Methods of polypeptide sequencing

Patent metadata

Field	Value
Publication number	US-2023016396-A1
Application number	US-202217848010-A
Country	US
Kind code	A1
Filing date	Jun 23, 2022
Priority date	Jun 28, 2021
Publication date	Jan 19, 2023
Grant date	—

Abstract

The present disclosure relates to methods and kits for performing an identification of a terminal amino acid residue of the polypeptide, or performing a polypeptide sequencing. The methods include a step of contacting the terminal amino acid residue of the polypeptide with a coupler, followed by attaching the coupler-polypeptide complex to the solid support and cleaving the coupler-polypeptide complex from the polypeptide, thereby isolating the terminal amino acid residue of the polypeptide from the remaining amino acid residues of the polypeptide in complex with the coupler, thereby enabling efficient identification of the terminal amino acid residue via recognition by binding agents capable of binding to the coupler-amino acid complex. In some embodiments, the coupler and the polypeptide are both associated with stabilizing components, and after binding of the coupler to the terminal amino acid of the polypeptide, tethering complex is formed between the stabilizing components releasably attached to the solid support.

First claim

1 . A method for identifying a polypeptide, comprising the steps of:
(a) providing a polypeptide and an associated recording tag attached to a solid support; (b) contacting the polypeptide with a coupler, wherein the coupler binds to a terminal amino acid of the polypeptide to form a coupler-polypeptide complex; (c) attaching the coupler to the solid support; (d) cleaving the peptide bond between the terminal amino acid and the penultimate terminal amino acid of the polypeptide within the coupler-polypeptide complex, thereby exposing a new terminal amino acid of the polypeptide and generating a coupler-amino acid complex attached to the solid support; (e) contacting the coupler-amino acid complex attached to the solid support with a binding agent capable of specific binding to the coupler-amino acid complex, wherein the binding agent comprises a coding tag with identifying information regarding the binding agent; (f) transferring the information of the coding tag of the binding agent to the recording tag to generate an extended recording tag; (g) releasing the coupler-amino acid complex from the solid support; (h) repeating steps (b) through (g) at least one more time; and (i) analyzing the extended recording tag after information transfer, thereby identifying at least a portion of the sequence of the polypeptide.

2 . The method of claim 1 , wherein the terminal amino acid is modified to produce a modified terminal amino acid before contacting the polypeptide with the coupler; at step (b) the coupler binds to the modified terminal amino acid; at step (d) a coupler-modified amino acid complex attached to the solid support is generated after cleavage of the peptide bond between the terminal amino acid and the penultimate terminal amino acid of the polypeptide; and at step (e) the binding agent is capable of binding to the coupler-modified amino acid complex.

3 . The method of claim 1 , wherein the coupler is releasably attached to the solid support.

4 . The method of claim 1 , wherein the polypeptide is further associated with a first stabilizing component and the coupler comprises a second stabilizing component; at step (c) after binding of the coupler to the terminal amino acid of the polypeptide, the first and second stabilizing components are linked together to form a tethering complex that comprises a first stabilizing component attached to the solid support and the second stabilizing component linked to the coupler-polypeptide complex; and at step (d) the coupler-amino acid complex generated after cleavage of the peptide bond between the terminal amino acid and the penultimate terminal amino acid of the polypeptide is releasably attached to the solid support via the tethering complex.

5 . The method of claim 4 , wherein the first stabilizing component is releasably associated with the polypeptide attached to the solid support.

6 . The method of claim 4 , wherein the stabilizing components are linked upon introduction of a linking agent that binds to the first stabilizing component and to the second stabilizing component.

7 . The method of claim 6 , wherein the linking agent is releasably associated with the first stabilizing component.

8 . The method of claim 6 , wherein the linking agent activates a stabilizing component.

9 . The method of claim 6 , wherein the linking agent comprises a linking polypeptide that binds to the first stabilizing component and to the second stabilizing component.

10 . The method of claim 4 , wherein the first or second stabilizing component comprises a polynucleotide; the stabilizing components are linked upon introduction of a linking agent that hybridizes to the polynucleotide of one of the stabilizing components.

11 . The method of claim 4 , wherein the first stabilizing component is the same as the second stabilizing component.

12 . The method of claim 4 , wherein the stabilizing components are linked upon introduction of a linking agent, the linking agent binds to the first stabilizing component and to the second stabilizing component, and the first stabilizing component has a lower affinity to the linking agent in comparison to an affinity of the second stabilizing component to the linking agent.

13 . The method of claim 1 , wherein the binding agent binds to the coupler-amino acid complex, but does not bind to the corresponding amino acid separated from the coupler-amino acid complex, or affinity of the binding agent for the corresponding amino acid separated from the coupler-amino acid complex is reduced compared to affinity of the binding agent for the coupler-amino acid complex by at least an order of magnitude.

14 . The method of claim 1 , wherein the extended recording tag after information transfer is analyzed using a nucleic acid sequencing method.

15 . The method of claim 1 , wherein the binding agent comprises an engineered carboxypeptidase.

16 . The method of claim 1 , wherein the coupler binds to a N-terminal amino acid (NTAA) of the polypeptide.

17 . The method of claim 1 , which further comprises releasing the coupler-amino acid complex from the solid support after transferring the information of the coding tag to the recording tag.

18 . A method for identifying a terminal amino acid of a polypeptide, comprising the steps of:
(a) providing a polypeptide and an associated recording tag attached to a solid support; (b) contacting the polypeptide with a coupler, wherein the coupler binds to a terminal amino acid of the polypeptide to form a coupler-polypeptide complex; (c) attaching the coupler to the solid support; (d) cleaving the peptide bond between the terminal amino acid and the penultimate terminal amino acid of the polypeptide within the coupler-polypeptide complex to generate a coupler-amino acid complex attached to the solid support; (e) contacting the coupler-amino acid complex with a binding agent capable of binding selectively to the coupler-amino acid complex, wherein the binding agent comprises a coding tag with identifying information regarding the binding agent; (f) transferring the information of the coding tag of the binding agent to the recording tag to generate an extended recording tag; and (g) analyzing the extended recording tag, thereby identifying the terminal amino acid of the polypeptide.

19 . A kit for analyzing a polypeptide, comprising:
a coupler, wherein the coupler is configured to bind to a terminal amino acid of the polypeptide to form a coupler-polypeptide complex; a reagent for cleaving the peptide bond between the terminal amino acid and the penultimate terminal amino acid of the polypeptide within the coupler-polypeptide complex to generate a coupler-amino acid complex; and a binding agent capable of binding to the coupler-amino acid complex.

Assignees

• Encodia Inc

Inventors

• Gunderson Kevin L
• Muranaka Norihito
• Shi Lei
• Verespy Iii Stephen

Classifications

• G01N33/582

  with fluorescent label · CPC title

• G01N33/6824 Primary

  involving N-terminal degradation, e.g. Edman degradation · CPC title

• C12N9/485 Primary

  Exopeptidases (3.4.11-3.4.19) · CPC title

• C12Y304/14

  Dipeptidyl-peptidases and tripeptidyl-peptidases (3.4.14) · CPC title

• G01N33/58

  involving labelled substances (G01N33/53 takes precedence) · CPC title