WO2020198264A1 - Clivages modifiés, utilisations et kits correspondants - Google Patents

Clivages modifiés, utilisations et kits correspondants Download PDF

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Publication number
WO2020198264A1
WO2020198264A1 PCT/US2020/024521 US2020024521W WO2020198264A1 WO 2020198264 A1 WO2020198264 A1 WO 2020198264A1 US 2020024521 W US2020024521 W US 2020024521W WO 2020198264 A1 WO2020198264 A1 WO 2020198264A1
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WIPO (PCT)
Prior art keywords
cleavase
amino acid
identity
modified
polypeptide
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PCT/US2020/024521
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English (en)
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WO2020198264A8 (fr
Inventor
Kevin Desai
Kevin L. GUNDERSON
Robert C. James
Lei Shi
Stephen VERESPY, III
Original Assignee
Encodia, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Priority to SG11202110373WA priority Critical patent/SG11202110373WA/en
Application filed by Encodia, Inc. filed Critical Encodia, Inc.
Priority to EP20779890.1A priority patent/EP3947667A4/fr
Priority to KR1020217033837A priority patent/KR102567902B1/ko
Priority to JP2021557332A priority patent/JP2022526939A/ja
Priority to AU2020247918A priority patent/AU2020247918B2/en
Priority to CN202080023620.0A priority patent/CN113993997A/zh
Priority to MX2021011726A priority patent/MX2021011726A/es
Priority to CA3134776A priority patent/CA3134776A1/fr
Publication of WO2020198264A1 publication Critical patent/WO2020198264A1/fr
Publication of WO2020198264A8 publication Critical patent/WO2020198264A8/fr
Priority to US17/213,169 priority patent/US11427814B2/en
Priority to US17/850,516 priority patent/US11788080B2/en
Priority to US18/098,647 priority patent/US20230257727A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/14—Dipeptidyl-peptidases and tripeptidyl-peptidases (3.4.14)
    • C12Y304/14004—Dipeptidyl-peptidase III (3.4.14.4)

Definitions

  • the present disclosure relates to modified cleavases for cleaving amino acids from peptides, polypeptides, and proteins, including modified peptides, polypeptides, and proteins. Also provided are methods of using the modified cleavases for treating polypeptides, and kits comprising the modified cleavase. In some embodiments, the methods and the kits also include other components for macromolecule sequencing and/or analysis.
  • Enzymes that are involved in degradation of peptides and proteins e.g., aminopeptidases, dipeptidyl peptidases, carboxypeptidases, endopeptidases, and others, hydrolyze peptide bonds (Sanderink et ah, J. Clin. Chem. Clin. Biochem. (1988) 26:795-807).
  • Various peptidases have been isolated and discovered in a number of organisms and from various tissues. Aminopeptidases naturally occur as monomeric and multimeric enzymes, and may be metal or ATP-dependent.
  • substrate-specific peptidases specifically remove one or two amino acid residues at a time from the amino-terminus of the peptide while others remove from the carboxy -terminus of the protein or peptide.
  • Natural aminopeptidases generally have limited specificity and eliminate amino acids in a processive manner, eliminating one amino acid one after another.
  • peptide degradation are useful for applications in protein analysis and/or sequencing.
  • peptide sequencing may involve Edman degradation to achieve stepwise degradation of the N-terminal amino acid (NTAA) on a peptide through a series of chemical modifications and downstream HPLC analysis or mass spectrometry analysis.
  • NTAA N-terminal amino acid
  • peptide sequencing may be limited, for example, typical Edman degradation requires deployment of high temperature and harsh chemical conditions (e.g ., strong acids; anhydrous TFA) for long incubation times.
  • Edman degradation may not be compatible with processes for protein analysis methods which may be sensitive to harsh chemical conditions, such as analysis methods which employ nucleic acids (e.g., DNA).
  • a modified cleavase comprising a mutation, e.g ., one or more amino acid modification(s) in an unmodified cleavase, wherein the modified cleavase is derived from a dipeptide cleavase and removes or is configured to remove a single labeled terminal amino acid from a polypeptide.
  • a modified cleavase comprising a mutation, e.g.
  • the modified cleavase is derived from a tripeptide cleavase and removes or is configured to remove a single labeled terminal amino acid from a polypeptide or a single labeled terminal dipeptide from a polypeptide.
  • the modified cleavase is configured to remove a single labeled terminal amino acid from the C-terminus or N-terminus of a polypeptide.
  • the modified cleavase is configured to remove a single labeled dipeptide (the terminal and penultimate terminal amino acids) from the C-terminus or N-terminus of a polypeptide.
  • the modified cleavase is derived from a wild-type or unmodified cleavase (e.g, a dipeptide cleavase or tripeptide cleavase).
  • the unmodified cleavase is a protein classified in EC 3.4.14, EC 3.4.15, MEROPS S8, MEROPS S9, MEROPS S33, MEROPS S46, MEROPS M49, or MEROPS S53, or a functional homolog or fragment thereof.
  • Also provided herein is a method of treating a polypeptide, comprising contacting the polypeptide with a modified cleavase comprising a mutation, e.g. , one or more amino acid modification(s) in an unmodified cleavase, wherein the modified cleavase is derived from a dipeptide cleavase and removes a single labeled terminal amino acid from a polypeptide.
  • a modified cleavase comprising a mutation, e.g. , one or more amino acid modification(s) in an unmodified cleavase, wherein the modified cleavase is derived from a dipeptide cleavase and removes a single labeled terminal amino acid from a polypeptide.
  • a method of treating a polypeptide comprising contacting the polypeptide with a modified cleavase comprising a mutation, e.g. , one or more amino acid modifications in an unmodified cleavase, wherein the modified cleavase is derived from a tripeptide cleavase and removes a single labeled terminal amino acid from a polypeptide or a single labeled terminal dipeptide from a polypeptide.
  • the modified cleavase removes a single labeled terminal amino acid from the C-terminus or N-terminus of the polypeptide treated with the modified cleavase.
  • the modified cleavase removes a single labeled dipeptide (the terminal and penultimate terminal amino acids) from the C-terminus or N- terminus of the polypeptide treated with the modified cleavase.
  • the modified cleavase is derived from a wild-type or unmodified cleavase (e.g, a dipeptide cleavase or tripeptide cleavase).
  • the unmodified cleavase is a protein classified in EC 3.4.14, EC 3.4.15, MEROPS S8, MEROPS S9, MEROPS S33, MEROPS S46, MEROPS M49, or MEROPS S53, or a functional homolog or fragment thereof.
  • the method further comprises contacting the polypeptide with a reagent for labeling the terminal amino acid of the polypeptide.
  • the method further comprises contacting the polypeptide with a binding agent capable of binding to the terminal amino acid of the polypeptide, wherein the binding agent comprises a coding tag with identifying information regarding the binding agent.
  • the method also further comprises transferring the identifying information of the coding tag to a recording tag attached to the polypeptide, thereby generating an extended recording tag(s) on the polypeptide. In some cases, the method further comprises removing the binding agent. In some further embodiments, the method further comprises analyzing the one or more extended recording tag(s). In some further embodiments, the method further comprises repeating some or all of the above steps one or more times.
  • a method for analyzing a polypeptide comprising the steps of: (a) contacting a polypeptide with a binding agent capable of binding to the terminal amino acid of the polypeptide, wherein the binding agent comprises a coding tag with identifying information regarding the binding agent; (b) transferring the identifying information of the coding tag to a recording tag associated with each of the polypeptide to generate an extended recording tag; (c) contacting the polypeptide with a reagent to label the terminal amino acid of the polypeptide; and (d) contacting the polypeptide with a modified cleavase comprising a mutation, e.g ., one or more amino acid modification(s), in an unmodified cleavase, wherein the modified cleavase is derived from a dipeptide cleavase and removes a single terminal amino acid labeled by the reagent in step (c) from the polypeptide; or the modified cleavase is derived from a tri
  • kits for treating a polypeptide comprising a modified cleavase comprising a mutation, e.g. , one or more amino acid modifications, in an unmodified cleavase and a reagent for labeling the terminal amino acid of the polypeptide.
  • the modified cleavase is derived from a dipeptide cleavase and removes or is configured to remove a single labeled terminal amino acid from a polypeptide or the modified cleavase is derived from a tripeptide cleavase and removes or is configured to remove a single labeled terminal amino acid from a polypeptide or a single labeled terminal dipeptide from a
  • the kit further comprises one or more binding agents, wherein each binding agent comprises a coding tag with identifying information regarding the binding agent.
  • the kit further comprises a reagent for transferring the identifying information of the coding tag to a recording tag attached to the polypeptide, wherein the transferring of the identifying information to the recording tag generates an extended recording tag on the polypeptide.
  • the kit further comprises one or more amplification reagent(s) for amplifying the extended recording tags.
  • the kit further comprises one or more substrates or supports.
  • the kit also comprises a reagent for nucleic acid sequencing analysis.
  • FIG. 1A-1B is a schematic depicting the removal of a single modified amino acid by exemplary modified cleavases as provided herein.
  • an exemplary unmodified cleavase derived from a dipeptide cleavase removes two amino acids from the N- terminus of the polypeptide, cleaving the bond between the penultimate (P2) and
  • an exemplary modified cleavase derived from a dipeptide cleavase removes a single labeled amino acid from the N-terminus of the polypeptide, cleaving the bond between the labeled terminal amino acid (PI; diamond indicates label) and the penultimate amino acid residue (P2).
  • an exemplary unmodified cleavase derived from a tripeptide cleavase removes three amino acids from the N- terminus of the polypeptide, cleaving the bond C-terminal to the antepenultimate amino acid on the N-terminus (between P3 and P4).
  • the modified tripeptide cleavase removes a single labeled amino acid from the N-terminus of the polypeptide, cleaving the bond between the labeled terminal amino acid (PI; diamonds indicates label) and the penultimate amino acid residue (P2).
  • the modified tripeptide cleavase removes a labeled dipeptide (P1-P2) from the N-terminus of the polypeptide, cleaving the bond between the penultimate terminal amino acid residue (P2) and the antepenultimate amino acid residue (P3).
  • FIG. 2A-2C is a schematic depicting a cycle of terminal amino acid removal using the modified cleavase and terminal amino acid labeling.
  • a polypeptide with a labeled N-terminal amino acid residue is cleaved at the bond between the terminal amino acid and penultimate amino acid by the modified cleavase and the labeled terminal amino acid is released.
  • the new terminal amino acid is labeled and the modified cleavase is able to recognize the new labeled terminal amino acid for further cleavage and release.
  • FIG. 3. depicts N-terminal amino acid (NTAA) conversion efficiency with different exemplary reagents for labeling the N-terminal amino acid.
  • N-G GRFSGIY (SEQ ID NO:40);
  • N-Terminal W (NT-W) WTQIFGA (SEQ ID NO: 41).
  • LC-MS was used to quantitate conversion efficiency.
  • FIG. 4A-4C depict results from a WebLogo analysis of sequence conservation of DAP BII homologs with 60% sequence similarity or identity.
  • the height of each stack indicates the sequence conservation at that position (measured in bits), and the height of symbols within the stack reflects the relative frequency of the corresponding amino acid at the indicated position (in reference to SEQ ID NO: 20).
  • modified cleavases comprising a mutation (e.g ., one or more modifications in an unmodified cleavase) and related methods of selecting, engineering, and using the modified cleavases.
  • methods for labeling a polypeptide e.g. with a chemical reagent
  • treating the labeled polypeptide with a modified cleavase to remove the labeled terminal amino acid from the polypeptide e.g. with a chemical reagent
  • kits comprising the modified cleavases.
  • the kits comprising the modified cleavase is used for treating peptides, polypeptides, and proteins, such as for sequencing and/or analysis.
  • protein analysis using the modified cleavase employs barcoding and nucleic acid encoding of molecular recognition events, and/or detectable labels.
  • the kits also include other components for treating the polypeptides, including tags (e.g, DNA tag or DNA recording tag), solid supports, and other reagents for preparing the polypeptides and other reagents for polypeptide analysis.
  • tags e.g, DNA tag or DNA recording tag
  • solid supports e.g., DNA tag or DNA recording tag
  • other reagents for preparing the polypeptides and other reagents for polypeptide analysis.
  • natural aminopeptidases may have limited specificity, and generically eliminate N-terminal amino acids in a processive manner, eliminating one amino acid off after another.
  • Some substrate-specific peptidases specifically remove one or two amino acid residues at a time from the amino-terminus or carboxy-terminus of peptides.
  • peptide sequencing may involve Edman degradation to achieve stepwise degradation of the N-terminal amino acid on a peptide through a series of chemical modifications and downstream HPLC analysis or mass spectrometry analysis.
  • Edman degradation peptide sequencing may be limited, for example, typical Edman degradation requires deployment of high temperature and harsh chemical conditions (e.g., strong acids; anhydrous TFA) for long incubation times.
  • Edman degradation may not be compatible with processes for protein analysis methods which may be sensitive to harsh chemical conditions, such as analysis methods which employ nucleic acids (e.g, DNA).
  • enzymatic methods for removing, eliminating, or cleaving amino acids from polypeptides may be desired.
  • modified peptidases that meet such needs.
  • enzymatic methods and reagents for removing amino acids from polypeptides are used for stepwise degradation of amino acids from polypeptides.
  • the removal of amino acids by the provided modified cleavase are suitable for cyclic removal of amino acids from the polypeptide.
  • the modified cleavase removes or is configured to remove a single labeled terminal amino acid from a polypeptide. In some embodiments, the modified cleavase removes a single labeled terminal amino acid from the C-terminus or N-terminus of a polypeptide. For example, the modified cleavase is configured to cleave the peptide bond between a terminal labeled amino acid residue and a penultimate terminal amino acid residue of the polypeptide. In some embodiments, the modified cleavase is configured to remove a single labeled dipeptide (the terminal and penultimate terminal amino acids) from the C-terminus or N- terminus of a polypeptide.
  • the modified cleavase is derived from a wild- type or unmodified cleavase (e.g ., a dipeptide cleavase or tripeptide cleavase).
  • the unmodified cleavase is a protein classified in EC 3.4.14, EC 3.4.15, MEROPS S8, MEROPS S9, MEROPS S33, MEROPS S46, MEROPS M49, or MEROPS S53, or a functional homolog or fragment thereof.
  • the modified cleavase is derived from a wild-type or unmodified cleavase (e.g., a dipeptidyl peptidase, a dipeptidyl aminopeptidase, a peptidyl-dipeptidase, or a dipeptidyl carboxypeptidase).
  • a wild-type or unmodified cleavase e.g., a dipeptidyl peptidase, a dipeptidyl aminopeptidase, a peptidyl-dipeptidase, or a dipeptidyl carboxypeptidase.
  • peptidases may be engineered to possess specific binding or catalytic activity to specific terminal amino acids only when modified with a label.
  • a cleavase may be engineered or modified, compared to a wild-type or unmodified cleavase, such than it only eliminates a terminal amino acid if it is labeled by a chemical label.
  • the modified cleavase eliminates only labeled amino acid(s) from the terminus of the polypeptide, and allows control of degradation in a desired manner.
  • the modified cleavase is configured to remove a single labeled terminal amino acid from the C-terminus or N-terminus of a polypeptide.
  • the modified cleavase is configured to remove a single labeled terminal dipeptide (the terminal and penultimate terminal amino acids) from the C-terminus or N-terminus of a polypeptide.
  • the modified cleavase is non-selective as to amino acid residue identity while being selective for the label (e.g, will remove any labeled terminal amino acid).
  • the modified cleavase exhibits some preference for certain amino acid residues or classes of amino acids (e.g. at the PI and/or P2 terminal positions of the polypeptide). In some cases, two or more modified cleavases with different preferences for certain amino acids (or classes of amino acids) may be used in combination.
  • the modified cleavase binds and removes amino acids from the N-terminus of the polypeptide. In some embodiments, the modified cleavase binds and removes amino acids from the C-terminus of the polypeptide.
  • known peptidases may be modified to achieve specific characteristics for binding and/or cleaving.
  • An example of a model of modifying the specificity of enzymatic N-terminal amino acid (NTAA) degradation involves a methionine aminopeptidase converted into a leucine aminopeptidase (Borgo et al., Protein Sci. (2014) 23(3):312-320).
  • aminopeptidase mutants were engineered to bind to and eliminate individual or small groups of labelled (biotinylated) NTAAs (see, PCT Publication No. W02010/065322).
  • modified cleavases which are selected or modified to remove terminal amino acids that are labeled, such as a chemically-modified (e.g ., PTC/DNP/acetyl/Cbz- modified) terminal amino acid on a polypeptide.
  • a wild-type cleavase is engineered (e.g., using structural -function based-design and/or directed evolution) to cleave or remove only an N-terminal amino acid having a PTC/DNP/acetyl/Cbz group present as the label.
  • the terminal amino acid to be removed is a Cbz labeled terminal amino acid.
  • the removed labeled terminal amino acid is removed as a single amino acid or as part of a dipeptide.
  • a compact monomeric metalloenzymatic aminopeptidase is engineered to recognize and eliminate isothiourea or Cbz labeled NTAAs.
  • the modified peptidase is a metallo-peptidase and requires a metal ion for activation.
  • the use of a monomeric metallo-aminopeptidase has two key advantages: 1) compact monomeric proteins are easier to display and screen using phage display; 2) a metallo- aminopeptidase has the unique advantage in that its activity can be turned on/off at will by adding or removing the appropriate metal cation.
  • the unmodified cleavase may be from any suitable organism.
  • the wild-type or unmodified cleavase is from a mammal, e.g., Homo sapiens, a fungus or yeast, e.g., Saccharomyces cerevisiae, or a bacterium, e.g., Bacteroides thetaiotaomicron, Porphyromonas gingivalis, Pseudomonas sp.,
  • Pseudoxanthomonas mexicana or Caldithrix abyssi are stable, robust, and active at room temperature and at or around pH 8.0, and thus compatible with mild conditions preferred for peptide analysis.
  • cyclic elimination or removal of amino acids is attained by engineering the peptidase (e.g, cleavase) to be active only in the presence of a terminal amino acid label.
  • the label is a chemical label.
  • the label is or comprises a blocked or labeled amino acid.
  • the label comprises an exogenous labeled or modified amino acid.
  • the peptidase may be engineered to be non-specific, such that it does not selectively recognize one particular amino acid over another, but recognizes any amino acid at the terminus that has a label.
  • the modified cleavase is selective for one or more, two or more, three or more, four or more, five or more, ten or more, fifteen or more, twenty or more etc. amino acids.
  • the provided modified cleavases are used for treating polypeptides obtained from a sample.
  • the sample and/or the polypeptide obtained from the sample is treated with other reagents for processing the polypeptides, such as digesting the polypeptides.
  • the polypeptides comprise a plurality of polypeptides obtained from a sample.
  • the sample is obtained from a subject.
  • antibody herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab') 2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g ., sdAb, sdFv, nanobody) fragments.
  • Fab fragment antigen binding
  • rlgG recombinant IgG
  • scFv single chain variable fragments
  • single domain antibodies e.g ., sdAb, sdFv, nanobody
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • the term“antibody” should be understood to encompass functional antibody fragments thereof.
  • the term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
  • An“individual” or“subject” includes a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats.
  • An“individual” or“subject” may include birds such as chickens, vertebrates such as fish and mammals such as mice, rats, rabbits, cats, dogs, pigs, cows, ox, sheep, goats, horses, monkeys and other non-human primates. In certain embodiments, the individual or subject is a human.
  • the term“sample” refers to anything which may contain an analyte for which an analyte assay is desired.
  • a“sample” can be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
  • the sample may be a biological sample, such as a biological fluid or a biological tissue.
  • biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like.
  • Biological tissues are aggregate of cells, usually of a particular kind together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cell(s).
  • the sample is a biological sample.
  • a biological sample of the present disclosure encompasses a sample in the form of a solution, a suspension, a liquid, a powder, a paste, an aqueous sample, or a non-aqueous sample.
  • a“biological sample” includes any sample obtained from a living or viral (or prion) source or other source of macromolecules and biomolecules, and includes any cell type or tissue of a subject from which nucleic acid, protein and/or other macromolecule can be obtained.
  • the biological sample can be a sample obtained directly from a biological source or a sample that is processed. For example, isolated nucleic acids that are amplified constitute a biological sample.
  • Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples from animals and plants and processed samples derived therefrom.
  • the sample can be derived from a tissue or a body fluid, for example, a connective, epithelium, muscle or nerve tissue; a tissue selected from the group consisting of brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, gland, and internal blood vessels; or a body fluid selected from the group consisting of blood, urine, saliva, bone marrow, sperm, an ascitic fluid, and subfractions thereof, e.g ., serum or plasma.
  • the terms“level” or“levels” are used to refer to the presence and/or amount of a target, e.g. , a substance or an organism that is part of the etiology of a disease or disorder, and can be determined qualitatively or quantitatively.
  • A“qualitative” change in the target level refers to the appearance or disappearance of a target that is not detectable or is present in samples obtained from normal controls.
  • A“quantitative” change in the levels of one or more targets refers to a measurable increase or decrease in the target levels when compared to a healthy control.
  • polypeptide encompasses peptides and proteins, and refers to a molecule comprising a chain of two or more amino acids joined by peptide bonds.
  • a polypeptide comprises 2 to 50 amino acids, e.g., having more than 20-30 amino acids.
  • a peptide does not comprise a secondary, tertiary, or higher structure.
  • the polypeptide is a protein.
  • a protein comprises 30 or more amino acids, e.g. having more than 50 amino acids.
  • a protein in addition to a primary structure, comprises a secondary, tertiary, or higher structure.
  • the amino acids of the polypeptides are most typically L-amino acids, but may also be D-amino acids, modified amino acids, amino acid analogs, amino acid mimetics, or any combination thereof.
  • Polypeptides may be naturally occurring, synthetically produced, or recombinantly expressed. Polypeptides may be synthetically produced, isolated, recombinantly expressed, or be produced by a combination of methodologies as described above. Polypeptides may also comprise additional groups modifying the amino acid chain, for example, functional groups added via post-translational modification.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the term also encompasses an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • amino acid refers to an organic compound comprising an amine group, a carboxylic acid group, and a side-chain specific to each amino acid, which serve as a monomeric subunit of a peptide.
  • An amino acid includes the 20 standard, naturally occurring or canonical amino acids as well as non-standard amino acids.
  • the standard, naturally-occurring 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 lie), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q or Gin), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Val), Tryptophan (W or Trp), and Tyrosine (Y or Tyr).
  • An amino acid may be an L-amino acid or a D-amino acid.
  • Non-standard amino acids may be modified amino acids, amino acid analogs, amino acid mimetics, non-standard proteinogenic amino acids, or non-proteinogenic amino acids that occur naturally or are chemically synthesized. Examples of non-standard amino acids include, but are not limited to, selenocysteine, pyrrolysine, and N-formylmethionine, b-amino acids, Homo-amino acids,
  • Proline and Pyruvic acid derivatives 3-substituted alanine derivatives, glycine derivatives, ring- substituted phenylalanine and tyrosine derivatives, linear core amino acids, N-methyl amino acids.
  • post-translational modification refers to modifications that occur on a peptide after its translation, e.g ., translation by ribosomes, is complete.
  • a post- translational modification may be a covalent chemical modification or enzymatic modification.
  • post-translation modifications include, but are not limited to, acylation, acetylation, alkylation (including methylation), biotinylation, butyrylation, carbamylation, carbonylation, deamidation, deiminiation, diphthamide formation, disulfide bridge formation, eliminylation, flavin attachment, formylation, gamma-carboxylation, glutamyl ati on, glycylation, glycosylation, glypiation, heme C attachment, hydroxylation, hypusine formation, iodination, isoprenyl ati on, lipidation, lipoylation, malonylation, methylation, myristolylation, oxidation, palmitoylation, pegylation, phosphopantetheinylation, phosphorylation, prenylation, propionylation, retinylidene Schiff base formation, S-glutathionylation, S-nitrosylation, S-sulfenylation,
  • a post-translational modification includes modifications of the amino terminus and/or the carboxyl terminus of a peptide.
  • Modifications of the terminal amino group include, but are not limited to, des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications.
  • Modifications of the terminal carboxy group include, but are not limited to, amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications (e.g., wherein lower alkyl is C 1 -C 4 alkyl).
  • a post-translational modification also includes modifications, such as but not limited to those described above, of amino acids falling between the amino and carboxy termini.
  • the term post-translational modification can also include peptide modifications that include one or more detectable labels.
  • binding agent refers to a nucleic acid molecule, a peptide, a polypeptide, a protein, carbohydrate, or a small molecule that binds to, associates, unites with, recognizes, or combines with a binding target, e.g, a polypeptide or a component or feature of a polypeptide.
  • a binding agent may form a covalent association or non-covalent association with the polypeptide or component or feature of a polypeptide.
  • a binding agent may also be a chimeric binding agent, composed of two or more types of molecules, such as a nucleic acid molecule-peptide chimeric binding agent or a carbohydrate-peptide chimeric binding agent.
  • a binding agent may be a naturally occurring, synthetically produced, or recombinantly expressed molecule.
  • a binding agent may bind to a single monomer or subunit of a polypeptide e.g ., a single amino acid of a polypeptide) or bind to a plurality of linked subunits of a polypeptide (e.g., a di-peptide, tri-peptide, or higher order peptide of a longer peptide, polypeptide, or protein molecule).
  • a binding agent may bind to a linear molecule or a molecule having a three-dimensional structure (also referred to as conformation).
  • an antibody binding agent may bind to linear peptide, polypeptide, or protein, or bind to a conformational peptide, polypeptide, or protein.
  • a binding agent may bind to an N-terminal peptide, a C-terminal peptide, or an intervening peptide of a peptide, polypeptide, or protein molecule.
  • a binding agent may bind to an N-terminal amino acid, C-terminal amino acid, or an intervening amino acid of a peptide molecule.
  • a binding agent may preferably bind to a chemically modified or labeled amino acid (e.g., an amino acid that has been labeled by a reagent comprising a compound of any one of Formula (I)-(IV) as described herein) over a non- modified or unlabeled amino acid.
  • a binding agent may preferably bind to an amino acid that has been labeled or modified over an amino acid that is unlabeled or unmodified.
  • a binding agent may bind to a post-translational modification of a peptide molecule.
  • a binding agent may exhibit selective binding to a component or feature of a polypeptide (e.g., a binding agent may selectively bind to one of the 20 possible natural amino acid residues and with bind with very low affinity or not at all to the other 19 natural amino acid residues).
  • a binding agent may exhibit less selective binding, where the binding agent is capable of binding or configured to bind to a plurality of components or features of a polypeptide (e.g., a binding agent may bind with similar affinity to two or more different amino acid residues).
  • a binding agent may comprise a coding tag, which may be joined to the binding agent by a linker.
  • linker refers to one or more of a nucleotide, a nucleotide analog, an amino acid, a peptide, a polypeptide, a polymer, or a non-nucleotide chemical moiety that is used to join two molecules.
  • a linker may be used to join a binding agent with a coding tag, a recording tag with a polypeptide, a polypeptide with a solid support, a recording tag with a solid support, etc.
  • a linker joins two molecules via enzymatic reaction or chemistry reaction (e.g, click chemistry).
  • ligand refers to any molecule or moiety connected to the compounds described herein.“Ligand” may refer to one or more ligands attached to a compound. In some embodiments, the ligand is a pendant group or binding site ( e.g ., the site to which the binding agent binds).
  • proteome can include the entire set of proteins, polypeptides, or peptides (including conjugates or complexes thereof) expressed by a genome, cell, tissue, or organism at a certain time, of any organism. In one aspect, it is the set of expressed proteins in a given type of cell or organism, at a given time, under defined conditions. Proteomics is the study of the proteome. For example, a“cellular proteome” may include the collection of proteins found in a particular cell type under a particular set of environmental conditions, such as exposure to hormone stimulation. An organism’s complete proteome may include the complete set of proteins from all of the various cellular proteomes. A proteome may also include the collection of proteins in certain sub-cellular biological systems.
  • proteome include subsets of a proteome, including but not limited to a kinome; a secretome; a receptome (e.g., GPCRome); an immunoproteome; a nutriproteome; a proteome subset defined by a post- translational modification (e.g., phosphorylation, ubiquitination, methylation, acetylation, glycosylation, oxidation, lipidation, and/or nitrosylation), such as a phosphoproteome (e.g, phosphotyrosine-proteome, tyrosine-kinome, and tyrosine-phosphatome), a glycoproteome, etc.; a proteome subset associated with a tissue or organ, a developmental stage, or a physiological or pathological condition; a proteome subset associated a cellular process, such as cell cycle, differentiation (or de).
  • a post- translational modification e.g., phosphorylation, ubiquitination, methyl
  • proteomics studies include the dynamic state of the proteome, continually changing in time as a function of biology and defined biological or chemical stimuli.
  • NTAA N-terminal amino acid
  • C-terminal amino acid C-terminal amino acid
  • the next amino acid is the n-1 amino acid, then the n-2 amino acid, and so on down the length of the peptide from the N-terminal end to C-terminal end.
  • an NTAA, CTAA, or both may be modified or labeled with a moiety or a chemical moiety.
  • barcode refers to a nucleic acid molecule of about 2 to about 30 bases (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases) providing a unique identifier tag or origin information for a polypeptide, a binding agent, a set of binding agents from a binding cycle, a sample
  • polypeptides a set of samples, polypeptides within a compartment (e.g., droplet, bead, or separated location), polypeptides within a set of compartments, a fraction of polypeptides, a set of polypeptide fractions, a spatial region or set of spatial regions, a library of polypeptides, or a library of binding agents.
  • a barcode can be an artificial sequence or a naturally occurring sequence. In certain embodiments, each barcode within a population of barcodes is different.
  • a portion of barcodes in a population of barcodes is different, e.g, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the barcodes in a population of barcodes is different.
  • a population of barcodes may be randomly generated or non-randomly generated.
  • a population of barcodes are error correcting barcodes. Barcodes can be used to computationally deconvolute the multiplexed sequencing data and identify sequence reads derived from an individual polypeptide, sample, library, etc.
  • a barcode can also be used for deconvolution of a collection of polypeptides that have been distributed into small
  • the peptide is mapped back to its originating protein molecule or protein complex.
  • the term“coding tag” refers to a polynucleotide with any suitable length, e.g, a nucleic acid molecule of about 2 bases to about 100 bases, including any integer including 2 and 100 and in between, that comprises identifying information for its associated binding agent.
  • A“coding tag” may also be made from a“sequenceable polymer” (see, e.g., Niu et ak, 2013, Nat. Chem. 5:282-292; Roy et ak, 2015, Nat. Commun. 6:7237; Lutz, 2015, Macromolecules 48:4759-4767; each of which are incorporated by reference in its entirety).
  • a coding tag may comprise an encoder sequence, which is optionally flanked by one spacer on one side or optionally flanked by a spacer on each side.
  • a coding tag may also be comprised of an optional UMI and/or an optional binding cycle-specific barcode.
  • a coding tag may be single stranded or double stranded.
  • a double stranded coding tag may comprise blunt ends, overhanging ends, or both.
  • a coding tag may refer to the coding tag that is directly attached to a binding agent, to a complementary sequence hybridized to the coding tag directly attached to a binding agent ( e.g ., for double stranded coding tags), or to coding tag information present in an extended recording tag.
  • a coding tag may further comprise a binding cycle specific spacer or barcode, a unique molecular identifier, a universal priming site, or any combination thereof.
  • spacer refers to a nucleic acid molecule of about 1 base to about 20 bases (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases) in length that is present on a terminus of a recording tag or coding tag.
  • a spacer sequence flanks an encoder sequence of a coding tag on one end or both ends. Following binding of a binding agent to a polypeptide, annealing between complementary spacer sequences on their associated coding tag and recording tag, respectively, allows transfer of binding information through a primer extension reaction or ligation to the recording tag, coding tag, or a di-tag construct.
  • Sp refers to spacer sequence complementary to Sp.
  • spacer sequences within a library of binding agents possess the same number of bases.
  • a common (shared or identical) spacer may be used in a library of binding agents.
  • a spacer sequence may have a“cycle specific” sequence in order to track binding agents used in a particular binding cycle.
  • the spacer sequence (Sp) can be constant across all binding cycles, be specific for a particular class of polypeptides, or be binding cycle number specific.
  • Polypeptide class-specific spacers permit annealing of a cognate binding agent’s coding tag information present in an extended recording tag from a completed binding/extension cycle to the coding tag of another binding agent recognizing the same class of polypeptides in a subsequent binding cycle via the class-specific spacers.
  • a spacer sequence may comprise sufficient number of bases to anneal to a complementary spacer sequence in a recording tag to initiate a primer extension (also referred to as polymerase extension) reaction, or provide a “splint” for a ligation reaction, or mediate a“sticky end” ligation reaction.
  • a spacer sequence may comprise a fewer number of bases than the encoder sequence within a coding tag.
  • the term "recording tag” refers to a moiety, e.g, a chemical coupling moiety, a nucleic acid molecule, or a sequenceable polymer molecule (see, e.g, Niu et ah, 2013, Nat. Chem. 5:282-292; Roy et ah, 2015, Nat. Commun. 6:7237; Lutz, 2015,
  • Identifying information can comprise any information characterizing a molecule such as information pertaining to sample, fraction, partition, spatial location, interacting neighboring molecule(s), cycle number, etc. Additionally, the presence of UMI information can also be classified as identifying information.
  • a binding agent after a binding agent binds to a polypeptide, information from a coding tag linked to a binding agent can be transferred to the recording tag associated with the polypeptide while the binding agent is bound to the polypeptide.
  • information from a recording tag associated with the polypeptide after a binding agent binds to a polypeptide, information from a recording tag associated with the polypeptide can be transferred to the coding tag linked to the binding agent while the binding agent is bound to the polypeptide.
  • a recoding tag may be directly linked to a polypeptide, linked to a polypeptide via a
  • a recording tag may be linked via its 5’ end or 3’ end or at an internal site, as long as the linkage is compatible with the method used to transfer coding tag information to the recording tag or vice versa.
  • a recording tag may further comprise other functional components, e.g., a universal priming site, unique molecular identifier, a barcode (e.g, a sample barcode, a fraction barcode, spatial barcode, a compartment tag, etc.), a spacer sequence that is complementary to a spacer sequence of a coding tag, or any combination thereof.
  • the spacer sequence of a recording tag is preferably at the 3’ -end of the recording tag in embodiments where polymerase extension is used to transfer coding tag information to the recording tag.
  • the term“primer extension”, also referred to as“polymerase extension”, refers to a reaction catalyzed by a nucleic acid polymerase (e.g., DNA polymerase) whereby a nucleic acid molecule (e.g, oligonucleotide primer, spacer sequence) that anneals to a complementary strand is extended by the polymerase, using the complementary strand as template.
  • a nucleic acid polymerase e.g., DNA polymerase
  • a nucleic acid molecule e.g, oligonucleotide primer, spacer sequence
  • the term“unique molecular identifier” or“UMI” refers to a nucleic acid molecule of about 3 to about 40 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, or 40 bases) in length providing a unique identifier tag for each macromolecule, polypeptide or binding agent to which the UMI is linked.
  • a polypeptide UMI can be used to computationally deconvolute sequencing data from a plurality of extended recording tags to identify extended recording tags that originated from an individual polypeptide.
  • a polypeptide UMI can be used to accurately count originating polypeptide molecules by collapsing NGS reads to unique UMIs.
  • a binding agent UMI can be used to identify each individual molecular binding agent that binds to a particular polypeptide. For example, a UMI can be used to identify the number of individual binding events for a binding agent specific for a single amino acid that occurs for a particular peptide molecule. It is understood that when UMI and barcode are both referenced in the context of a binding agent or polypeptide, that the barcode refers to identifying information other that the UMI for the individual binding agent or polypeptide (e.g ., sample barcode, compartment barcode, binding cycle barcode).
  • universal priming site or“universal primer” or “universal priming sequence” refers to a nucleic acid molecule, which may be used for library amplification and/or for sequencing reactions.
  • a universal priming site may include, but is not limited to, a priming site (primer sequence) for PCR amplification, flow cell adaptor sequences that anneal to complementary oligonucleotides on flow cell surfaces enabling bridge
  • Universal priming sites can be used for other types of amplification, including those commonly used in conjunction with next generation digital sequencing.
  • extended recording tag molecules may be circularized and a universal priming site used for rolling circle amplification to form DNA nanoballs that can be used as sequencing templates (Drmanac et al., 2009, Science 327:78-81).
  • recording tag molecules may be circularized and sequenced directly by polymerase extension from universal priming sites (Korlach et al., 2008, Proc. Natl. Acad. Sci. 105: 1176-1181).
  • forward when used in context with a“universal priming site” or“universal primer” may also be referred to as “5”’ or“sense”.
  • reverse when used in context with a“universal priming site” or “universal primer” may also be referred to as“3”’ or“antisense”.
  • extended recording tag refers to a recording tag to which information of at least one binding agent’s coding tag (or its complementary sequence) has been transferred following binding of the binding agent to a polypeptide.
  • Information of the coding tag may be transferred to the recording tag directly (e.g., ligation) or indirectly (e.g., primer extension).
  • Information of a coding tag may be transferred to the recording tag enzymatically or chemically.
  • An extended recording tag may comprise binding agent information of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
  • the base sequence of an extended recording tag may reflect the temporal and sequential order of binding of the binding agents identified by their coding tags, may reflect a partial sequential order of binding of the binding agents identified by the coding tags, or may not reflect any order of binding of the binding agents identified by the coding tags.
  • the coding tag information present in the extended recording tag represents with at least 25%, 30%, 35% , 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity the polypeptide sequence being analyzed.
  • errors may be due to off-target binding by a binding agent, or to a“missed” binding cycle (e.g ., because a binding agent fails to bind to a polypeptide during a binding cycle, because of a failed primer extension reaction), or both.
  • extended coding tag refers to a coding tag to which information of at least one recording tag (or its complementary sequence) has been transferred following binding of a binding agent, to which the coding tag is joined, to a polypeptide, to which the recording tag is associated.
  • Information of a recording tag may be transferred to the coding tag directly (e.g., ligation), or indirectly (e.g., primer extension).
  • Information of a recording tag may be transferred enzymatically or chemically.
  • an extended coding tag comprises information of one recording tag, reflecting one binding event.
  • the term“di-tag” or“di-tag construct” or“di-tag molecule” refers to a nucleic acid molecule to which information of at least one recording tag (or its complementary sequence) and at least one coding tag (or its complementary sequence) has been transferred following binding of a binding agent, to which the coding tag is joined, to a polypeptide, to which the recording tag is associated.
  • Information of a recording tag and coding tag may be transferred to the di-tag indirectly (e.g., primer extension).
  • Information of a recording tag may be transferred enzymatically or chemically.
  • a di-tag comprises a UMI of a recording tag, a compartment tag of a recording tag, a universal priming site of a recording tag, a UMI of a coding tag, an encoder sequence of a coding tag, a binding cycle specific barcode, a universal priming site of a coding tag, or any combination thereof.
  • solid support As used herein, the term“solid support”,“solid surface”, or“solid substrate”, or “sequencing substrate”, or“substrate” refers to any solid material, including porous and non- porous materials, to which a polypeptide can be associated directly or indirectly, by any means known in the art, including covalent and non-covalent interactions, or any combination thereof.
  • a solid support may be two-dimensional (e.g., planar surface) or three-dimensional (e.g., gel matrix or bead).
  • a solid support can be any support surface including, but not limited to, a bead, a microbead, an array, a glass surface, a silicon surface, a plastic surface, a filter, a membrane, a PTFE membrane, a PTFE membrane, a nitrocellulose membrane, a nitrocellulose-based polymer surface, nylon, a silicon wafer chip, a flow through chip, a flow cell, a biochip including signal transducing electronics, a channel, a microtiter well, an ELISA plate, a spinning interferometry disc, a nitrocellulose membrane, a nitrocellulose-based polymer surface, a polymer matrix, a nanoparticle, or a microsphere.
  • Materials for a solid support include but are not limited to acrylamide, agarose, cellulose, dextran, nitrocellulose, glass, gold, quartz, polystyrene, polyethylene vinyl acetate, polypropylene, polyester, polymethacrylate, polyacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, poly vinyl alcohol (PVA), Teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid,
  • Solid supports further include thin film, membrane, bottles, dishes, fibers, woven fibers, shaped polymers such as tubes, particles, beads, microspheres, microparticles, or any combination thereof.
  • the bead can include, but is not limited to, a ceramic bead, polystyrene bead, a polymer bead, a polyacrylate bead, a methylstyrene bead, an agarose bead, a cellulose bead, a dextran bead, an acrylamide bead, a solid core bead, a porous bead, a paramagnetic bead, a glass bead, a controlled pore bead, a silica-based bead, or any combinations thereof.
  • a bead may be spherical or an irregularly shaped.
  • a bead or support may be porous.
  • a bead’s size may range from nanometers, e.g., 100 nm, to millimeters, e.g., 1 mm.
  • beads range in size from about 0.2 micron to about 200 microns, or from about 0.5 micron to about 5 micron.
  • 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, 10.5, 15, or 20 pm in diameter.
  • “a bead” solid support may refer to an individual bead or a plurality of beads.
  • the solid surface is a nanoparticle.
  • the nanoparticles range in size from about 1 nm to about 500 nm in diameter, for example, between about 1 nm and about 20 nm, between about 1 nm and about 50 nm, between about 1 nm and about 100 nm, between about 10 nm and about 50 nm, between about 10 nm and about 100 nm, between about 10 nm and about 200 nm, between about 50 nm and about 100 nm, between about 50 nm and about 150, between about 50 nm and about 200 nm, between about 100 nm and about 200 nm, or between about 200 nm and about 500 nm in diameter.
  • the nanoparticles can be about 10 nm, about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 300 nm, or about 500 nm in diameter. In some embodiments, the nanoparticles are less than about 200 nm in diameter.
  • nucleic acid molecule or“polynucleotide” refers to a single- or double-stranded polynucleotide containing deoxyribonucleotides or ribonucleotides that are linked by 3’ -5’ phosphodiester bonds, as well as polynucleotide analogs.
  • a nucleic acid molecule includes, but is not limited to, DNA, RNA, and cDNA.
  • a polynucleotide analog may possess a backbone other than a standard phosphodiester linkage found in natural
  • Polynucleotides and, optionally, a modified sugar moiety or moieties other than ribose or deoxyribose contain bases capable of hydrogen bonding by Watson- Crick base pairing to standard polynucleotide bases, where the analog backbone presents the bases in a manner to permit such hydrogen bonding in a sequence-specific fashion between the oligonucleotide analog molecule and bases in a standard polynucleotide.
  • polynucleotide analogs include, but are not limited to xeno nucleic acid (XNA), bridged nucleic acid (BNA), glycol nucleic acid (GNA), peptide nucleic acids (PNAs), yPNAs, morpholino polynucleotides, locked nucleic acids (LNAs), threose nucleic acid (TNA), T -O-Methyl polynucleotides, 2'-0-alkyl ribosyl substituted polynucleotides, phosphorothioate
  • a polynucleotide analog may possess purine or pyrimidine analogs, including for example, 7-deaza purine analogs, 8-halopurine analogs, 5-halopyrimidine analogs, or universal base analogs that can pair with any base, including hypoxanthine, nitroazoles, isocarbostyril analogues, azole carboxamides, and aromatic triazole analogues, or base analogs with additional functionality, such as a biotin moiety for affinity binding.
  • the nucleic acid molecule or oligonucleotide is a modified oligonucleotide.
  • the nucleic acid molecule or oligonucleotide is a DNA with pseudo-complementary bases, a DNA with protected bases, an RNA molecule, a BNA molecule, an XNA molecule, a LNA molecule, a PNA molecule, a gRNA molecule, or a morpholino DNA, or a combination thereof.
  • the nucleic acid molecule or oligonucleotide is backbone modified, sugar modified, or nucleobase modified.
  • the nucleic acid molecule or oligonucleotide has nucleobase protecting groups such as Alloc, electrophilic protecting groups such as thiranes, acetyl protecting groups, nitrobenzyl protecting groups, sulfonate protecting groups, or traditional base-labile protecting groups.
  • nucleic acid sequencing means the determination of the order of nucleotides in a nucleic acid molecule or a sample of nucleic acid molecules.
  • next generation sequencing refers to high-throughput sequencing methods that allow the sequencing of millions to billions of molecules in parallel.
  • next generation sequencing methods include sequencing by synthesis, sequencing by ligation, sequencing by hybridization, polony sequencing, ion semiconductor sequencing, and pyrosequencing.
  • primers By attaching primers to a solid substrate and a complementary sequence to a nucleic acid molecule, a nucleic acid molecule can be hybridized to the solid substrate via the primer and then multiple copies can be generated in a discrete area on the solid substrate by using polymerase to amplify (these groupings are sometimes referred to as polymerase colonies or polonies).
  • a nucleotide at a particular position can be sequenced multiple times ( e.g ., hundreds or thousands of times) - this depth of coverage is referred to as "deep sequencing.”
  • high throughput nucleic acid sequencing technology include platforms provided by Illumina, BGI, Qiagen, Thermo-Fisher, 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 (See e.g., Service, Science (2006) 311 :1544-1546).
  • single molecule sequencing or “third generation sequencing” refers to next-generation sequencing methods wherein reads from single molecule sequencing instruments are generated by sequencing of a single molecule of DNA. Unlike next generation sequencing methods that rely on amplification to clone many DNA molecules in parallel for sequencing in a phased approach, single molecule sequencing interrogates single molecules of DNA and does not require amplification or synchronization. Single molecule sequencing includes methods that need to pause the sequencing reaction after each base incorporation ('wash-and-scan' cycle) and methods which do not need to halt between read steps.
  • analyzing means to identify, detect, quantify, characterize, distinguish, or a combination thereof, all or a portion of the components of the polypeptide. For example, analyzing a peptide, polypeptide, or protein includes determining all or a portion of the amino acid sequence (contiguous or non-continuous) of the peptide.
  • Analyzing a polypeptide also includes partial identification of a component of the polypeptide.
  • partial identification of amino acids in the polypeptide protein sequence can identify an amino acid in the protein as belonging to a subset of possible amino acids. Analysis typically begins with analysis of the n NTAA, and then proceeds to the next amino acid of the peptide (i.e., n-1 , n-2 , n-3 , and so forth). This is accomplished by elimination of the n NTAA, thereby converting the n-1 amino acid of the peptide to an N-terminal amino acid (referred to herein as the“ n-1 NTAA”).
  • Analyzing the peptide may also include determining the presence and frequency of post-translational modifications on the peptide, which may or may not include information regarding the sequential order of the post-translational modifications on the peptide. Analyzing the peptide may also include determining the presence and frequency of epitopes in the peptide, which may or may not include information regarding the sequential order or location of the epitopes within the peptide. Analyzing the peptide may include combining different types of analysis, for example obtaining epitope information, amino acid sequence information, post-translational modification information, or any combination thereof.
  • wild-type or“native”
  • biological materials such as nucleic acid molecules and proteins (e.g., cleavase) refers to those which are found in nature and not modified by human intervention.
  • modified as used in reference to nucleic acid molecules and proteins, e.g, a modified cleavase created by human intervention.
  • the variant, mutant or modified cleavase is a polypeptide having an altered amino acid sequence, relative to an unmodified or wild-type cleavase.
  • the variant or modified cleavase is a polypeptide which differs from a wild-type cleavase sequence by one or more amino acid substitutions, deletions, additions, or combinations thereof.
  • a variant, mutant or modified cleavase can contain 1, 2, 3,
  • a variant or modified cleavase polypeptide generally exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a corresponding wild-type or unmodified cleavase.
  • Non-naturally occurring amino acids as well as naturally occurring amino acids are included within the scope of permissible substitutions or additions.
  • a variant, mutant or modified cleavase is not limited to any variant, mutant or modified cleavase made or generated by a particular method of making and includes, for example, a variant, mutant or modified cleavase made or generated by genetic selection, protein engineering, directed evolution, de novo recombinant DNA techniques, or combinations thereof.
  • a mutant, variant or modified cleavase polypeptide is altered in primary amino acid sequence by substitution, addition, or deletion of amino acid residues.
  • the term "variant" in the context of variant or modified cleavase is not be construed as imposing any condition for any particular starting composition or method by which the variant or modified cleavase is created.
  • variant or modified cleavase denotes a composition and not necessarily a product produced by any given process.
  • a variety of techniques including genetic selection, protein engineering, recombinant methods, chemical synthesis, or combinations thereof, may be employed.
  • nucleotides or amino acid positions “correspond to” nucleotides or amino acid positions in a disclosed sequence refers to nucleotides or amino acid positions identified upon alignment with the disclosed sequence based on structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm.
  • corresponding residues can be determined by alignment with a reference sequence, such as set forth in SEQ ID NOs: 5-8, 10- 16, 20, by structural alignment methods as described herein. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides.
  • domain refers to a portion of a molecule, such as a protein or encoding nucleic acid, that is structurally and/or functionally distinct from other portions of the molecule and is identifiable.
  • domains include those portions of a polypeptide chain that can form an independently folded structure within a protein made up of one or more structural motifs and/or that is recognized by virtue of a functional activity, such as binding activity.
  • a protein can have one, or more than one, distinct domains.
  • a domain can be identified, defined or distinguished by homology of the primary sequence or structure to related family members, such as homology to motifs.
  • a domain can be distinguished by its function, such as an ability to interact with a molecule, such as a cognate binding partner.
  • a domain independently can exhibit a biological function or activity such that the domain independently or fused to another molecule can perform an activity, such as, for example, binding.
  • a domain can be a linear sequence of amino acids or a non-linear sequence of amino acids.
  • Many polypeptides contain a plurality of domains. Such domains are known, and can be identified by those of skill in the art. For exemplification herein, definitions are provided, but it is understood that it is well within the skill in the art to recognize particular domains by name. If needed, appropriate software can be employed to identify domains.
  • sequence identity refers to the sequence identity between genes or proteins at the nucleotide or amino acid level, respectively.
  • sequence identity is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at nucleotide level.
  • the protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned.
  • nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned. Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA.
  • the BLAST algorithm calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences.
  • the software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website.
  • alkyl refers to and includes saturated linear and branched univalent hydrocarbon structures and combination thereof, having the number of carbon atoms designated (i.e., Ci-Cio or Ci-io means one to ten carbons). Particular alkyl groups are those having 1 to 20 carbon atoms (a“C1-C20 alkyl”).
  • alkyl groups are those having 1 to 8 carbon atoms (a“Ci-Cs alkyl”), 3 to 8 carbon atoms (a“C3-C8 alkyl”), 1 to 6 carbon atoms (a“C1-C6 alkyl”), 1 to 5 carbon atoms (a“C1-C5 alkyl”), or 1 to 4 carbon atoms (a “C1-C4 alkyl”), unless otherwise specified
  • alkyl include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.
  • the alkenyl group may be in“cis” or“trans” configurations, or alternatively in“E” or“Z” configurations.
  • alkenyl groups are those having 2 to 20 carbon atoms (a“C2-C20 alkenyl”), having 2 to 8 carbon atoms (a“C2-C8 alkenyl”), having 2 to 6 carbon atoms (a“C2-C6 alkenyl”), or having 2 to 4 carbon atoms (a“C2-C4 alkenyl”).
  • alkenyl examples include, but are not limited to, groups such as ethenyl (or vinyl), prop-l-enyl, prop-2-enyl (or allyl), 2-methylprop-l-enyl, but-l-enyl, but-2-enyl, but-3-enyl, buta-l,3-dienyl, 2-methylbuta-l,3-dienyl, homologs and isomers thereof, and the like.
  • groups such as ethenyl (or vinyl), prop-l-enyl, prop-2-enyl (or allyl), 2-methylprop-l-enyl, but-l-enyl, but-2-enyl, but-3-enyl, buta-l,3-dienyl, 2-methylbuta-l,3-dienyl, homologs and isomers thereof, and the like.
  • aminoalkyl refers to an alkyl group that is substituted with one or more -NH2 groups. In certain embodiments, an aminoalkyl group is substituted with one, two, three, four, five or more -NEE groups. An aminoalkyl group may optionally be substituted with one or more additional substituents as described herein.
  • aryl or“Ar” refers to an unsaturated aromatic carbocyclic group having a single ring ( e.g ., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl) which condensed rings may or may not be aromatic.
  • the aryl group contains from 6 to 14 annular carbon atoms.
  • An aryl group having more than one ring where at least one ring is non-aromatic may be connected to the parent structure at either an aromatic ring position or at a non-aromatic ring position.
  • an aryl group having more than one ring where at least one ring is non-aromatic is connected to the parent structure at an aromatic ring position.
  • phenyl is a preferred aryl group.
  • arylalkyl refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
  • arylalkyl include, but are not limited to, benzyl, 2- phenylethyl, 3- phenylpropyl, 2-naphth-2-ylethyl, and the like.
  • cycloalkyl refers to and includes cyclic univalent hydrocarbon structures, which may be fully saturated, mono- or polyunsaturated, but which are non-aromatic, having the number of carbon atoms designated (e.g, C1-C10 means one to ten carbons). Cycloalkyl can consist of one ring, such as cyclohexyl, or multiple rings, such as adamantly, but excludes aryl groups. A cycloalkyl comprising more than one ring may be fused, spiro or bridged, or combinations thereof. In some embodiments, the cycloalkyl is a cyclic hydrocarbon having from 3 to 13 annular carbon atoms.
  • the cycloalkyl is a cyclic hydrocarbon having from 3 to 8 annular carbon atoms (a "C 3 -C 8 cycloalkyl").
  • cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1- cyclohexenyl, 3-cyclohexenyl, cycloheptyl, norbomyl, and the like.
  • the“halogen” represents chlorine, fluorine, bromine, or iodine.
  • the term“halo” represents chloro, fluoro, bromo, or iodo.
  • haloalkyl refers to an alkyl group as described above, wherein one or more hydrogen atoms on the alkyl group have been replaced by a halo group.
  • groups include, without limitation, fluoroalkyl groups, such as fluoroethyl, trifluoromethyl, difluoromethyl, trifluoroethyl and the like.
  • heteroaryl refers to and includes unsaturated aromatic cyclic groups having from 1 to 10 annular carbon atoms and at least one annular heteroatom, including but not limited to heteroatoms such as nitrogen, oxygen and sulfur, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. It is understood that the selection and order of heteroatoms in a heteroaryl ring must conform to standard valence requirements and provide an aromatic ring character, and also must provide a ring that is sufficiently stable for use in the reactions described herein.
  • a heteroaryl ring has 5-6 ring atoms and 1-4 heteroatoms, which are selected from N, O and S unless otherwise specified; and a bicyclic heteroaryl group contains two 5-6 membered rings that share one bond and contain at least one heteroatom and up to 5 heteroatoms selected from N, O and S as ring members.
  • a heteroaryl group can be attached to the remainder of the molecule at an annular carbon or at an annular heteroatom, in which case the heteroatom is typically nitrogen.
  • Heteroaryl groups may contain additional fused rings ( e.g from 1 to 3 rings), including additionally fused aryl, heteroaryl, cycloalkyl, and/or heterocyclyl rings.
  • heteroaryl groups include, but are not limited to, pyrazolyl, imidazolyl, triazolyl, pyrrolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl, thiophenyl, furanyl, thiazolyl, and the like.
  • heterocycle refers to a saturated or an unsaturated non-aromatic group having from 1 to 10 annular carbon atoms and from 1 to 4 annular heteroatoms, such as nitrogen, sulfur or oxygen, and the like, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized.
  • a heterocyclyl group may have a single ring or multiple condensed rings, but excludes heteroaryl groups.
  • a heterocycle comprising more than one ring may be fused, spiro or bridged, or any combination thereof.
  • one or more of the fused rings can be aryl or heteroaryl.
  • heterocyclyl groups include, but are not limited to, tetrahydropyranyl, dihydropyranyl, piperidinyl, piperazinyl, pyrrolidinyl, thiazolinyl, thiazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, 2,3-dihydrobenzo[b]thiophen-2-yl, 4- amino-2-oxopyrimidin-l(2H)-yl, and the like.
  • substituted means that the specified group or moiety bears one or more substituents in place of a hydrogen atom of the unsubstituted group, including, but not limited to, substituents such as alkoxy, acyl, acyloxy, carbonylalkoxy, acylamino, amino, aminoacyl, aminocarbonylamino, aminocarbonyloxy, cycloalkyl, cycloalkenyl, aryl, heteroaryl, aryloxy, cyano, azido, halo, hydroxyl, nitro, carboxyl, thiol, thioalkyl, cycloalkyl, cycloalkenyl, alkyl, alkenyl, alkynyl, heterocyclyl, aralkyl, aminosulfonyl, sulfonylamino, sulfonyl, oxo, carbonylalkylenealkoxy and the like.
  • substituents such as alkoxy, acy
  • unsubstituted means that the specified group bears no substituents.
  • optionally substituted means that the specified group is unsubstituted or substituted by one or more substituents and thus includes both substituted and unsubstituted versions of the group. Where the term“substituted” is used to describe a structural system, the substitution is meant to occur at any valency-allowed position on the system.
  • a compound can exist in more than one tautomeric form, typically one tautomer is depicted or described, and the structure is understood to represent each stable tautomer as well as mixtures of the tautomers.
  • guanidine groups and heteroaryl groups substituted by hydroxyl or amine groups are often able to exist in multiple tautomers, and the description or depiction of one tautomer is understood to include the other tautomers of the same compound.
  • aspects and embodiments of the invention described herein include“consisting” and/or“consisting essentially of’ aspects and embodiments.
  • sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4,
  • a modified cleavase comprising a mutation, e.g ., one or more amino acid modification(s) in an unmodified cleavase, wherein the modified cleavase is derived from a dipeptide cleavase and removes or is configured to remove a single labeled terminal amino acid from a polypeptide.
  • a modified cleavase comprising a mutation, e.g.
  • the modified cleavase is derived from a tripeptide cleavase and removes or is configured to remove a single labeled terminal amino acid from a polypeptide or a single labeled terminal dipeptide from a polypeptide.
  • the modified cleavase is configured to remove a single labeled terminal amino acid from the C-terminus or N-terminus of a polypeptide.
  • the modified cleavase is configured to remove a single labeled dipeptide (the terminal and penultimate terminal amino acids) from the C-terminus or N-terminus of a polypeptide.
  • the modified cleavase is derived from a wild-type or unmodified cleavase (e.g, a dipeptide cleavase or tripeptide cleavase).
  • the terminal labeled amino acid residue is an N-terminal amino acid.
  • the terminal labeled amino acid residue is a C-terminal amino acid.
  • the removed labeled terminal amino acid is removed as a single amino acid or as part of a dipeptide.
  • a labeled amino acid is a terminal amino acid that is modified by treating with a chemical reagent.
  • the removed single labeled terminal amino acid or single labeled terminal dipeptide comprises an amide bond.
  • the modified cleavase comprises an active site that interacts with the amide bond (e.g ., amide bond between the terminal labeled amino acid residue and a penultimate terminal amino acid residue of the polypeptide).
  • the mutation is or comprises an amino acid substitution, deletion, addition, or any combinations thereof.
  • the modified cleavase exhibits activity that is different from the activity of the unmodified or wild-type cleavase.
  • “Unmodified cleavase” or“wild-type cleavase” as used herein refers to any natural or wild-type exopeptidase that possesses catalytic activity to remove a dipeptide or tripeptide from the terminus of a polypeptide (e.g., from the C- terminus or N-terminus of a polypeptide).
  • the unmodified or wild-type cleavase may be an exopeptidase that catalyzes the cleavage of a penultimate peptide bond to release a dipeptide from the peptide chain or that catalyzes the cleavage of an antepenultimate peptide bond to release a tripeptide from the peptide chain.
  • the unmodified or wild-type cleavase may be a proteolytic enzyme such as an aminopeptidase or a carboxypeptidase.
  • the unmodified cleavase described herein may be used to refer to a protein classified by the Enzyme Commission (EC) as EC 3.4.14, EC 3.4.15, MEROPS S8, MEROPS S9, MEROPS S33, MEROPS S46, MEROPS M49, or MEROPS S53, or a functional homolog or fragment thereof.
  • the unmodified cleavase described herein may be used to refer to a dipeptidyl peptidase, a dipeptidyl aminopeptidase, a peptidyl-dipeptidase, a dipeptidyl carboxypeptidase, a sedolisin, or a tripeptidyl peptidase.
  • A“modified cleavase” or“variant cleavase” refers to any exopeptidase that has been modified from a unmodified or wild-type cleavase as described.
  • the modified or variant cleavase may be derived from an unmodified or wild-type dipeptide cleavase (e.g. a dipeptidyl peptidase, a dipeptidyl aminopeptidase, a peptidyl-dipeptidase, a dipeptidyl carboxypeptidase) or from a unmodified or wild-type tripeptide cleavase (e.g. a sedolisin, or a tripeptidyl peptidase).
  • dipeptide cleavase e.g. a dipeptidyl peptidase, a dipeptidyl aminopeptidase, a peptidyl-dipeptidase, a dipeptidyl carboxypeptidase
  • a modified cleavase derived from a dipeptide cleavase removes only a labeled PI terminal amino acid from the peptide at a time.
  • a modified cleavase derived from a tripeptide cleavase removes a labeled PI terminal amino acid from the peptide at a time or a labeled P1-P2 dipeptide from the peptide at a time (see e.g, FIG. 1A-1B) [0086]
  • the modified cleavase removes a labeled terminal amino acid, e.g ., a labeled N-terminal amino acid (NTAA).
  • the modified cleavase removes a labeled C-terminal amino acid (CTAA).
  • CAA C-terminal amino acid
  • the removed labeled terminal amino acid is removed as a single amino acid or as part of a dipeptide.
  • the modified cleavase derived from the dipeptide cleavase or tripeptide cleavase is configured to cleave the peptide bond between a terminal labeled amino acid residue and a penultimate terminal amino acid residue of the polypeptide.
  • the modified cleavase derived from the tripeptide cleavase is configured to cleave the peptide bond between a penultimate terminal labeled amino acid residue and a antepenultimate terminal amino acid residue of the polypeptide.
  • the unmodified or wild-type cleavase is a protein classified in EC 3.4.14, EC 3.4.15, MEROPS S8, MEROPS S9, MEROPS S33, MEROPS S46, MEROPS M49, or MEROPS S53, or a homolog thereof.
  • the unmodified or wild-type cleavase is a protein provided in Tables 1, 2, 3, 4, 5, 6, 7, and 8A-8B.
  • the modified cleavase is derived from a protein classified EC 3.4.14, EC 3.4.15, MEROPS S8, MEROPS S9, MEROPS S33, MEROPS S46, MEROPS M49, or MEROPS S53, or a functional homolog or fragment thereof, (as provided in Tables 1, 2, 3, 4, 5, 6, 7, and 8A- 8B )
  • peptidases e.g ., cleavases
  • Some peptidases cleave dipeptides from a range of tripeptides to decapeptides.
  • Cleavases as described herein refer to enzymes that are classified under the Enzyme Commission (EC) Class 3 of hydrolases.
  • Dipeptidyl peptidases and tripeptidyl peptidases are a class of exopeptidases which digest dipeptides (two amino acid residues, P1-P2) or tripeptides (three amino acid residues, P1-P2-P3) from the N-terminal end of a peptide, typically in a processive manner.
  • Peptidyl dipeptidases also known as dipeptidyl carboxypeptidases (EC 3.4.15; Table 2) act from the C-terminal end in removing dipeptides in a processive manner.
  • the unmodified or wild- type cleavase is an exoaminopeptidase or an exopeptidase.
  • the unmodified or wild-type cleavase is a metallopeptidase, e.g ., a zinc-dependent metallopeptidase or a zinc- dependent hydrolase.
  • the unmodified or wild-type cleavase is a serine exopeptidase or a serine protease.
  • DPPs typically recognize the N-terminal alpha amine, and cleave the peptide bond between the penultimate and antepenultimate amino acid residues of a polypeptide (P2-P3). See e.g.
  • Tripeptidyl Peptidases A, B, and C are classified in the MEROPS family S33 (MEROPS S33.002, S33.006, S33.007).
  • Sedolisins also known as serine-carboxyl peptidases, are proteolytic enzymes MEROPS family of peptidases, S53 ( See e.g. , Wlodawer et al., Acta Biochim Pol. 2003;50(1):81-102).
  • the modified cleavase exhibits activity including the removal of a terminal amino acid from polypeptides or proteins (e.g, from the N-terminus or C- terminus).
  • the terminal amino acid may be removed from the polypeptide as a single amino acid or as part of a dipeptide.
  • the peptidase activity is capable of removing the amino acid Xaai and/or Xaa2 from the terminus of a peptide, polypeptide, or protein, wherein Xaa may represent any amino acid residue selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
  • the modified cleavase is partially specific or selective. In some aspects, the modified cleavase preferentially cleaves or removes some amino acids at the PI or P2 position of the peptide over others. In some cases, the modified cleavase preferentially cleaves or removes a class of amino acids over others, e.g., preferentially removing hydrophobic amino acids over other classes of amino acids. In some aspects, the modified cleavase may also have a preference for one or more amino acids at the second, third, fourth, fifth, etc. positions from the terminal amino acid. In some cases, the modified cleavase exhibits specificity to subsets of amino acids and preferentially removes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or more specific terminal amino acid over others.
  • the modified cleavase is a polypeptide having an altered amino acid sequence, relative to an unmodified or wild-type cleavase.
  • the modified cleavase is a polypeptide which differs from a wild-type cleavase sequence by one or more amino acid substitutions, deletions, additions, or combinations thereof.
  • a variant or modified cleavase can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
  • the variant or modified cleavase polypeptide generally exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • the wild-type or unmodified cleavase comprises the amino acid sequence of any one of SEQ ID NO: 5-8, 10-16, 20, a mature sequence thereof, or a portion thereof containing the active site.
  • the variant or modified cleavase polypeptide generally exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a wild-type or unmodified cleavase set forth in SEQ ID NOs: 5-8, 10-16, 20, or a homolog thereof.
  • the unmodified or reference cleavase polypeptide generally exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the sequences set forth in SEQ ID NOs: 5-8 and 10-16, 20.
  • corresponding residues can be determined by alignment of a reference sequence with a sequence provided herein (for example, sequences set forth in SEQ ID NOs: 5-8, 10-16, 20, or a functional homolog or fragment thereof) using known alignment methods. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides. In some cases, while the numbering of the residues provided herein may differ from a reference sequence, using the alignment method will allow determination of corresponding residues.
  • the modified cleavase comprises a mutation, e.g ., one or more amino acid modification(s), in an unmodified cleavase, wherein the unmodified cleavase is a dipeptidyl peptidase 3.
  • Dipeptidyl peptidase 3 also known as dipeptidyl peptidase III, dipeptidyl aminopeptidase III, dipeptidyl arylamidase III, enkephalinase B, red cell
  • angiotensinase DPP3, or DPP III
  • DPP3 is a metalloproteinase (zinc-dependent) that sequentially removes dipeptides (two amino acid residues) from the N-terminus of short peptides.
  • Wild-type or unmodified DPP3 is classified in the M49 family (MEROPS database identifier M49.001).
  • the unmodified dipeptidyl peptidase 3 exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to UniProt Accession No.
  • DPP3 preferentially digests peptides that are 3 to 10 amino acids in length.
  • DPP3 harbors a unique HEXXGH catalytic motif (SEQ ID NO: 1). Both histidines in this motif along with the glutamate residue of a second conserved EEXRAE/D motif are involved in zinc coordination (SEQ ID NO: 2).
  • SEQ ID NO: 2 HEXXGH catalytic motif
  • C-terminal peptide modifications do not affect the activity of DPP3 enzymes. See Kumar et ak, Sci Rep. (2016) 6:23787.
  • N-terminal tyrosine is structurally similar to a phenylisothiocyanate (PITC), nitro-PITC, sulfo-PITC, or a phenylisocyanate version of these modifiers, and these substrate-bound structures may be useful for a targeted active-site design approach.
  • PITC phenylisothiocyanate
  • nitro-PITC nitro-PITC
  • sulfo-PITC sulfo-PITC
  • a phenylisocyanate version of these modifiers e.g., a modified cleavase derived from a dipeptidyl peptidase 3 that cleaves labeled terminal amino acid residues, e.g, labeled N-terminal amino acid residues.
  • the modified cleavase comprises a mutation, e.g, one or more amino acid modification(s), in an unmodified cleavase, wherein the unmodified cleavase is a dipeptidyl peptidase 5.
  • Dipeptidyl peptidase 5 is also known as allergen Tri m 4 ( Trichophyton mentagrophytes ), allergen Tri r 4 ( Trichophyton rubrum), allergen Tri 14
  • Wild-type or unmodified dipeptidyl peptidase 5 is classified in the peptidase family S9 (MEROPS database identifier S09.012). Wild-type or unmodified dipeptidyl peptidase 5 has been observed to catalyze the hydrolysis of X- Ala, His-Ser, and Ser- Tyr dipeptides at a neutral pH optimum ( See e.g. , Beauvais et al., J Biol Chem. 1997;
  • Wild-type or unmodified dipeptidyl peptidase 5 is described as a secreted dipeptidyl peptidase which contains the consensus sequences of the catalytic site of the nonclassical serine proteases. In some cases, the unmodified cleavase exhibits at least 50%,
  • the mutations e.g. , one or more amino acid modifications (e.g., substitutions, deletions, additions) in the modified cleavase is in reference to the amino acid sequence set forth in reference to numbering of SEQ ID NO: 10 or 16.
  • the modified cleavase comprises a mutation, e.g, one or more amino acid modification(s), in an unmodified cleavase, wherein the unmodified cleavase is a dipeptidyl peptidase 7 (DPP7). Wild-type or unmodified DPP7 is classified in S46 protease family (MEROPS database identifier S46.001).
  • Wild-type or unmodified DPP7 has been observed to catalyze the removal of dipeptides from the N-terminus of oligopeptides, including a broad specificity for both aliphatic and aromatic residues in the PI position, with glycine or proline being not acceptable in this position ( See e.g, Banbula et al., J. Biol. Chem. 2001, 276:6299-6305).
  • DPP7 has been shown to exhibit activity for cleaving the synthetic substrates Met-Leu-methylcoumaryl-7-amide (Met-Leu-MCA), Leu-Arg-MCA, and Lys-Ala-MCA (Rouf et al., FEBS Open Bio. 2013; 3:177-83).
  • the unmodified cleavase exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
  • the mutations e.g, one or more amino acid modification(s) (e.g., substitutions, deletions, additions) in the modified cleavase is in reference to the amino acid sequence set forth in reference to numbering of SEQ ID NO: 11.
  • the modified cleavase comprises a mutation, e.g, one or more amino acid modification(s), in an unmodified cleavase, wherein the unmodified cleavase is a dipeptidyl peptidase 11.
  • Dipeptidyl peptidase 11 is also known as Asp/Glu-specific dipeptidyl-peptidase or DPP11. Wild-type or unmodified dipeptidyl peptidase 11 is classified in S46 protease family (MEROPS database identifier S46.002), and shares 38.7% sequence identity with dipeptidyl peptidase 7.
  • Wild-type or unmodified dipeptidyl peptidase 11 has been observed to catalyze the removal of dipeptides from the N-terminus of oligopeptides, including removing dipeptides from oligopeptides with the penultimate N-terminal Asp and Glu and has a P2-position preference to hydrophobic residues (See e.g., Ohara-Nemoto et al., J Biol Chem. 2011; 286(44):38115-27).
  • the unmodified cleavase exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to UniProt Accession No. B2RID1 or F8WQK8 as set forth in SEQ ID NO: 12 and 14, respectively.
  • the mutations, e.g, one or more amino acid modifications (e.g., substitutions, deletions, additions) in the modified cleavase is in reference to the amino acid sequence set forth in reference to numbering of SEQ ID NO: 12.
  • the mutations, e.g, one or more amino acid modifications e.g.,
  • substitutions, deletions, additions) in the modified cleavase is in reference to the amino acid sequence set forth in reference to numbering of SEQ ID NO: 14.
  • the modified cleavase comprises a mutation, e.g, one or more amino acid modification(s), in an unmodified cleavase, wherein the unmodified cleavase is a dipeptidyl aminopeptidase BII (DAP BII or dipeptidyl peptidase BII).
  • DAP BII dipeptidyl aminopeptidase BII
  • Wild-type or unmodified DAP BII catalyzes the removal of dipeptides from the amino terminus of peptides (See e.g, Ogasawara et al., J. Bacterid. 1996, 178:6288-6295); Sakamoto et al., Scientific Reports 2014, 4:4977).
  • DAP BII is a serine protease that belongs to the serine peptidase family S46 (MEROPS database identifier S46.003).
  • the amino acid sequence of the catalytic unit of DAP BII exhibits significant similarity to those classified in the clan PA endopeptidases.
  • the unmodified cleavase exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to UniProt Accession No. V5YM14 as set forth in SEQ ID NO: 13.
  • the modified cleavase contains one or more amino acids modifications in the catalytic domain of an unmodified DAP BII (e.g, residues 1-252 and residues 550 to 698 of SEQ ID NO: 13).
  • the mutations, e.g, one or more amino acid modifications (e.g., substitutions, deletions, additions) in the modified cleavase is in reference to the amino acid sequence set forth in reference to numbering of SEQ ID NO: 13.
  • the modified cleavase is derived from DAP BII and removes or is configured to remove a labeled terminal single amino acid from a polypeptide.
  • the modified cleavase has one or more amino acid modifications (e.g.
  • the modified cleavase comprises one or more amino acid modifications in an unmodified cleavase, corresponding to position(s) 126, 188, 189, 190, 191, 192, 196, 238, 302, 306, 307, 310, 525, 528, 546, 604, 650, 651, 665, and/or 692, with reference to numbering of SEQ ID NO: 13, and comprises an amino acid sequence that exhibits at least 30 % identity, at least 40 % identity, at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, or at least 90 % or more identity to any of SEQ ID NOs: 17-19, 23-28, or 31-39.
  • the modified cleavase has one or more amino acid modifications (e.g. substitutions, deletions, additions, or combinations thereof) in an unmodified DAP BII cleavase or fragment thereof corresponding to any one or more of position(s) 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, and/or 202, with reference to numbering of SEQ ID NO: 13.
  • the modified cleavase has one or more amino acid modifications (e.g. substitutions, deletions, additions, or
  • the modified cleavase has one or more amino acid modifications (e.g. substitutions, deletions, additions, or combinations thereof) in an unmodified DAP BII cleavase or fragment thereof corresponding to any one or more of position(s) 191, 192, 196, 306, and/or 650, with reference to numbering of SEQ ID NO: 13.
  • the modified cleavase has one or more amino acid modifications (e.g.
  • the modified cleavase has one or more amino acid modifications (e.g . substitutions, deletions, additions, or combinations thereof) in an unmodified DAP BII cleavase or fragment thereof corresponding to any one or more of position(s) 310, 651, 655, and/or 656 with reference to numbering of SEQ ID NO: 13.
  • the modified cleavase has one or more amino acid modifications (e.g.
  • the modified cleavase is derived from DAP BII and removes or is configured to remove a labeled terminal single amino acid from a polypeptide.
  • the modified cleavase has one or more amino acid modifications (e.g.
  • the modified cleavase has one or more amino acid substitutions selected from the group consisting of A126T, D188V, I189A, D190S, N191C, N191F, N191L, N191M, N191R, N191S, N191T, N191V, W192F, W192G, W192L, R196H, R196K, R196S, R196T, R196V, G238V, A302W, N306A, N306G, N306R, N306S, T307K, N310D, N310G, N310K, N310L, N525K, A528V, F546L, A604V, D650A, D650G, D650S, G651H, G651T, G651V, G651Y, S655G, S655T, V656E, V656G, V656S, K665I, and K69
  • N191T/R196H/N306A/D650G N191M/R196H/N306A/D650G, N191V/N306A/D650S, or N191S/N306G/D650S.
  • the modified cleavase has an amino acid sequence that has at least 20 % identity, at least 30 % identity, at least 40 % identity, at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, or at least 90 % or more identity to any of SEQ ID NOs: 17-19, 23-28, 31-39 or a specific binding fragment thereof.
  • the specific binding fragment has a length ranging from about 10 amino acids to about 400 amino acids, from about 10 amino acids to about 300 amino acids, from about 10 amino acids to about 200 amino acids, from about 10 amino acids to about 100 amino acids, or from about 10 amino acids to about 50 amino acids.
  • the modified cleavase contains one or more of the amino acid substitutions provided in SEQ ID NO: 17-19, 23-28, or 31-39.
  • the modified cleavase comprises the sequence of amino acids set forth in any of SEQ ID NO: 17-19, 23-28, 31-39, or a sequence of amino acids that exhibits at least 95% sequence identity to any of SEQ ID NOs: 17-19, 23-28, 31-39, or a specific binding fragment thereof.
  • the modified cleavase comprises one or more amino acid modifications in an unmodified cleavase, corresponding to position(s) 188, 189, 190, 191, 192, 196, 302, 306, 310, and/or 650, with reference to numbering of SEQ ID NO: 13, and has an amino acid sequence that has at least 30 % identity, at least 40 % identity, at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, or at least 90 % or more identity to any of SEQ ID NOs: 17-19, 23-28, 31-39.
  • the modified cleavase comprises one or more amino acid modifications in an unmodified cleavase, corresponding to position(s) 188, 189, 190, 191, 192, 196, 302, 306, 310, and/or 650, with reference to numbering of SEQ ID NO: 13, and has an amino acid sequence that has at least 30 % identity, at least 40 % identity, at least 50
  • cleavase comprises one or more amino acid modifications in an unmodified cleavase, corresponding to position(s) 191, 192, 196, 306, and/or 650, with reference to numbering of SEQ ID NO: 13, and has an amino acid sequence that has at least 30 % identity, at least 40 % identity, at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, or at least 90 % or more identity to any of SEQ ID NOs: 17-19, 23-28, or 31-39.
  • the modified cleavase has the substrate specificity of any one of the sequences in SEQ ID NOs: 17-19, 23-28, or 31-39.
  • the modified cleavase has the cleaving activity of any one of the sequences in SEQ ID NOs: 17-19, 23-28, or 31-39.
  • the modified cleavase has an amino acid sequence that comprises a catalytic domain with at least 20 % identity, at least 30 % identity, at least 40 % identity, at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, or at least 90 % or more identity with the catalytic domain of any of SEQ ID NOs: 17- 19, 23-28, or 31-39.
  • the modified cleavase has an amino acid sequence that comprises an amine binding site with at least 20 % identity, at least 30 % identity, at least 40 % identity, at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, or at least 90 % or more identity with the amine binding site of any of SEQ ID NOs: 17-19, 23-28, or 31-39.
  • the modified cleavase has an amino acid sequence that comprises a loop domain with at least 20 % identity, at least 30 % identity, at least 40 % identity, at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, or at least 90 % or more identity with the loop domain of any of SEQ ID NOs: 17-19, 23-28, or 31-39.
  • a desired modified cleavase may exhibit reduced bias towards specific amino acids in the PI or P2 position of the polypeptide.
  • such modified cleavases may be obtained by targeting the PI or PI pocket of the wildtype or unmodified enzyme in genetic selection.
  • the residues N310, G651, S655, and/or V656 with reference to numbering of SEQ ID NO: 13 may be targeted to reduce bias.
  • the residues N215, W216, R220, N330, and/or D674 with reference to numbering of SEQ ID NO: 13 may be targeted to reduce bias.
  • Table 9 also provides exemplary sequences by reference to SEQ ID NO for exemplary modified cleavases.
  • the modified cleavase contains one or more of the amino acid substitutions provided in Table 9.
  • the removed amino acid is labeled or modified by a chemical reagent or enzymatic reagent.
  • the labeled amino acid is removed as a single terminal amino acid or as part of a dipeptide.
  • the label is or includes a modifier or label that“mimics” the size/shape of a terminal amino acid (e.g ., an N- terminal amino acid).
  • the removed amino acid is labeled with an amino acid (e.g., an exogenous amino acid), a modified amino acid, a portion of an amino acid, a blocked or protected amino acid, or any combinations thereof.
  • the label attached to the terminal amino acid is an N-terminal blocked (devoid of alpha amine) amino acid.
  • selection of the appropriate label with an appropriately engineered or modified cleavase derived from a dipeptide cleavase enables removal of a single labeled terminal amino acid residue.
  • selection of the appropriate label with an appropriately engineered or modified cleavase derived from a tripeptide cleavase enables cleavage of a labeled terminal dipeptide.
  • the active site and/or amino acid binding site(s) of the unmodified cleavase is modified.
  • the modified cleavase comprises a modification or mutation within its substrate binding site, at the boundary of the substrate binding site, in the catalytic domain, in the PI or P2 pocket, in a chymotrypsin fold, at an amine binding site, in the loop domain, or a combination thereof.
  • the label-Pl replaces the native Pl- P2 residue of the peptide, such that cleavage in the modified cleavase then occurs between PI and P2 (modified cleavase derived from a dipeptide cleavase) rather than between P2 and P3 (native dipeptide cleavase) (See e.g. , FIG. 1A).
  • the label-Pl replaces the native P1-P2-P3 residue of the peptide or portion thereof, such that cleavage in the modified cleavase then occurs between PI and P2 (modified cleavase derived from a tripeptide cleavase) rather than between P3 and P4 (native tripeptide cleavase) (See e.g. , FIG. IB top right).
  • the label-Pl-P2 replaces the native P1-P2-P3 residue of the peptide or portion thereof, such that cleavage in the modified cleavase then occurs between P2 and P3 (modified cleavase derived from a tripeptide cleavase) rather than between P3 and P4 (native tripeptide cleavase) (See e.g., FIG. IB bottom right).
  • the modified cleavase is derived from a dipeptide cleavase (e.g, a dipeptidyl peptidase, a dipeptidyl aminopeptidase, a peptidyl-dipeptidase, or a dipeptidyl carboxypeptidase) and one of the spaces of the unmodified cleavase that fits one residue of the dipeptide for removal is configured in the modified cleavase to fit a label instead (e.g. a chemical label or a chemical modification).
  • a label e.g. a chemical label or a chemical modification
  • the modified cleavase is derived from a tripeptide cleavase (e.g, a sedolisin or a tripeptidyl peptidase) and one or two of the spaces of the unmodified cleavase that fits one or two residue(s) of the tripeptide for removal is configured in the modified cleavase to fit a label instead (e.g. a chemical label or a chemical modification).
  • a label e.g. a chemical label or a chemical modification
  • the modified cleavase comprises an amino acid mutation (e.g, modifications, substitutions, deletions, additions, or combinations thereof) compared to the wild-type cleavase polypeptide in the chymotrypsin fold of the cleavase.
  • the modified cleavase comprises an amino acid mutation (e.g, modifications, substitutions, deletions, additions, or combinations thereof) compared to the wild-type cleavase polypeptide in at an amine binding site.
  • the modified cleavase comprises an amino acid mutation (e.g, modifications, substitutions, deletions, additions, or combinations thereof) compared to the wild-type cleavase polypeptide in the loop domain.
  • the modified cleavase comprises an amino acid mutation (e.g, modifications, substitutions, deletions, additions, or combinations thereof) compared to the wild-type cleavase polypeptide for improving accessibility to the active site of the modified cleavase.
  • the modified cleavase exhibits greater accessibility of the substrate (e.g, polypeptide) to the active site compared to the unmodified cleavase.
  • the modified cleavase may allow larger substrates to access the active site.
  • the modified cleavase comprises an amino acid mutation (e.g, modifications, substitutions, deletions, additions, or combinations thereof) compared to the wild-type cleavase polypeptide in the active site or catalytic domain of the cleavase or the binding pockets of the cleavase.
  • the modified cleavase comprises an amino acid mutation (e.g ., substitutions, deletions, additions, or combinations thereof) compared to the wild-type cleavase polypeptide in the hinge region of the cleavase.
  • the modified cleavase comprises an amino acid mutation compared to the wild-type cleavase polypeptide in the binding cleft of the cleavase. In some cases, the modified cleavase comprises an amino acid mutation compared to the wild-type cleavase polypeptide in the inter-lobe cleft of the cleavase. In some embodiments, the modified cleavase comprises an amino acid mutation compared to the wild-type cleavase polypeptide in the alpha amine binding region of the cleavase. For example, the modified cleavase exhibits reduced alpha amine binding compared to the wild-type cleavase polypeptide. See e.g., Kumar et al., Sci Rep. (2016) 6:23787.
  • the modified cleavase exhibits altered activity, substrate binding capability, or cleavage characteristics compared to the unmodified cleavase.
  • the modified cleavase is modified in the catalytic motif or catalytic domain of the unmodified cleavase (e.g, the HEXXGH catalytic motif as set forth in SEQ ID NO: 1).
  • the mutations e.g, one or more amino acid modifications (e.g, substitutions, deletions, additions) corresponds to position(s) 316, 391, or 394 with reference to numbering of SEQ ID NO: 5.
  • modifications corresponds to position(s) amino acid residues 419, 420, 421, 422, 423, 424, 425, 426, or a combination thereof, with reference to numbering of SEQ ID NO: 5.
  • the unmodified cleavase is a metallopeptidase.
  • the modified cleavase is a metallopeptidase.
  • the modified cleavase is a zinc-dependent metallopeptidase or a zinc-dependent hydrolase or derived from such.
  • Some known metallopeptidase are characterized by the presence of a conventional catalytic signature motif HEXXH. In some aspects, the two His residues of the HEXXH motif contribute to coordinate the divalent metal ion (e.g, Zn 2+ , Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ ).
  • the modified cleavase requires the presence of or contact with specific metal ions (e.g, zinc ions, chloride ions) for activation.
  • function of the modified cleavase can be modulated or controlled by the presence or absence of metal ions, or by contacting with metal chelating agents.
  • the modified cleavase exhibits altered binding affinity and/or specificity to specific substrates compared to the unmodified cleavase.
  • the modified cleavase exhibits increased binding affinity and/or specificity for labeled terminal amino acids compared to the unmodified cleavase.
  • the modified cleavase exhibits decreased binding affinity and/or specificity for a substrate compared to the unmodified cleavase.
  • the modified cleavase exhibits one or more desired
  • the modified cleavase can be removed or released from the polypeptide at a desired rate.
  • a reaction with a modified cleavase can be enhanced by recruiting the modified cleavase to the labeled terminal amino acid.
  • one or more modified cleavases can be recruited to the labeled terminal amino acid of the polypeptide via hybridization of complementary universal priming sequences a DNA tag or sequence associated with the modified cleavase and a DNA tag or sequence associated with the polypeptide to be treated with the modified cleavase(s).
  • This hybridization step may improve the effective affinity of the modified cleavase for the labeled terminal amino acid (e.g ., NTAA).
  • the labeled terminal amino acid e.g ., NTAA
  • the labeled terminal amino acid after the labeled terminal amino acid is removed, it may diffuse away, and the associated modified cleavase can be removed by stripping the hybridized DNA tag.
  • the modified cleavase is attached to an anchoring sequence.
  • the modified cleavase is attached to the anchoring sequence directly or indirectly.
  • the anchoring sequence is complementary to a sequence attached to the polypeptides.
  • the anchoring sequence is a universal sequence or a universal DNA tag.
  • the polypeptide is also attached to a universal sequence.
  • the anchoring sequence on the modified cleavase brings the enzyme in proximity to the polypeptide.
  • the anchoring sequence brings the enzyme in proximity or co-localizes the modified cleavase to the polypeptide. In some embodiments, this co-localization of the modified cleavase and the polypeptide aids in binding and/or removal of the single labeled amino acid or dipeptide from said polypeptide.
  • recruitment of one or more modified cleavases to the terminal amino acid of the polypeptide may be enhanced via a chimeric modified cleavase and a chimeric terminal amino acid modifier, wherein the chimeric modified cleavase and chimeric terminal amino acid (e.g. NTAA) modifier each comprise a moiety capable of a binding reaction with each other (e.g, biotin-streptavidin).
  • a modified cleavase may be a low affinity enzyme (> mM Kd) and it is recruited to labeled NTAA associated with a biotin using a streptavidin-chimeric modified cleavase.
  • the efficiency of modified cleavase to remove labeled terminal amino acid(s) can be improved due to the increase in effective local concentration as a result of the biotin-strepavidin interaction. In some cases, this approach effectively increases the affinity KD from mM to subpicomolar.
  • a number of other bioconjugation recruitment strategies can also be employed.
  • An azide modified PITC is commercially available (4-Azidophenyl isothiocyanate, Sigma), allowing a number of simple transformations of azide-PITC into other bioconjugates of PITC, such as biotin-PITC via a click chemistry reaction with alkyne-biotin.
  • after the labeled terminal amino acid is removed it may diffuse away with the associated modified cleavase from the
  • the modified cleavase can be a single polypeptide chain or a multimer (dimers or higher order multimers) of at least two polypeptide chains.
  • monomeric, dimeric, and higher order multimeric modified cleavase polypeptides are within the scope of the defined term.
  • Multimeric polypeptides can be homomultimeric (of identical polypeptide chains) or heteromultimeric (of non-identical polypeptide chains).
  • the modified cleavase is a monomeric enzyme.
  • the modified cleavase is a fusion molecule or a chimeric molecule.
  • the modified cleavase may be attached or associated, directly or indirectly via a linker, to a oligonucleotide.
  • the modified cleavase may be joined to a moiety such as a
  • the terminal amino acid of a peptide removed by the modified cleavase is labeled or modified.
  • a label can comprise any suitable material or moiety. Any suitable molecule or materials may be employed for this purpose, including proteins, amino acids, nucleic acids, carbohydrates, chemical moieties, and small molecules.
  • a suitable label is capable of fitting in the binding pocket of the modified cleavase.
  • the labeling of a terminal amino acid is performed in a manner that is nucleic acid-compatible (e.g the labeling is performed in a manner that is not damaging to nucleic acids).
  • a suitable label enables the modified cleavase to remove a single labeled terminal amino acid residue or dipeptide from the polypeptide.
  • the terminal amino acid of the polypeptides may be labeled by any suitable methods.
  • the terminal amino acid is labeled chemically or enzymatically.
  • the terminal amino acid is labeled by a reagent that is or comprises a chemical agent, an enzyme, and/or a biological agent.
  • the terminal amino acid is labeled with a chemical label or moiety.
  • the terminal amino acid is labeled with a blocked or modified amino acid.
  • a precursor polypeptide e.g ., an unlabeled polypeptide
  • a reagent for labeling the terminal amino acid of the precursor polypeptide is contacted with a reagent for labeling the terminal amino acid of the precursor polypeptide to provide a polypeptide prepared for treatment with the modified cleavase.
  • the contacting of the precursor polypeptide with the reagent for labeling the terminal amino acid is performed prior to contacting the polypeptide with a modified cleavase.
  • the modified cleavase is contacted with a polypeptide that has been labeled or modified.
  • the contacting of the precursor polypeptide with the reagent for labeling the terminal amino acid and contacting the polypeptide with a modified cleavase are performed
  • the amino acid for removal by the modified cleavase is labeled with a chemical label.
  • the amino acid for removal by the modified cleavase is labeled with a chemical reagent.
  • the labeling of a terminal amino acid by treating with a chemical reagent is performed in a manner that is nucleic acid-compatible (e.g., the labeling is performed under conditions that is not damaging to nucleic acids).
  • the modified cleavase removes amino acid(s) that are labeled, such as a chemically-modified or labeled (e.g, PTC/DNP/acetyl/Cbz-modified or labeled) amino acids on a polypeptide.
  • the removed amino acid is a single labeled terminal amino acid.
  • the removed amino acid is part of a terminal dipeptide.
  • the modified cleavase e.g. dipeptide cleavase
  • the amino acid for removal by the modified cleavase which may be the single terminal amino acid or the terminal amino acid of a dipeptide to be removed by the cleavase, is labeled with a reagent selected from the group consisting of a phenyl isothiocyanate (PITC), a nitro-PITC, a sulfo-PITC, a phenyl isocyanate (PIC), a nitro- PIC, a sulfo-PIC, benzyloxycarbonyl chloride or carbobenzoxy chloride (Cbz-Cl), N- (Benzyloxycarbonyloxy)succinimide (Cbz-OSu or Cbz-O-NHS), a carboxyl-activated amino- blocked amino acid (e.g.
  • a reagent selected from the group consisting of a phenyl isothiocyanate (PITC), a nitro-PITC, a sulfo-PITC,
  • guanidinylation reagent a thioacylation reagent, a thioacetylation reagent, a thiobenzylation reagent, a diheterocyclic methanimine reagent, or a derivative thereof.
  • the terminal amino acid for removal by the modified cleavase which may be the terminal amine of a dipeptide to be removed by the cleavase, is labeled with an anhydride or derivative thereof.
  • the reagent for labeling the amino acid for removal by the modified cleavase is selected from the group consisting of: S- Acetylmercaptosuccinic anhydride, cis-Aconitic anhydride, 4-Amino-l,8-naphthalic anhydride, endo-Bicyclo[2.2.2]oct-5-ene-2,3-dicarboxylic anhydride, 5-Bromoisatoic anhydride,
  • Tetrachlorophthalic anhydride Tetrafluorophthalic anhydride, 3,4,5,6-Tetrahydrophthalic anhydride, 3,3-Tetramethyleneglutaric anhydride, Trimellitic anhydride chloride, and 2- (Triphenylphosphoranylidene)succinic anhydride.
  • 2- (Triphenylphosphoranylidene)succinic anhydride See e.g. Staiger et al., J. Org. Chem. 1959, 24, 9, 1214-1219; Jiang et al. J. Org. Chem. 2019, 84, 4, 2022-2031; U.S. Patent No. 9,867,883.
  • a polypeptide in preparation for treatment with a modified cleavase of the invention, is treated with a chemical reagent that comprises an isatoic anhydride, an isonicotinic anhydride, an azaisatoic anhydride, a succinic anhydride, or a derivative of one of these, and the terminal amino acid of the polypeptide is modified, or labeled, by the chemical reagent.
  • a chemical reagent that comprises an isatoic anhydride, an isonicotinic anhydride, an azaisatoic anhydride, a succinic anhydride, or a derivative of one of these.
  • labeling of a terminal amino acid of a polypeptide to be treated with a modified cleavase of the invention include:
  • G'-G 4 are each independently selected from CH, CX, and N;
  • R represents the side chain of an amino acid, e.g. one of the side chains of the 20 common amino acids
  • R 2 is independently at each occurrence selected from H and C1-C2 alkyl
  • R 3 is independently at each occurrence selected from C1-C2 alkyl
  • Ar is independently selected at each occurrence from phenyl, pyridinyl, pyrimidinyl, pyridazinyl, and pyrazinyl, each of which is optionally substituted by one or two groups selected from halo, CN, NO2, C1-C2 alkyl, C1-C2 haloalkyl, C1-C2 haloalkoxy, and -OR 2 ; and
  • PP represents a portion of a polypeptide, particularly the portion of a polypeptide being prepared for treatment with a modified cleavase of the invention excluding the N-terminal amino acid.
  • the compound of Formula (C) is typically a polypeptide for use in the methods of the invention, and R represents the side chain of the terminal amino acid of the polypeptide.
  • the terminal amino acid shown in Formula (C) is in the L-configuration when R is not H.
  • the compounds of Formula (B) are polypeptides, sometimes referred to as labeled polypeptides, that have been prepared for use in the modified cleavase reactions described herein.
  • the amino acid for removal by the modified cleavase is labeled with an exemplary reagent derived from an isatoic anhydride, an isonicotinic anhydride or an azaisatoic anhydride, especially compounds of Formula (A) as described herein.
  • the amino acid for removal by the modified cleavase is labeled with an exemplary reagent selected from the list consisting of N-Methyl-isatoic anhydride, N-acetyl-isatoic anhydride, 4-carboxylic acid isatoic anhydride, 5-methoxy-isatoic anhydride, 5-nitro-isatoic anhydride, 4-chloro-isatoic anhydride, 4-fluoro-isatoic anhydride, 6-fluoro-isatoic anhydride, N- benzyl -isatoic anhydride, 4-trifluoromethyl-isatoic anhydride, 5-trifluoromethyl-isatoic anhydride, 4-nitro-isatoic anhydride, 4-methoxy-isatoic anhydride, and 5-Amino-2-fluoro- isonicotinic anhydride (6-fluoro-lH-pyrido[3,4-d][l,
  • the labeled amino acid or dipeptide removed by the action of a modified cleavase of the invention comprises an optionally substituted benzamide, typically one derived from any of the optionally substituted isatoic anhydrides disclosed herein, including a compound of Formula (B) as described herein.
  • the polypeptide in preparation for treatment with a modified cleavase of the invention, is treated with a chemical reagent that comprises a succinic anhydride, a phthalic anhydride, a pyrazinedicarboxylic anhydride, or a derivative of one of these, and the terminal amino acid is modified, or labeled, by the chemical reagent.
  • a chemical reagent that comprises a succinic anhydride, a phthalic anhydride, a pyrazinedicarboxylic anhydride, or a derivative of one of these, and the terminal amino acid is modified, or labeled, by the chemical reagent.
  • n 0 or 1
  • Ring Cy represents a 5- or 6-membered ring or an 8-10 membered bicyclic ring that may be absent or present; when present, ring Cy may be saturated, unsaturated, or aromatic, and the dashed bond may be a single bond, double bond, or aromatic bond;
  • Cy when Cy is present, it may be a carbocyclic ring, or it may contain one or two heteroatoms selected from N, O and S as ring members;
  • Ring Cy when Ring Cy is present, it is optionally substituted with one to six groups (or with one to four groups when Cy is aromatic) selected from halo, CN, NCh, C 1 -C 2 alkyl, Ci- C 2 haloalkyl, C 1 -C 2 haloalkoxy, and -OR 4 ;
  • the dashed bond when ring Cy is absent, the dashed bond may be a single bond or a double bond, and the dashed bond is optionally substituted by one or two groups selected from halo, CN, C 1 -C 2 alkyl, C1-C2 haloalkyl, C1-C2 haloalkoxy, CO2R 4 , and -OR 4 ;
  • R represents the side chain of an amino acid, e.g. one of the side chains of the 20 common amino acids
  • R 4 is independently selected at each occurrence from H, C 1 -C 2 alkyl, and C 1 -C 2 haloalkyl;
  • R 5 is independently selected at each occurrence from H, halo, C 1 -C 2 alkyl, C 1 -C 2 haloalkyl, C1-C2 alkoxy, and C1-C2 haloalkoxy;
  • PP represents a portion of a polypeptide, particularly the portion of a polypeptide being prepared for treatment with a modified cleavase of the invention excluding the N- terminal amino acid.
  • the compound of Formula (C) is typically a polypeptide for use in the methods of the invention, and R represents the side chain of the terminal amino acid of the polypeptide.
  • ring Cy is absent when n is 1.
  • ring Cy is present and is a phenyl ring or a 2,3-pyrazine ring, each of which is optionally substituted as described above, and n is 0.
  • the terminal amino acid shown in Formula (C) is in the L-configuration when R is not H.
  • the compounds of Formula (E) are polypeptides, sometimes referred to as labeled polypeptides, that have been prepared for use in the modified cleavase reactions described herein.
  • the amino acid for removal by the modified cleavase is labeled with an exemplary reagent derived from succinic anhydride, or a compound of Formula (D) wherein ring Cy is absent and the dashed bond represents a single bond.
  • the reagent is 3,6, difluorophthalic anhydride, 2,3 pyrazinedicarboxylic anhydride, or succinic anhydride.
  • the removed labeled amino acid or dipeptide comprises 4-carboxybutylamide.
  • a polypeptide in preparation for treatment with a modified cleavase of the invention, is treated with any suitable chemical reagent that is capable of forming an amide bond with the a-amine of the polypeptide N-terminus.
  • a number of chemical reagents react with terminal amines of the polypeptide to form a modified polypeptide with an amide bond linking the polypeptide to the modification; this N-terminal modified polypeptide can be a substrate for a modified cleavase.
  • Chemical reagents that react with amines to form an amide bond are known from the field of peptide coupling, including but not limited to: acyl halides (chlorides, fluorides, bromides), acyl imidazoles, O-acyl isoureas, activated esters [N- hydroxysuccinimide (NHS or HOSu),N-hydroxysulfosuccinimide (sulfo-NHS) p-nitrophenyl (PNP), Pentafluorophenyl (Pfp), 4-sulfo-2,3,5,6,-tetrafluorophenyl, 2,4,5-trichlorophenol, N- hydroxy-5-norbomene-2,3-dicarboximide (HONB), 3 -hydroxy-4-oxo-3, 4-dihydro- 1,2,3 - benzotriazine (HODhbt), hydroxybenzotriazole (HOBt), l-hydroxy-7-azabenzotriazole
  • An example of labeling a polypeptide with a PNP ester is provided with 4-Nitrophenyl Anthranilate which can be used to label a polypeptide under the following conditions: 4-Nitrophenol anthranilate (PNP A) is dissolved in DMSO at 100 mM; and PNPA used at 10 mM with 1 mM peptide in lXPBS (pH 8.5) or 100 mM NaHC03 carbonate buffer (pH 8.5) in 10% DMSO for 37 °C for 1 hr.
  • PNP A 4-Nitrophenol anthranilate
  • PNPA used at 10 mM with 1 mM peptide in lXPBS (pH 8.5) or 100 mM NaHC03 carbonate buffer (pH 8.5) in 10% DMSO for 37 °C for 1 hr.
  • the resulting peptide product generated is equivalent to labeling a peptide with isatoic anhydride, and generates a 2-aminobenzamide-modified peptide suitable as a substrate for a modified cleavase (e.g., derived from DAP BII) as illustrated in Table 11.
  • a modified cleavase e.g., derived from DAP BII
  • a polypeptide in preparation for treatment with a modified cleavase of the invention, is treated with a chemical reagent that comprises an amine-protected activated ester to form an amide bond and the terminal amino acid of the polypeptide is modified, or labeled, by the chemical reagent.
  • This modified polypeptide can then be further appropriately treated to remove the designated protecting group, yielding a modified polypeptide for treatment with a modified cleavase.
  • G 3 -G 4 are each independently selected from CH, CX, and N;
  • R represents the side chain of an amino acid, e.g. one of the side chains of the 20 common amino acids
  • R 2 is independently at each occurrence selected from H and C1-C2 alkyl
  • R 3 is independently at each occurrence selected from C1-C2 alkyl;
  • Ar is independently selected at each occurrence from phenyl, pyridinyl, pyrimidinyl, pyridazinyl, and pyrazinyl, each of which is optionally substituted by one or two groups selected from halo, CN, NO2, C1-C2 alkyl, C1-C2 haloalkyl, C1-C2 haloalkoxy, and -OR 2 ;
  • -NRkPG can be replaced by -N3;
  • PG is H or a nitrogen protecting groups which may be selected from
  • a polypeptide in preparation for treatment with a modified cleavase of the invention, is treated with 4-Nitrophenyl Anthranilate.
  • the chemical reagent for modifying or labeling the amino acid for removal by the modified cleavase is one or more of any of the compounds of Formula (A) or (D), described herein, or a salt or conjugate thereof.
  • the chemical reagent for modifying or labeling the amino acid for removal by the modified cleavase is one or more of any of the compounds of Formula (I), (II), (III), (IV), or (AB), described herein, or a salt or conjugate thereof.
  • the reagent for modifying or labeling the amino acid for removal by the modified cleavase comprises a compound selected from the group consisting of a compound of Formula (I):
  • R 1 and R 2 are each independently H, Ci- 6 alkyl, cycloalkyl, -C(0)R a , -C(0)OR b ,
  • R a , R b , and R c are each independently H, Ci- 6 alkyl, Ci- 6 haloalkyl, arylalkyl, aryl, or heteroaryl, wherein the Ci- 6 alkyl, Ci- 6 haloalkyl, arylalkyl, aryl, and heteroaryl are each unsubstituted or substituted;
  • R 3 is heteroaryl, -NR d C(0)0R e , or -SR f , wherein the heteroaryl is unsubstituted or substituted;
  • R d , R e , and R f are each independently H or Ci- 6 alkyl.
  • R 3 when R 1 and R 2 are not both H. In some embodiments of Formula (I), both R 1 and R 2 are H. In some embodiments, neither R 1 nor R 2 are H. In some embodiments, one of R 1 and R 2 is Ci- 6 alkyl. In some embodiments, one of R 1 and R 2 is H, and the other is Ci- 6 alkyl, cycloalkyl, -C(0)R a , -C(0)0R b , or -S(0) 2 R C . In some embodiments, one or both of R 1 and R 2 is Ci- 6 alkyl. In some embodiments, one or both of R 1 and R 2 is cycloalkyl.
  • R 1 and R 2 is -C(0)R a . In some embodiments, one or both of R 1 and R 2 is -C(0)0R b . In some embodiments, one or both of R 1 and R 2 is -S(0) 2 R C . In some embodiments, one or both of R 1 and R 2 is -S(0) 2 R C , wherein R c is
  • Ci- 6 alkyl Ci- 6 haloalkyl, arylalkyl, aryl, or heteroaryl.
  • R 1 is
  • R 2 is In some embodiments, both R 1 and R 2 are
  • R 3 is a monocyclic heteroaryl group. In some embodiments of Formula (I), R 3 is a 5- or 6-membered monocyclic heteroaryl group. In some embodiments of Formula (I), R 3 is a 5- or 6-membered monocyclic heteroaryl group containing one or more N. Preferably, R 3 is selected from pyrazole, imidazole, triazole and tetrazole, and is linked to the amidine of Formula (I) via a nitrogen atom of the pyrazole, imidazole, triazole or tetrazole ring, and R 3 is optionally substituted by a group
  • R 3 is , wherein Gi is N, CH, or CX where X is halo, C 1-3 alkyl, C 1-3 haloalkyl, or nitro. In some embodiments, R 3 is or where X is Me, F, Cl, CF 3 , or NO 2 . In some embodiments, R 3 is wherein Gi is N or CH. In some embodiments, R 3 is . In some embodiments, R 3 is a bicyclic heteroaryl group. In some embodiments, R 3 is a 9- or 10-
  • N ⁇ membered bicyclic heteroaryl group.
  • R 3 is or N
  • the compound of Formula (I) is . In some embodiments, the compound of Formula (I) is .
  • the compound of Formula (I) is not
  • the compound of Formula (I) is selected from the group consisting of:
  • the chemical reagent additionally comprises Mukaiyama’s reagent (2-chloro-l-methylpyridinium iodide).
  • the reagent comprises at least one compound of Formula (I) and Mukaiyama’s reagent.
  • the chemical reagent comprising a cyanamide derivative is used to label one or more amino acids of the polypeptide.
  • a cyanamide derivative See, e.g., Kwon et ah, Org. Lett.
  • the chemical reagent comprises a compound selected from the group consisting of a compound of Formula (II):
  • R 4 is H, Ci- 6 alkyl, cycloalkyl, -C(0)R g , or -C(0)OR g ;
  • R g is H, Ci- 6 alkyl, C2-6alkenyl, Ci- 6 haloalkyl, or arylalkyl, wherein the Ci- 6 alkyl, C2- 6 alkenyl, Ci- 6 haloalkyl, and arylalkyl are each unsubstituted or substituted.
  • a reagent comprising an isothiocyanate derivative is used to label the terminal amino acid (e.g., NTAA) of a polypeptide.
  • NTAA terminal amino acid
  • the chemical reagent comprises a compound selected from the group consisting of a compound of Formula (III):
  • R 5 is Ci- 6 alkyl, C2-6alkenyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • Ci- 6 alkyl, C2-6alkenyl, cycloalkyl, heterocyclyl, aryl or heteroaryl are each unsubstituted or substituted with one or more groups selected from the group consisting of halo, -NR R', -S(0) 2 R J , or heterocyclyl;
  • R h , R 1 , and R 1 are each independently H, Ci- 6 alkyl, Ci- 6 haloalkyl, arylalkyl, aryl, or heteroaryl, wherein the Ci- 6 alkyl, Ci- 6 haloalkyl, arylalkyl, aryl, and heteroaryl are each unsubstituted or substituted.
  • R 5 is substituted phenyl. In some embodiments, R 5 is substituted phenyl substituted with one or more groups selected from halo, -NR R', -S(0) 2 R i , or heterocyclyl. In some embodiments, R 5 is unsubstituted Ci- 6 alkyl. In some embodiments, R 5 is substituted Ci- 6 alkyl. In some embodiments, R 5 is substituted Ci- 6 alkyl, substituted with one or more groups selected from halo, -NR R', -S(0) 2 R J , or heterocyclyl. In some embodiments, R 5 is unsubstituted C2-6alkenyl.
  • R 5 is C2-6alkenyl. In some embodiments, R 5 is substituted C2-6alkenyl, substituted with one or more groups selected from halo, -NR R', -S(0) 2 R i , or heterocyclyl. In some embodiments, R 5 is unsubstituted aryl. In some embodiments, R 5 is substituted aryl. In some embodiments, R 5 is aryl, substituted with one or more groups selected from halo, -NR R', -S(0) 2 R J , or heterocyclyl. In some embodiments, R 5 is unsubstituted cycloalkyl. In some embodiments, R 5 is substituted cycloalkyl. In some embodiments, R 5 is cycloalkyl, substituted with one or more groups selected from halo,
  • R 5 is unsubstituted heterocyclyl. In some embodiments, R 5 is substituted heterocyclyl. In some embodiments, R 5 is heterocyclyl, substituted with one or more groups selected from halo, -NR R', -S(0) 2 R J , or heterocyclyl. In some embodiments, R 5 is unsubstituted heteroaryl. In some embodiments, R 5 is substituted heteroaryl. In some embodiments, R 5 is heteroaryl, substituted with one or more groups selected from halo, -NR R', -S(0) 2 R i , or heterocyclyl.
  • the compound of Formula (III) is trimethyl silyl isothiocyanate (TMSITC) or pentafluorophenyl isothiocyanate (PFPITC).
  • the compound is not trifluoromethyl isothiocyanate, allyl isothiocyanate, dimethylaminoazobenzene isothiocyanate, 4-sulfophenyl isothiocyanate, 3- pyridyl isothiocyanate, 2-piperidinoethyl isothiocyanate, 3-(4-morpholino) propyl
  • the reagent is or comprises an alkyl amine.
  • the reagent additionally comprises DIPEA, trimethylamine, pyridine, and/or N- methylpiperidine.
  • the reagent additionally comprises pyridine and triethylamine in acetonitrile.
  • the reagent additionally comprises N- methylpiperidine in water and/or methanol.
  • the polypeptide is also contacted with a carbodiimide compound.
  • the chemical reagent comprises a carbodiimide derivative (See, e.g., Chi et ah, 2015, Chem. Eur. J 2015, 21, 10369-10378, incorporated by reference in their entireties).
  • the NTAA of a polypeptide is labeled via acylation.
  • acylation See, e.g., Protein Science (1992), I, 582-589, incorporated by reference in their entireties).
  • the chemical reagent comprises a compound selected from the group consisting of a compound of Formula (IV):
  • R 8 is halo or -OR m ;
  • R m is H, Ci- 6 alkyl, or heterocyclyl
  • R 9 is hydrogen, halo, or Ci- 6 haloalkyl.
  • R 8 is halo. In some embodiments, R 8 is
  • R 9 is hydrogen. In some embodiments, R 9 is halo, such as bromo.
  • the compound of Formula (IV) is selected from acetyl chloride, acetyl anhydride, and acetyl-NHS. In some embodiments, the compound is not acetyl anhydride or acetyl-NHS.
  • the polypeptide is also contacting with a peptide coupling reagent.
  • the peptide coupling reagent is a carbodiimide compound.
  • the carbodiimide compound is diisopropylcarbodiimide (DIC) or l-ethyl-3- (3-dimethylaminopropyl)carbodiimide (EDC).
  • the method includes contacting with at least one compound of Formula (I) and a carbodiimide compounds, such as DIC or EDC.
  • the chemical reagent comprises a metal complex.
  • a metal complex See, e.g., Bentley et al., Biochem. J 7973(135), 507-511; Bentley et al., Biochem. J 1976(153), 137- 138; Huo et al., J. Am. Chem. Soc. 2007, 139, 9819-9822; Wu et al., J. Am. Chem. Soc. 2016, 138(44), 14554-14557 incorporated by reference in their entireties).
  • the metal complex is a metal directing/chelating group.
  • the metal complex comprises one or more ligands chelated to a metal center.
  • the ligand is a monodentate ligand. In some embodiments, the ligand is a bidentate or polydentate ligand. In some embodiments, the metal complex comprises a metal selected from the group consisting of Co, Cu, Pd, Pt, Zn, and Ni.
  • the chemical reagent comprises a conjugate of Formula
  • the reagent used to modify the terminal amino acid of a polypeptide comprises a compound of Formula (I), Formula (I), Formula (II), Formula (III), or Formula (IV).
  • the reagent used to modify the terminal amino acid of a polypeptide comprises a compound of Formula (I), Formula (I), Formula (I), Formula (II), Formula (III), or Formula (IV).
  • the chemical reagent comprises a conjugate of Formula (I)-Q, Formula (II)-Q, Formula (III)-Q, or Formula (IV)-Q, wherein Formula (I)-(IV) are as defined above, and Q is a ligand.
  • the ligand Q is a pendant group or binding site (e.g, the site to which the binding agent binds).
  • the polypeptide binds covalently to a binding agent.
  • the polypeptide comprises a terminal amino acid which includes a ligand group that is capable of covalent binding to a binding agent.
  • the polypeptide comprises a labeled NTAA with a compound of Formula (I)-Q, Formula (II)-Q, Formula (III)-Q, or Formula (IV)-Q, wherein the Q binds covalently to a binding agent.
  • a coupling reaction is carried out to create a covalent linkage between the polypeptide and the binding agent (e.g, a covalent linkage between the ligand Q and a functional group on the binding agent).
  • the chemical reagent comprises a conjugate of Formula
  • the chemical reagent comprises a conjugate of Formula
  • R 4 is as defined above, and Q is a ligand.
  • the chemical reagent comprises a conjugate of Formula
  • R 5 is as defined above and Q is a ligand.
  • the chemical reagent comprises a conjugate of Formula (IV)-Q
  • R 8 and R 9 are as defined above and Q is a ligand.
  • Q is a fluorophore.
  • Q is selected from a lanthanide, europium, terbium, XL665, d2, quantum dots, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, fluorescein, rhodamine, eosin, Texas red, cyanine, indocarbocyanine, ocacarbocyanine, thiacarbocyanine, merocyanine, pyridyloxadole, benzoxadiazole, cascade blue, nile red, oxazine 170, acridine orange, proflavin, auramine, malachite green crystal violet, porphine phtalocyanine, and bilirubin.
  • reagents used in labeling the terminal amino acid or amino acid for removal by the modified cleavase with more than one label are provided in other aspects.
  • labeling the terminal amino acid (e.g., NTAA) or amino acid for removal by the modified cleavase includes using a first reagent and a second reagent.
  • the terminal amino acid is concurrently or sequentially labeled with the first reagent and the second reagent.
  • the first reagent comprises a compound selected from the group consisting of a compound of Formula (I), (II), (III), and (IV), or a salt or conjugate thereof, as described herein.
  • the second reagent comprises a compound of Formula (Va) or (Vb):
  • R 13 is H, Ci- 6 alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl, wherein the Ci- 6 alkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl are each unsubstituted or substituted; or
  • R 13 is Ci- 6 alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl, each of which is unsubstituted or substituted;
  • X is a halogen
  • R 13 is H. In some embodiments, R 13 is methyl. In some embodiments, R 13 is ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, pentyl, or hexyl. In some embodiments, R 13 is Ci- 6 alkyl, which is substituted. In some embodiments, R 13 is Ci- 6 alkyl, which is substituted with aryl, heteroaryl, cycloalkyl, or heterocyclyl. In some embodiments, R 13 is Ci- 6 alkyl, which is substituted with aryl. In some embodiments, R 13 is - CH 2 CH 2 Ph, -CH 2 Ph, -CH(CH )Ph, or -CH(CH )Ph.
  • R 13 is methyl. In some embodiments, R 13 is ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, pentyl, or hexyl. In some embodiments, R 13 is Ci- 6 alkyl, which is substituted. In some embodiments, R 13 is Ci- 6 alkyl, which is substituted with aryl, heteroaryl, cycloalkyl, or heterocyclyl. In some embodiments, R 13 is Ci- 6 alkyl, which is substituted with aryl. In some embodiments, R 13 is -CH 2 CH 2 Ph, -CH 2 Ph, -CH(CH 3 )Ph, or - CH(CH )Ph.
  • the reagent for modifying or labeling the terminal amino acid or amino acid for removal by the modified cleavase comprises formaldehyde. In some embodiments, the reagent for modifying or labeling the terminal amino acid comprises methyl iodide.
  • the polypeptide is also contacted with a reducing agent.
  • the reducing agent comprises a borohydride, such as NaBFLt, KBFE, ZnBFLt, NaBFFCN or LiBu 3 BH.
  • the reducing agent comprises an aluminum or tin compound, such as LiAlFLt or SnCl.
  • the reducing agent comprises a borane complex, such as B 2 H 6 and dimethyamine borane.
  • the reagent additionally comprises NaBFECN.
  • the reagents that may be used to label the terminal amino acid include: 4-sulfophenyl isothiocyanate (sulfo-PITC), 4-nitrophenyl isothiocyanate (nitro-PITC), 3-pyridyl isothiocyanate (PYITC), a phenyl isocyanate (PIC), a nitro-PIC, a sulfo-PIC, a carboxyl -activated amino-blocked amino acid, an anhydride (e.g., an isatoic anhydride, an isonicotinic anhydride, an azaisatoic anhydride, a succinic anhydride), 2- piperidinoethyl isothiocyanate (PEITC), 3-(4-morpholino) propyl isothiocyanate (MPITC), 3- (diethylamino)propyl isothiocyanate
  • thioacylation reagent a thioacetylation reagent, and/or a thiobenzylation reagent.
  • Many of these reagents are unreactive or minimally reactive with DNA including PITC, nitro-PITC, sulfo- PITC, PYITC, and guanidinylation reagents ( e.g ., PCA compounds). If the amino acid is blocked to labeling, there are a number of approaches to unblock the terminus, such as removing N-acetyl blocks with acyl peptide hydrolase (APH) (Farries, Harris et ah, 1991, Eur. J. Biochem. 196:679-685).
  • APH acyl peptide hydrolase
  • Dansyl chloride reacts with the free amine group of a peptide to yield a dansyl derivative of the NTAA.
  • DNFB and SNFB react the a-amine groups of a peptide to produce DNP-NTAA, and SNP-NTAA, respectively. Additionally, both DNFB and SNFB also react with the with e-amine of lysine residues. DNFB also reacts with tyrosine and histidine amino acid residues.
  • SNFB has better selectivity for amine groups than DNFB (Carty et ak, J Biol Chem (1968) 243(20): 5244-5253).
  • lysine e-amines are pre-blocked with an organic anhydride prior to polypeptide protease digestion into peptides.
  • Isothiocyanates in the presence of ionic liquids, have been shown to have enhanced reactivity to primary amines.
  • Ionic liquids are excellent solvents (and serve as a catalyst) in organic chemical reactions and can enhance the reaction of isothiocyanates with amines to form thioureas.
  • ionic liquids may act as absorbers of microwave radiation to further enhance reactivity (Martinez -Palou, J. Mex. Chem. Soc (2007) 51(4): 252-264).
  • An example is the use of the ionic liquid 1 -butyl-3 -methyl-imidazolium tetraflouorab orate
  • the peptide may be labeled by treating with a chemical reagent comprising a compound of Formula (AB) as shown in the scheme below:
  • the peptide treated with a chemical reagent to modify the N-terminal amino acid (NTAA) of peptides is treated with a diheterocyclic methanimine reagent.
  • the reagent for modifying or labeling the terminal amino acid or amino acid for removal by the modified cleavase comprises a compound of Formula (AB):
  • R 2 is H, R 4 , OH, OR 4 , NH 2 , or -NHR 4 ;
  • R 4 is Ci - 6 alkyl, which is optionally substituted with one or two members selected from halo, C 1-3 alkyl, C 1-3 alkoxy, C 1-3 haloalkyl, phenyl, 5-membered heteroaryl, and 6-membered heteroaryl, wherein each phenyl, 5-membered heteroaryl, and 6-membered heteroaryl is optionally substituted with one or two members selected from halo, -OH, C 1-3 alkyl, C 1-3 alkoxy, C 1-3 haloalkyl, NO 2 , CN, COOR”, and CON(R”) 2 ,
  • each R is independently H or C1-3 alkyl
  • ring A and ring B are each independently a 5-membered heteroaryl ring containing up to three N atoms as ring members and each is optionally fused to an additional phenyl or a 5- 6 membered heteroaryl ring, and wherein the 5-membered heteroaryl ring and optional fused phenyl or 5-6 membered heteroaryl ring are each optionally substituted with one or two groups selected from Ci-4 alkyl, Ci-4 alkoxy, -OH, halo, Ci-4 haloalkyl, NO2, COOR, COMO, -SO2R*, - MO, phenyl, and 5-6 membered heteroaryl;
  • each R is independently selected from H and C1-3 alkyl optionally substituted with OH, OR*, -MO, -NHR*, or -MOO;
  • each R* is C1-3 alkyl, optionally substituted with OH, oxo, C 1-2 alkoxy, or CN; wherein two R, or two R”, or two R* on the same N can optionally be taken together to form a 4-7 membered heterocyclic ring, optionally containing an additional heteroatom selected from N, O and S as a ring member, and optionally substituted with one or two groups selected from halo, C 1-2 alkyl, OH, oxo, C i-2 alkoxy, or CN.
  • Ring A and Ring B are not both unsubstituted imidazole, and that Ring A and Ring B are not both unsubstituted benzotri azole;
  • R 2 is H or R 4 .
  • the 5-membered heteroaryl group when present, can be a 5-membered ring comprising one to three heteroatoms selected from N, O and S as ring members, and the 6- membered heteroaryl group when present can be a 6-membered ring comprising one to three nitrogen atoms as ring members.
  • neither ring A nor ring B is unsubstituted imidazole or unsubstituted benzotri azole.
  • R 2 is H. In some of these embodiments, neither ring A nor ring B is unsubstituted imidazole or unsubstituted benzotri azole.
  • Ring A and Ring B are different. In some embodiments, Ring A and Ring B are the same. Specific compounds of this embodiment include:
  • each 5-6 membered heteroaryl ring is independently selected and contains 1 or 2 heteroatoms selected from N, O and S as ring members.
  • each 5-membered heteroaryl group present can be a 5-membered ring comprising one or two heteroatoms selected from N, O and S as ring members
  • the 6-membered heteroaryl group can be a 6-membered ring comprising one to two nitrogen atoms as ring members.
  • Ring A and Ring B are selected from:
  • each R x , R y and R z is independently selected from H, halo, C1-2 alkyl, C1-2 haloalkyl, NO2, S0 2 (Ci- 2 alkyl), COOR # , C(0)N(R # ) 2 , and phenyl optionally substituted with one or two groups selected from halo, C1-2 alkyl, Ci-2 haloalkyl, NO2, S0 2 (C 1-2 alkyl), COOR # , and C(0)N(R # ) 2 ,
  • R x , R y or R z on adjacent atoms of a ring can optionally be taken together to form a phenyl group, 5-membered heteroaryl group, or 6-membered heteroaryl group fused to the ring, and the fused phenyl, 5-membered heteroaryl, or 6-membered heteroaryl group can optionally be substituted with one or two groups selected from halo, C1-2 alkyl, Ci- 2 haloalkyl, N0 2 , S0 2 (Ci- 2 alkyl), COOR # , and C(0)N(R # ) 2 ;
  • each R # is independently H or C1-2 alkyl; and wherein two R# on the same nitrogen can optionally be taken together to form a 4-7 membered heterocycle optionally containing an additional heteroatom selected from N, O and S as a ring member, wherein the 4-7 membered heterocycle is optionally substituted with one or two groups selected from halo, OH, OMe, Me, oxo, NH2, NHMe and MVfe;
  • each 5-membered heteroaryl group present can be a 5- membered ring comprising one to three heteroatoms selected from N, O and S as ring members
  • the 6-membered heteroaryl group can be a 6-membered ring comprising one to three nitrogen atoms as ring members.
  • Ring A and Ring B are the same and are selected from:
  • the label is any label that is capable of being recognized or bound by the modified cleavase.
  • the terminal amino acid label is or includes a label that“mimics” the size/shape of a terminal amino acid (e.g ., an N-terminal amino acid).
  • the label is a modified amino acid, a portion of an amino acid, a protected amino acid, a blocked amino acid, or any combination thereof.
  • the label attached to the terminal amino acid is an N-terminal blocked (devoid of alpha amine) amino acid.
  • the label comprises an amino acid that is any amino acid, naturally occurring or synthetic.
  • the label is a synthesized blocked amino acid.
  • any suitable methods of introducing amino acid blocking groups may be used.
  • blocking groups are introduced by reacting an amino acid with a reagent to introduce the blocking group. Any suitable activated and blocked amino acid may be used to label the terminal amino acid. See e.g., Kruse et ah, J. Org. Chem. (1985) 50(15):2792-2794.
  • a synthesized label that comprises one or more exogenous amino acids may be used as a label and attached to the polypeptide for treatment with the modified cleavase.
  • amino acid blocking groups may be mono- or di-valent, suitable groups including acyl groups, for example lower alkanoyl such as acetyl, substituted lower alkanoyl, e.g ., lower haloalkanoyl such as chloroacetyl, aryl-lower alkanoyl such as phenylacetyl, and aroyl such as benzoyl or phthaloyl; lower alkoxycarbonyl groups such as ethoxycarbonyl, isobutyloxycarbonyl or t-butyloxycarbonyl and substituted lower alkanoyl groups, for example lower alkanoyl such as acetyl, substituted lower alkanoyl, e.g ., lower haloalkanoyl such as chloroacetyl, aryl-lower alkanoyl such as phenylacetyl, and aroyl such as benzoyl or phthaloyl; lower alkoxycarbony
  • alkoxycarbonyl groups e.g. , lower haloalkoxy carbonyl such as 2, 2, 2-tri chi oroethoxy carbonyl; aryl-lower alkoxycarbonyl groups such as benzyloxycarbonyl; sulphonyl groups, for example lower alkyl sulphonyl such as methanesulphonyl and arylsulphonyl such as benzenesulphonyl or p-toluenesulphonyl; ylidene groups formed by reaction with aldehydes and ketones which form Schiffs bases, for example benzaldehyde, salicaldehyde or acetcacetic ester; and divalent groups such that the nitrogen atom forms part of a dihydropyridine ring, such protecting groups being obtained by, for example, reaction with formaldehyde and a b-ketoester, e.g. , acetoacetic ester.
  • protecting groups being obtained by,
  • the labeling of a peptide for removal includes attaching a label that comprises an amino acid with a label.
  • labeled amino acids are commercially available, such as Cbz-amino acids (e.g, N- CBZ-DL-serine, N-CBZ-L-aspartic acid) or N-protected amino acids.
  • the labeling of a terminal amino acid for removal includes attaching a Cbz-amino acid as a label to the terminal amino acid for removal.
  • the labeling includes attaching a label that comprises two amino acids with a label.
  • a synthesized label that includes -Xaa-Xaa-label (e.g., Z-gly-gly-osu) may be used as a label and attached to the peptide for treatment with the modified cleavase.
  • the label comprises an amino acid with an Ac protected amine.
  • the label comprises an amino acid that is Fmoc, Boc, or Cbz protected.
  • the labeled amino acid comprises an amino acid with an amine that is dialkyl.
  • the carboxylic acid of the amino acid of the label is free and the amino acid is coupled using standard peptide coupling reagents such as HATU + DIEA or EDC + DIEA + HOBt.
  • the label comprises an amino acid that could be D or L chirality.
  • the modified cleavase may remove a dipeptide from the polypeptide that includes one amino acid of the polypeptide and one exogenous amino acid that was added as part of the label.
  • the exogenous amino acid Xaa may represent any amino acid residue selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
  • a label comprising Xaa-label is added to the polypeptide at the terminus to form -P3-P2-Pl-Xaa- label.
  • An exemplary modified cleavase is then contacted with the labeled polypeptide to cleave a dipeptide comprising -PI -Xaa-label from the polypeptide, thereby removing the PI amino acid from the N-terminus of the polypeptide.
  • a modified cleavase derived from a unmodified or wild-type tripeptide cleavase may remove a tripeptide comprising P2-P1 -Xaa-label from the polypeptide, thereby removing the PI and P2 amino acids from the N- terminus of the polypeptide.
  • the modified cleavase may remove a tripeptide from the polypeptide that includes one amino acid of the polypeptide and two exogenous amino acid that was added as part of the label. For example, if a polypeptide at the N-terminus has -P3-P2-P1, a label comprising Xaa-Xaa-label is added to the polypeptide at the terminus to form -P3-P2-P1 -Xaa-Xaa-label.
  • An exemplary modified cleavase is then contacted with the labeled polypeptide to cleave a tripeptide comprising -Pl- Xaa-Xaa-label from the polypeptide, thereby removing the PI amino acid from the N-terminus of the polypeptide.
  • the modified cleavase provided herein can be made, isolated, engineered, or selected for using any suitable methods.
  • the variant or modified cleavase polypeptide is altered in primary amino acid sequence compared to the wild- type or unmodified cleavase by introducing one or more substitutions, additions, or deletions of amino acid residues.
  • the modified cleavase is derived from a wild-type or unmodified cleavase (e.g., a dipeptidyl peptidase, a dipeptidyl aminopeptidase, a peptidyl- dipeptidase, a dipeptidyl carboxypeptidase, a sedolisin, a tripeptidyl peptidase, or a protein classified in EC 3.4.14, EC 3.4.15, MEROPS S8, MEROPS S9, MEROPS S33, MEROPS S46, MEROPS M49, or MEROPS S53, or a functional homolog or fragment thereof) via engineering and genetic selection.
  • a wild-type or unmodified cleavase e.g., a dipeptidyl peptidase, a dipeptidyl aminopeptidase, a peptidyl- dipeptidase, a dipeptidyl carboxypeptidase, a
  • the modified cleavase is engineered using a rational design approach for select activities, substrate binding capability, or other cleaving
  • a rational design approach is based on crystal structure of the unmodified cleavase. In some examples, the rational design approach is based on crystal structure of the unmodified cleavase with substrates to identify target amino acid residues for modification. In some cases, the modifications may be targeted at residues of specific domains of the unmodified cleavase ( See e.g ., Sakamoto et al., Scientific Reports 2014, 4:4977). In some embodiments, a rational design is used to engineer a modified cleavase with modified amino acids in the substrate binding domain of the unmodified cleavase. In some embodiments, the mutations, e.g.
  • one or more amino acid modifications corresponds to position(s) 316, 391, 394, or a combination thereof, with reference to numbering of SEQ ID NO: 5 or the sequence of a human dipeptidyl peptidase 3 (DPP3) or a homolog thereof.
  • a rational design is used to engineer a modified cleavase that is able to bind or cleave polypeptides of increased length compared to an unmodified cleavase.
  • the mutations e.g, one or more amino acid modifications (e.g,
  • substitutions, additions, deletions corresponds to position(s) amino acid residues 419, 420, 421, 422, 423, 424, 425, 426, or a combination thereof, with reference to numbering of SEQ ID NO:
  • a rational design is used to engineer a modified cleavase with modified amino acids in the hinge region of the unmodified cleavase.
  • the genetic selection or other engineering methods are designed to identify modified cleavases that are active on labeled polypeptides (e.g. chemically labeled polypeptides). In some embodiments, the genetic selection or other engineering methods are designed to identify modified cleavases that are active on modified or labeled polypeptides having a labeled N-terminal amino acid. In some cases, the size or other characteristics of the moiety or label on the labeled polypeptide is considered in the design of the genetic selection or other engineering methods to obtain a desired modified cleavase.
  • labeled polypeptides e.g. chemically labeled polypeptides
  • the genetic selection or other engineering methods are designed to identify modified cleavases that are active on modified or labeled polypeptides having a labeled N-terminal amino acid. In some cases, the size or other characteristics of the moiety or label on the labeled polypeptide is considered in the design of the genetic selection or other engineering methods to obtain a desired modified cleavase.
  • polypeptides and the description of domains thereof are theoretically derived based on homology analysis and alignments with similar molecules. Thus, the exact locus can vary, and is not necessarily the same for each protein.
  • the specific domain such as specific binding domain, loop domain, or other functional domain
  • amino acids for modification in a wildtype dipeptide cleavase can be chosen using analysis of crystal structure of the wild-type cleavase (e.g . wildtype DAP BII) and its substrate to identify contact residues and other residues at the protein interaction interface. This analysis can be performed for example, using Rosetta software suite for macromolecular modeling (Das et al., Annu Rev Biochem (2008) 77:363-382). In some embodiments, using the selected target residues for modification, an alignment of wildtype cleavase sequences of other organisms can be used to identify conserved residues (Crooks et al., Genome Res (2004) 14(6): 1188-1190).
  • conserved target residues or corresponding residues in homologs can be modified.
  • the identified contact residues or other residues of interest are modified to introduce new functions.
  • FIG. 4A-4C a WebLogo analysis of DAP BII homologs with 60% sequence similarity or identity showed sequence conservation across various residues. For example, sequence conservation was observed for residues at the amine binding sites of DAP BII including positions N215, W216, R220, N330, and D674 in reference to the wildtype DAP BII sequence set forth in SEQ ID NO: 20.
  • sequence conservation was observed for residues at the amine binding sites of DAP BII including positions G207, K208, F209, G210, G211, D212, 1213, D214, N215, W216, M217, W218, P219, R220, H221, T222, G223, A224, F225, A226, A326, and/or N334, in reference to the wildtype DAP BII sequence set forth in SEQ ID NO: 20.
  • a rational design approach for engineering DAP BII may be used to target domains or residues such that the resulting modified cleavase removes or is configured to remove a labeled N-terminal amino acid (NTAA) using crystal structures of DAP BII in complex with substrates (Sakamoto et al., Scientific Reports 2014, 4:4977).
  • the DAP BII structure in complex with a peptide substrate at the residues N191, W192, R196, N306, and D650 (based on the sequence of the protein set forth in SEQ ID NO: 13; UniProt Accession No. V5YM14) interacts with the peptide N-terminal amine group.
  • a loop of approximately 20 residues makes contact with the N-terminal residue and penultimate residue of a bound peptide substrate.
  • it may be desired to modify the specificity of the unmodified or wildtype cleavase See e.g ., Sakamoto et al., Scientific Reports 2014, 4:4977).
  • residues in the SI subsite or pocket of DAP BII can be targeted to engineer a modified cleavase with preferred specificity (e.g., reduced specificity for a specific amino acid residue at the PI position of the polypeptide treated with the modified cleavase).
  • the modified cleavase comprises mutations, e.g, one or more amino acid modifications (e.g, substitutions, additions, deletions) corresponding to position(s) D627, 1628, G630, A648, G651, S655, M669, or a combination thereof, with reference to numbering of SEQ ID NO: 13.
  • a modified cleavase variant can be identified using a genetic screen.
  • the genetic screen uses a cell-based system.
  • the genetic screen uses prokaryotic cells, such as E. coli strains including E. coli variants or mutants.
  • the genetic screen uses eukaryotic cells, such as yeast two-hydrid systems.
  • the genetic selection is designed to select for modified cleavases with desired characteristics for binding of substrates, cleaving, and/or removal of labeled terminal amino acids.
  • carrying out a genetic selection screen involves preparing various cleavase genes (e.g, a dipeptidyl peptidase, a dipeptidyl aminopeptidase, a peptidyl- dipeptidase, a dipeptidyl carboxypeptidase, a sedolisin, or a tripeptidyl peptidase) for expression.
  • various cleavase genes e.g, a dipeptidyl peptidase, a dipeptidyl aminopeptidase, a peptidyl- dipeptidase, a dipeptidyl carboxypeptidase, a sedolisin, or a tripeptidyl peptidase
  • a plasmid or cosmid containing nucleic acid sequences encoding mutated or modified cleavase polypeptides is readily constructed using standard techniques well known in the art.
  • the expression of any of the cleavases may further include a signal sequence.
  • a signal sequence may be useful for purification purposes.
  • a periplasm targeting sequence such as PelB can be included in the expression construct.
  • Recombinant vectors can be generated using any of the recombinant techniques known in the art.
  • the vectors can include a prokaryotic origin of replication and/or a gene whose expression confers a detectable or selectable marker for propagation and/or selection in prokaryotic systems.
  • the DNA constructs may be introduced into an appropriate host.
  • prokaryotic hosts can be used including bacteria such as E. coli., Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc.
  • Various techniques may be employed, such as protoplast fusion, calcium phosphate precipitation, electroporation or other conventional techniques. After the fusion, the cells are grown in media and screened for appropriate activities.
  • libraries of mutated cleavase genes can be generated by error prone PCR or rational mutagenesis using the crystal structure of the cleavase as a guide, or a combination thereof. Other suitable methods for generating mutations or generating a library may also be used.
  • a library of mutated cleavase genes can be subsequently cloned into a vector and transformed into an E. coli auxotroph strain (available from CSSC E. coli Genetic Stock Center at Yale - https://cgsc2.biology.yale.edu/).
  • the screen involves isolating colonies growing on the selection media and extracting and analyzing plasmid DNA to identify modified cleavase polypeptides that remove a labeled terminal amino acid or labeled terminal dipeptide from a polypeptide.
  • a screen can be performed to identify and isolate a modified cleavase that cleaves or is configured to cleave a polypeptide with a labeled amino acid (e.g ., a PITC-labeled NTAA or a Cbz-labeled NTAA).
  • the genetic screen is aimed at selecting for the binding of the label but not for a specific amino acid, therefore, the screen uses polypeptides with various labeled terminal amino acids.
  • selecting a modified cleavase further includes purifying, characterizing, assessing and/or optimizing of the activity of the modified cleavase.
  • the modified cleavase may be isolated and purified in accordance with conventional methods, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like.
  • a genetics screen or other selection methods can also be used to select for and obtain modified cleavases with an altered active site of the cleavase or with altered binding pockets of the cleavase.
  • genetics screen or selection methods can also be used to select for and obtain modified cleavases with an altered hinge region of the cleavase.
  • genetic screen or selection methods can also be used to select for and obtain modified cleavases with an altered binding cleft of the cleavase.
  • genetic screen or selection methods can also be used to select for and obtain modified cleavases with an altered inter-lobe cleft of the cleavase.
  • genetic screen or selection methods can also be used to select for and obtain modified cleavases with an altered alpha amine binding region of the cleavase.
  • the modified cleavase exhibits reduced alpha amine binding compared to the wild-type cleavase polypeptide.
  • a genetic screen or other selection methods can also be used to select for and obtain modified cleavases configured to remove a labeled terminal amino acid from polypeptides of various lengths.
  • porin size in the E. coli outer membrane limits the peptide length that can be uptaken. In some embodiments, this length limitation is overcome by briefly treating E. coli with Tris-EDTA or the small molecule
  • the modified cleavase is capable of cleaving or is configured to remove amino acids from polypeptides that are greater than 5 amino acids in length, greater than 6 amino acids in length, greater than 7 amino acids in length, greater than 8 amino acids in length, greater than 9 amino acids in length, greater than 10 amino acids in length, greater than 15 amino acids in length, greater than 20 amino acids in length, greater than 25 amino acids in length, or greater than 30 amino acids in length.
  • the modified dipeptidyl peptidase is capable of cleaving or is configured to remove amino acids from polypeptides that are less than 30 amino acids in length, less than 40 amino acids in length, less than 50 amino acids in length, less than 75 amino acids in length, less than 100 amino acids in length, less than 200 amino acids in length, less than 300 amino acids in length, less than 400 amino acids in length, less than 500 amino acids in length, less than 600 amino acids in length, less than 700 amino acids in length, less than 800 amino acids in length, less than 900 amino acids in length, or less than 1000 amino acids in length.
  • the modified cleavase is capable of cleaving or is configured to remove amino acids from polypeptides that are between 5 to 100 amino acids in length, between 10 to 100 amino acids in length, between 20 to 100 amino acids in length, between 30 to 100 amino acids in length, between 5 to 50 amino acids in length, between 10 to 50 amino acids in length, between 20 to 50 amino acids in length, between 30 to 50 amino acids in length, between 5 to 30 amino acids in length, between 10 to 30 amino acids in length, between 20 to 30 amino acids in length, between 10 to 20 amino acids in length.
  • the modified cleavase is capable of cleaving or is configured to remove amino acids from polypeptides that are between 50 to 1000 amino acids in length, between 100 to 1000 amino acids in length, between 300 to 1000 amino acids in length, between 500 to 1000 amino acids in length, between 10 to 500 amino acids in length, between 50 to 500 amino acids in length, between 100 to 500 amino acids in length, or between 200 to 500 amino acids in length.
  • the modified cleavase is capable or configured to remove amino acids from partial or digested proteins and polypeptides ( e.g ., protein or polypeptide fragments).
  • the modified cleavase is capable or configured to remove amino acids from whole or undigested proteins and polypeptides.
  • the modified cleavase removes the single terminal amino acid or terminal dipeptide by contacting the polypeptide with a modified cleavase for less than 5 minutes, less than 10 minutes, less than 20 minutes, less than 30 minutes, less than 40 minutes, less than 50 minutes, less than 60 minutes, less than 2 hours, less than 5 hours, less than 8 hours, or less than 10 hours.
  • the modified cleavase achieves a yield of polypeptides with the terminal amino acid removed of >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95%, >99% or more by treating the polypeptide with the modified cleavase for about less than 15 minutes. In some embodiments, the modified cleavase achieves a yield of polypeptides with the terminal amino acid removed of >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95%, >99% or more by treating the polypeptide with the modified cleavase for about less than 30 minutes.
  • the modified cleavase achieves a yield of polypeptides with the terminal amino acid removed of >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95%, >99% or more by treating the polypeptide with the modified cleavase for about less than 45 minutes.
  • the modified cleavase achieves a yield of polypeptides with the terminal amino acid removed of >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95%, >99% or more by treating the polypeptide with the modified cleavase for about less than 1 hour. In some embodiments, the modified cleavase achieves a yield of polypeptides with the terminal amino acid removed of >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95%, >99% or more by treating the polypeptide with the modified cleavase for about less than 2 hours.
  • the modified cleavase achieves a yield of polypeptides with the terminal amino acid removed of >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95%, >99% or more by treating the polypeptide with the modified cleavase for about less than 5 hours.
  • the modified dipeptide or tripeptide cleavase is capable of cleaving terminal amino acids or functions at a temperature of higher than about 10° C, higher than about 20° C higher than about 30° C , or higher than about 40° C.
  • the modified dipeptide or tripeptide cleavase is capable of cleaving terminal amino acids or functions at a temperature of about 10° C to 20° C, about 10° C to 30° C, about 10° C to 40° C, about 10° C to 50° C, about 10° C to 60° C, about 10° C to 70° C, about 10° C to 80° C, about 10° C to 90° C or about 10° C to 100° C; about 20° C to 30° C, about 20° C to 40° C, about 20° C to 50° C, about 20° C to 60° C, about 20° C to 70° C, about 20° C to 80° C, about 20° C to 90° C, or about 20° C to 100° C; about 30° C to 40° C, about 30° C to 50° C, about 30° C to 60° C; about 50° C to 70° C, about 50° C to 80° C, about 50° C to 90° C, or about 50° C to 100° C.
  • the modified cleavase is capable of cleaving terminal amino acids at a temperature at which the secondary structure of the polypeptide is disrupted. In some embodiments, the modified cleavase functions at about 20 to 25° C. In some embodiments, the method includes contacting the modified cleavase with the polypeptide while applying heating. In some embodiments, the heating is achieved by applying microwave energy. In some embodiments of any of the methods provided herein, the contacting of the modified cleavase with the polypeptide to remove a terminal amino acid is performed in the presence of microwave energy.
  • the DNA molecules and recombinant expression vectors are isolated from the genetic engineering and selection methods described.
  • a host cell comprising the DNA molecule is also provided.
  • a method of producing a modified or variant cleavase comprising introducing the nucleic acid molecule according to any one of the embodiments described herein or vector according to any one of the embodiments described herein into a host cell under conditions to express the protein in the cell. Also provided herein are methods for producing any of the modified cleavases provided herein including: cultivating a transformed host cell under conditions suitable for expression of the modified cleavase, and separating, purifying and/or recovering the mutant organism expressing the modified cleavase.
  • a host cell comprising a DNA molecule encoding a modified cleavase.
  • the host cell comprises a recombinant expression vector for expressing a modified cleavase.
  • the method further includes isolating or purifying the variant or modified cleavase from the cell.
  • an engineered cell expressing the variant or modified cleavase polypeptide according to any one of the embodiments described herein or the nucleic acid molecule encoding a variant or modified cleavase described herein, or the vector according to any one of the embodiments described herein.
  • the variant or modified cleavase polypeptide contains a signal peptide.
  • the present disclosure relates to the treatment of polypeptides with any of the modified cleavases provided herein.
  • the labeled terminal amino acid is removed from a polypeptide (including a partial or fragmented polypeptide).
  • the terminal amino acid is removed from a polypeptide that has a length of greater than 4 amino acids, greater than 5 amino acids, greater than 6 amino acids, greater than 7 amino acids, greater than 8 amino acids, greater than 9 amino acids, greater than 10 amino acids, greater than 11 amino acids, greater than 12 amino acids, greater than 13 amino acids, greater than 14 amino acids, greater than 15 amino acids, greater than 20 amino acids, greater than 25 amino acids, or greater than 30 amino acids.
  • the length of the polypeptide is greater than 10 amino acids.
  • the terminal amino acid is removed from a polypeptide that has a length of less than 30 amino acids, less than 40 amino acids, less than 50 amino acids, less than 75 amino acids, less than 100 amino acids, less than 200 amino acids, less than 300 amino acids, less than 400 amino acids, less than 500 amino acids, less than 600 amino acids, less than 700 amino acids, less than 800 amino acids, less than 900 amino acids, or less than 1000 amino acids.
  • the terminal amino acid is removed from a polypeptide that has a length of between 5 to 100 amino acids, between 10 to 100 amino acids, between 20 to 100 amino acids, between 30 to 100 amino acids, between 5 to 50 amino acids, between 10 to 50 amino acids, between 20 to 50 amino acids, between 30 to 50 amino acids, between 5 to 30 amino acids, between 10 to 30 amino acids, between 20 to 30 amino acids, between 10 to 20 amino acids.
  • the terminal amino acid is removed from a polypeptide that has a length of between 50 to 1000 amino acids, between 100 to 1000 amino acids, between 300 to 1000 amino acids, between 500 to 1000 amino acids, between 10 to 500 amino acids, between 50 to 500 amino acids, between 100 to 500 amino acids, or between 200 to 500 amino acids.
  • the terminal amino acid is removed from a partial or digested protein and polypeptide (e.g ., a polypeptide fragment). In some embodiments, the terminal amino acid is removed from a whole or undigested protein and polypeptide.
  • a polypeptide treated with the modified cleavases provided herein and according the methods disclosed herein may be obtained from a suitable source or sample, including but not limited to: biological samples, such as cells (both primary cells and cultured cell lines), cell lysates or extracts, cell organelles or vesicles, including exosomes, tissues and tissue extracts; biopsy; fecal matter; bodily fluids (such as blood, whole blood, serum, plasma, urine, lymph, bile, cerebrospinal fluid, interstitial fluid, aqueous or vitreous humor, colostrum, sputum, amniotic fluid, saliva, anal and vaginal secretions, perspiration and semen, a transudate, an exudate (e.g., fluid obtained from an abscess or any other site of infection or inflammation) or fluid obtained from a joint (normal joint or a joint affected by disease such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis) of
  • the polypeptide is a protein or a protein complex.
  • a polypeptide may comprise L-amino acids, D-amino acids, or both.
  • a polypeptide may comprise a standard, naturally occurring amino acid, a modified amino acid (e.g, post-translational modification), an amino acid analog, an amino acid mimetic, or any combination thereof.
  • the polypeptide is naturally occurring, synthetically produced, or recombinantly expressed.
  • the polypeptide may further comprise a post-translational modification.
  • Standard, naturally occurring 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 lie), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q or Gin), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Val), Tryptophan (W or Trp), and Tyrosine (Y or Tyr).
  • Non-standard amino acids include selenocysteine,
  • pyrrolysine and N-formylmethionine
  • b-amino acids Homo-amino acids
  • Proline and Pyruvic acid derivatives 3-substituted Alanine derivatives
  • Glycine derivatives Ring-substituted Phenylalanine and Tyrosine Derivatives
  • Linear core amino acids and N-methyl amino acids.
  • a post-translational modification (PTM) of a polypeptide may be a covalent modification or enzymatic modification.
  • post-translation modifications include, but are not limited to, acylation, acetylation, alkylation (including methylation), biotinylation, butyrylation, carbamylation, carbonylation, deamidation, deiminiation, diphthamide formation, disulfide bridge formation, eliminylation, flavin attachment, formylation, gamma-carboxylation, glutamyl ati on, glycylation, glycosylation (e.g., N-linked, O-linked, C-linked,
  • phosphoglycosylation glypiation, heme C attachment, hydroxylation, hypusine formation, iodination, isoprenyl ati on, lipidation, lipoylation, malonylation, methylation, myristolylation, oxidation, palmitoylation, pegylation, phosphopantetheinylation, phosphorylation, prenylation, propionylation, retinylidene Schiff base formation, S-glutathionylation, S-nitrosylation, S- sulfenylation, selenation, succinylation, sulfmation, ubiquitination, and C-terminal amidation.
  • a post-translational modification includes modifications of the amino terminus and/or the carboxyl terminus of a peptide, polypeptide, or protein.
  • Modifications of the terminal amino group include, but are not limited to, des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications.
  • Modifications of the terminal carboxy group include, but are not limited to, amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications (e.g., wherein lower alkyl is C1-C4 alkyl).
  • a post-translational modification also includes modifications, such as but not limited to those described above, of amino acids falling between the amino and carboxy termini of a peptide, polypeptide, or protein.
  • Post-translational modification can regulate a protein’s“biology” within a cell, e.g., its activity, structure, stability, or localization.
  • Phosphorylation is the most common post-translational modification and plays an important role in regulation of protein, particularly in cell signaling (Prabakaran et ah, (2012) Wiley Interdiscip Rev Syst Biol Med 4: 565-583).
  • the addition of sugars to proteins, such as glycosylation has been shown to promote protein folding, improve stability, and modify regulatory function.
  • the polypeptide can be fragmented.
  • the fragmented polypeptide can be obtained by fragmenting a polypeptide, protein or protein complex from a sample, such as a biological sample.
  • the polypeptide, protein or protein complex can be fragmented by any means known in the art, including fragmentation by a protease or endopeptidase.
  • fragmentation of a polypeptide, protein or protein complex is targeted by use of a specific protease or endopeptidase.
  • a specific protease or endopeptidase binds and cleaves at a specific consensus sequence (e.g ., TEV protease which is specific for ENLYFQ ⁇ S consensus sequence).
  • fragmentation of a peptide, polypeptide, or protein is non-targeted or random by use of a non-specific protease or endopeptidase.
  • a non-specific protease may bind and cleave at a specific amino acid residue rather than a consensus sequence (e.g., proteinase K is a non-specific serine protease).
  • Proteinases and endopeptidases are well known in the art, and examples of such that can be used to cleave a protein or polypeptide into smaller peptide fragments include proteinase K, trypsin, chymotrypsin, pepsin, thermolysin, thrombin, Factor Xa, furin, endopeptidase, papain, pepsin, subtilisin, elastase, enterokinase, GenenaseTM I, Endoproteinase LysC, Endoproteinase AspN, Endoproteinase GluC, etc. (Granvogl et ak, (2007) Anal Bioanal Chem 389: 991-1002).
  • a peptide, polypeptide, or protein is fragmented by proteinase K, or optionally, a thermolabile version of proteinase K to enable rapid inactivation.
  • Proteinase K is quite stable in denaturing reagents, such as urea and SDS, enabling digestion of completely denatured proteins.
  • the polypeptide is contacted with one or more enzymes in addition to a modified cleavase to eliminate the NTAA (e.g, a proline aminopeptidase to remove an N-terminal proline, if present).
  • the additional enzyme eliminates an NTAA from the polypeptide that is a proline.
  • the enzyme is a proline aminopeptidase, a proline iminopeptidase (PIP), or a pyroglutamate aminopeptidase (pGAP).
  • PIP proline iminopeptidase
  • pGAP pyroglutamate aminopeptidase
  • one or more modified cleavases are used in combination with other enzymes to treat the polypeptides.
  • the polypeptide is first contacted with a proline aminopeptidase under conditions suitable to remove an N-terminal proline, if present.
  • Chemical reagents can also be used to digest proteins into peptide fragments.
  • a chemical reagent may cleave at a specific amino acid residue (e.g., cyanogen bromide hydrolyzes peptide bonds at the C-terminus of methionine residues).
  • Chemical reagents for fragmenting polypeptides or proteins into smaller peptides include cyanogen bromide (CNBr), hydroxylamine, hydrazine, formic acid, BNPS-skatole [2-(2-nitrophenylsulfenyl)-3- methylindole], iodosobenzoic acid, vNTCB +Ni (2-nitro-5-thiocyanobenzoic acid), etc.
  • some polypeptides can be treated with a reagent for enzymatic or chemical elimination.
  • the resulting polypeptide fragments are approximately the same desired length, e.g ., from about 10 amino acids to about 70 amino acids, from about 10 amino acids to about 60 amino acids, from about 10 amino acids to about 50 amino acids, about 10 to about 40 amino acids, from about 10 to about 30 amino acids, from about 20 amino acids to about 70 amino acids, from about 20 amino acids to about 60 amino acids, from about 20 amino acids to about 50 amino acids, about 20 to about 40 amino acids, from about 20 to about 30 amino acids, from about 30 amino acids to about 70 amino acids, from about 30 amino acids to about 60 amino acids, from about 30 amino acids to about 50 amino acids, or from about 30 amino acids to about 40 amino acids.
  • a elimination reaction may be monitored, preferably in real time, by spiking the protein or polypeptide sample with a short test FRET (fluorescence resonance energy transfer) polypeptide comprising a peptide sequence containing a proteinase or endopeptidase elimination site.
  • FRET fluorescence resonance energy transfer
  • a fluorescent group and a quencher group are attached to either end of the peptide sequence containing the elimination site, and fluorescence resonance energy transfer between the quencher and the fluorophore leads to low fluorescence.
  • the quencher and fluorophore are separated giving a large increase in fluorescence.
  • An elimination reaction can be stopped when a certain fluorescence intensity is achieved, allowing a reproducible elimination end point to be achieved.
  • polypeptides of the present disclosure are joined to a surface of a solid support (also referred to as“substrate surface”).
  • the polypeptides are joined to a solid support prior to contacting with the modified cleavase.
  • the modified cleavase removes a labelled terminal amino acid from a polypeptide that is join (directly or indirectly) to a solid support.
  • the labelled terminal amino acid is removed as a single amino acid or as part of a dipeptide.
  • the solid support can be any porous or non-porous support surface including, but not limited to, a bead, a microbead, an array, a glass surface, a silicon surface, a plastic surface, a filter, a membrane, a PTFE membrane, a PTFE membrane, a nitrocellulose membrane, a nitrocellulose-based polymer surface, nylon, a silicon wafer chip, a flow cell, a flow through chip, a biochip including signal transducing electronics, a microtiter well, an ELISA plate, a spinning interferometry disc, a nitrocellulose membrane, a nitrocellulose-based polymer surface, a nanoparticle, or a microsphere.
  • Materials for a solid support include but are not limited to acrylamide, agarose, cellulose, dextran, nitrocellulose, glass, gold, quartz, polystyrene, polyethylene vinyl acetate, polypropylene, polyester, polymethacrylate, polyacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, poly vinyl alcohol (PVA), Teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid,
  • Solid supports further include thin film, membrane, bottles, dishes, fibers, woven fibers, shaped polymers such as tubes, particles, beads, microparticles, or any combination thereof.
  • the bead can include, but is not limited to, a polystyrene bead, a polymer bead, a polyacrylate bead, a methylstyrene bead, an agarose bead, a cellulose bead, a dextran bead, an acrylamide bead, a solid core bead, a porous bead, a paramagnetic bead, glass bead, a controlled pore bead, a silica-based bead, or any combinations thereof.
  • a solid support is a bead, which may refer to an individual bead or a plurality of beads.
  • the bead is compatible with a selected next generation sequencing platform that will be used for downstream analysis (e.g ., SOLiD or 454).
  • a solid support is an agarose bead, a paramagnetic bead, a polystyrene bead, a polymer bead, an acrylamide bead, a solid core bead, a porous bead, a glass bead, or a controlled pore bead.
  • a bead may be coated with a binding functionality (e.g., amine group, affinity ligand such as streptavidin for binding to biotin labeled polypeptide, antibody) to facilitate binding to a polypeptide.
  • a binding functionality e.g., amine group, affinity ligand such as streptavidin for binding to biotin labeled polypeptide, antibody
  • Proteins, polypeptides, or peptides can be joined to the solid support, directly or indirectly, by any means known in the art, including covalent and non-covalent interactions, or any combination thereof (see, e.g., Chan et al., 2007, PLoS One 2:el 164; Cazalis et al., Bioconj. Chem. 15: 1005-1009; Soellner et al., 2003, J. Am. Chem. Soc. 125: 11790-11791; Sun et al., 2006, Bioconjug. Chem. 17-52-57; Decreau et al., 2007, J. Org. Chem.
  • the peptide may be joined to the solid support by a ligation reaction.
  • the solid support can include an agent or coating to facilitate joining, either direct or indirectly, the peptide to the solid support.
  • Any suitable molecule or materials may be employed for this purpose, including proteins, nucleic acids, carbohydrates and small molecules.
  • the agent is an affinity molecule.
  • the agent is an azide group, which group can react with an alkynyl group in another molecule to facilitate association or binding between the solid support and the other molecule.
  • Proteins, polypeptides, or peptides can be joined to the solid support using methods referred to as“click chemistry.” For this purpose, any reaction which is rapid and substantially irreversible can be used to attach proteins, polypeptides, or peptides to the solid support.
  • Exemplary reactions include the copper catalyzed reaction of an azide and alkyne to form a triazole (Huisgen 1, 3 -dipolar cycloaddition), strain-promoted azide alkyne cycloaddition (SPAAC), reaction of a diene and dienophile (Diels-Alder), strain-promoted alkyne-nitrone cycloaddition, reaction of a strained alkene with an azide, tetrazine or tetrazole, alkene and azide [3+2] cycloaddition, alkene and tetrazine inverse electron demand Diels-Alder (IEDDA) reaction ( e.g ., m-tetrazine (mTet) or phenyl tetrazine (pTet) and trans-cyclooctene (TCO); or pTet and an alkene), alkene and te
  • Exemplary displacement reactions include reaction of an amine with: an activated ester; an N-hydroxysuccinimide ester; an isocyanate; an isothioscyanate, an aldehyde, an epoxide, or the like.
  • the polypeptide and solid support are joined by a functional group capable of formation by reaction of two complementary reactive groups, for example a functional group which is the product of one of the foregoing“click” reactions.
  • functional group can be formed by reaction of an aldehyde, oxime, hydrazide, alkyne, amine, azide, acylazide, acylhalide, nitrile, nitrone, sulfhydryl, disulfide, sulfonyl halide, isothiocyanate, imidoester, activated ester ( e.g ., N-hydroxysuccinimide ester, pentynoic acid STP ester), ketone, a,b-unsaturated carbonyl, alkene, maleimide, a-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biot
  • the functional group comprises an alkene, ester, amide, thioester, disulfide, carbocyclic, heterocyclic or heteroaryl group. In further embodiments, the functional group comprises an alkene, ester, amide, thioester, thiourea, disulfide, carbocyclic, heterocyclic or heteroaryl group. In other embodiments, the functional group comprises an amide or thiourea. In some more specific embodiments, functional group is a triazolyl functional group, an amide, or thiourea functional group.
  • iEDDA click chemistry is used for immobilizing polypeptides to a solid support since it is rapid and delivers high yields at low input
  • m-tetrazine rather than tetrazine is used in an iEDDA click chemistry reaction, as m-tetrazine has improved bond stability.
  • phenyl tetrazine pTet is used in an iEDDA click chemistry reaction.
  • the substrate surface is functionalized with TCO, and the recording tag-labeled protein, polypeptide, peptide is immobilized to the TCO coated substrate surface via an attached m-tetrazine moiety.
  • polypeptides are immobilized to a surface of a solid support by its C-terminus, N-terminus, or an internal amino acid, for example, via an amine, carboxyl, or sulfydryl group.
  • Standard activated supports used in coupling to amine groups include CNBr-activated, NHS-activated, aldehyde-activated, azlactone-activated, and CDI- activated supports.
  • Standard activated supports used in carboxyl coupling include carbodiimide- activated carboxyl moieties coupling to amine supports. Cysteine coupling can employ maleimide, idoacetyl, and pyridyl disulfide activated supports.
  • An alternative mode of peptide carboxy terminal immobilization uses anhydrotrypsin, a catalytically inert derivative of trypsin that binds peptides containing lysine or arginine residues at their C-termini without cleaving them.
  • a polypeptide is immobilized to a solid support via covalent attachment of a solid surface bound linker to a lysine group of the protein, polypeptide, or peptide.
  • a polypeptide is first labeled with a DNA tag, and the chimeric DNA-polypeptide molecule is immobilized to a solid support via nucleic acid hybridization and ligation to a DNA sequence attached to the solid support.
  • protein and polypeptide fragmentation into peptides can be performed before or after attachment of a DNA tag or DNA recording tag.
  • a sample of polypeptides can undergo protein fractionation methods prior to attachment to a solid support, where proteins or peptides are separated by one or more properties such as cellular location, molecular weight, hydrophobicity, or isoelectric point, or protein enrichment methods.
  • protein enrichment methods may be used to select for a specific protein or peptide (see, e.g., Whiteaker et al., (2007) Anal. Biochem.
  • a particular class or classes of proteins such as immunoglobulins, or immunoglobulin (Ig) isotypes such as IgG, can be affinity enriched or selected for analysis.
  • immunoglobulin molecules analysis of the sequence and abundance or frequency of hypervariable sequences involved in affinity binding are of particular interest, particularly as they vary in response to disease progression or correlate with healthy, immune, and/or disease phenotypes. Overly abundant proteins can also be subtracted from the sample using standard immunoaffmity methods.
  • Depletion of abundant proteins can be useful for plasma samples where over 80% of the protein constituent is albumin and immunoglobulins.
  • Several commercial products are available for depletion of plasma samples of overly abundant proteins, such as PROTIA and PROT20 (Sigma-Aldrich).
  • the methods provided herein may be performed on polypeptides that have been normalized.
  • subtraction of certain protein species e.g., highly abundant proteins from the sample is performed. This can be
  • a protein sample dynamic range can be modulated by fractionating the protein sample using standard fractionation methods, including electrophoresis and liquid chromatography (Zhou et al., Anal Chem (2012) 84(2): 720-734), or partitioning the fractions into compartments (e.g., droplets) loaded with limited capacity protein binding beads/resin (e.g. hydroxylated silica particles) (McCormick, Anal Biochem (1989) 181(1): 66-74) and eluting bound protein. Excess protein in each compartmentalized fraction is washed away.
  • standard fractionation methods including electrophoresis and liquid chromatography (Zhou et al., Anal Chem (2012) 84(2): 720-734), or partitioning the fractions into compartments (e.g., droplets) loaded with limited capacity protein binding beads/resin (e.g. hydroxylated silica particles) (McCormick, Anal Biochem (1989) 181(1): 66-74) and eluting bound
  • electrophoretic methods include capillary electrophoresis (CE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (CITP), free flow
  • electrophoresis gel-eluted liquid fraction entrapment electrophoresis
  • liquid chromatography protein separation methods include reverse phase (RP), ion exchange (IE), size exclusion (SE), hydrophilic interaction, etc.
  • compartment partitions include emulsions, droplets, microwells, physically separated regions on a flat substrate, etc.
  • Exemplary protein binding beads/resins include silica nanoparticles derivatized with phenol groups or hydroxyl groups (e.g., StrataClean Resin from Agilent Technologies, RapidClean from LabTech, etc.). By limiting the binding capacity of the beads/resin, highly-abundant proteins eluting in a given fraction will only be partially bound to the beads, and excess proteins removed.
  • the modified cleavase comprises a mutation, e.g, one or more amino acid modifications in an unmodified cleavase, wherein the modified cleavase is derived from a dipeptide cleavase and removes a single labeled terminal amino acid from a polypeptide.
  • the modified cleavase is derived from a tripeptide cleavase and removes a single labeled terminal amino acid from a polypeptide or a single labeled terminal dipeptide from a polypeptide.
  • polypeptides are contacted with any one or more of the modified cleavases as described in Section I.
  • the method further comprises contacting the polypeptide with a reagent for labeling the terminal amino acid.
  • the contacting with the reagent for labeling the terminal amino acid is with any one or more of the reagents described in Section I. A.
  • one or more cycles of contacting the polypeptide with the modified cleavase and contacting with a reagent to label the terminal amino acid is performed, such as in a cyclic manner as depicted in FIG. 2A-2C.
  • the polypeptide is bound to a support.
  • the method includes joining the polypeptides to a solid support ( e.g ., directly or indirectly).
  • the removal of NTAA from a peptide using the provided modified cleavases can be combined with a chemical method for removing the NTAA from a peptide, such as described in PCT publication number WO 2019/089846.
  • the removed labeled terminal amino acid is removed as a single amino acid or as part of a dipeptide.
  • the modified cleavases provided herein can be used for treating polypeptides to be analyzed and/or sequenced.
  • the methods are for determining the sequence of at least a portion of the polypeptide.
  • the provided methods can be used in the context of a degradation-based polypeptide sequencing assay.
  • the method may include performing any of the methods as described in International Patent Publication No. WO 2017/192633.
  • the sequence of the polypeptide is analyzed by construction of an extended recording tag (e.g., DNA sequence) representing the polypeptide sequence, such as an extended recording tag.
  • the methods provided herein apply to or can be used in combination with a ProteoCode assay.
  • the provided modified cleavase provides certain advantages.
  • the recognition and removal of labeled amino acids may provide a pause to amino acid removal as compared to an enzyme which removes unlabeled amino acids, which may continuously remove amino acids from the polypeptide before other steps of the assay can be performed (e.g, binding of the NTAA by a binding agent and recording information of the NTAA to a recording tag).
  • the modified cleavase removes the NTAA only after a labeling step has occurred.
  • the modified cleavase provides control over the removal of amino acids compared to the unmodified cleavase which removes unlabeled amino acids.
  • a method comprising the modified cleavase is conducted in the absence of a condition that degrades nucleic acids (e.g, DNA, such as a recording tag). In some embodiments, the method comprising the modified cleavase is conducted in the absence of a chemical condition that degrades nucleic acids. In some embodiments, the method comprising the modified cleavase is conducted in conditions compatible with a degradation-based polypeptide sequencing assay (e.g, the methods as described in International Patent Publication No. WO 2017/192633). In some cases, the method comprising the modified cleavase is conducted in the presence of conditions compatible with nucleic acids.
  • a degradation-based polypeptide sequencing assay e.g, the methods as described in International Patent Publication No. WO 2017/192633.
  • the method comprising the modified cleavase is conducted in the absence of a strong acid or a strong base.
  • the strong acid is a strong anhydrous acid.
  • the method comprising the modified cleavase is conducted in the absence of anhydrous TFA.
  • the method includes contacting the polypeptide with more than one modified cleavase.
  • various modified cleavases may exhibit different characteristics, for example, binding preferences for polypeptides and/or differences in cleaving amino acids.
  • different modified cleavases may be used in any of the described methods, as a mixture of enzymes or each separately.
  • the different modified cleavases are contacted with polypeptides simultaneously or sequentially.
  • the polypeptide is contacted with one or more additional enzymes to eliminate the NTAA (e.g ., a proline aminopeptidase to remove an N-terminal proline, if present).
  • the methods of the invention may include optionally treating the polypeptides with an enzyme to remove one or more NTAAs (e.g., proline aminopeptidase) before, during, or after treatment with any of the provided chemical reagents for labeling the NTAA.
  • the methods of the invention may include optionally treating the polypeptides with an enzyme to remove one or more NTAAs (e.g., proline aminopeptidase) before, during, or after treatment with any of the provided modified cleavases.
  • the enzyme eliminates an NTAA from the polypeptide that is a proline.
  • the enzyme is a proline aminopeptidase, a proline iminopeptidase (PIP), or a pyroglutamate aminopeptidase (pGAP).
  • PIP proline iminopeptidase
  • pGAP pyroglutamate aminopeptidase
  • one or more modified cleavases are used in combination with other enzymes to treat the polypeptides.
  • the modified cleavase and/or other enzymes are provided as a cocktail.
  • the method further comprises contacting the polypeptide with a one or more binding agents capable of binding to the terminal amino acid of the polypeptide, wherein each binding agent comprises a coding tag with identifying information regarding the binding agent.
  • the binding agent may bind to a labeled terminal amino acid of the polypeptide.
  • the method further comprises transferring the identifying information of the coding tag to a recording tag attached to the polypeptide, thereby generating an extended recording tag on the polypeptide.
  • the method further comprises removing or releasing the one or more binding agents from the polypeptide.
  • one or more steps of contacting the polypeptide with various reagents is repeated in a cyclic manner.
  • a method for analyzing a polypeptide comprising the steps of: (a) contacting a polypeptide with a binding agent capable of binding to the terminal amino acid of the polypeptide, wherein each binding agent comprises a coding tag with identifying information regarding the binding agent; (b) transferring the identifying information of the coding tag to a recording tag associated with each of the polypeptides to generate an extended recording tag; (c) contacting the polypeptide with a reagent to label the terminal amino acid of the polypeptide; and (d) contacting the polypeptide with a modified cleavase comprising a mutation, e.g ., one or more amino acid modifications in an unmodified cleavase, wherein the modified cleavase removes a single terminal amino acid labeled by the reagent in step (c) from the polypeptide.
  • the removed terminal amino acid may be removed as a single amino acid residue or as part of a dipeptide.
  • steps (a)-(d) are repeated for“n” binding cycles, wherein the information of each coding tag of each binding agent that binds to the polypeptide is transferred to the extended recording tag generated from the previous binding cycle to generate an nth order extended recording tag.
  • the method further comprises (bl) removing or releasing the one or more binding agents from the plurality of polypeptides.
  • the polypeptides includes a plurality of polypeptides.
  • the polypeptide is contacted with a plurality of binding agents.
  • the polypeptide is contacted with two or more binding agents.
  • step (a) is performed before step (b); step (a) is performed before step (c); step (a) is performed before step (d); step (b) is performed before step (c); step (b) is performed before step (d); step (c) is performed before step (a); step (c) is performed before step (b); and/or step (c) is performed before step (d).
  • the steps are performed in the order: (a), (b), (c), and (d).
  • the steps are performed in the order: (c), (a), (b), and (d).
  • the method further comprises (e) analyzing the nth order extended recording tag.
  • the method further comprises removing the one or more binding agents.
  • step (bl) is performed after step (a); step (bl) is performed after step (b); step (bl) is performed before step (c); and/or step (bl) is performed before step (d).
  • the treatment and analysis of the polypeptides is as follows: a large collection of polypeptides (e.g ., 50 million - 1 billion or more) from a proteolytic digest are immobilized randomly on a single molecule sequencing substrate (e.g., beads) at an appropriate intramolecular spacing. In some cases, the polypeptides are attached to recording tags.
  • the terminal amino acid (e.g, N-terminal amino acid) of each peptide is labeled (e.g, PTC, modified-PTC, Cbz, DNP, SNP, acetyl, guanidinyl, amino guanidinyl, heterocyclic methanimine).
  • labeling of the terminal amino acid can be performed as a later step.
  • the labeled N-terminal amino acid (e.g, PITC-NTAA, Cbz- NTAA, DNP-NTAA, SNP-NTAA, acetyl-NTAA, guanidinylated-NTAA, heterocyclic methanimine-NTAA) of each immobilized peptide is bound by the cognate NTAA binding agent which is attached to a coding tag, and information from the coding tag associated with the bound NTAA binding agent is transferred to the recording tag associated with the immobilized peptide, thereby generating an extended recording tag.
  • the one or more bindings agents is removed or released from the polypeptides.
  • the labeled NTAA is removed by contacting with a modified cleavase which is capable of removing a single amino acid that is labeled from the polypeptide.
  • a modified cleavase which is capable of removing a single amino acid that is labeled from the polypeptide.
  • One or more cycles of the labeling, contacting with the binding agent, transferring identifying information, and removal of the single amino acid can be performed.
  • the final extended recording tag is optionally flanked by universal priming sites to facilitate downstream amplification and/or DNA sequencing.
  • the forward universal priming site e.g, Illumina’s P5-S1 sequence
  • the reverse universal priming site e.g, Illumina’s P7-S2’ sequence
  • the addition of forward and reverse priming sites can be done independently of a binding agent.
  • the order of the steps in the process for a degradation- based peptide or polypeptide sequencing assay can be reversed or be performed in various orders.
  • the terminal amino acid labeling can be conducted before and/or after the polypeptide is bound to the binding agent.
  • contacting with the one or more binding agents is before contacting the polypeptide with the reagent for labeling the terminal amino acid.
  • contacting with the one or more binding agents is before contacting the polypeptide with the modified cleavase to remove the labeled terminal amino acid.
  • the terminal amino acid labeling can be conducted before or after the polypeptide is bound to a support.
  • the terminal amino acid removal can be conducted before and/or after the polypeptide is bound to the binding agent.
  • the contacting of the polypeptides with the reagent for labeling the terminal amino acid is before the contacting with the binding agent and the contacting with the one or more binding agents is before the contacting of the polypeptides with the modified cleavase.
  • transferring of the identifying information is performed after the contacting of the polypeptide with the one or more binding agents and before the contacting of the polypeptide with the modified cleavase.
  • removing the one or more binding agents is after the transferring of identifying information from the coding tag to a recording tag associated with each of the polypeptides to generate an extended recording tag. In some of any such embodiments, removing the one or more binding agents is before contacting the polypeptides with a reagent to label the terminal amino acid of the polypeptide. In some embodiments, removing the one or more binding agents is before contacting the polypeptide with a modified cleavase.
  • any of the steps of the provided methods for treating the proteins or polypeptides can be reversed or be performed in various orders.
  • the methods provided comprise contacting polypeptides with the modified cleavase and optionally other reagents for polypeptide analysis.
  • the protein or polypeptide is labeled with DNA recording tags through standard amine coupling chemistries.
  • the e-amino group (e.g. , of lysine residues) and the N-terminal amino group are particularly susceptible to labeling with amine-reactive coupling agents, depending on the pH of the reaction (Mendoza et al., Mass Spectrom Rev (2009) 28(5): 785- 815).
  • the recording tag is comprised of a reactive moiety (e.g., for conjugation to a solid surface, a multifunctional linker, or a polypeptide), a linker, a universal priming sequence, a barcode (e.g., compartment tag, partition barcode, sample barcode, fraction barcode, or any combination thereof), an optional UMI, and a spacer (Sp) sequence for facilitating information transfer to/from a coding tag.
  • a reactive moiety e.g., for conjugation to a solid surface, a multifunctional linker, or a polypeptide
  • a linker e.g., a universal priming sequence
  • a barcode e.g., compartment tag, partition barcode, sample barcode, fraction barcode, or any combination thereof
  • an optional UMI e.g., an optional UMI
  • a spacer (Sp) sequence for facilitating information transfer to/from a coding tag.
  • the Sp sequence can serve as an overhang of 1-8 bases.
  • the protein can be first labeled with a universal DNA tag, and the barcode-Sp sequence (representing a sample, a compartment, a physical location on a slide, etc.) are attached to the protein later through and enzymatic or chemical coupling step.
  • a universal DNA tag comprises a short sequence of nucleotides that are used to label a polypeptide and can be used as point of attachment for a barcode ( e.g ., compartment tag, recording tag, etc.).
  • a recording tag may comprise at its terminus a sequence complementary to the universal DNA tag.
  • a universal DNA tag is a universal priming sequence.
  • the annealed universal DNA tag may be extended via primer extension, transferring the recording tag information to the DNA tagged protein.
  • the protein is labeled with a universal DNA tag prior to proteinase digestion into peptides.
  • the universal DNA tags on the labeled peptides from the digest can then be converted into an informative and effective recording tag.
  • protein and polypeptide fragmentation into peptides can be performed before or after attachment of a DNA tag or DNA recording tag.
  • At least one recording tag is associated or co-localized directly or indirectly with the polypeptide and joined to the solid support.
  • a recording tag may comprise DNA, RNA, or polynucleotide analogs including PNA, gPNA, GNA, HNA, BNA, XNA, TNA, or a
  • a recording tag may be single stranded, or partially or completely double stranded.
  • a recording tag may have a blunt end or overhanging end.
  • identifying information of the binding agent’s coding tag is transferred to the recording tag to generate an extended recording tag. Further extensions to the extended recording tag can be made in subsequent binding cycles.
  • a recording tag can be joined to the solid support, directly or indirectly (e.g., via a linker), by any means known in the art, including covalent and non-covalent interactions, or any combination thereof.
  • the recording tag may be joined to the solid support by a ligation reaction.
  • the solid support can include an agent or coating to facilitate joining, either direct or indirectly, of the recording tag, to the solid support.
  • the co-localization of a polypeptide and associated recording tag is achieved by conjugating polypeptide and recording tag to a bifunctional linker attached directly to the solid support surface (Steinberg et al. (2004) Biopolymers 73:597-605).
  • a trifunctional moiety is used to derivatize the solid support (e.g ., beads), and the resulting bifunctional moiety is coupled to both the polypeptide and recording tag.
  • the co-localization of a polypeptide and associated recording tag is achieved by coupling the polypeptide to the associated DNA recording tag and ligating the chimera to a DNA decorated solid support surface.
  • Methods and reagents e.g., click chemistry reagents and photoaffmity labelling reagents
  • click chemistry reagents and photoaffmity labelling reagents such as those described for attachment of polypeptides and solid supports, may also be used for attachment of recording tags.
  • a single recording tag is attached to a polypeptide, preferably via the attachment to a de-blocked N- or C-terminal amino acid.
  • multiple recording tags are attached to the polypeptide, preferably to the lysine residues or peptide backbone.
  • a polypeptide labeled with multiple recording tags is fragmented or digested into smaller peptides, with each peptide labeled on average with one recording tag.
  • a polypeptide is first labeled with a DNA recording tag, and the chimeric DNA-polypeptide molecule is immobilized to a solid support via nucleic acid hybridization and ligation to a DNA sequence attached to the solid support.
  • a recording tag comprises an optional, unique molecular identifier (UMI), which provides a unique identifier tag for each polypeptide to which the UMI is associated with.
  • UMI can be about 3 to about 40 bases, or a subrange thereof, e.g, about 3 to about 30 bases, about 3 to about 20 bases, or about 3 to about 10 bases, or about 3 to about 8 bases.
  • a UMI is about 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases, 19 bases, 20 bases, 25 bases, 30 bases, 35 bases, or 40 bases in length.
  • a UMI can be used to de-convolute sequencing data from a plurality of extended recording tags to identify sequence reads from individual polypeptides.
  • each polypeptide is associated with a single recording tag, with each recording tag comprising a unique UMI.
  • multiple copies of a recording tag are associated with a single polypeptide, with each copy of the recording tag comprising the same UMI.
  • a UMI has a different base sequence than the spacer or encoder sequences within the binding agents’ coding tags to facilitate distinguishing these components during sequence analysis.
  • a recording tag comprises a barcode, e.g ., other than the UMI if present.
  • a barcode is a nucleic acid molecule of about 3 to about 30 bases, or a subrange thereof, e.g. , about 3 to about 25 bases, about 3 to about 20 bases, about 3 to about 10 bases, about 3 to about 10 bases, about 3 to about 8 bases in length.
  • a barcode is about 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases, 20 bases, 25 bases, or 30 bases in length.
  • a barcode allows for multiplex sequencing of a plurality of samples or libraries.
  • a barcode may be used to identify a partition, a fraction, a compartment, a sample, a spatial location, or library from which the polypeptide derived. Barcodes can be used to de-convolute multiplexed sequence data and identify sequence reads from an individual sample or library. For example, a barcoded bead is useful for methods involving emulsions and partitioning of samples, e.g., for purposes of partitioning the proteome.
  • a barcode can represent a compartment tag in which a compartment, such as a droplet, microwell, physical region on a solid support, etc. is assigned a unique barcode.
  • a compartment such as a droplet, microwell, physical region on a solid support, etc.
  • the association of a compartment with a specific barcode can be achieved in any number of ways such as by encapsulating a single barcoded bead in a compartment, e.g, by direct merging or adding a barcoded droplet to a compartment, by directly printing or injecting a barcode reagent to a compartment, etc.
  • the barcode reagents within a compartment are used to add
  • compartment-specific barcodes to the polypeptide or fragments thereof within the compartment.
  • the barcodes can be used to map analysed peptides back to their originating protein molecules in the compartment. This can greatly facilitate protein identification. Compartment barcodes can also be used to identify protein complexes.
  • multiple compartments that represent a subset of a population of compartments may be assigned a unique barcode representing the subset.
  • a barcode may be a sample identifying barcode.
  • a sample barcode is useful in the multiplexed analysis of a set of samples in a single reaction vessel or
  • polypeptides from many different samples can be labeled with recording tags with sample-specific barcodes, and then all the samples pooled together prior to immobilization to a solid support, cyclic binding, and recording tag analysis.
  • the samples can be kept separate until after creation of a DNA- encoded library, and sample barcodes attached during PCR amplification of the DNA-encoded library, and then mixed together prior to sequencing. This approach could be useful when assaying analytes (e.g ., proteins) of different abundance classes.
  • the sample can be split and barcoded, and one portion processed using binding agents to low abundance analytes, and the other portion processed using binding agents to higher abundance analytes.
  • this approach helps to adjust the dynamic range of a particular protein analyte assay to lie within the“sweet spot” of standard expression levels of the protein analyte.
  • polypeptides from multiple different samples are labeled with recording tags containing sample-specific barcodes.
  • the multi-sample barcoded polypeptides can be mixed together prior to a cyclic binding reaction.
  • RPPA digital reverse phase protein array
  • a recording tag comprises a universal priming site, e.g., a forward or 5’ universal priming site.
  • a universal priming site is a nucleic acid sequence that may be used for priming a library amplification reaction and/or for sequencing.
  • a universal priming site may include, but is not limited to, a priming site for PCR amplification, flow cell adaptor sequences that anneal to complementary oligonucleotides on flow cell surfaces (e.g., Illumina next generation sequencing), a sequencing priming site, or a combination thereof.
  • a universal priming site can be about 10 bases to about 60 bases.
  • a universal priming site comprises an Illumina P5 primer (5’-AATGATACGGCGACCACCGA- 3’ - SEQ ID NO:3) or an Illumina P7 primer (5’ -C AAGC AGAAGACGGC ATACGAGAT - 3’
  • a recording tag comprises a spacer at its terminus, e.g,
  • spacer sequence in the context of a recording tag includes a spacer sequence that is identical to the spacer sequence associated with its cognate binding agent, or a spacer sequence that is complementary to the spacer sequence associated with its cognate binding agent.
  • the terminal, e.g ., 3’, spacer on the recording tag permits transfer of identifying information of a cognate binding agent from its coding tag to the recording tag during the first binding cycle (e.g., via annealing of complementary spacer sequences for primer extension or sticky end ligation).
  • the spacer sequence is about 1-20 bases in length or a subrange thereof, e.g, about 2-12 bases in length, or 5-10 bases in length.
  • the length of the spacer may depend on factors such as the temperature and reaction conditions of the primer extension reaction for transferring coding tag information to the recording tag.
  • the recording tag does not comprise a spacer.
  • the spacer sequence in the recording is designed to have minimal complementarity to other regions in the recording tag; likewise, the spacer sequence in the coding tag should have minimal complementarity to other regions in the coding tag.
  • the spacer sequence of the recording tags and coding tags should have minimal sequence complementarity to components such unique molecular identifiers, barcodes (e.g., compartment, partition, sample, spatial location), universal primer sequences, encoder sequences, cycle specific sequences, etc. present in the recording tags or coding tags.
  • polypeptides share a common spacer sequence.
  • the recording tags associated with a library of polypeptides have binding cycle specific spacer sequences that are complementary to the binding cycle specific spacer sequences of their cognate binding agents, which can be useful when using non-concatenated extended recording tags.
  • the collection of extended recording tags can be concatenated.
  • the bead solid supports each bead comprising on average one or fewer than one polypeptide per bead, each polypeptide having a collection of extended recording tags that are co-localized at the site of the polypeptide, are placed in an emulsion.
  • the emulsion is formed such that each droplet, on average, is occupied by at most 1 bead.
  • the DNA recording tag is comprised of a universal priming sequence (Ul), one or more barcode sequences (BCs), and a spacer sequence (Spl) specific to the first binding cycle.
  • binding agents employ DNA coding tags comprised of an Spl complementary spacer, an encoder barcode, and optional cycle barcode, and a second spacer element (Sp2).
  • the utility of using at least two different spacer elements is that the first binding cycle selects one of potentially several DNA recording tags and a single DNA recording tag is extended resulting in a new Sp2 spacer element at the end of the extended DNA recording tag.
  • binding agents contain just the Sp2’ spacer rather than Spl’ . In this way, only the single extended recording tag from the first cycle is extended in subsequent cycles.
  • the second and subsequent cycles can employ binding agent specific spacers.
  • a recording tag comprises from 5’ to 3’ direction: a universal forward (or 5’) priming sequence, a UMI, and a spacer sequence.
  • a recording tag comprises from 5’ to 3’ direction: a universal forward (or 5’) priming sequence, an optional UMI, a barcode (e.g ., sample barcode, partition barcode, compartment barcode, spatial barcode, or any combination thereof), and a spacer sequence.
  • a recording tag comprises from 5’ to 3’ direction: a universal forward (or 5’) priming sequence, a barcode (e.g., sample barcode, partition barcode, compartment barcode, spatial barcode, or any combination thereof), an optional UMI, and a spacer sequence.
  • UMIs may be constructed by“chemical ligating” together sets of short word sequences (4-15mers), which have been designed to be orthogonal to each other (Spiropulos and Heemstra 2012).
  • a DNA template is used to direct the chemical ligation of the “word” polymers.
  • the DNA template is constructed with hybridizing arms that enable assembly of a combinatorial template structure simply by mixing the sub-components together in solution.
  • the size of the word space can vary from 10’s of words to 10,000’ s or more words or a subrange thereof.
  • the words are chosen such that they differ from one another to not cross hybridize, yet possess relatively uniform hybridization conditions.
  • These UMI sequences will be appended to the polypeptide at the single molecule level.
  • the diversity of UMIs exceeds the number of molecules of polypeptides to which the UMIs are attached.
  • the UMI uniquely identifies the polypeptide of interest.
  • the use of combinatorial word UMFs facilitates readout on high error rate sequencers, (e.g., nanopore sequencers, nanogap tunneling sequencing, etc.) since single base resolution is not required to read words of multiple bases in length.
  • Combinatorial word approaches can also be used to generate other identity-informative components of recording tags or coding tags, such as compartment tags, partition barcodes, spatial barcodes, sample barcodes, encoder sequences, cycle specific sequences, and barcodes.
  • Methods relating to nanopore sequencing and DNA encoding information with error-tolerant words (codes) are known in the art (see, e.g., Kiah et al., 2015, Codes for DNA sequence profiles. IEEE International
  • an extended recording tag, an extended coding tag, or a di-tag construct in any of the embodiments described herein is comprised of identifying components (e.g., UMI, encoder sequence, barcode, compartment tag, cycle specific sequence, etc.) that are error correcting codes.
  • the error correcting code is selected from: Hamming code, Lee distance code, asymmetric Lee distance code, Reed-Solomon code, and Levenshtein-Tenengolts code.
  • the current or ionic flux profiles and asymmetric base calling errors are intrinsic to the type of nanopore and biochemistry employed, and this information can be used to design more robust DNA codes using the aforementioned error correcting approaches.
  • the identifying components of a coding tag, recording tag, or both are capable of generating a unique current or ionic flux or optical signature, wherein the analysis step of any of the methods provided herein comprises detection of the unique current or ionic flux or optical signature in order to identify the identifying components.
  • the identifying components are selected from an encoder sequence, barcode, UMI, compartment tag, cycle specific sequence, or any combination thereof.
  • all or a substantial amount of the polypeptides within a sample are labeled with a recording tag. Attaching of the recording tag to the polypeptides may occur before or after immobilization of the polypeptides to a solid support.
  • a subset of polypeptides within a sample are labeled with recording tags.
  • a subset of polypeptides from a sample undergo targeted (analyte specific) labeling with recording tags.
  • Targeted recording tag labeling of proteins may be achieved using target protein-specific binding agents (e.g., antibodies, aptamers, etc.) that are linked a short target-specific DNA capture probe, e.g., analyte-specific barcode, which anneal to complementary target-specific bait sequence, e.g., analyte-specific barcode, in recording tags.
  • the recording tags comprise a reactive moiety for a cognate reactive moiety present on the target protein (e.g., click chemistry labeling, photoaffmity labeling).
  • recording tags may comprise an azide moiety for interacting with alkyne-derivatized proteins, or recording tags may comprise a benzophenone for interacting with native proteins, etc.
  • the DNA capture probe may be designed to contain uracil bases, which are then targeted for digestion with a uracil-specific excision reagent (e.g ., USERTM), and the target- protein specific binding agent may be dissociated from the target protein.
  • a uracil-specific excision reagent e.g ., USERTM
  • antibodies specific for a set of target proteins can be labeled with a DNA capture probe that hybridizes with recording tags designed with complementary bait sequence.
  • Sample-specific labeling of proteins can be achieved by employing DNA-capture probe labeled antibodies hybridizing with complementary bait sequence on recording tags comprising of sample-specific barcodes.
  • target protein-specific aptamers are used for targeted recording tag labeling of a subset of proteins within a sample.
  • a target specific-aptamer is linked to a DNA capture probe that anneals with complementary bait sequence in a recording tag.
  • the recording tag comprises a reactive chemical or photo-reactive chemical probes (e.g. benzophenone (BP)) for coupling to the target protein having a corresponding reactive moiety.
  • BP benzophenone
  • the aptamer binds to its target protein molecule, bringing the recording tag into close proximity to the target protein, resulting in the coupling of the recording tag to the target protein.
  • Photoaffmity (PA) protein labeling using photo-reactive chemical probes attached to small molecule protein affinity ligands has been previously described (Park, Koh et al. 2016).
  • Typical photo-reactive chemical probes include probes based on benzophenone (reactive diradical, 365 nm), phenyldiazirine (reactive carbon, 365 nm), and phenylazide (reactive nitrene free radical, 260 nm), activated under irradiation wavelengths as previously described (Smith et al., Future Med Chem. (2015) 7(2): 159-183).
  • target proteins within a protein sample are labeled with recording tags comprising sample barcodes using the method disclosed by Li et al., in which a bait sequence in a benzophenone labeled recording tag is hybridized to a DNA capture probe attached to a cognate binding agent (e.g., nucleic acid aptamer (Li et al., Angew Chem Int Ed Engl (2013) 52(36): 9544-9549).
  • a cognate binding agent e.g., nucleic acid aptamer (Li et al., Angew Chem Int Ed Engl (2013) 52(36): 9544-9549).
  • a cognate binding agent e.g., nucleic acid aptamer (Li et al., Angew Chem Int Ed Engl (2013) 52(36): 9544-9549).
  • a cognate binding agent e.g., nucleic acid aptamer (Li et al., Angew Chem
  • the two moieties can be covalently linked, using a linker that is designed to be cleaved and release the binding agent once the captured target protein (or other polypeptide) is covalently linked to the recording tag.
  • a suitable linker can be attached to various positions of the recording tag, such as the 3’ end, or within the linker attached to the 5’ end of the recording tag.
  • Recording tags can be attached to the protein, polypeptide, or peptides pre- or post-immobilization to the solid support.
  • proteins, polypeptides, or peptides can be first labeled with recording tags and then immobilized to a solid surface via a recording tag comprising at two functional moieties for coupling.
  • One functional moiety of the recording tag couples to the protein, and the other functional moiety immobilizes the recording tag-labeled protein to a solid support.
  • polypeptides are immobilized to a solid support prior to labeling of the proteins, polypeptides or peptides with recording tags.
  • proteins can first be derivatized with reactive groups such as click chemistry moieties. The activated protein molecules can then be attached to a suitable solid support and then labeled with recording tags using the complementary click chemistry moiety.
  • proteins derivatized with alkyne and mTet moieties may be immobilized to beads derivatized with azide and TCO and attached to recording tags labeled with azide and TCO.
  • the surface of a solid support is passivated (blocked) to minimize non-specific absorption to binding agents.
  • A“passivated” surface refers to a surface that has been treated with outer layer of material to minimize non-specific binding of a binding agent.
  • Methods of passivating surfaces include standard methods from the fluorescent single molecule analysis literature, including passivating surfaces with polymer like polyethylene glycol (PEG) (Pan et al., 2015, Phys. Biol. 12:045006), polysiloxane (e.g., Pluronic F-127), star polymers (e.g., star PEG) (Groll et al., 2010, Methods Enzymol.
  • passivating agents can be employed as well including surfactants like Tween-20, polysiloxane in solution (Pluronic series), poly vinyl alcohol, (PVA), and proteins like BSA and casein.
  • surfactants like Tween-20, polysiloxane in solution (Pluronic series), poly vinyl alcohol, (PVA), and proteins like BSA and casein.
  • density of proteins, polypeptide, or peptides can be titrated on the surface or within the volume of a solid substrate by spiking a competitor or“dummy” reactive molecule when immobilizing the proteins, polypeptides or peptides to the solid substrate.
  • the polypeptides can be spaced appropriately to reduce the occurrence of or prevent a cross-binding or inter-molecular event, e.g ., where a binding agent binds to a first polypeptides and its coding tag information is transferred to a recording tag associated with a neighboring polypeptides rather than the recording tag associated with the first polypeptide.
  • a binding agent binds to a first polypeptides and its coding tag information is transferred to a recording tag associated with a neighboring polypeptides rather than the recording tag associated with the first polypeptide.
  • the density of functional coupling groups e.g, TCO
  • multiple polypeptides are spaced apart on the surface or within the volume (e.g., porous supports) of a solid support at a distance of about 50 nm to about 500 nm, or a subrange thereof, e.g, or about 50 nm to about 400 nm, or about 50 nm to about 300 nm, or about 50 nm to about 200 nm, or about 50 nm to about 100 nm.
  • multiple polypeptides are spaced apart on the surface of a solid support with an average distance of at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 250 nm, at least 300 nm, at least 350 nm, at least 400 nm, at least 450 nm, or at least 500 nm. In some embodiments, multiple polypeptides are spaced apart on the surface of a solid support with an average distance of at least 50 nm.
  • polypeptides are spaced apart on the surface or within the volume of a solid support such that, empirically, the relative frequency of inter- to intra-molecular events is ⁇ 1 : 10; ⁇ 1 : 100; ⁇ 1 : 1,000; or ⁇ 1 : 10,000.
  • a suitable spacing frequency can be determined empirically using a functional assay (see, Example 31 of
  • PEG-5000 (MW ⁇ 5000) is used to block the interstitial space between peptides on the substrate surface (e.g., bead surface).
  • the peptide is coupled to a functional moiety that is also attached to a PEG-5000 molecule.
  • this is accomplished by coupling a mixture of NHS-PEG-5000-TCO + NHS-PEG-5000-Methyl to amine-derivatized beads.
  • TCO vs. methyl is titrated to generate an appropriate density of functional coupling moieties (TCO groups) on the substrate surface; the methyl-PEG is inert to coupling.
  • the effective spacing between TCO groups can be calculated by measuring the density of TCO groups on the surface.
  • the mean spacing between coupling moieties (e.g ., TCO) on the solid surface is at least 50 nm, at least 100 nm, at least 250 nm, or at least 500 nm.
  • the excess NEE groups on the surface are quenched with a reactive anhydride (e.g. acetic or succinic anhydride).
  • a reactive anhydride e.g. acetic or succinic anhydride
  • Other MW PEGs can also be used for passivation from MW - 300 Da to over 50 kDa.
  • the spacing is accomplished by titrating the ratio of available attachment molecules on the substrate surface.
  • the substrate surface e.g., bead surface
  • the substrate surface is functionalized with a carboxyl group (COOH) which is treated with an activating agent (e.g., activating agent is EDC and Sulfo-NHS).
  • an activating agent e.g., activating agent is EDC and Sulfo-NHS.
  • the substrate surface e.g., bead surface
  • a mixture of mPEG redesign-NH2 and NH2-PEG memo-mTet is added to the activated beads (wherein n is any number, such as 1-100).
  • the ratio between the mPEG 3 -NH 2 (not available for coupling) and NH2- PEG24-mTet (available for coupling) is titrated to generate an appropriate density of functional moieties available to attach the analyte on the substrate surface.
  • the mean spacing between coupling moieties (e.g., NH 2 -PEG 4 -mTet) on the solid surface is at least 50 nm, at least 100 nm, at least 250 nm, or at least 500 nm.
  • the ratio of NH 2 -PEG memo-mTet to mPEG 3 -NH2 is about or greater than 1: 1000, about or greater than 1: 10,000, about or greater than 1: 100,000, or about or greater than 1: 1,000,000.
  • the capture nucleic acid attaches to the NH2-PEG friction-mTet.
  • the polypeptide(s) and/or the recording tag(s) are immobilized on a substrate or support at a density such that the interaction between (i) a coding agent bound to a first polypeptide (particularly, the coding tag in that bound coding agent), and (ii) a second polypeptide and/or its recording tag, is reduced, minimized, or completely eliminated. Therefore, false positive assay signals resulting from“intermolecular” engagement can be reduced, minimized, or eliminated.
  • the density of the polypeptides and/or the recording tags on a substrate is determined for each type of polypeptide. For example, the longer a denatured polypeptide chain is, the lower the density should be in order to reduce, minimize, or prevent “intermolecular” interactions. In certain aspects, increasing the spacing between the polypeptide molecules and/or the recording tags (i.e., lowering the density) increases the signal to background ratio of the presently disclosed assays.
  • the polypeptide molecules and/or the recording tags are deposited or immobilized on a substrate at any suitable average density, e.g ., at an average density of about 0.0001 molecule/pm 2 , 0.001 molecule/pm 2 , 0.01 molecule/pm 2 , 0.1
  • molecule/pm 2 1 molecule/pm 2 , about 2 molecules/pm 2 , about 3 molecules/pm 2 , about 4 molecules/pm 2 , about 5 molecules/pm 2 , about 6 molecules/pm 2 , about 7 molecules/pm 2 , about 8 molecules/pm 2 , about 9 molecules/pm 2 , or about 10 molecules/pm 2 .
  • the polypeptide(s) and/or the recording tag(s) are deposited or immobilized at an average density of about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, or about 200 molecules/pm 2 on a substrate.
  • the polypeptide(s) and/or the recording tag(s) are deposited or immobilized at an average density of about 1 molecule/mm 2 , about 10 molecules/mm 2 , about 50 molecules/mm 2 , about 100 molecules/mm 2 , about 150 molecules/mm 2 , about 200 molecules/mm 2 , about 250
  • molecules/mm 2 about 300 molecules/mm 2 , about 350 molecules/mm 2 , 400 molecules/mm 2 , about 450 molecules/mm 2 , about 500 molecules/mm 2 , about 550 molecules/mm 2 , about 600 molecules/mm 2 , about 650 molecules/mm 2 , about 700 molecules/mm 2 , about 750
  • the polypeptide(s) and/or the recording tag(s) are deposited or immobilized on a substrate at an average density between about l x lO 3 and about 0 5 10 4 molecules/mm 2 , between about 0.5x l0 4 and about l x lO 4 molecules/mm 2 , between about l x lO 4 and about 0.5x 10 s molecules/mm 2 , between about 0.5x 10 s and about l x lO 5 molecules/mm 2 , between about l x lO 5 and about 0.5x 10 6 molecules/mm 2 , or between about 0.5x 10 6 and about 1 c 10 6 molecules/mm 2 .
  • the average density of the polypeptide(s) and/or the recording tag(s) deposited or immobilized on a substrate can be, for example, between about 1 molecule/cm 2 and about 5 molecules/cm 2 , between about 5 and about 10 molecules/cm 2 , between about 10 and about 50 molecules/cm 2 , between about 50 and about 100 molecules/cm 2 , between aboutlOO and about 0.5x l0 3 molecules/cm 2 , between about 0.5x l0 3 and about l x lO 3 molecules/cm 2 , l x lO 3 and about 0.5x l0 4 molecules/cm 2 , between about 0 5 10 4 and about 1 10 4 molecules/cm 2 , between about 1 10 4 and about 0.5x 10 s molecules/cm 2 , between about 0.5x 10 s and about 1 x 10 s molecules/cm 2 , between about 1 x 10 s and about 0.5x l0 6 molecules/cm 2 ,
  • an extended recording tag may comprise information from a binding agent’s coding tag representing each binding cycle performed. However, an extended recording tag may also experience a“missed” binding cycle, e.g., because a binding agent fails to bind to the polypeptide, because the coding tag was missing, damaged, or defective, because the primer extension reaction failed. Even if a binding event occurs, transfer of information from the coding tag to the recording tag may be incomplete or less than 100% accurate, e.g. , because a coding tag was damaged or defective, because errors were introduced in the primer extension reaction). Thus, an extended recording tag may represent 100%, or up to 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 65%, 55%,
  • the coding tag information present in the extended recording tag may have at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity the corresponding coding tags.
  • a binding agent may bind to an NTAA, a CTAA, an intervening amino acid, dipeptide (sequence of two amino acids), tripeptide (sequence of three amino acids), or higher order peptide of a peptide molecule.
  • each binding agent in a library of binding agents selectively binds to a particular amino acid, for example one of the twenty standard naturally occurring amino acids.
  • the standard, naturally- occurring 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 lie), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q or Gin), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Val), Tryptophan (W or Trp), and Tyrosine (Y or Tyr).
  • the binding agent binds to an unmodified or native amino acid. In some examples, the binding agent binds to an unmodified or native dipeptide (sequence of two amino acids), tripeptide (sequence of three amino acids), or higher order peptide of a peptide molecule.
  • a binding agent may be engineered for high affinity for a native or unmodified NTAA, high specificity for a native or unmodified NTAA, or both. In some embodiments, binding agents can be developed through directed evolution of promising affinity scaffolds using phage display.
  • a binding agent may bind to an N-terminal peptide, a C-terminal peptide, or an intervening peptide of a peptide, polypeptide, or protein molecule.
  • a binding agent may bind to an N-terminal amino acid, C-terminal amino acid, or an intervening amino acid of a peptide molecule.
  • a binding agent may bind to an N-terminal or C-terminal diamino acid moiety.
  • a binding agent may preferably bind to a chemically modified or labeled amino acid.
  • a binding agent may preferably bind to an amino acid that has been functionalized with an acetyl moiety, Cbz moiety, guanyl moiety, dansyl moiety, PTC moiety, DNP moiety, SNP moiety, heterocyclic methanimine moiety, etc., over an amino acid that does not possess said moiety.
  • an extended recording tag may comprise information from multiple coding tags representing multiple, successive binding events.
  • a single, concatenated extended recording tag can be representative of a single polypeptide.
  • transfer of coding tag information to a recording tag also includes transfer to an extended recording tag as would occur in methods involving multiple, successive binding events.
  • the binding event information is transferred from a coding tag to a recording tag in a cyclic fashion.
  • Cross-reactive binding events can be informatically filtered out after sequencing by requiring that at least two different coding tags, identifying two or more independent binding events, map to the same class of binding agents (cognate to a particular protein).
  • An optional sample or compartment barcode can be included in the recording tag, as well an optional UMI sequence.
  • the coding tag can also contain an optional UMI sequence along with the encoder and spacer sequences. Universal priming sequences may also be included in extended recording tags for amplification and NGS sequencing.
  • Coding tag information associated with a specific binding agent may be transferred to a recording tag using a variety of methods.
  • information of a coding tag is transferred to a recording tag via primer extension (Chan, McGregor et al. 2015).
  • a spacer sequence on the 3’-terminus of a recording tag or an extended recording tag anneals with complementary spacer sequence on the 3’ terminus of a coding tag and a polymerase (e.g ., strand-displacing polymerase) extends the recording tag sequence, using the annealed coding tag as a template.
  • oligonucleotides complementary to coding tag encoder sequence and 5’ spacer can be pre-annealed to the coding tags to prevent hybridization of the coding tag to internal encoder and spacer sequences present in an extended recording tag.
  • the 3’ terminal spacer, on the coding tag, remaining single stranded, preferably binds to the terminal 3’ spacer on the recording tag.
  • a nascent recording tag can be coated with a single stranded binding protein to prevent annealing of the coding tag to internal sites.
  • the nascent recording tag can also be coated with RecA (or related homologues such as uvsX) to facilitate invasion of the 3’ terminus into a completely double stranded coding tag (Bell et al., 2012, Nature 491 :274-278).
  • RecA or related homologues such as uvsX
  • This configuration prevents the double stranded coding tag from interacting with internal recording tag elements, yet is susceptible to strand invasion by the RecA coated 3’ tail of the extended recording tag (Bell, et al., 2015, Elife 4: e08646).
  • the presence of a single-stranded binding protein can facilitate the strand displacement reaction.
  • a DNA polymerase that is used for primer extension possesses strand-displacement activity and has limited or is devoid of 3’-5 exonuclease activity.
  • Several of many examples of such polymerases include Klenow exo- (Klenow fragment of DNA Pol 1), T4 DNA polymerase exo-, T7 DNA polymerase exo (Sequenase 2.0), Pfu exo-, Vent exo-, Deep Vent exo-, Bst DNA polymerase large fragment exo-, Bca Pol, 9°N Pol, and Phi29 Pol exo-.
  • the DNA polymerase is active at room temperature and up to 45°C.
  • thermophilic polymerase in another embodiment, a“warm start” version of a thermophilic polymerase is employed such that the polymerase is activated and is used at about 40°C-50°C.
  • An exemplary warm start polymerase is Bst 2.0 Warm Start DNA Polymerase (New England Biolabs).
  • Additives useful in strand-displacement replication include any of a number of single-stranded DNA binding proteins (SSB proteins) of bacterial, viral, or eukaryotic origin, such as SSB protein of E. coli, phage T4 gene 32 product, phage T7 gene 2.5 protein, phage Pf3 SSB, replication protein A RPA32 and RPA14 subunits (Wold, 1997); other DNA binding proteins, such as adenovirus DNA-binding protein, herpes simplex protein ICP8, BMRF1 polymerase accessory subunit, herpes virus UL29 SSB-like protein; any of a number of replication complex proteins known to participate in DNA replication, such as phage T7 helicase/primase, phage T4 gene 41 helicase, E. coli Rep helicase, E. coli recBCD helicase, recA, E. coli and eukaryotic topoisomerases (Annu Rev Biochem. (2001) 70:
  • the extended recording tag (with or without a non-templated adenosine base) can anneal to the coding tag and undergo primer extension.
  • polymerase extension buffers are comprised of 40-120 mM buffering agent such as Tris-Acetate, Tris-HCl, HEPES, etc. at a pH of 6-9.
  • Self-priming/mis-priming events initiated by self-annealing of the terminal spacer sequence of the extended recording tag with internal regions of the extended recording tag may be minimized by including pseudo-complementary bases in the recording/extended recording tag (Lahoud et al., Nucleic Acids Res. (2008) 36:3409-3419), (Hoshika et al., Angew Chem Int Ed Engl (2010) 49(32): 5554-5557). Pseudo-complementary bases show significantly reduced hybridization affinities for the formation of duplexes with each other due the presence of chemical modification. However, many pseudo-complementary modified bases can form strong base pairs with natural DNA or RNA sequences.
  • the coding tag spacer sequence is comprised of multiple A and T bases, and commercially available pseudo complementary bases 2-aminoadenine and 2-thiothymine are incorporated in the recording tag using phosphoramidite oligonucleotide synthesis. Additional pseudocomplementary bases can be incorporated into the extended recording tag during primer extension by adding pseudo complementary nucleotides to the reaction (Gamper et al., Biochemistry. (2006) 45(22):6978- 86).
  • competitor also referred to as blocking
  • blocking oligonucleotides complementary to recording tag spacer sequences
  • blocking oligonucleotides are relatively short. Excess competitor oligonucleotides are washed from the binding reaction prior to primer extension, which effectively dissociates the annealed competitor oligonucleotides from the recording tags, especially when exposed to slightly elevated temperatures (e.g 30-50 °C).
  • Blocking oligonucleotides may comprise a terminator nucleotide at its 3’ end to prevent primer extension.
  • the coding tag may comprise a hairpin.
  • the hairpin comprises mutually complementary nucleic acid regions are connected through a nucleic acid strand.
  • the nucleic acid hairpin can also further comprise 3' and/or 5' single-stranded region(s) extending from the double-stranded stem segment.
  • the hairpin comprises a single strand of nucleic acid.
  • the annealing of the spacer sequence on the recording tag to the complementary spacer sequence on the coding tag is metastable under the primer extension reaction conditions (i.e., the annealing Tm is similar to the reaction temperature). This allows the spacer sequence of the coding tag to displace any blocking oligonucleotide annealed to the spacer sequence of the recording tag.
  • transfer of PNAs can be accomplished with chemical ligation using published techniques.
  • the structure of PNA is such that it has a 5’ N-terminal amine group and an unreactive 3’ C-terminal amide.
  • Chemical ligation of PNA requires that the termini be modified to be chemically active. This is typically done by derivatizing the 5’ N- terminus with a cysteinyl moiety and the 3’ C-terminus with a thioester moiety.
  • Such modified PNAs easily couple using standard native chemical ligation conditions (Roloff et al., (2013) Bioorgan. Med. Chem. 21 :3458-3464).
  • coding tag information can be transferred using topoisomerase.
  • Topoisomerase can be used be used be used to ligate a topo-charged 3’ phosphate on the recording tag to the 5’ end of the coding tag, or complement thereof (Shuman et al., 1994, J.
  • an extended recording tag comprises coding tag information relating to amino acid sequence and post-translational modifications of the polypeptide.
  • detection of internal post-translationally modified amino acids e.g ., phosphorylation, glycosylation, succinylation, ubiquitination, S-Nitrosylation, methylation, N-acetylation, lipidation, etc.
  • terminal amino acids e.g., NTAA or CTAA.
  • a peptide is contacted with binding agents for PTM modifications, and associated coding tag information are transferred to the recording tag.
  • the PTM modifying groups can be removed before detection and transfer of coding tag information for the primary amino acid sequence using N-terminal or C- terminal degradation methods.
  • resulting extended recording tags indicate the presence of post-translational modifications in a peptide sequence, though not the sequential order, along with primary amino acid sequence information.
  • an ensemble of recording tags may be employed per polypeptide to improve the overall robustness and efficiency of coding tag information transfer.
  • the use of an ensemble of recording tags associated with a given polypeptide rather than a single recording tag improves the efficiency of library construction due to potentially higher coupling yields of coding tags to recording tags, and higher overall yield of libraries.
  • the yield of a single concatenated extended recording tag is directly dependent on the stepwise yield of concatenation, whereas the use of multiple recording tags capable of accepting coding tag information does not suffer the exponential loss of concatenation.
  • the bound binding agent and annealed coding tag can be removed following primer extension by using highly denaturing conditions (e.g., 0 1 0.2 N NaOH, 6M Urea, 2.4 M guanidinium isothiocyanate, 95% formamide, etc.).
  • highly denaturing conditions e.g., 0 1 0.2 N NaOH, 6M Urea, 2.4 M guanidinium isothiocyanate, 95% formamide, etc.
  • the methods for analyzing a polypeptide provided in the present disclosure comprise multiple binding cycles, where the polypeptide is contacted with a plurality of binding agents, and successive binding of binding agents transfers historical binding information in the form of a nucleic acid based coding tag to at least one recording tag associated with the polypeptide. In this way, a historical record containing information about multiple binding events is generated in a nucleic acid format.
  • the concentration of the binding agents in a solution is controlled to reduce background and/or false positive results of the assay.
  • the concentration of a binding agent can be at any suitable concentration, e.g ., at about 0.0001 nM, about 0.001 nM, about 0.01 nM, about 0.1 nM, about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 20 nM, about 50 nM, about 100 nM, about 200 nM, about 500 nM, or about 1000 nM.
  • the concentration of a soluble conjugate used in the assay is between about 0.0001 nM and about 0.001 nM, between about 0.001 nM and about 0.01 nM, between about 0.01 nM and about 0.1 nM, between about 0.1 nM and about 1 nM, between about 1 nM and about 2 nM, between about 2 nM and about 5 nM, between about 5 nM and about 10 nM, between about 10 nM and about 20 nM, between about 20 nM and about 50 nM, between about 50 nM and about 100 nM, between about 100 nM and about 200 nM, between about 200 nM and about 500 nM, between about 500 nM and about 1000 nM, or more than about 1000 nM.
  • n NTAA is eliminated as described herein.
  • the use of the modified cleavase in a polypeptide analysis assay is for removal of the labeled NTAA.
  • two or more any of the modified cleavases described in Section I can be used in combination to remove the labeled NTAA.
  • a sample can be treated with a mixture of modified cleavase enzymes to achieve removal of various NTAAs in the peptides in the sample. Removal of the n labeled NTAA by contacting with the modified cleavase converts the n-1 amino acid of the peptide to an N-terminal amino acid, which is referred to herein as an n-1 NTAA.
  • a second binding agent is contacted with the peptide and binds to the n-1 NTAA, and the second binding agent’s coding tag information is transferred to the first order extended recording tag thereby generating a second order extended recording tag (e.g ., for generating a concatenated n th order extended recording tag representing the peptide), or to a different recording tag (e.g., for generating multiple extended recording tags, which collectively represent the peptide).
  • Elimination of the n-1 labeled NTAA by a modified cleavase converts the n-2 amino acid of the peptide to an N-terminal amino acid, which is referred to herein as n-2 NTAA.
  • n“order” when used in reference to a binding agent, coding tag, or extended recording tag refers to the n binding cycle, wherein the binding agent and its associated coding tag is used or the n binding cycle where the extended recording tag is created.
  • steps including the NTAA in the described exemplary approach can be performed instead with a CTAA.
  • contacting of the first binding agent and second binding agent to the polypeptide, and optionally any further binding agents are performed at the same time.
  • the first binding agent and second binding agent, and optionally any further order binding agents can be pooled together, for example to form a library of binding agents.
  • the first binding agent and second binding agent, and optionally any further order binding agents, rather than being pooled together are added simultaneously to the polypeptide.
  • a library of binding agents comprises at least 20 binding agents that selectively bind to the 20 standard, naturally occurring amino acids.
  • the length of the final extended recording tags generated by the methods described herein is dependent upon multiple factors, including the length of the coding tag (e.g ., encoder sequence and spacer), the length of the recording tag (e.g., unique molecular identifier, spacer, universal priming site, bar code), the number of binding cycles performed, and whether coding tags from each binding cycle are transferred to the same extended recording tag or to multiple extended recording tags.
  • the coding tag e.g ., encoder sequence and spacer
  • the length of the recording tag e.g., unique molecular identifier, spacer, universal priming site, bar code
  • the number of binding cycles performed e.g., unique molecular identifier, spacer, universal priming site, bar code
  • coding tags from each binding cycle are transferred to the same extended recording tag or to multiple extended recording tags.
  • the coding tag information on the final extended recording tag which represents the peptide’s binding agent history, is 10 bases x number of degradation cycles.
  • the tag can be capped by addition of a universal reverse priming site via ligation, primer extension or other methods known in the art.
  • the universal forward priming site in the recording tag is compatible with the universal reverse priming site that is appended to the final extended recording tag.
  • a universal reverse priming site is an Illumina P7 primer (5’- CAAGCAGAAGACGGCATACGAGAT - 3’ - SEQ ID NO:4) or an Illumina P5 primer (5’- AATGATACGGCGACCACCGA-3’ - SEQ ID NO:3).
  • cycle-specific barcodes present in the coding tag sequence allows an advantage to the assay.
  • Suitable sequencing methods for use in the invention include, but are not limited to, sequencing by hybridization, sequencing by synthesis technology (e.g ., HiSeqTM and SolexaTM, Illumina), SMRTTM (Single Molecule Real Time) technology ( Pacific Biosciences), true single molecule sequencing (e.g., HeliScopeTM, Helicos Biosciences), massively parallel next generation sequencing (e.g., SOLiDTM, Applied Biosciences; Solexa and HiSeqTM, Illumina), massively parallel semiconductor sequencing (e.g., Ion Torrent), and pyrosequencing technology (e.g., GS FLX and GS Junior Systems, Roche/454), and nanopore sequence (e.g., Oxford Nanopore Technologies).
  • sequencing by synthesis technology e.g ., HiSeqTM and SolexaTM, Illumina
  • SMRTTM Single Molecule Real Time
  • true single molecule sequencing e.g., HeliScopeTM, Helicos Bioscience
  • a library of extended recording tags, extended coding tags, or di-tags may be amplified in a variety of ways.
  • a library of extended recording tags, extended coding tags, or di-tags may undergo exponential amplification, e.g., via PCR or emulsion PCR. Emulsion PCR is known to produce more uniform amplification (Hori, Fukano et ak, Biochem Biophys Res Commun (2007) 352(2): 323-328).
  • a library of extended recording tags, extended coding tags, or di-tags may undergo linear amplification, e.g., via in vitro transcription of template DNA using T7 RNA polymerase.
  • a 20 m ⁇ PCR reaction volume is set up using an extended recording tag library eluted from ⁇ 1 mg of beads ( ⁇ 10 ng), 200 mM dNTP, 1 mM of each forward and reverse amplification primers, 0.5 m ⁇ (1U) of Phusion Hot Start enzyme (New England Biolabs) and subjected to the following cycling conditions: 98° C for 30 sec followed by 20 cycles of 98° C for 10 sec, 60° C for 30 sec, 12° C for 30 sec, followed by 12° C for 7 min, then hold at 4° C.
  • the library of extended recording tags, extended coding tags, or di-tags can undergo target enrichment.
  • target enrichment can be used to selectively capture or amplify extended recording tags representing polypeptides of interest from a library of extended recording tags, extended coding tags, or di-tags before sequencing.
  • target enrichment for protein sequencing is challenging because of the high cost and difficulty in producing highly-specific binding agents for target proteins. In some cases, antibodies are notoriously non-specific and difficult to scale production across thousands of proteins.
  • representations of the peptides of interest are used in the hybrid capture assay.
  • sequential rounds or enrichment can also be carried out, with the same or different bait sets.
  • Targeted enrichment methods can also be used in a negative selection mode to selectively remove extended recording tags, extended coding tags, or di-tags from a library before sequencing.
  • biotinylated bait in the example described above using biotinylated bait
  • oligonucleotides and streptavidin coated beads the supernatant is retained for sequencing while the bait-oligonucleotide:extended recording tag, extended coding tag, or di-tag hybrids bound to the beads are not analysed.
  • extended recording tags, extended coding tags, or di-tags that can be removed are those representing over abundant polypeptide species, e.g., for proteins, albumin, immunoglobulins, etc.
  • a competitor oligonucleotide bait hybridizing to the target but lacking a biotin moiety, can also be used in the hybrid capture step to modulate the fraction of any particular locus enriched.
  • the competitor oligonucleotide bait competes for hybridization to the target with the standard biotinylated bait effectively modulating the fraction of target pulled down during enrichment.
  • the ten orders dynamic range of protein expression can be compressed by several orders using this competitive suppression approach, especially for the overly abundant species such as albumin.
  • the fraction of library elements captured for a given locus relative to standard hybrid capture can be modulated from 100% down to 0% enrichment.
  • a library of extended recording tags, extended coding tags, or di-tags is concatenated by ligation or end-complementary PCR to create a long DNA molecule comprising multiple different extended recorder tags, extended coding tags, or di-tags, respectively (Du et al., (2003) BioTechniques 35:66-72; Muecke et al., (2008) Structure 16:837- 841; U.S. Patent No. 5,834,252, each of which is incorporated by reference in its entirety).
  • This embodiment is preferable for nanopore sequencing in which long strands of DNA are analyzed by the nanopore sequencing device.
  • direct single molecule analysis is performed on an extended recording tag, extended coding tag, or di-tag (see, e.g., Harris et al., (2008) Science 320: 106-109).
  • the extended recording tags, extended coding tags, or di-tags can be analysed directly on the solid support, such as a flow cell or beads that are compatible for loading onto a flow cell surface (optionally microcell patterned), wherein the flow cell or beads can integrate with a single molecule sequencer or a single molecule decoding instrument.
  • hybridization of several rounds of pooled fluorescently-labelled of decoding oligonucleotides can be used to ascertain both the identity and order of the coding tags within the extended recording tag.
  • the coding tag sequence can be optimized for the particular sequencing analysis platform.
  • the sequencing platform is nanopore sequencing.
  • the sequencing platform has a per base error rate of > 1%, > 5%, > 10%, >15%, > 20%, > 25%, or > 30%.
  • the barcode sequences e.g., encoder sequences
  • the barcode sequences can be designed to be optimally electrically distinguishable in transit through a nanopore.
  • the spacer element can be designed to adopt a secondary structure such as a G-quartet, which will transiently stall the extended recording tag, extended coding tag, or di-tag as it passes through the nanopore enabling readout of the adjacent encoder sequence (Shim et al., Nucleic Acids Res (2009)
  • the methods disclosed herein can be used for analysis, including detection, quantitation and/or sequencing, of a plurality of polypeptides simultaneously (multiplexing).
  • Multiplexing refers to analysis of a plurality of polypeptides in the same assay.
  • the plurality of polypeptides can be derived from the same sample or different samples.
  • the plurality of polypeptides can be derived from the same subject or different subjects.
  • the plurality of polypeptides that are analyzed can be different polypeptides, or the same polypeptide derived from different samples.
  • Sample multiplexing can be achieved by upfront barcoding of recording tag labeled polypeptide samples. Each barcode represents a different sample, and samples can be pooled prior to cyclic binding assays or sequence analysis. In this way, many barcode-labeled samples can be simultaneously processed in a single tube.
  • This approach is a significant improvement on immunoassays conducted on reverse phase protein arrays (RPPA) (Akbani et al., Mol Cell Proteomics (2014) 13(7): 1625-1643; Creighton et al., Drug Des Devel Ther (2015) 9: 3519-3527; Nishizuka et al., Drug Metab Pharmacokinet (2016) 31(1): 35-45).
  • RPPA reverse phase protein arrays
  • the present disclosure essentially provides a highly digital sample and analyte multiplexed alternative to the RPPA assay with a simple workflow.
  • kits comprising one or more modified cleavases comprising a mutation, e.g ., one or more amino acid modifications in an unmodified cleavase and a reagent for or labeling the terminal amino acid of the polypeptide.
  • the modified cleavase is derived from a dipeptide cleavase and removes a single labeled terminal amino acid from a polypeptide or the modified cleavase is derived from a tripeptide cleavase and removes a single labeled terminal amino acid from a polypeptide or a single labeled terminal dipeptide from a polypeptide.
  • kits also include other components for treating the polypeptides and analysis of the polypeptides, including tags (e.g., a DNA tag or a DNA recording tag), solid supports, and other reagents for preparing the polypeptides and reagents for polypeptide analysis.
  • tags e.g., a DNA tag or a DNA recording tag
  • solid supports e.g., a DNA tag or a DNA recording tag
  • other reagents for preparing the polypeptides and reagents for polypeptide analysis.
  • the kit comprises more than one modified cleavase.
  • a variety of modified cleavases may exhibit different characteristics, for example, preferences for binding polypeptides and/or cleaving amino acids.
  • two or more modified cleavases may be included in the kit as a mixture of enzymes or separately with each modified cleavase in a container.
  • the different modified cleavases are contacted with polypeptides simultaneously or sequentially.
  • the kit also comprises one or more additional enzymes to eliminate the NTAA (e.g, a proline aminopeptidase).
  • the additional enzyme is a proline aminopeptidase, a proline iminopeptidase (PIP), or a pyroglutamate aminopeptidase (pGAP).
  • PIP proline iminopeptidase
  • pGAP pyroglutamate aminopeptidase
  • one or more modified cleavases are provided in combination with other enzymes in the kit.
  • the modified cleavase and other enzymes are provided as a cocktail in the kit.
  • the kit comprises a library of binding agents.
  • the two or more binding agents may be provided in individual containers or as a mixture in a container.
  • the kit further includes a reagent for transferring the identifying information of the coding tag to a recording tag attached to the polypeptide, wherein the transferring of the identifying information to the recording tag generates an extended recording tag on the polypeptide.
  • the reagent for transferring identifying information is a chemical ligation reagent or a biological ligation reagent.
  • the kit further includes an amplification reagent for amplifying the extended recording tags.
  • the kit further comprises substrates selected from the group consisting of a bead, a porous bead, a magnetic bead, a paramagnetic bead, a porous matrix, an array, a surface, a glass surface, a silicon surface, a plastic surface, a slide, a filter, nylon, a chip, a silicon wafer chip, a flow through chip, a biochip including signal transducing electronics, a well, a microtitre well, a plate, an ELISA plate, a disc, a spinning interferometry disc, a membrane, a nitrocellulose membrane, a nitrocellulose-based polymer surface, a nanoparticle (e.g ., comprising a metal such as magnetic nanoparticles (FesCri), gold
  • nanoparticles and/or silver nanoparticles
  • quantum dots a nanoshell, a nanocage, a
  • the kit includes one or more reagents for nucleic acid sequence analysis.
  • the reagent for sequence analysis is for use in sequencing by synthesis, sequencing by ligation, sequencing by hybridization, polony sequencing, ion semiconductor sequencing, pyrosequencing, single molecule real-time sequencing, nanopore- based sequencing, or direct imaging of DNA using advanced microscopy, or any combination thereof.
  • kit components any of the above-mentioned kit components, and any molecule, molecular complex or conjugate, reagent (e.g ., chemical or biological reagents, including modified cleavases), agent, structure (e.g., support, surface, particle, or bead), reaction intermediate, reaction product, binding complex, or any other article of manufacture disclosed and/or used in the exemplary kits and methods, may be provided separately or in any suitable combination in order to form a kit.
  • the kit may optionally comprise instructions for using the modified cleavase.
  • a modified cleavase comprising a mutation, e.g, one or more amino acid modification(s), in an unmodified cleavase, wherein:
  • the modified cleavase is derived from a dipeptide cleavase and removes or is configured to remove a single labeled terminal amino acid from a polypeptide;
  • modified cleavase of any one of embodiments 1-3 wherein the modified cleavase comprises an active site that interacts with the amide bond between the terminal labeled amino acid residue and a penultimate terminal amino acid residue of the polypeptide.
  • unmodified cleavase is selected from the group consisting of a metallopeptidase, a zinc-dependent metallopeptidase, or a zinc-dependent hydrolase.
  • modified cleavase of any one of embodiments 1-17 wherein the modified cleavase comprises an amino acid sequence that exhibits at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, or at least 90 % or more identity with the unmodified cleavase.
  • modified cleavase of any one of embodiments 1-28 wherein the modified cleavase comprises an amino acid sequence that exhibits at least 20 % identity, at least 30 % identity, at least 40 % identity, at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, or at least 90 % or more identity to any of SEQ ID NOs: 17-19, 23-28, 31-39, or a specific binding fragment thereof.
  • modified cleavase of any one of embodiments 1-29 comprising the sequence of amino acids set forth in any of SEQ ID NOs: 17-19, 23-28, or a sequence of amino acids that exhibits at least 95% sequence identity to any of SEQ ID NOs: 17-19, 23-28, 31-39, or a specific binding fragment thereof.
  • modified cleavase of any one of embodiments 1-30 wherein the modified cleavase comprises one or more amino acid modifications in an unmodified cleavase, corresponding to position(s) 126, 188, 189, 190, 191, 192, 196, 238, 302, 306, 307, 310, 525, 528, 546, 604, 650, 651, 655, 656, 665, and/or 692, with reference to numbering of SEQ ID NO: 13. 32.
  • N191T/R196H/N306A/D650G N191M/R196H/N306A/D650G, N191V/N306A/D650S, or N191 S/N306G/D650S.
  • modified cleavase of any one of embodiments 1-37 wherein the modified cleavase comprises an amino acid sequence that comprises a loop domain with at least 20 % identity, at least 30 % identity, at least 40 % identity, at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, or at least 90 % or more identity with the loop domain of any of SEQ ID NOs: 17-19, 23-28, or 31-39.
  • modified cleavase of any one of embodiments 1-38 wherein the modified cleavase comprises one or more amino acid modifications in an unmodified cleavase, corresponding to position(s) 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, and/or 202, with reference to numbering of SEQ ID NO: 13.
  • modified cleavase of any one of embodiments 1-38 wherein the modified cleavase comprises one or more amino acid modifications in an unmodified cleavase, corresponding to position(s) 191, 192, 196, 306, 310, 627, 628, 630, 648, 650, 651, 655, 656, and/or 669 with reference to numbering of SEQ ID NO: 13.
  • a method of treating a polypeptide comprising contacting the polypeptide with a modified cleavase comprising a mutation, e.g ., one or more amino acid modification(s), in an unmodified cleavase, wherein:
  • the modified cleavase is derived from a dipeptide cleavase and removes or is configured to remove a single labeled terminal amino acid from a polypeptide;
  • the modified cleavase is derived from a tripeptide cleavase and removes or is configured to remove a single labeled terminal amino acid from a polypeptide or a single labeled terminal dipeptide from a polypeptide.
  • modified cleavase comprises an active site that interacts with the amide bond between the terminal labeled amino acid residue and a penultimate terminal amino acid residue of the polypeptide.
  • cleavase is a dipeptidyl peptidase, a dipeptidyl aminopeptidase, a peptidyl-dipeptidase, a dipeptidyl carboxypeptidase, a sedolisin, or a tripeptidyl peptidase.
  • cleavase is a dipeptidyl peptidase 3, dipeptidyl peptidase 5, dipeptidyl peptidase 7, dipeptidyl peptidase 11, dipeptidyl aminopeptidase BII, or dipeptidyl peptidase BII.
  • NAA N-terminal amino acid
  • CAA C-terminal amino acid
  • modified cleavase comprises an amino acid sequence that exhibits at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, or at least 90 % or more identity with the unmodified cleavase.
  • any one of embodiments 43-54 wherein the length of the polypeptide is greater than 4 amino acids, greater than 5 amino acids, greater than 6 amino acids, greater than 7 amino acids, greater than 8 amino acids, greater than 9 amino acids, greater than 10 amino acids, greater than 11 amino acids, greater than 12 amino acids, greater than 13 amino acids, greater than 14 amino acids, greater than 15 amino acids, greater than 20 amino acids, greater than 25 amino acids, or greater than 30 amino acids.
  • modified cleavase is derived from a dipeptidyl aminopeptidase BII or dipeptidyl peptidase BII as provided in SEQ ID NO: 13 or 20.
  • modified cleavase comprises an amino acid sequence that exhibits at least 20 % identity, at least 30 % identity, at least 40 % identity, at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, or at least 90 % or more identity to any of SEQ ID NOs: 17-19, 23-28, 31-39, or a specific binding fragment thereof.
  • modified cleavase comprises one or more amino acid modifications in an unmodified cleavase, corresponding to position(s) 126, 188, 189, 190, 191, 192, 196, 238, 302, 306, 307, 310, 525, 528, 546, 604, 650, 651, 655, 656, 665, and/or 692, with reference to numbering of SEQ ID NO: 13.
  • N191T/R196H/N306A/D650G N191M/R196H/N306A/D650G, N191V/N306A/D650S, or N191S/N306G/D650S.
  • the modified cleavase comprises an amino acid sequence that comprises an amine binding site with at least 20 % identity, at least 30 % identity, at least 40 % identity, at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, or at least 90 % or more identity with the amine binding site of any of SEQ ID NOs: 17-19, 23-28, or 31-39.
  • modified cleavase comprises an amino acid sequence that comprises a loop domain with at least 20 % identity, at least 30 % identity, at least 40 % identity, at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, or at least 90 % or more identity with the loop domain of any of SEQ ID NOs: 17-19, 23-28, or 31-39.
  • modified cleavase comprises one or more amino acid modifications in an unmodified cleavase, corresponding to position(s) 188, 189, 190, 191, 192, 302, and/or 310, with reference to numbering of SEQ ID NO: 13.
  • modified cleavase comprises one or more amino acid modifications in an unmodified cleavase, corresponding to position(s) 191, 192, 196, 306, 310, 627, 628, 630, 648, 650, 651, 655, 656, and/or 669 with reference to numbering of SEQ ID NO: 13.
  • a chemical reagent is selected from the group consisting of a phenyl isothiocyanate (PITC), a nitro-PITC, a sulfo- PITC, a phenyl isocyanate (PIC), a nitro-PIC, a sulfo-PIC, Cbz-Cl (benzyl chloroformate) or Cbz-OSu (benzyloxycarbonyl N-succinimide), a carboxyl-activated amino-blocked amino acid, a l-fluoro-2, 4-dinitrobenzene (Sanger’s reagent, DNFB), dansyl chloride (DNS-C1, or 1- dimethylaminonaphthalene-5-sulfonyl chloride), 4-sulfonyl-2-nitrofluorobenzene (SNFB), an anhydride, 2-Pyridinecarboxaldehyde, 2-
  • Pentafluorophenylisothiocyanate 4-(Trifluoromethoxy)-phenylisothiocyanate, 4- (Trifluoromethyl)-phenylisothiocyanate, 3-(Carboxylic acid)-phenylisothiocyanate, 3- (Trifluoromethyl)-phenylisothiocyanate, 1-Naphthylisothiocyanate, N-nitroimidazole-1- carboximidamide, N,N’ -Bis(pivaloyl)- lH-pyrazole- 1 -carboxamidine, N,N’ - Bis(benzyloxycarbonyl)-lH-pyrazole-l-carboxamidine, an acetylating reagent, a
  • the contacting with the reagent for labeling the terminal amino acid is before the contacting with the binding agent
  • the contacting with the binding agent is before the contacting of the polypeptide with the modified cleavase.
  • a method for analyzing a polypeptide comprising the steps of:
  • binding agent capable of binding to the terminal amino acid of the polypeptide, wherein the binding agent comprises a coding tag with identifying information regarding the binding agent;
  • the modified cleavase is derived from a dipeptide cleavase and removes or is configured to remove a single terminal amino acid labeled by the reagent in step (c) from the polypeptide; or
  • the modified cleavase is derived from a tripeptide cleavase and removes or is configured to remove a single terminal amino acid or a single terminal dipeptide labeled by the reagent in step (c) from the polypeptide.
  • 105. The method of embodiment 104, wherein the modified cleavase derived from the dipeptide cleavase or tripeptide cleavase is configured to cleave the peptide bond between a terminal labeled amino acid residue and a penultimate terminal amino acid residue of the polypeptide.
  • cleavase is a dipeptidyl peptidase, a dipeptidyl aminopeptidase, a peptidyl-dipeptidase, a dipeptidyl carboxypeptidase, a sedolisin, or a tripeptidyl peptidase.
  • cleavase is a dipeptidyl peptidase 3, dipeptidyl peptidase 5, dipeptidyl peptidase 7, dipeptidyl peptidase 11, dipeptidyl aminopeptidase BII, or dipeptidyl peptidase BII.
  • binding agent comprises two or more binding agents.
  • step (a) is performed before step (b);
  • step (a) is performed before step (c);
  • step (a) is performed before step (d);
  • step (b) is performed before step (c);
  • step (b) is performed before step (d);
  • step (bl) is performed after step (a);
  • step (bl) is performed after step (b);
  • step (bl) is performed before step (c);
  • step (bl) is performed before step (d);
  • step (c) is performed before step (a);
  • step (c) is performed before step (b);
  • step (c) is performed before step (d).
  • the recording tag is a DNA molecule, an RNA molecule, a PNA molecule, a BNA molecule, an XNA, molecule, an LNA molecule, a gRNA molecule, or a combination thereof.
  • the solid support is a bead, a porous bead, a porous matrix, an array, a glass surface, a silicon surface, a plastic surface, a filter, a membrane, nylon, a silicon wafer chip, a flow through chip, a biochip including signal transducing electronics, a microtitre well, an ELISA plate, a spinning interferometry disc, a nitrocellulose membrane, a nitrocellulose-based polymer surface, a nanoparticle, or a microsphere.
  • the solid support comprises a polystyrene bead, a polyacrylate bead, a polymer bead, an agarose bead, a cellulose bead, a dextran bead, an acrylamide bead, a solid core bead, a porous bead, a paramagnetic bead, a glass bead, a controlled pore bead, a silica-based bead, or any combinations thereof.
  • the binding agent is an aminopeptidase or variant, mutant, or modified protein thereof; an aminoacyl tRNA synthetase or variant, mutant, or modified protein thereof; an anticalin or variant, mutant, or modified protein thereof; a ClpS, ClpS2, or variant, mutant, or modified protein thereof; a UBR box protein or variant, mutant, or modified protein thereof; or a modified small molecule that binds amino acid(s), i.e. vancomycin or a variant, mutant, or modified molecule thereof; or an antibody or binding fragment thereof; or any combination thereof.
  • analyzing the one or more extended recording tags comprises a nucleic acid sequencing method.
  • nucleic acid sequencing method is sequencing by synthesis, sequencing by ligation, sequencing by hybridization, polony sequencing, ion semiconductor sequencing, or pyrosequencing.
  • nucleic acid sequencing method is single molecule real-time sequencing, nanopore-based sequencing, or direct imaging of DNA using advanced microscopy.
  • kits for treating a polypeptide comprising:
  • modified cleavase comprising a mutation, e.g. , one or more amino acid modification(s), in an unmodified cleavase, wherein: the modified cleavase is derived from a dipeptide cleavase and removes or is configured to remove a single labeled terminal amino acid from a polypeptide; or
  • the modified cleavase is derived from a tripeptide cleavase and removes or is configured to remove a single labeled terminal amino acid from a polypeptide or a single labeled terminal dipeptide from a polypeptide;
  • kit of embodiment 146 wherein the modified cleavase derived from the dipeptide cleavase or tripeptide cleavase is configured to cleave the peptide bond between a terminal labeled amino acid residue and a penultimate terminal amino acid residue of the polypeptide.
  • kit of any one of embodiments 146-148, wherein the modified cleavase comprises an active site that interacts with the amide bond between the terminal labeled amino acid residue and a penultimate terminal amino acid residue of the polypeptide.
  • cleavase is a dipeptidyl peptidase, a dipeptidyl aminopeptidase, a peptidyl-dipeptidase, a dipeptidyl carboxypeptidase, a sedolisin, or a tripeptidyl peptidase.
  • kit of any one of embodiments 146-152, wherein the unmodified cleavase is a dipeptidyl peptidase 3, dipeptidyl peptidase 5, dipeptidyl peptidase 7, dipeptidyl peptidase 11, dipeptidyl aminopeptidase BII, or dipeptidyl peptidase BII.
  • NTAA N-terminal amino acid
  • CTAA C-terminal amino acid
  • the modified cleavase does not cleave the peptide bond between a penultimate terminal amino acid residue and an antepenultimate terminal amino acid residue of the polypeptide.
  • kit of any one of embodiments 146-156, wherein the modified cleavase comprises an amino acid sequence that exhibits at least 50 % identity, at least 60 % identity, at least 70 % identity, at least 80 % identity, or at least 90 % or more identity with the unmodified cleavase.
  • polypeptide is greater than 4 amino acids, greater than 5 amino acids, greater than 6 amino acids, greater than 7 amino acids, greater than 8 amino acids, greater than 9 amino acids, greater than 10 amino acids, greater than 11 amino acids, greater than 12 amino acids, greater than 13 amino acids, greater than 14 amino acids, greater than 15 amino acids, greater than 20 amino acids, greater than 25 amino acids, or greater than 30 amino acids.
  • polypeptide is greater than 10 amino acids.

Abstract

L'invention concerne des compositions et procédés d'inhibition, de traitement et/ou de prévention de la myélopathie dégénérative (DM) ou de la sclérose latérale amyotrophique (SLA). Selon la présente invention, des molécules d'acide nucléique pour inhiber l'expression du gène de superoxyde dismutase 1 (SOD) sont fournies. Dans un mode de réalisation particulier, les molécules d'acide nucléique comprennent un domaine de recuit lié de manière fonctionnelle à au moins un domaine effecteur, le domaine de recuit s'hybridant au pré-ARNm de SOD (par exemple, canin ou humain) et le domaine effecteur s'hybridant au snARN U1 du snRNP U1 .
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KR1020217033837A KR102567902B1 (ko) 2019-03-26 2020-03-24 변형된 클리바아제, 이의 용도 및 관련 키트
JP2021557332A JP2022526939A (ja) 2019-03-26 2020-03-24 修飾された切断酵素、その使用、および関連キット
AU2020247918A AU2020247918B2 (en) 2019-03-26 2020-03-24 Modified cleavases, uses thereof and related kits
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CA3134776A CA3134776A1 (fr) 2019-03-26 2020-03-24 Clivages modifies, utilisations et kits correspondants
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US17/850,516 US11788080B2 (en) 2019-03-26 2022-06-27 Modified cleavases, uses thereof and related kits
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CN114460052A (zh) * 2022-01-11 2022-05-10 武汉理工大学 一种基于荧光碳量子点直接检测丙酮酸钠浓度的方法

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WO2020198264A8 (fr) 2021-01-07
MX2021011726A (es) 2021-10-22
CA3134776A1 (fr) 2020-10-01
JP2022526939A (ja) 2022-05-27
EP3947667A1 (fr) 2022-02-09
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KR102567902B1 (ko) 2023-08-18
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