monomer detection (e.g., by having multiple detectable copies of the first polymerizable molecule). In some cases, the plurality of first polymerizable molecules include a plurality of nucleic acid barcode molecules with different sequences. In such cases, the different sequences can represent different monomer types (e.g., 20 proteinogenic amino acids, post-translational modifications, etc.). The different sequences can also include unique priming regions, for example, for annealing or coupling to an activator (e.g., coupling to a binding agent), which also includes a unique priming sequence. For example, each binding agent type having specificity for one or more monomers can include an activator with a unique priming sequence; when providing a plurality of first polymerizable molecules with different sequences, the plurality of first polymerizable molecules can be selectively bound to different binding agents based on the matching or complementarity of the unique priming sequences.

[0181] Uncoupling monomers, binding agents: In some cases, after the first polymerizable molecule is coupled to the second polymerizable molecule, the monomer can be uncoupled from the monomer-capture moiety complex or substrate. Uncoupling can be performed chemically, mechanically or enzymatically. For example, a monomer can be coupled to the capture moiety via a connecting nucleic acid molecule (e.g., a joint comprising a monomer reactive group and a connecting nucleic acid molecule). The connecting nucleic acid molecule can include a cleavage site, such as a restriction site, and can be uncoupled by enzymatic cleavage at the cleavage site using, for example, a restriction endonuclease. Alternatively or additionally, the capture moiety or any polymerizable molecule can include a cleavage site that allows the monomer to be uncoupled from a portion of the capture moiety. In a non-limiting example, examples of cleavage enzymes include glycosylases (e.g., uracil glycosylases), restriction endonucleases, micrococcal nucleases, transposases, Cas proteins (e.g., Cas9), Argonaut endonucleases, etc. Beneficially, removing the monomer from the monomer-capture moiety complex can allow the capture moiety to be available for subsequent reactions or iterations, or prevent additional binding agents (e.g., in a single cycle or subsequent iterations) from binding to the capture moiety, which can help reduce erroneous or repeated coupling of the polymerizable molecules of the additional binding agents to the capture moiety. Alternatively or additionally, uncoupling can occur using a stimulus (e.g., a light stimulus (such as UV, γ, X-ray irradiation), a thermal stimulus, a chemical stimulus, etc.). In some cases, the linker can comprise a cleavable group, and application of an appropriate stimulus can result in cleavage of the linker, as described elsewhere herein.

[0182] Alternatively or additionally, the monomer can be altered so that it cannot be detected by the binding agent, for example, to prevent the binding agent from binding to the cleaved monomer in subsequent iterations of cleavage, coupling to the capture moiety, and contact with the additional binding agent. For example, the monomer can be contacted with a blocking agent or derivatized so that the binding agent no longer recognizes the derivatized form. Such blocking strategies can be useful in eliminating the need to detect or transfer information from the polymerizable molecule to which the binding agent is coupled, thereby removing the need for the cleaved monomer. Other strategies for inhibiting binding of the binding agent to the cleaved monomer are described elsewhere herein.

[0183] Similarly, in some cases, the binding agent can be removed from the monomer-capture moiety complex by any useful or convenient operation, such as after the polymerizable molecule is coupled. Removal of the binding agent can be performed using chemical or enzymatic methods, such as using chemical denaturants, detergents, acidic or alkaline conditions, heat or proteases. Alternatively or additionally, if the polymerizable molecule is coupled to the binding agent, the polymerizable molecule can be removed from the binding agent, such as via cleavage or restriction sites and the use of a cleavage enzyme (e.g., UDG, restriction enzyme), chemical cleavage, photolysis or other methods. In some cases, the polymerizable molecule is coupled to the binding agent via non-covalent interactions, such as desthiobiotin-avidin; therefore, uncoupling of the polymerizable molecule from the binding agent can be achieved by competitively replacing desthiobiotin using a competitor, such as biotin of higher affinity.

[0184] Figure 1A An example workflow for analyzing a polymeric analyte is shown. In workflow 100, a polymeric analyte 103 is provided, sequentially disassembled into individual monomers via contact with a capture moiety and cleavage, and the individual cleaved monomers are contacted with a binding agent comprising a polymerizable molecule that identifies or encodes the binding agent. The polymerizable molecule is coupled to an additional polymerizable molecule, and the monomer is cleaved from the capture moiety. Figure 1A In sub-figure A, substrate 101 is coupled to a polymer analyte 103 (e.g., a peptide to be sequenced), a capture portion 105 (e.g., a nucleic acid primer), and an additional polymerizable molecule 107 (e.g., another nucleic acid primer). In some cases, the capture portion 105 and the additional polymerizable molecule 107 are the same molecule (e.g., comprising the same sequence). The polymer analyte can be contacted with a bifunctional linker 109, which comprises an amino acid coupling group (e.g., PITC) and a click chemistry portion (e.g., an azide). In some cases, the bifunctional linker 109 is coupled to a terminal monomer (e.g., a terminal amino acid such as an N-terminal amino acid (NTAA)). Such coupling can be performed using a chemical reaction, such as a chemical reaction of PITC with a terminal amino acid to generate a phenylthiocarbamoyl-derivatized amino acid. In Figure 1A In panel B, a linker nucleic acid molecule 111 comprising a click chemistry moiety (e.g., an alkyne such as DBCO) is reacted with and covalently linked to a bifunctional linker 109. In some cases, pre-coupled linker nucleic acid molecule 111 and bifunctional linker 109 are provided (see, e.g., Figure 2 ).exist Figure 1AIn panel C, the linker nucleic acid molecule 111 is coupled to the capture moiety 105, which can be mediated by hybridization of the linker nucleic acid molecule 111 to the capture moiety 105 (hybridization not shown) or by using a splint oligonucleotide 113 comprising a sequence complementary to the sequence of the linker nucleic acid molecule 111 and the capture moiety 105. In some cases (not shown), the linker nucleic acid molecule 111 comprises a self-splint sequence, such that the linker nucleic acid molecule 111 can be coupled to the capture moiety 105 in the absence of a separate splint molecule (see Figure 24 ). A ligase can be used to covalently link the linking nucleic acid molecule 111 to the capture moiety 105. Alternatively, the linking nucleic acid molecule 111 can comprise a first reactive portion (not shown) that can react with a second reactive portion (not shown) of the capture moiety 105. Figure 1A In panel D, the system is subjected to conditions sufficient to cleave the terminal monomer (e.g., amino acid) from the polymer analyte 103 (e.g., peptide). The conditions may include performing an Edman degradation reaction, such as using acid and heat to cleave the terminal amino acid. Cleavage of the monomer from the polymer analyte results in coupling of the cleaved monomer to the bifunctional linker 109, the connecting nucleic acid molecule 111, and the capture moiety 105. Figure 1A In sub-figure E, a binding agent 115 (e.g., an antibody, antibody fragment, nanobody, scFv) comprising another polymerizable molecule 117 (e.g., a nucleic acid molecule) is provided. The binding agent 115 can specifically bind to a monomer (e.g., to a single amino acid), a monomer-linker complex (e.g., PITC-amino acid), or a monomer-linker-capture moiety complex. The polymerizable molecule 117 of the binding agent can contain identity information about the binding agent or the specific monomer (e.g., a single amino acid) to which the binding agent binds, such as a barcode sequence. The polymerizable molecule 117 of the binding agent can contain additional sequences such as UMIs, restriction sites, transposition sites, sequences representing cycle or iteration numbers, or other functional sequences, such as sequencing sites or sequences (e.g., P5 or P7 sequences), primer binding sequences, read sequences (e.g., R1 or R2 sequences). The polymerizable molecule 117 of the binding agent can be coupled to the additional polymerizable molecule 107 coupled to the substrate 101. In some cases, an extension reaction can be performed (e.g., using a polymerase) to copy the sequence of the polymerizable molecule 117 of the binding agent to additional polymerizable molecules 107 coupled to the substrate 101. Alternatively, the polymerizable molecule 117 of the binding agent can be chemically (e.g., via click chemistry) or enzymatically (e.g., using a ligase or enzyme analog such as a ribozyme or deoxyribozyme) linked to the additional polymerizable molecule 107. Figure 1AIn sub-graph F, the monomer can be decoupled (e.g., removed or cleaved) from the capture portion 105. For example, all or a portion of the monomer, bifunctional linker 109, and connecting nucleic acid molecule 111 can be cut (depicted as a star). Cutting can be performed chemically, mechanically, or enzymatically. In the example of enzymatic cutting, the connecting nucleic acid molecule 111 can contain a restriction site or other cutting site (e.g., uracil), and cutting occurs by introducing a restriction enzyme or cutting enzyme (e.g., uracil DNA glycosylase) to cut the restriction / cutting site. The workflow 100 can then be iterated or repeated to sequence all or a portion of the polymer analyte 103.

[0185] Figure 1B An example workflow for analyzing polymeric analytes using a binding agent comprising an activator (e.g., a polymerizable molecule) is schematically shown. Figure 1A Partial workflow shown in sub-figures AD, thereby generating a cleaved monomeric analyte coupled to a bifunctional linker 109, a capture moiety 105, and in some cases, a connecting nucleic acid molecule 111. A binding agent 115 comprising an activated polymerizable molecule 123 (e.g., an activating nucleic acid molecule) is contacted with the monomeric analyte and can specifically bind to the monomeric analyte (e.g., to a single amino acid) or a portion thereof, or the binding agent 115 can semi-specifically bind to multiple different monomers or portions thereof. A polymerizable molecule 119 (e.g., a nucleic acid barcode molecule) that can contain identification information (e.g., amino acid type) of the monomer can be provided and coupled to the activated polymerizable molecule 123 via hybridization (not shown) or using a splint molecule 121 that contains a sequence complementary to a portion of the activated polymerizable molecule 123, the polymerizable molecule 118, and optionally the additional polymerizable molecule 107. The polymerizable molecule 119 is optionally covalently coupled to an additional polymerizable molecule 107, such as via a linkage, and the binding agent 115 and activated polymerizable molecule 123 can be removed to repeat the workflow for additional monomers of the polymer analyte.

[0186] In some cases, the activated polymerizable molecule 123 comprises a unique initiating sequence, and the splint molecule 121 comprises a unique initiating complementary sequence that can anneal to the unique initiating sequence of the polymerizable molecule 123. Thus, by designing unique sequences of the activated polymerizable molecule 123, the splint molecule 121, and the polymerizable molecule 119 comprising the recognition information, multiplexing of multiple monomer types using multiple binding agents can be achieved.

[0187] Figure 1C-1D Another example workflow for analyzing polymeric analytes using hybridization chain reaction (HCR) to generate polymerizable molecules containing encoded information is schematically shown. Figure 1CIn panel A, a binding agent 115 is provided that is coupled to an activated polymerizable molecule (e.g., a nucleic acid molecule) comprising a first sequence 104 and a second sequence 102. Figure 1C In sub-figure B, two hairpin oligonucleotides (oligonucleotide A and oligonucleotide B) are provided. The first hairpin molecule oligonucleotide A comprises sequence 104' (complement of the first sequence 104), 102' (complement of the second sequence 102), 106, and 102 annealed to 102'. The second hairpin molecule oligonucleotide B comprises flap sequences 108, 106', 102', 104, and 102 annealed to 102'. In some cases, in the absence of an activation sequence, oligonucleotide A does not bind or hybridize to oligonucleotide B. Oligonucleotide A hybridizes with the activated polymerizable molecule through complementary base pairing to form an oligonucleotide A-activated polymerizable complex, which can trigger hybridization of oligonucleotide B. Figure 1C In panel C, oligonucleotide B is annealed to oligonucleotide A-activated polymerizable complex. In some cases, oligonucleotide A, oligonucleotide B, or both hairpin molecules contain a barcode sequence segment. After hybridization, the flap sequence 108 of oligonucleotide B contains a single-stranded overhang. Figure 1C In panel D, multiple rounds of HCR can be performed to anneal additional copies of oligonucleotide A and oligonucleotide B to generate adapter sequences of oligonucleotide A and oligonucleotide B molecules. Figure 1C In panel E, the binding agent 115 can be contacted with the monomeric analyte (e.g., as in workflow 100 and Figure 1A AD), the analyte is coupled to a capture moiety 105 on a substrate, which also comprises a plurality of additional polymerizable molecules 107. The flap sequence 108 of the adapter sequence can be coupled to one or more additional polymerizable molecules 107, for example, via hybridization or splinting. Alternatively or additionally, an extension reaction can be performed to couple oligonucleotide B to the additional polymerizable molecules 107.

[0188] like Figure 1CIn the context of the present invention, the first polymerizable molecule (for example, comprising the coding information of a binding agent or a monomer type) can refer to any single or combination of the first hairpin molecule (oligonucleotide A), the second hairpin molecule (oligonucleotide B) or other polymerizable molecules. For example, the first polymerizable molecule can include the first hairpin molecule or the first polymerizable molecule can include the second hairpin molecule. In another example, the first polymerizable molecule can include the connection sequence of the first hairpin molecule and the second hairpin molecule. Any number of different hairpin molecules can be used for HCR, such as 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 or more number of hairpin molecules. Similarly, the connection sequence of hairpin molecule can include any useful number of hairpin molecules, such as at least 2, at least 5, at least 10, at least 20, at least 50, at least 100 or more number of hairpin molecules.

[0189] As described elsewhere herein, it will be understood that the order of operations can be changed. For example, before carrying out HCR reaction, binding agent can be contacted with monomeric analytes. In another example, HCR reaction can be carried out before binding agent is contacted with monomeric analytes, for example, to generate a barcoded library of binding agent before contacting with monomeric analytes.

[0190] Figure 1D Schematically shows Figure 1C Iterations of the workflow described in . Figure 1D In sub-graph A, multiple oligonucleotide B molecules are connected via flap sequence ( Figure 1C 108) is coupled to an additional polymerizable molecule 107. Figure 1D In panel B, cleavage or removal of a portion of the oligonucleotide B molecule (e.g., sequences 102 and 106) can be performed. Optionally, monomeric analytes can also be removed from the substrate, for example, via cleavage of a linked nucleic acid molecule or capture moiety. Figure 1D In panel C, the next (e.g., n-1) monomer is locally tethered to the substrate via a capture moiety, a bifunctional linker, and a connecting nucleic acid molecule, as shown in FIG. Figure 1A As described in . Additional binding agents are provided that can recognize n-1 monomers that can be coupled to additional adapter sequences. The additional adapter sequences can include sequences that are different from the (first) binding agent. In some examples, additional adapter sequences can be generated using the same activated polymerizable molecule comprising sequences 104 and 102, oligonucleotide A and oligonucleotide C, which can include a barcode sequence segment that is different from oligonucleotide B. Like oligonucleotide B, oligonucleotide C also includes a flap sequence that enables oligonucleotide C to be coupled to a cleaved oligonucleotide B molecule. In Figure 1DIn panel D, additional adapter sequence flaps are coupled to one or more cleaved oligonucleotide B molecules, for example, via hybridization, splinting, or extension reactions, thereby generating a stacked polymerizable molecule comprising a first barcode sequence segment (oligonucleotide B) containing the identity of the first (terminal) monomer and a second barcode sequence segment containing the identity of the next (n-1) monomer. Additional iterations of the workflow can be performed, for example, until all monomers have been locally tethered, cleaved, and contacted with a binder.

[0191] In some cases, the activator includes an anchoring nucleic acid molecule, which can indirectly facilitate the coupling of the first polymerizable molecule and the second polymerizable molecule. Figure 1E An example of a binding agent comprising a polymerizable molecule and an activator is schematically shown. The binding agent 115 can include one or more polymerizable molecules 117 for identifying the binding agent or its homologous molecule (e.g., monomer type), and an activator 123 comprising an anchoring nucleic acid molecule. The anchoring nucleic acid molecule can include a region that can be coupled to the substrate-bound polymerizable molecule 107. In some cases, the anchoring nucleic acid molecule includes a self-splinting region that can partially hybridize to the substrate-bound polymerizable molecule 107. Optional connections can be made to covalently link the activator 123 to the substrate-bound polymerizable molecule 107. Before, during, or after the activator 123 is coupled to the substrate-bound polymerizable molecule 107, the polymerizable molecule 117 coupled to the binding agent can be coupled to another substrate-bound polymerizable molecule. Alternatively or additionally, polymerizable molecules 117 can be coupled to substrate-bound polymerizable molecules comprising encoded polymerizable molecules from a previous round or cycle or iteration, thereby generating stacked polymerizable molecules comprising encoded information from multiple rounds or cycles.

[0192] Figure 1FSchematically illustrated are example activators comprising enzymes.Binding agent 115 can be coupled to activator 123, and activator 123 can include any useful enzyme, such as peroxidase, ligase, kinase, phosphatase, phosphorylase or nuclease, such as Cas protein, restriction endonuclease. In some examples, enzyme includes peroxidase (for example, horseradish peroxidase or APEX) or other color-producing or fluorogenic enzymes, which can generate direct readout products (for example, chemiluminescence, luminescence, fluorescence, etc.). Product can be detected using standard imaging or fluorescence or luminescence detection methods. Alternatively or additionally, enzyme can promote the coupling of polymerizable molecules, for example, encoding (first) polymerizable molecules such as identification binding agent or monomer type nucleic acid barcode molecules coupled to a second polymerizable molecule, for example, substrate-bound nucleic acid molecules. For example, a peroxidase can be used to locally couple a click chemistry functionalized tyramine (e.g., an azide-tyramine) to a substrate (e.g., containing a tyrosine or free radical acceptor) or a substrate-bound (second) polymerizable molecule adjacent to a monomeric analyte (e.g., a fluorescein-conjugated nucleic acid molecule, as described in Wilbanks et al. 2021. Chembiochem. 22(8): 1400-1404, incorporated herein by reference, or a tyrosine-containing polymerizable molecule); subsequently, a coding (first) polymerizable molecule comprising a complementary click chemistry moiety (e.g., DBCO) can be conjugated to the tyramide-modified substrate-bound (second) polymerizable molecule. Alternatively or additionally, the tyramide-conjugated coding (first) polymerizable molecule (e.g., a nucleic acid barcode molecule) can be directly coupled to a substrate-bound (second) polymerizable molecule (e.g., a fluorescein-conjugated nucleic acid molecule or a tyrosine-containing polymerizable molecule) via a peroxidase coupled to a binding agent.

[0193] In another example, activator includes ligase, which can be used to connect coding (first) polymerizable molecule to substrate-bound (second) polymerizable molecule, optionally using splint molecules. In another example, enzyme can be used to process or activate substrate-bound (second) polymerizable molecule or the first polymerizable molecule, so as to enable coupling. For example, substrate-bound (second) polymerizable molecule can include restriction sites or cleavage sites that can be cut using restriction enzymes or other cutting enzymes, so as to reveal coding (first) polymerizable molecule, such as via coupling sites that can be coupled to each other or with splint molecule hybridization. In some cases, in the absence of activation (for example, cutting) operation, the first and second polymerizable molecules may not be able to be coupled to each other. In other examples, enzyme can include kinase, such as T7 polynucleotide kinase, which can phosphorylate the first or second polymerizable molecule, so as to allow the first polymerizable molecule to be connected to the second polymerizable molecule.

[0194] Figure 2Example linkers that can be used to sequence polymeric analytes such as peptides are schematically shown. Figure 2 Sub-figure A shows a bifunctional linker 203 (e.g., 1-(but-3-yn-1-yl)-4-phenylisothiocyanate, also referred to herein as alkyne-PITC or PITC-alkyne), comprising an amino acid reactive portion (e.g., PITC) and an alkyne click chemistry portion that can react with a polymerizable molecule 201 (e.g., a linker nucleic acid molecule) comprising a complementary azide click chemistry portion. The bifunctional linker can also comprise a spacer portion, such as an alkyl chain of any length (ethyl is depicted), a polymer of any length (e.g., PEG), etc. The spacer portion can be positioned between the amino acid reactive portion and the click chemistry portion. Figure 2 Sub-figure B shows the product of the click chemistry cycloaddition reaction between an azide and an alkyne group to generate a linker molecule comprising a polymerizable molecule and an amino acid reactive portion. The conjugation of the polymerizable molecule 201 to the bifunctional linker 203 can occur at any useful or convenient step. In an alternative example (not shown), the bifunctional linker 203 can comprise an azide group, such as 1-(2-azidoethyl)-4-phenylisothiocyanate (also referred to herein as azide-PITC or PITC-azide), which can react with the polymerizable molecule 201 comprising the alkyne portion.

[0195] Iteration: In some cases, one or more of the operations described herein can be iterated or repeated. Iterative operations can allow for the sequential processing, analysis, or identification of individual monomers of a polymer analyte, which can allow for the reconstruction of the entire polymer analyte. For example, with reference to Figure 1, the operation of workflow 100 can be performed to encode the identity of a terminal amino acid (e.g., NTAA) (e.g., via a polymerizable molecule 117 of a binding agent) onto an additional polymerizable molecule 107. The operation of workflow 100 can then be repeated to encode the identity of the n-1 terminal amino acid, the n-2 terminal amino acid, the n-3 terminal amino acid, etc., until all or part of the peptide is sequenced. Encoding can occur on the same (additional) polymerizable molecule 107, for example, generating a stacked polymerizable molecule comprising multiple polymerizable molecules from multiple binding agents, or encoding can occur on additional polymerizable molecules (not shown) present on a substrate. In the former case, in some cases, the polymerizable molecule of the second (or third, fourth, fifth, ... nth) cycle can be configured to couple only to the first (or second, third, fourth, ... n-1th) polymerizable molecule. For example, a first circulating binder polymerizable molecule can comprise a unique binding sequence that is not present on another polymerizable (or capture) molecule of the substrate, and a second circulating binder polymerizable molecule can bind to this binding sequence. Thus, a second circulating binder polymerizable molecule can only bind to the first circulating binder polymerizable molecule and not to any other polymerizable (or capture) molecule of the substrate. In the event that no binding occurs (an "ineffective" event), a bridging polymerizable molecule can be provided that encodes an ineffective binding event but comprises a unique binding sequence so that subsequent rounds can continue even if the binder does not bind to the cleaved monomer.

[0196] In the case where one or more additional polymerizable molecules are used, the polymerizable molecules 117 of the binder can additionally contain time information about the number of cycles or iterations so that the order of individual monomers can be determined. For example, for a given peptide, the terminal amino acid can be coupled to a capture moiety and cleaved, and then contacted with a binder containing a barcode sequence that identifies (i) the identity of the amino acid (e.g., any of the twenty proteinogenic amino acids) and (ii) the cycle number (e.g., cycle 1). The information encoded by the barcode sequence can be transferred to an adjacent (additional) polymerizable molecule 107 via coupling and optional copying. After cleavage of a monomer from the capture moiety (e.g., as Figure 1A(shown in subfigure F), workflow 100 can be repeated for the n-1 terminal amino acid, which can again be coupled to a capture moiety, cleaved, and contacted with a binding agent comprising an additional barcode sequence that identifies (i) the identity of the amino acid (e.g., any of the twenty proteinogenic amino acids) and (ii) the cycle number (e.g., cycle 2). The information encoded by the additional barcode sequence can be transferred to the same (additional) polymerizable molecule 107 or to an additional polymerizable molecule present on the substrate (not shown). In the former case, the polymerizable molecule can thus contain information regarding: (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), etc.

[0197] Alternatively, time information can be provided separately. For example, before, during or after the first polymerizable molecule is coupled to the second polymerizable molecule, a time barcode can be provided, which can be coupled to the first polymerizable molecule, the second polymerizable molecule or both. The time barcode can include any useful reagent, including nucleic acid molecules, peptides, lipids, carbohydrates, enzymes (e.g., chromogenic or luciferase) or ribozymes or deoxyribozymes, fluorophores, dyes, intercalators, dideoxynucleotides, fluorescent nucleic acid molecules or nucleotides, radioisotopes, mass tags or other detectable markers that can indicate the time or cycle (or iteration) number it provides. In some cases, the time barcode includes a cycle-specific nucleic acid barcode molecule that can be coupled to the first polymerizable molecule (including the identity of the monomer) or the terminal polymerizable molecule of the stacked polymerizable molecule, which includes polymerizable molecules from multiple rounds or iterations. The time barcode can include any additional useful functional sequences, such as primer sites, sequencing sites, restriction sites, abasic or cleavable sites, etc. In some cases, a temporal barcode may comprise an amplification site that allows for bridge amplification of the temporal barcode and optional coupled polymerizable molecules with other capture or polymerizable molecules.

[0198] One or more operations may be repeated in a given workflow. For example, referring to workflow 100, Figure 1A Any process in sub-graphs AF can be iterated or repeated. In some cases, multiple rounds of binding agent coupling can be performed (e.g., Figure 1A E), optionally with a wash operation, which can result in (i) multiple polymerizable molecules (e.g., 107) containing the same encoding information of the polymerizable molecule 117 of the binding agent, (ii) the same polymerizable molecule (e.g., 107) containing multiple copies of the same polymerizable molecule 117 of the binding agent, or (iii) both. This multi-cycle encoding method can be useful, for example, in improving the sensitivity or accuracy of detecting single monomers.

[0199] Identification of polymerizable molecules: Polymerizable molecules can be sequenced to determine the identity of individual monomers (e.g., amino acids). For example, monomers can be cleaved from the capture moiety (e.g., Figure 1A F) or after any number of iterations of workflow 100, the polymerizable molecules containing the binder information and, therefore, the identity of the monomer can be removed from the substrate and prepared for sequencing (e.g., DNA sequencing, NGS). Removal of the polymerizable molecules can be achieved using any useful method, such as chemical or enzymatic cleavage. In some cases, for example, prior to removing the polymerizable molecules containing the monomer information, any excess or uncoupled polymerizable molecules or capture moieties can be removed. For example, again referring to Figure 1A Subgraph B and Figure 1A Panel E, the coupling event can generate a double-stranded or partially double-stranded molecule; thus, a single-stranded polymerizable molecule or capture portion that does not contain monomers or additional polymerizable molecules (e.g., from a binding agent) can be digested using an enzyme such as a Type II restriction endonuclease, S1 endonuclease.

[0200] Alternatively or additionally, the polymerizable molecule is amplified (e.g., using a nucleic acid amplification method such as polymerase chain reaction (PCR), isothermal amplification, ligation-mediated amplification, transcription-based amplification, etc.) to generate an amplicon for sequencing. Amplification can be performed, for example, using a capture moiety or a polymerizable molecule as a primer binding site. Alternatively or additionally, an adapter sequence comprising a primer binding site can be added to the polymerizable molecule. Any number of useful preparation operations can be performed, such as purification or enrichment, cleanup, nucleic acid reaction (e.g., connection, extension, amplification, marker cleavage, restriction enzyme cleavage, enzyme treatment (e.g., using exonucleases, RNA enzymes, CRISPR, Argonaut, terminal transferases)), fragmentation, barcoding, addition of adapters, enzyme treatment, etc. In some cases, the polymerizable molecule or a substrate comprising the polymerizable molecule can be filtered based on any useful feature or property. Filtering based on features or properties can achieve higher accuracy or lower noise by removing low-quality molecules or enriching high-quality polymerizable molecules before sequencing. For example, polymerizable molecules or substrates (e.g., beads or particles) containing polymerizable molecules can be filtered by size or length, number, presence of specific sequences (e.g., primer sequences, sequences of interest), GC content, polarity, polarization, birefringence, fluorescence (or other optical properties), anisotropy, charge, secondary structure (e.g., hairpins), or other useful indicators, features, or properties, or combinations thereof. Such filtering or enrichment can be performed using any suitable method, such as affinity or hybridization methods (e.g., bead-based affinity sequence or hybridization assays that can enrich for specific sequences), chromatography, size-based filtration, electrophoresis, electrofocusing, optoelectronics, digital fluidics, magnetic-activated sorting, fluorescence-activated sorting, flow cytometry, or other suitable techniques.

[0201] Sequencing can be performed using commercially available nanopore systems (e.g., Oxford Nanopore Technologies, Genia Technologies, NobleGen, or Quantum Biosystem) or other sequencing and next-generation sequencing systems (e.g., Illumina, BGI, Qiagen, ThermoFisher, PacBio, and Roche), including formats such as parallel bead arrays, sequencing by synthesis, sequencing by ligation (e.g., SOLiD), capillary electrophoresis, electronic microchips, "biochips," microarrays, parallel microchips, single molecule arrays, and Sanger sequencing, as described elsewhere herein.

[0202] Sequencing can output the identity of the polymerizable molecule or the sequence of the polymerizable molecules coupled together. For example, referring again to Figure 1, after one or more iterations of the workflow 100, the polymerizable molecule 107 can include the nucleic acid sequence of the polymerizable molecule 117 of the binder (or its complement), or a stack of polymerizable molecules obtained from multiple rounds of binding of the binder to its target monomer or monomer-capture moiety complex (or its complement). Thus, sequencing the polymerizable molecule 107 can produce sequencing reads that identify the nucleic acid sequence of the polymerizable molecule 117 of the binder and the information encoded therein, such as the cycle number and identity of the binder or monomer (e.g., one of the 20 proteinogenic amino acids). In the case where the polymerizable molecule 117 of the binder includes a nucleic acid molecule that encodes additional information (e.g., including a barcode sequence, a UMI, cycle information, spatial information, etc.), multiple types of information can be revealed from the nucleic acid sequencing of the polymerizable molecule 107.

[0203] Sequencing reads can be assembled using a de novo method to identify peptides or proteins. For example, a common barcode sequence can be used to mark the fragmented peptides produced from a common parent protein, as described elsewhere herein. Therefore, it is possible to assemble the inferred peptide reads based on a common barcode sequence, amino acid identity, and, if applicable, the number of cycles. Error reads can be identified by probabilistic modeling of read accuracy, thereby generating a fragment peptide sequence (contig) with a reconstruction of the possible gap of a lost or unidentified round / amino acid. The alternative option for de novo read reconstruction can adopt an end-to-end, unsupervised machine learning-based reconstruction of peptide reads. This option can adopt a machine learning algorithm, such as a model based on deep learning, which uses the NGS sequencing read associated with the parent protein / peptide barcode as its input, and outputs the possible reconstruction of peptide reads (contigs). The training of the model can be performed using known protein / peptide standards with protein sequencing operations. De novo reconstruction can output the reconstructed fragment peptide sequence (contig), with probability assigned to each amino acid and the assembled peptide sequence. In some cases, fixed length nucleotide string (k-mer) or De Brujin method can be used for peptide sequence reconstruction.For example, the read segment produced from each polymerizable molecule can be decomposed into a shorter fixed length nucleotide string sequence.The fixed length nucleotide string sequence from the read segment pool can be assembled into a longer contig sequence.De Brujin diagram can be generated, for example, to represent splice variants, post-translational modifications or other protein variants.Isoforms (isoforms) can be assembled, and expression levels can be determined using Bayesian methods.The assembled isoforms of proteins can be evaluated and error corrected, for example, by comparing with the standard protein incorporated into the sample, and evaluating the missing segments, incorrect or redundant assembly, uniform coverage, etc. of the sequence.

[0204] Alternatively, the identity of the polymerizable molecule can be obtained without using a sequencing method. For example, a probe can be used to couple to a specific region of the polymerizable molecule. The probe can include a nucleic acid probe with a probe sequence that can be used to specifically detect a monomer type. In one such example, the polymer analyte comprises a peptide, and a single amino acid (monomer unit) can be coupled to a capture portion and cleaved from the peptide. The monomer-capture portion complex can be contacted with a binding agent (e.g., an antibody, nanobody, scFv) containing a nucleic acid barcode molecule (polymerizable molecule) that identifies the binding agent. The binding agent may be specific for an amino acid (e.g., one of the 20 proteinogenic amino acids), and thus the nucleic acid barcode molecule encodes a specific amino acid. Therefore, a nucleic acid probe having a complementary sequence to the nucleic acid barcode molecule of the binding agent can be used to identify the presence of the binding agent (e.g., via in situ hybridization). In some cases, the probe can include a detectable label or portion, such as a fluorophore, a radioisotope, a mass tag, etc. For example, a hybridization-based assay such as SeqFISH or Nanostring can be performed to detect or assay a specific region of the polymerizable molecule to determine its identity. In other examples, amplification-based methods can be used to determine the presence and identity of polymerizable molecules. For example, PCR or nested PCR methods can be used to selectively detect specific sequences of polymerizable molecules.

[0205] Alternatively or additionally, the binding agent may comprise a detectable label or portion. For example, the binding agent may include a fluorophore, a radioisotope, a mass label, a chromogenic enzyme (e.g., horseradish peroxidase), etc., which may be detected using appropriate imaging techniques. Different binding agents (e.g., binding agents that recognize different monomers or amino acids) may be labeled with different markers (e.g., different fluorophores), which may be used to identify the presence of a monomer or amino acid. In some examples, single molecule imaging (e.g., total internal reflection, confocal, wide field of view, or super-resolution microscopy (e.g., PALM, STORM, STED)) may be used to detect binding agents labeled with fluorophores.

[0206] In some cases, a substrate comprising a polymerizable molecule (e.g., Figures 1A-1F) can be provided on an array for sequencing. For example, a plurality of beads comprising a polymerizable molecule encoding amino acids of a plurality of peptides can be provided on an array for sequencing. In one such example, a plurality of beads can be coupled directly or indirectly to an additional substrate (e.g., a planar substrate such as a microscope slide or a multi-well plate), and can be sequenced using an image-based sequencing method (e.g., using synthetic sequencing or in situ hybridization probes and a single-molecule resolution imaging system), amplification-based sequencing, or both. A plurality of beads can be coupled to an additional substrate using any suitable technique, such as nucleic acid attachment using polymerizable molecules or capture molecules, magnetic attachment (using a magnetic field and magnetic beads), optoelectronics, digital microfluidics, applied electric fields, gravity sedimentation, centrifugation, capillary forces, hydrogen bonds, electrostatic interactions, or other suitable methods.

[0207] Advantageously, the methods and systems described herein can be particularly useful in achieving highly accurate sequencing of polymer analytes (e.g., peptides) or identification of single monomers (e.g., amino acids). As described herein, the sequencing methods disclosed herein provide for local tethering and cleavage of monomers, coupling a binder comprising a polymerizable molecule with encoding information to the cleaved monomer, and coupling the polymerizable molecule to other local polymerizable molecules for transferring the encoding information, which can then be used to identify (e.g., via sequencing) each monomer. Because the methods outlined herein include removing the monomer and tethering it locally, followed by coupling the binder, local environmental issues (variability in binder specificity caused by different adjacent amino acids) are avoided, highly accurate readings of single monomers (e.g., amino acids) with single-molecule sensitivity can be achieved.

[0208] For example, the method outlined herein can achieve individual reading accuracy, for example, the probability of correctly identifying a single monomer (for example, amino acid) is greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90% or higher. Similarly, when performing multiple iterations of operation, individual reading accuracy can remain relatively constant. In other words, individual reading accuracy can not be based on how many rounds or iterations of order-checking have been performed and substantial changes. For example, for 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, at least 500, at least 1000 times or more iterations of order-checking of a single monomer (for example, amino acid) for identifying a given polymer analyte, high individual reading accuracy can be obtained. For peptides, the method provided herein can allow single amino acid to be distinguished from other amino acids with a high degree of specificity. For example, using the methods described herein, at least one single amino acid residue can be distinguished from the other 19 proteinogenic amino acid residues, or in some cases, from amino acids comprising post-translational modifications or unnatural amino acids. The methods described herein can allow for the specific identification of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or all 20 proteinogenic amino acids.

[0209] In some cases, individual read accuracy can be defined by a probability function. For example, individual read accuracy can represent the probability of correctly identifying all or a subset of amino acids of a peptide. Therefore, in some contexts, individual read accuracy can be described as P (amino acid A, position X) = number of correctly identified / (number of correctly identified + incorrect or failed identifications). The number of incorrect or failed identifications can include, for example, incorrect amino acid identification (incorrect A), insertion (incorrect X) or deletion (incorrect X), or a combination thereof. In one such example, the insertion / deletion in a given cycle ("cycle N") can result in a failure or incorrect reading, but subsequent cycles can be accurate, so P = (N-1) / N.

[0210] In some cases, individual reading accuracy can be characterized by the probability of correctly identifying the class or group of amino acids. For example, the class or group of amino acids can be classified based on physical, chemical, biological, physicochemical or other properties. For example, aliphatic side chains (for example, G, A, V, L, I), hydroxyl side chains or sulfur / selenium side chains (for example, S, C, U, T, M), aromatic side chains (for example, F, Y, W), basic side chains (for example, H, K, R), acidic side chains (for example, D, E, N, Q) can each form a class of amino acids. In another example, positively charged side chains (for example, arginine, histidine, lysine), negatively charged side chains (aspartic acid, glutamic acid), polar uncharged side chains (for example, serine, threonine, asparagine, glutamine), hydrophobic side chains (for example, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan) can each form a class of amino acids. In some examples, some types of post-translational modifications can form a class of amino acids. A class of amino acids can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more members, and the class comprising any number of identical or different number members can be used to characterize individual reading accuracy. For example, the first class of amino acids can include 2 kinds of amino acid types (for example, leucine and isoleucine), and the second class of amino acids can include 3 kinds of amino acid types (for example, tryptophan, phenylalanine and tyrosine). In this type of example, if the output leucine is read when the actual residue is isoleucine, then the first time reading is still counted as correctly identified. Similarly, if the output tryptophan is read when the actual residue is phenylalanine or tyrosine, then this reading is also counted as correctly identified.

[0211] Relative (e.g., positional) individual read accuracy can also be attributed to individual monomeric or polymeric analytes. Relative individual read accuracy can be the relative positioning accuracy of each monomer in the sequence, but not the absolute position. For example, for a peptide comprising the sequence AGN, if A is first correctly identified, G is not identified, and then N is correctly identified, then a relative individual read accuracy or correct identification of the order of the amino acid (A and N) subset can be assigned (e.g., 100%) even if not every single amino acid is identified. In such examples, the relative individual read accuracy relies on the correct identification of the order or relative positioning of the amino acid subset of the peptide, but does not necessarily rely on the absolute position of each identified amino acid.

[0212] Fingerprint analysis: The methods described herein can be used for complete de novo protein or peptide sequencing (e.g., identifying each amino acid in a peptide) or for protein fingerprint analysis (e.g., identifying only a subset of the amino acid types in a peptide and inferring from a reference database or mapping the identified amino acids to a reference database to identify the peptide or protein). For fingerprint analysis, a subset of amino acids can be identified, for example, using the methods described herein, without the need for specific binding agents for all 20 proteinogenic amino acids. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 different binding agents with single or multiple amino acid specificity can be sufficient to determine the identity of a protein or peptide. For a known proteome database, a reference-based reconstruction can be performed by simulating the NGS reads generated from the possible peptide sets in the workflow. For each possible peptide, the simulation can generate an NGS read that mimics the output of the protein sequence system. Next, the real (experimental) NGS reads from the run can be matched to the simulated reads of the candidate peptides from the database based on likelihood. This results in reconstructed, fragmented peptide sequences (contigs) with probabilities assigned to the assembled peptide sequence.

[0213] In some fingerprint analysis methods, not all amino acids need to be identified to accurately identify a protein. By correctly identifying only a subset of amino acids, the mapping accuracy or accuracy of correctly identifying a protein (e.g., using a protein database) can be, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more. For example, to identify a protein, only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids may need to be identified. In some cases, the individual read accuracy of a single amino acid does not need to be highly accurate in order to accurately identify a protein. For example, the individual read accuracy of a set of single amino acids can average about 50%, about 60%, about 70%, about 80%, about 90%, or the like, so as to produce a correct identification of a protein or peptide with an accuracy or confidence of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more.

[0214] In some cases, in order to accurately identify proteins, the individual reading accuracy of a single amino acid does not need to be higher than a threshold percentage. For example, a binding agent may off-target bind with chemically similar residues (e.g., a binding agent for leucine may off-target bind with isoleucine), which will result in poor individual reading accuracy, but still allow the correct identification of peptides. In such examples, it is not necessary to identify an exact amino acid (e.g., leucine), but the position (e.g., "X" position) or relative position may be expressed as one of multiple amino acids (e.g., one of leucine or isoleucine). Proteins can then be identified using context clues and homology with respect to a reference protein database, knowing that the X amino acid is one of multiple amino acids (e.g., one of leucine or isoleucine). For example, knowing exact NTAA, exact n-1NTAA, and X amino acid (as one of multiple amino acids) may be sufficient to correctly identify proteins. Therefore, even if individual reading accuracy is not high, the accuracy of correctly identifying peptides can also be high.

[0215] High-throughput sequencing / parallelization:Methods described herein can be carried out in a high-throughput format of parallelization.Such parallelization can be by making the substrate comprising multiple polymer analytes be coupled thereto, and iteratively perform operations (for example, monomer is coupled to capture portion, cutting, contacted with binding agent or binding agent library, coupling polymerizable molecule, optionally monomer is cut from capture portion) across substrate to realize.Methods described herein can allow 1, 10, 100, 1,000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000 or more polymer analytes parallel analysis.In some cases, binding agent library can be used to identify different monomer types (for example, different amino acids of peptide analytes or derived amino acids), so that different polymer analytes (for example, different peptides) can be processed on a single substrate, thereby increasing the multiplexing or detection of multiple different analytes.

[0216] Binding agent libraries can be used to identify different monomers and contribute to high-throughput readout. As described herein, binding agent libraries can include binding agents that can identify single monomers (e.g., single cleaved amino acids) or multiple monomers (e.g., multiple cleaved amino acids). In some cases, binding agents with different levels of specificity can be used sequentially or in order, which can help make the binding agent with less specificity more specific based only on the sequence provided. For example, a first binding agent can be able to specifically bind to a first monomer analyte and a second binding agent can be able to bind to both the first monomer analyte and the second monomer analyte. A first binding agent can be provided and contacted with the first monomer analyte and the second monomer analyte. Since the first binding agent has specificity for the first monomer analyte, the first binding agent will uniquely bind to the first monomer analyte. A second binding agent can subsequently be provided; however, since the first monomer analyte is bound to the first binding agent, the second binding agent may not be able to approach (e.g., spatially blocked) the first monomer analyte. Therefore, the second binding agent can only bind to the second monomer analyte. Thus, identification of the first and second binding agents (eg, by detecting a label / tag or by sequencing a polymerizable molecule coupled to the binding agent and optionally transferred to a substrate) can allow identification of the first and second monomeric analytes.

[0217] Figure 3 Schematically illustrates an order-dependent binding scheme for a library of binding agents, and an example of how less specific binding agents (e.g., binding agents that can bind to multiple monomer types) can be optimized and made more specific based on the binding order of each binding agent in the library. A substrate 310 is provided, which is coupled to multiple cleaved monomers (e.g., terminal amino acids cleaved from a single peptide). In this example, the substrate includes, from left to right, cleaved glycine (G), cleaved leucine (L), cleaved tyrosine (Y), cleaved proline (P), cleaved histidine (H), cleaved isoleucine (I), and cleaved cysteine ​​(C). The binding agent library (frame) can include binding agents (e.g., antibodies) with different specificities. Binder 301 has specificity for cysteine ​​(C), binder 302 has specificity for tyrosine (Y), binder 303 has specificity for isoleucine (I), and binder 304 has specificity for histidine (H). The binder 305 may bind to Y and P, the binder 306 may bind to L and I, and the binder 307 may bind to G, L, and Y.

[0218] In process 320, binding agents (301, 302, 303, and 304) with a single specificity are provided and allowed to bind to a specific cleaved monomer specific for each. Subsequently, in process 330, binding agents 305 and 306 with lower specificity (e.g., binding to more than one cleaved monomer) are provided. Binding agent 305, which binds to Y and P, binds to P because the Y residue is already bound and is unavailable due to the inaccessibility caused by binding agent 302. Similarly, binding agent 306, which binds to L and I, binds to L because I is already bound and is unavailable due to binding agent 303. In process 340, binding agent 307 is provided that can bind to G, L, and Y. However, because all G, L, and Y residues are bound by other binding agents and are therefore unavailable, binding agent 307 specifically binds to G. Subsequent downstream analysis, such as identifying each binding agent (e.g., via imaging or sequencing of binding agent polymerizable molecules coupled to a substrate, not shown), can reveal the identity of each binding agent and, therefore, the monomeric analyte. For example, even though binder 307 can bind three different monomeric analytes (G, L, Y), the identity of G can be determined by the presence of binder 307 due to the sequential / ordered approach provided by the binder (e.g., blocking by binding to L and Y, leaving only G).

[0219] In some cases, it may be useful to barcode the polymer analyte prior to processing. The barcode sequence can be attached to the polymer analyte at a single location (e.g., at the end), multiple locations, adjacent to the polymer analyte (e.g., on a substrate), etc., as described elsewhere herein. For example, a peptide can be labeled with a nucleic acid barcode molecule at the N-terminus, C-terminus, or internal amino acid. The nucleic acid barcode sequence can contain information or be unique to a partition or compartment, sample, peptide, etc., such that each unique barcode sequence can be traced back (e.g., after nucleic acid sequencing or other detection methods) to the origin partition or compartment, sample, peptide, etc.

[0220] Alternatively or additionally, the capture portion or polymerizable molecule may comprise a barcode sequence. The barcode sequence may be specific to a particular partition, sample, or spatial position. For example, a substrate may include multiple individually addressable units. The polymerizable unit or capture portion of each individually addressable unit may include a unique barcode (e.g., a spatial barcode) specific to the individually addressable unit. The polymer analyte may be coupled to the substrate so that each individually addressable unit contains an average of no more than one polymer analyte. For example, such a distribution of polymer analytes may be obtained using a limiting dilution method (e.g., diluting the polymer analyte to reduce the number of polymer analytes that may be attached to a given individually addressable unit) or by introducing a chaotropic agent (e.g., guanidine, formamide, urea). The polymer analyte may be distributed across the individually addressable unit according to a Poisson distribution. Therefore, for a given substrate, approximately 6%, 10%, 18%, 20%, 30%, 36%, 40% or 50% of the individually addressable units may contain one or less polymer analytes.

[0221] Order of Operations: It will be understood that the operations set forth in the methods described herein can be performed in any useful or convenient order, and in some cases, some operations can be optional. For example, in some cases, coupling of the monomer to the capture moiety can occur before, during, or after cleavage of the monomer from the polymer analyte. In another example, the substrate can be provided with a cleaved monomer coupled thereto such that cleavage of the monomer from the polymer analyte is avoided. In another example, where a linker is used to couple to a monomer (e.g., an amino acid) and a capture moiety or substrate, the linker can comprise a monomer coupling group and subsequently react with a substrate binding group (e.g., an oligonucleotide); alternatively, the linker can be provided with a substrate binding group as part of the linker (e.g., pre-conjugated to the substrate binding group).

[0222] Additional manipulations can be performed at any useful or convenient step, such as before providing the polymer analyte (eg, peptide) coupled to the substrate or after one or more processing manipulations (eg, after coupling of the polymerizable molecule).

[0223] Modified polymer analytes and polymerizable molecules

[0224] The present disclosure also provides methods for modifying polymeric analytes or monomers of polymeric analytes (e.g., amino acids of a peptide) and polymerizable molecules described herein. Such modifications can be useful, for example, to render the monomer more resistant to certain reaction conditions (e.g., Edman degradation), to increase or decrease the binding affinity of a binding agent to the modified monomer, to aid in docking or association of the modified monomer with an enzyme (e.g., a protease, a cleavage enzyme, a binding agent, or an enzyme analog, such as a ribozyme or deoxyribozyme), or for other purposes.

[0225] In some cases, polymer analytes such as peptides can be modified to make the peptide or constituent amino acids more resistant to the reaction conditions for cleaving amino acids from the peptide. As described elsewhere herein, the Edman degradation reaction can be used to cleave terminal amino acids from peptides; such reaction conditions can include the use of strong acids (e.g., trifluoroacetic acid) or elevated temperatures, which can result in changes in amino acid residues or post-translational modifications (PTMs). Therefore, peptides can be pretreated to protect amino acid residues or PTMs. For example, peptides can be alkylated, for example, using 4-vinylpyridine, iodoacetamide, which can be used to prevent oxidation of cysteine ​​residues. Peptides can be acetylated, for example, O-acetylated to form esters such as acetyl chloride, which can be used to prevent dehydration, racemization, or destruction of serine or threonine derivatization (e.g., PTH forms). Peptides can be subjected to β-elimination of phosphate, followed by Michael addition of sulfhydryl groups (e.g., as described in Knight et al. 2003. Nature Biotechnology 21, 1047-1054, which is incorporated herein by reference) to detect phosphorylation events. The peptides can be contacted with phenyl isothiocyanate, acetic anhydride or other amine reactive groups to protect lysine residues. Additional examples of peptide processing for Edman degradation can be found in Tarr, Methods of Protein Microcharacterization. pp 155-194 (which is incorporated herein by reference).

[0226] The polymer analyte or monomer can be modified to affect the interaction of the binding agent with the polymer analyte or monomer. Controlling the interaction of the binding agent with the polymer analyte or monomer (e.g., cleaved monomer) can allow modification or control of the identification of single monomers (e.g., amino acids of a peptide). For example, a binding agent comprising a polymerizable molecule with identification information (e.g., a nucleic acid barcode sequence) can be blocked or inhibited from recognizing or binding to a monomer (e.g., cleaved monomer); therefore, the identification information of the polymerizable molecule may not be transferred to adjacent polymerizable molecules, and therefore the monomer will not be identified as present. Such methods of blocking or inhibiting binding of binding agents can be beneficial for sequential analysis of single monomers of polymer analytes, for example, removing their signal or detectability after a monomer has been detected and preventing false positives in subsequent iterations.

[0227] In some cases, blocking of the binding agent can be performed by changing the chemical composition of the cleaved monomer (e.g., amino acid) to prevent recognition and coupling of the binding agent. Such chemical composition changes can be performed by derivatizing the cleaved monomer, adding chemical groups to the cleaved monomer, or performing other chemical treatments (e.g., adding or removing groups) from the cleaved monomer. For example, as described herein, the peptide can be contacted with a linker comprising (i) an amino acid reactive portion (e.g., PITC) and (ii) a capture portion coupling group, which can allow the terminal amino acid (e.g., NTAA) to be cleaved from the peptide simultaneously or sequentially and tethered to the capture portion (e.g., coupled to a substrate). The resulting monomer-capture portion complex can comprise a derivatized amino acid, such as a PTC-derived amino acid, to which the binding agent can be coupled. Blocking of the binding agent can be achieved by attaching a blocker (e.g., a chemical group or adduct) to the monomer-capture portion complex; for example, the monomer can be conjugated to a synthetic polymer (e.g., PEG), a nucleic acid molecule, a fluorophore, a quencher, a nanotube, a nanoparticle, a small molecule, a polypeptide or protein, a fatty acid chain, or other large sterically hindered molecules. The blocker can be attached to the monomer using chemical methods (e.g., reaction with an amino acid, such as via a photoreaction) or enzymatic methods (e.g., using a methyltransferase, tRNA synthetase, acetyltransferase, etc.). The blocker can be capable of reacting with a derivatized amino acid (e.g., a PTC-derived amino acid) or a derivative thereof, such as ATZ, PTH, or other derivatized forms of an amino acid. Alternatively or additionally, the blocker can be capable of reacting with another portion of the monomer or with a capture moiety.

[0228] Conjugation of the blocker to the cleaved monomer can be achieved using chemical or enzymatic methods. Chemical methods can involve the use of one or more linkers (e.g., heterobifunctional linkers) to connect the blocker to the cleaved monomer. Alternatively or additionally, enzymes such as engineered tRNA synthetases, methyltransferases, acetyltransferases, etc. can be used to add moieties to the cleaved monomer.

[0229] In some cases, blocking binding of a binder to a monomer can be achieved by derivatizing the cleaved monomer. For example, a binder may be specific and bind to the PTC-derived form of an amino acid, but not to the ATZ- or PTH-derived forms of the amino acid. Thus, derivatization of a PTC-derived amino acid to the ATZ- or PTH-derived form can inhibit or prevent binding of the binder. Alternatively, a binder may be specific and bind to the ATZ-derived form, while derivatization of the amino acid to the PTH- or PTC-derived form can prevent binding of the binder to the cleaved, derivatized monomer. In one example, a cleaved PTC-derived amino acid that has come into contact with a binder can be converted to the PTH-derived form by reaction with amine-PEG-biotin, which will prevent further recognition by the binder.

[0230] Alternatively or additionally, the binding agent may comprise a quenchable moiety. For example, the binding agent may comprise a quenchable fluorophore or dye (e.g., Black Hole Quencher TM The fluorophore or dye can be detected using an appropriate imaging technique (e.g., total internal reflection fluorescence microscopy, super-resolution imaging), and the presence of a fluorescent signal can indicate the presence of a specific monomer (e.g., a cleaved PTC-derived amino acid). After detection, the quenchable moiety can be quenched or photobleached so that any residual binder from an earlier cycle is undetectable in subsequent cleavage iterations or cycles and monomer detection. In an example, the cleaved PTH-derived amino acid can be detected via fluorescence microscopy using a binder that is converted to a PTH-derived form by reaction with an amine-quencher (e.g., an amine-PEG-quencher), which helps prevent fluorescence emission.

[0231] It will be appreciated that the binding of a binding agent to a cleaved monomer (e.g., a monomer-capture moiety complex) can also be enhanced using any of the above methods. For example, a PTC-derived amino acid can be further derivatized into another form (e.g., ATZ or PTH) that increases or enhances recognition or binding of the binding agent. In another example, an adduct or chemical moiety can be added to the cleaved monomer to increase or improve the interaction of the binding agent with the cleaved monomer. Such adducts or chemical moieties can be added to select monomer types (e.g., a subset of amino acids, such as amino acids with similar characteristics or properties) or indiscriminately to all monomer types (e.g., all amino acid types). For example, a peptide can be contacted with 3-arylpropionitrile (APN), which can react with cysteine ​​residues and optionally can tag cysteine ​​residues. Cysteine-APN specific binding agents can be used for improved detection of cysteine ​​residues.

[0232] Figure 4-8 Examples of modifications that can affect the monomer or cleaved monomer to which the binding agent is coupled are schematically shown. Figure 4 Schematic diagram of an example of a post-translational modification that can be modified to improve or enhance the recognition and binding of a binding agent (e.g., an antibody or antibody fragment) to a PTM. In one such example (bottom sub-figure), the peptide can comprise a phosphorylated amino acid (a naturally occurring PTM). The peptide can be subjected to a β-elimination followed by a Michael addition of a sulfhydryl group to produce a modified amino acid. The peptide can then be subjected to conditions sufficient to tether the NTAA to the capture moiety, cleave the NTAA from the peptide, and contact the binding agent.

[0233] Figure 5Schematic diagram of an example of a derivatization process or the addition of a chemical moiety to a monomer to alter binding agent coupling. A linker 501 comprising a monomer reactive group can react with a monomer 503 to generate a linker-monomer complex 505a. The linker-monomer complex 505a can be contacted with a binding agent 507 that specifically recognizes the linker-monomer complex 505a. The linker-monomer complex 505a can then be derivatized to generate a derivatized linker-monomer complex 505b that is not recognized by the binding agent 507. Alternatively or additionally, the linker-monomer complex 505a can be coupled to a chemical moiety (e.g., an adduct or blocking group) to generate a blocked linker-monomer complex 505c that is also not recognized by the binding agent 507. In some embodiments, the linker 501 comprises an amino acid reactive group (e.g., PITC) and is coupled to the terminal amino acid (monomer 503) of a peptide (not shown) to generate a PTC-derived amino acid (linker-monomer complex 505a). The PTC-derivatized amino acids can be derivatized (e.g., using heat, acid) to generate ATZ-derivatized amino acids or PTH-derivatized amino acids (derivatized linker-monomer complex 505b). Binding agent 507 can specifically recognize the PTC-derivatized amino acids, but not the ATZ- or PTH-derivatized forms.

[0234] Figure 6Schematically illustrated is an example workflow for sequencing polymer analytes, including blocking operations. In workflow 600, similar to the workflow 100 in Fig. 1, a polymer analyte 603 is provided, which is sequentially disassembled into single monomers via contact with and cutting of the capture portion, and a single cut monomer is contacted with a binding agent, which optionally comprises a polymerizable molecule for identifying or encoding a binding agent. The polymerizable molecule of the binding agent can be coupled with additional polymerizable molecules, and subsequently monomers can be cut from the capture portion to repeat the process. Substrate 601 is coupled to polymer analyte 603 (for example, peptide to be sequenced), capture portion 605 (for example, nucleic acid primers, click chemistry moieties such as alkynes) and other capture portion 607 (for example, additional polymerizable molecules such as nucleic acid primers or click chemistry moieties). In some cases, capture portion 605 and other capture portion 607 are identical molecules. Polymer analyte 603 can be contacted with bifunctional linker 609, and this bifunctional linker comprises amino acid coupling group (for example, PITC) and capture part coupling group (for example, nucleic acid molecule or click chemistry part such as azide).In process 610, bifunctional linker 609 is coupled to terminal monomer (for example, terminal amino acid such as N-terminal amino acid (NTAA)).In process 620, bifunctional linker 609 is coupled to capture part 605, and this can be mediated by hybridization of complementary nucleic acid molecules, using splint oligonucleotide or click chemistry (for example, the cycloaddition reaction of azide and alkyne).In process 630, this system is subjected to the condition that is enough to cut terminal monomer (for example, amino acid) from polymer analyte 603 (for example, peptide).Condition can include carrying out Edman degradation reaction.Cutting monomer from polymer analyte causes through cutting monomer to be coupled to bifunctional linker 609 and capture part 605, for example, generates monomer-capture part complex.In process 640, binding agent 615 is provided. The binding agent may comprise a detectable moiety (e.g., a fluorophore) that can be detected using an appropriate detection technique (e.g., spectroscopy, microscopy), or the binding agent may comprise another polymerizable molecule (e.g., a nucleic acid molecule) that encodes information about the identity of the binding agent (not shown). The binding agent 615 may be specific for a monomer (e.g., for a single amino acid) or for a monomer-linker complex (e.g., PITC-amino acid). In the case where the binding agent comprises a polymerizable molecule, the polymerizable molecule may comprise additional sequences such as barcode sequences, UMIs, restriction sites, transposition sites, sequencing sites (e.g., P5 or P7 sequences), sequences representing cycle or iteration numbers, or other functional sequences. The polymerizable molecule of the binding agent may be coupled to an additional polymerizable molecule 607 coupled to the substrate 601. In some cases, an extension reaction (e.g., using a polymerase) may be performed to copy the sequence of the polymerizable molecule of the binding agent to an additional capture moiety 607 coupled to the substrate 601.Alternatively, the polymerizable molecule of the binding agent can be chemically (e.g., via complementary click chemistry) or enzymatically (e.g., using a ligase) attached to the additional polymerizable molecule 607. In process 650, the monomer-capture moiety complex can be modified, such as additional derivatization or coupling to a blocking moiety 617, which can prevent coupling of the binding agent 615 or other additional binding agents. In some embodiments, the monomer can be decoupled (e.g., removed or cleaved) from the capture moiety 605, for example, using chemical, mechanical, or enzymatic methods. In process 660, the workflow 600 can then be iterated or repeated to sequence all or a portion of the polymer analyte 603.

[0235] Figure 7 Schematically illustrates an example of treating a cut monomer to prevent binding of a binding agent. Polymer analyte 703 (e.g., a peptide) can be treated as described herein so that the terminal monomer is coupled to the capture portion to generate a monomer-capture portion complex 711. The monomer-capture portion complex 711 can be contacted with a binding agent that recognizes at least a portion of the monomer-capture portion complex (not shown). The binding agent can be detected (e.g., the binding agent can include a fluorophore that can be detected using imaging or microscopy or via a polymerizable molecular method as described herein). Subsequently, in order to prevent the heavy binding or detection of the binding agent or additional binding agent, or to prevent the off-target binding of additional binding agents with different affinities, the monomer-capture portion complex 711 can be treated using various methods. In some examples, the monomer-capture portion complex can be converted (e.g., derivatized) using a chemical or enzymatic method so that the binding agent does not recognize or bind to the converted or derived form. In another example, a chemical group or part (e.g., an adduct, a bulky blocking group) can be added to the monomer-capture portion complex, which can spatially inhibit the binding of the binding agent. In another example, the monomer-capture moiety complex can be detected using a binding agent comprising a fluorophore; after detection, a quenchable moiety can be added to the monomer-capture moiety complex (e.g., via reaction with an amine-quenchable fluorophore). The quenchable moiety can quench the signal of any binding agent within the vicinity of the monomer-capture moiety complex, such that the monomer-capture moiety complex will not be detected in subsequent iterations of sequencing.

[0236] Polymer analytes can also be modified to promote the cutting of monomers therefrom. As described herein, in some cases, the cutting of monomers from polymer analytes can be mediated by a cutting enzyme or a ribozyme or a deoxyribozyme. The affinity of the cutting enzyme (or ribozyme or deoxyribozyme) to the monomer (e.g., NTAA or CTAA) can be enhanced or regulated by adding a chemical group or part to generate a modified monomer. For example, NTAA can be contacted with an amino acid reactant, such as thiocyanate, SNFB, dansyl chloride, DNFB, which can change the chemical structure of NTAA. Alternatively or additionally, the monomer can be modified with a hapten, a guanidino group, a thiobenzoylating agent, a thioacetylating agent, a thioacylating agent, etc. Adding a chemical group or part can improve or enhance the binding or recognition of the cutting enzyme (or ribozyme or deoxyribozyme) to the modified monomer. The cutting enzyme (or ribozyme or deoxyribozyme) can be a naturally occurring or engineered protein, such as an aminopeptidase, a carboxypeptidase, a tRNA synthetase or other peptides modified to identify and cut a monomer or a modified monomer.

[0237] Systems, compositions, kits: Also provided herein are systems, compositions, and kits for processing polymer analytes. The systems of the present disclosure may include a sequencing instrument configured to receive a polymerizable molecule or stacked polymerizable molecules, as described herein, and provide sequencing reads of the polymerizable molecule or stacked polymerizable molecules. Alternatively or additionally, the systems of the present disclosure may be configured to process polymer analytes or sequence polymer analytes. The system may include a capture portion and a binding agent comprising a first polymerizable molecule coupled thereto. The system may be configured to provide a polymer analyte, a capture portion, and a binding agent; cut a monomer from the polymer analyte; couple the monomer to the capture portion to generate a monomer-capture portion complex; and contact the monomer-capture portion complex with a binding agent. Accordingly, the systems of the present disclosure may include any useful device or tool, including but not limited to mixers, liquid handlers, vortexers, centrifuges, heating or cooling elements, mechanical stages, microfluidic chambers, and fluid controls.

[0238] The compositions of the present disclosure may include any useful items or reagents for processing polymer analytes or sequencing polymer analytes. Compositions may include capture moieties configured to be coupled to monomers of polymer analytes and binding agents comprising polymerizable molecules coupled thereto. Compositions may further include substrates, linkers (e.g., polymer analytes, polymerizable molecules or monomers coupled to substrates), cutting agents, linkers (e.g., ligases), additional polymerizable molecules, reagents for reacting, or combinations thereof. In some cases, compositions include substrates comprising additional polymerizable molecules and capture moieties coupled thereto.

[0239] The kits disclosed herein may include any useful reagents for processing, analyzing, or sequencing polymer analytes. The kits may include reagents for providing polymer analytes and capture moieties, reagents for coupling polymer analytes and capture moieties to substrates, reagents for cutting polymer analyte monomers, reagents for coupling monomers to capture moieties to generate monomer-capture moiety complexes, reagents for providing binding agents comprising polymerizable molecules, reagents for coupling polymerizable molecules to second polymerizable molecules, or combinations thereof. The kits may further include reagents for removing or uncoupling monomers from monomer-capture moiety complexes. In some cases, the kits may further include reagents for removing or uncoupling polymerizable molecules or stacked polymerizable molecules from substrates, or the kits may include reagents for performing nucleic acid extension reactions. The kit can include any relevant reagents, for example, buffers, detergents, chelating agents, cofactors, enzymes, ribozymes or deoxyribozymes, acids, bases, salts, metal ions, primers, nucleic acid molecules, nucleotides, proteins, polynucleotides, binding agents (e.g., antibodies, aptamers, nanobodies, antibody fragments), lipids, carbohydrates, ribozymes, riboswitches, probes, fluorophores, oxidants, reducing agents, nuclease or protease inhibitors, dyes, organic molecules, inorganic molecules, emulsifiers, surfactants, stabilizers, polymers, water, small molecules, therapeutic agents, radioactive substances, preservatives or other useful agents. The kit of the present disclosure can also provide instructions for use of the kit contents.

[0240] Terminal monomer cleavage

[0241] Also provided herein are methods for processing polymeric analytes, such as peptides, comprising sequentially degrading individual amino acids from the termini of the peptide, including standard and modified Edman degradation. In standard Edman degradation, the N-terminal amino acid is reacted with phenyl isothiocyanate (PITC) under mild alkaline conditions to form a phenylthiocarbamoyl (PTC) derivative. The PTC-derived amino acid is treated with an acid (e.g., TFA) to generate a cleaved cyclic 2-anilino-5(4)-thiazolinone (ATZ)-derived amino acid, leaving a new N-terminus on the remaining peptide. The ATZ-derived amino acid can be converted to a phenylthiohydantoin (PTH) derivative and analyzed, for example, by reverse phase HPLC or mass spectrometry. The process can be iterated until all or part of the amino acids comprising the peptide sequence have been removed and identified from the N-terminus.

[0242] In view of the harsh rea...

Claims

1. A method for sequencing peptides with single-molecule sensitivity, the method comprising: (a) providing the peptide, wherein the peptide comprises a plurality of amino acids; as well as (b) sequencing the peptide to determine the identity and order of at least a subset of the plurality of amino acids, Wherein the individual read accuracy of the amino acids is greater than 50% for at least 5 different amino acids.

2. The method of claim 1, wherein (b) comprises: (i) cleaving the amino acid from the peptide; (ii) coupling the amino acid to a capture moiety to form an amino acid-capture moiety complex; (iii) contacting the amino acid-capture moiety complex with a binding agent; (iv) providing a first polymerizable molecule; and (v) coupling the first polymerizable molecule to a second polymerizable molecule.

3. The method of claim 2, wherein (iv) occurs during (iii).

4. The method of claim 2 or 3, wherein during (iv), the binding agent is not coupled to the first polymerizable molecule.

5. The method of claim 4, wherein the binding agent comprises an activator, wherein the activator promotes coupling of the first polymerizable molecule to the second polymerizable molecule.

6. The method of claim 5, wherein the activating agent comprises an activated polymerizable molecule, and further comprising, after (iv), coupling the first polymerizable molecule to the activated polymerizable molecule.

7. The method of claim 6, wherein the coupling is mediated using a splint molecule comprising a sequence complementary to at least a portion of (i) the first polymerizable molecule, (ii) the second polymerizable molecule, and (iii) the activated polymerizable molecule.

8. The method of claim 6, wherein the first polymerizable molecule and the activated polymerizable molecule are nucleic acid molecules, and wherein the coupling of the first polymerizable molecule to the activated polymerizable molecule occurs via a hybridization chain reaction (HCR).

9. The method of claim 5, wherein the activator comprises an enzyme.

10. The method of claim 9, wherein the enzyme comprises a peroxidase, a Cas protein, a ligase, a kinase, or a restriction enzyme.

11. The method of claim 10, wherein the enzyme comprises a kinase, wherein the kinase is configured to phosphorylate the second polymerizable molecule, thereby allowing the first polymerizable molecule to attach to the second polymerizable molecule.

12. The method of claim 2, wherein the binding agent is indirectly coupled to the first polymerizable molecule.

13. The method of claim 12, wherein the binding agent comprises an activated polymerizable molecule, and wherein the first polymerizable molecule is coupled to the activated polymerizable molecule.

14. The method of claim 2, wherein the binding agent comprises an activator, wherein the activator is configured to promote coupling of the first polymerizable molecule to the second polymerizable molecule.

15. The method of claim 14, wherein the activator comprises an enzyme or a nucleic acid molecule.

16. The method of claim 15, wherein the activating agent comprises an anchoring nucleic acid molecule, wherein the anchoring nucleic acid molecule is configured to couple to an additional second polymerizable molecule adjacent to the second polymerizable molecule.

17. The method of claim 15, wherein the activator comprises an enzyme selected from the group consisting of a peroxidase, a Cas protein, a ligase, a kinase, and a restriction enzyme.

18. The method of claim 17, wherein the enzyme is a kinase, and further comprising using the kinase to phosphorylate the second polymerizable molecule, thereby allowing the first polymerizable molecule to couple to the second polymerizable molecule.

19. The method of claim 17, wherein the enzyme is horseradish peroxidase (HRP) or ascorbate peroxidase (APEX), wherein the HRP or the APEX activates the first polymerizable molecule or the second polymerizable molecule, thereby coupling the first polymerizable molecule to the second polymerizable molecule.

20. The method of claim 17, wherein the enzyme is a restriction enzyme, and wherein the second polymerizable molecule comprises a restriction recognition site, and further comprising cleaving the second polymerizable molecule with the restriction enzyme prior to the coupling of the first polymerizable molecule to the second polymerizable molecule.

21. The method of claim 2, wherein the binding agent comprises an activating nucleic acid molecule, and wherein the first polymerizable molecule is a nucleic acid molecule, and further comprising, prior to (iii), coupling the first polymerizable molecule to the activating nucleic acid sequence.

22. The method of claim 21, further comprising generating the first polymerizable molecule via a hybridization chain reaction (HCR).

23. The method of claim 22, wherein the generating comprises (A) providing a first hairpin molecule and a second hairpin molecule; (B) coupling the first hairpin molecule to the activating nucleic acid sequence; and (C) coupling the second hairpin molecule to the first hairpin molecule, thereby generating the first polymerizable molecule coupled to the activating nucleic acid molecule.

24. The method of claim 23, wherein after (C), the first polymerizable molecule comprises a flap sequence, and further comprising using the flap sequence to couple the first polymerizable molecule to the second polymerizable molecule.

25. The method of any one of claims 2-24, further comprising repeating (i)-(v) at least once.

26. The method of claim 1, wherein (b) comprises: (i) cleaving the amino acid from the peptide; (ii) coupling the amino acid to a capture moiety to form an amino acid-capture moiety complex; (iii) contacting the amino acid-capture moiety complex with a binding agent, wherein the binding agent includes an activator; and (iv) detecting a product from the activator.

27. A method for processing a polymer analyte, the method comprising: (a) providing the polymer analyte and a capture moiety, wherein the polymer analyte comprises a plurality of monomers; (b) cleaving a monomer of the plurality of monomers from the polymer analyte; (c) coupling the monomer to the capture moiety to generate a monomer-capture moiety complex; (d) after (b) and (c), contacting the monomer-capture moiety complex with a binding agent comprising an activator; (e) providing a first polymerizable molecule; as well as (f) using the activator to couple the first polymerizable molecule to the second polymerizable molecule.

28. The method of claim 27, wherein during (e), the binding agent is not coupled to the first polymerizable molecule.

29. The method of claim 27, wherein the binding agent is indirectly coupled to the first polymerizable molecule.

30. The method of any one of claims 27-29, wherein the activating agent comprises an activated polymerizable molecule, and further comprising coupling the first polymerizable molecule to the activated polymerizable molecule.

31. The method of claim 30, wherein the first polymerizable molecule, the second polymerizable molecule, and the activated polymerizable molecule are nucleic acid molecules, and wherein the coupling is mediated using a splint molecule comprising a sequence complementary to at least a portion of (i) the first polymerizable molecule, (ii) the second polymerizable molecule, and (iii) the activated polymerizable molecule.

32. The method of any one of claims 27-31, wherein the first polymerizable molecule comprises a nucleic acid molecule, and further comprising generating the first polymerizable molecule via a hybridization chain reaction (HCR).

33. A method as claimed in claim 32, wherein the generating comprises (A) providing a first hairpin molecule and a second hairpin molecule; (B) coupling the first hairpin molecule to the activated polymerizable molecule; and (C) coupling the second hairpin molecule to the first hairpin molecule, thereby generating the first polymerizable molecule coupled to the activated polymerizable molecule.

34. The method of claim 33, wherein after (C), the first polymerizable molecule comprises a flap sequence, and further comprising using the flap sequence to couple the first polymerizable molecule to the second polymerizable molecule.

35. The method of claim 33, further comprising coupling an additional first hairpin molecule to the first polymerizable molecule and coupling an additional second hairpin molecule to the additional first hairpin molecule, thereby generating additional first polymerizable molecules coupled to the first polymerizable molecule.

36. The method of claim 35, further comprising coupling the additional first polymerizable molecule to an additional second polymerizable molecule.

37. The method of claim 33, further comprising repeating (b)-(f), wherein the repeating comprises generating additional first polymerizable molecules by repeating (A)-(C).

38. The method of claim 37, wherein the repetition of (A)-(C) comprises providing additional hairpin molecules, wherein at least a subset of the additional hairpin molecules comprises a sequence that is different from the first hairpin molecule or the second hairpin molecule.

39. The method of claim 38, wherein the subset of the additional hairpin molecules comprises a sequence configured to couple to at least a portion of the first polymerizable molecule.

40. The method of claim 32, further comprising cleaving a portion of the first polymerizable molecule.

41. The method of claim 27, wherein the activator comprises an anchor nucleic acid molecule, wherein the anchor nucleic acid molecule is configured to couple to an additional second polymerizable molecule adjacent to the second polymerizable molecule.

42. The method of claim 41, wherein the first polymerizable molecule is coupled to the binding agent.

43. The method of claim 27, wherein the activator comprises an enzyme selected from the group consisting of a peroxidase, a Cas protein, a ligase, a kinase, and a restriction enzyme.

44. The method of claim 43, wherein the enzyme is a kinase, and further comprising using the kinase to phosphorylate the second polymerizable molecule, thereby allowing the first polymerizable molecule to be coupled to the second polymerizable molecule.

45. The method of claim 43, wherein the enzyme is horseradish peroxidase (HRP) or ascorbate peroxidase (APEX), wherein the HRP or the APEX activates the first polymerizable molecule or the second polymerizable molecule, thereby coupling the first polymerizable molecule to the second polymerizable molecule.

46. ​​The method of claim 43, wherein the enzyme is a restriction enzyme, and wherein the second polymerizable molecule comprises a restriction recognition site, and further comprising cleaving the second polymerizable molecule with the restriction enzyme prior to the coupling of the first polymerizable molecule to the second polymerizable molecule.

47. A method for processing an analyte, the method comprising: (a) providing (i) the analyte, (ii) a binding agent comprising an activator, (iii) a first polymerizable molecule, and (iv) a second polymerizable molecule, wherein the analyte has a size of <10 nm; (b) contacting the analyte with the binding agent; and (c) using the activator to couple the first polymerizable molecule to the second polymerizable molecule.

48. The method of claim 47, wherein the binding agent is not coupled to the first polymerizable molecule.

49. The method of claim 47, wherein the binding agent is indirectly coupled to the first polymerizable molecule.

50. The method of claim 47, wherein the activating agent comprises an activated polymerizable molecule, and further comprising coupling the first polymerizable molecule to the activated polymerizable molecule.

51. The method of claim 50, wherein the first polymerizable molecule, the second polymerizable molecule, and the activated polymerizable molecule are nucleic acid molecules, and wherein the coupling is mediated using a splint molecule comprising a sequence complementary to at least a portion of (i) the first polymerizable molecule, (ii) the second polymerizable molecule, and (iii) the activated polymerizable molecule.

52. The method of any one of claims 47-51, wherein the first polymerizable molecule comprises a nucleic acid molecule, and further comprising generating the first polymerizable molecule via a hybridization chain reaction (HCR).

53. A method as claimed in claim 52, wherein the generating comprises (A) providing a first hairpin molecule and a second hairpin molecule; (B) coupling the first hairpin molecule to the activated polymerizable molecule; and (C) coupling the second hairpin molecule to the first hairpin molecule, thereby generating the first polymerizable molecule coupled to the activated polymerizable molecule.

54. The method of claim 53, wherein after (C), the first polymerizable molecule comprises a flap sequence, and further comprising using the flap sequence to couple the first polymerizable molecule to the second polymerizable molecule.

55. The method of any one of claims 47-54, wherein the activating agent comprises an anchoring nucleic acid molecule, wherein the anchoring nucleic acid molecule is configured to couple to an additional second polymerizable molecule adjacent to the second polymerizable molecule.

56. The method of claim 55, wherein the first polymerizable molecule is coupled to the binding agent.

57. The method of any one of claims 47-56, wherein the activator comprises an enzyme selected from the group consisting of a peroxidase, a Cas protein, a ligase, a kinase, and a restriction enzyme.

58. The method of claim 57, wherein the enzyme is a kinase, and further comprising using the kinase to phosphorylate the second polymerizable molecule, thereby allowing the first polymerizable molecule to couple to the second polymerizable molecule.

59. The method of claim 58, wherein the enzyme is horseradish peroxidase (HRP) or ascorbate peroxidase (APEX), wherein the HRP or the APEX activates the first polymerizable molecule or the second polymerizable molecule, thereby coupling the first polymerizable molecule to the second polymerizable molecule.

60. The method of claim 58, wherein the enzyme is a restriction enzyme, and wherein the second polymerizable molecule comprises a restriction recognition site, and further comprising cleaving the second polymerizable molecule with the restriction enzyme prior to the coupling of the first polymerizable molecule to the second polymerizable molecule.

61. A method for processing an analyte, the method comprising: (a) providing (i) the analyte, (ii) a binding agent, and (iii) a first polymerizable molecule, wherein the first polymerizable molecule is not coupled to the binding agent; and (iv) a second polymerizable molecule, wherein the analyte has a size of <10 nm; (b) contacting the analyte with the binding agent; and (c) coupling the first polymerizable molecule to the second polymerizable molecule.

62. The method of claim 61, wherein the binding agent comprises an activated polymerizable molecule, and further comprising coupling the first polymerizable molecule to the activated polymerizable molecule.

63. The method of claim 62, wherein the first polymerizable molecule, the second polymerizable molecule, and the activated polymerizable molecule are nucleic acid molecules, and wherein the coupling is mediated using a splint molecule comprising a sequence complementary to at least a portion of (i) the first polymerizable molecule, (ii) the second polymerizable molecule, and (iii) the activated polymerizable molecule.

64. The method of any one of claims 61-63, wherein the first polymerizable molecule comprises a nucleic acid molecule, and further comprising generating the first polymerizable molecule via a hybridization chain reaction (HCR).

65. A method as claimed in claim 64, wherein the generating comprises (A) providing a first hairpin molecule and a second hairpin molecule; (B) coupling the first hairpin molecule to the activated polymerizable molecule; and (C) coupling the second hairpin molecule to the first hairpin molecule, thereby generating the first polymerizable molecule coupled to the activated polymerizable molecule.

66. The method of claim 65, wherein after (C), the first polymerizable molecule comprises a flap sequence, and further comprising using the flap sequence to couple the first polymerizable molecule to the second polymerizable molecule.

67. The method of any one of claims 61-66, wherein the binding agent comprises an anchoring nucleic acid molecule, wherein the anchoring nucleic acid molecule is configured to couple to an additional second polymerizable molecule adjacent to the second polymerizable molecule.

68. The method of any one of claims 61-67, wherein the binding agent comprises an enzyme coupled thereto, wherein the enzyme is selected from the group consisting of: a peroxidase, a Cas protein, a ligase, a kinase, and a restriction enzyme.

69. The method of claim 68, wherein the enzyme is a kinase, and further comprising using the kinase to phosphorylate the second polymerizable molecule, thereby allowing the first polymerizable molecule to couple to the second polymerizable molecule.

70. The method of claim 68, wherein the enzyme is horseradish peroxidase (HRP) or ascorbate peroxidase (APEX), wherein the HRP or the APEX activates the first polymerizable molecule or the second polymerizable molecule, thereby coupling the first polymerizable molecule to the second polymerizable molecule.

71. The method of claim 68, wherein the enzyme is a restriction enzyme, and wherein the second polymerizable molecule comprises a restriction recognition site, and further comprising cleaving the second polymerizable molecule with the restriction enzyme prior to the coupling of the first polymerizable molecule to the second polymerizable molecule.

72. The method of any one of claims 61-71, wherein the analyte comprises a monomer unit of a polymer.

73. The method of claim 72, wherein the polymer is a peptide and the monomer units comprise amino acids.

74. The method of claim 73, wherein at least one amino acid of the peptide comprises a post-translational modification.

75. The method of any one of claims 61-74, further comprising, prior to (a), providing a polymer comprising a plurality of monomers, coupling a monomer of the plurality of monomers to a substrate, and cleaving the monomer from the polymer to produce the analyte coupled to the substrate.

76. The method of claim 75, wherein the polymer comprises a peptide and the monomer comprises an amino acid or a modified amino acid.

77. The method of claim 75, wherein the coupling is mediated by a linker.

78. The method of claim 77, wherein the linker comprises a first reactive moiety capable of coupling to the monomer and a second reactive moiety capable of coupling to the substrate.

79. A method comprising: (a) providing (i) a peptide comprising a plurality of amino acids, wherein an amino acid in the plurality of amino acids comprises a non-naturally occurring modification of the plurality of amino acids, (ii) a substrate coupled to the peptide, and (iii) a linker; (b) using the linker to couple the amino acid or another amino acid from the plurality of amino acids to the substrate to produce a substrate-coupled amino acid; as well as (c) after (b), cleaving the substrate-coupled amino acid from the peptide, thereby producing (i) the removed substrate-coupled amino acid and (ii) the remaining portion of the peptide.

80. A method for sequencing a peptide, the method comprising: (a) providing a substrate comprising the peptide and a nucleic acid molecule; (b) providing a linker, wherein the linker comprises an amino acid reactive group and a substrate tethering portion; (c) coupling the linker to the terminal amino acid of the peptide and the substrate; (d) cleaving the terminal amino acid, thereby providing a cleaved amino acid-linker complex coupled to the substrate; (e) contacting the cleaved amino acid-linker complex with a binding agent comprising an activator; (f) providing a nucleic acid barcode molecule; as well as (g) using the activator to couple the nucleic acid barcode molecule to the nucleic acid molecule.

81. The method of claim 80, wherein the substrate comprises a capture moiety, wherein the substrate tethering moiety is configured to couple to the capture moiety.

82. The method of claim 81, wherein the capture portion comprises an additional nucleic acid molecule, wherein the linker comprises a connecting nucleic acid molecule, and wherein in (c), the connecting nucleic acid molecule is coupled to the additional nucleic acid molecule.

83. The method of claim 81, wherein the capture moiety comprises a first click chemistry moiety and wherein the linker comprises a second click chemistry moiety, and wherein the coupling in (c) comprises reacting the first click chemistry moiety with the second click chemistry moiety.

84. The method of any one of claims 80-83, wherein the activating agent comprises an activated nucleic acid molecule, and further comprising coupling the nucleic acid barcode molecule to the activated polymerizable molecule.

85. The method of claim 84, wherein the coupling is mediated using a splint molecule comprising a sequence complementary to at least a portion of (i) the nucleic acid barcode molecule, (ii) the nucleic acid molecule, and (iii) the activation nucleic acid molecule.

86. The method of any one of claims 80-85, further comprising generating the nucleic acid barcode molecule via hybridization chain reaction (HCR).

87. A method as described in claim 86, wherein the generating comprises (A) providing a first hairpin molecule and a second hairpin molecule; (B) coupling the first hairpin molecule to the activator; and (C) coupling the second hairpin molecule to the first hairpin molecule, thereby generating the nucleic acid barcode molecule.

88. The method of claim 87, wherein the activator comprises an activating nucleic acid molecule.

89. The method of claim 87 or 88, wherein after (C), the nucleic acid barcode molecule comprises a flap sequence, and wherein (g) uses the flap sequence to mediate coupling of the nucleic acid barcode molecule to the nucleic acid molecule.

90. The method of claim 87, wherein the nucleic acid barcode molecule comprises the first hairpin molecule or the second hairpin molecule.

91. The method of any one of claims 80-90, wherein the activator comprises an anchor nucleic acid molecule, wherein the anchor nucleic acid molecule is configured to couple to an additional nucleic acid molecule adjacent to the nucleic acid molecule.

92. The method of claim 80, wherein the nucleic acid barcode molecule is coupled to the binding agent.

93. The method of any one of claims 80-92, wherein the activator comprises an enzyme selected from the group consisting of a peroxidase, a Cas protein, a ligase, a kinase, and a restriction enzyme.

94. The method of claim 93, wherein the enzyme is a kinase, and further comprising using the kinase to phosphorylate the nucleic acid molecule, thereby allowing the nucleic acid barcode molecule to be coupled to the nucleic acid molecule.

95. The method of claim 93, wherein the enzyme is horseradish peroxidase (HRP) or ascorbate peroxidase (APEX), wherein the HRP or the APEX activates the nucleic acid barcode molecule or the nucleic acid molecule, thereby coupling the nucleic acid barcode molecule to the nucleic acid molecule.

96. The method of claim 93, wherein the enzyme is a restriction enzyme, and wherein the nucleic acid molecule comprises a restriction recognition site, and further comprising cleaving the nucleic acid molecule with the restriction enzyme prior to the coupling of the nucleic acid barcode molecule to the nucleic acid molecule.

97. The method of any one of claims 80-96, wherein at least one amino acid of the peptide comprises a post-translational modification.

98. The method of any one of claims 80-97, wherein the coupling in (c) is mediated by a linker.

99. A method for characterizing a plurality of analytes, the method comprising: (a) providing a first analyte, a second analyte, a first binding agent capable of coupling to the first analyte and not coupling to the second analyte, and a second binding agent capable of coupling to both the first analyte and the second analyte; (b) contacting the first analyte and the second analyte with the first binding agent; (c) after (b), contacting the first analyte and the second analyte with the second binding agent; (d) identifying the first binding agent and the second binding agent; as well as (e) using the first binding agent and the second binding agent identified in (d) to identify the first analyte and the second analyte.

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