Methods for stable complex formation and related kits

Patent metadata

Field	Value
Publication number	US-11169157-B2
Application number	US-202117149324-A
Country	US
Kind code	B2
Filing date	Jan 14, 2021
Priority date	Jan 7, 2020
Publication date	Nov 9, 2021
Grant date	Nov 9, 2021

Abstract

The present disclosure relates to methods and kits for forming a stable complex comprising a binding agent and a target (e.g., a macromolecule). In some embodiments, the target comprises a peptide, a polypeptide, or a protein to be analyzed. In some embodiments, the present disclosure relates to formation of a stable complex comprising a binding agent and a target (e.g., a macromolecule) to be analyzed in a method which employs barcoding and nucleic acid encoding of molecular recognition events, and/or detectable labels. Provided herein is also a programmable system for information transfer comprising one or more adaptor molecules.

First claim

What is claimed is:

1. A method for analyzing a macromolecule comprising the steps of:
(a) providing the macromolecule joined to a support, wherein the macromolecule is associated with a first stabilizing component;
(b) binding a binding agent to the macromolecule by contacting the macromolecule with the binding agent capable of specifically binding to the macromolecule, wherein the binding agent is associated with a second stabilizing component, and the binding of the binding agent to the macromolecule does not depend on the presence of the first stabilizing component and the second stabilizing component;
(c) after binding of the binding agent to the macromolecule, linking the first and second stabilizing components together via a linking agent to form a stable complex comprising the binding agent, the macromolecule and the stabilizing components, wherein the first stabilizing component and the second stabilizing component are linked upon introduction of the linking agent that comprises a polypeptide that binds to the first stabilizing component and to the second stabilizing component; and
(d) analyzing the macromolecule by obtaining information about the binding agent bound to the macromolecule.

2. The method of claim 1 , wherein no covalent bonds are formed during formation of the stable complex.

3. The method of claim 1 , wherein the stabilizing components are linked upon introduction to light.

4. The method of claim 1 , wherein the first or second stabilizing component comprises a polynucleotide.

5. The method of claim 1 , wherein the first stabilizing component is the same as the second stabilizing component.

6. The method of claim 1 , wherein 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.

7. The method of claim 1 , wherein the method comprises contacting a plurality of binding agents with a single macromolecule, or contacting a plurality of binding agents with a plurality of macromolecules, and wherein at least one binding agent of the plurality of binding agents is capable of binding to the macromolecule and each binding agent of the plurality of binding agents is associated with the second stabilizing component.

8. The method of claim 1 , wherein the binding agent is fluorescently labeled to enable detection of the contact between the macromolecule and the binding agent; and analyzing the macromolecule comprises detecting the fluorescence from the binding agent after contacting the macromolecule.

9. The method of claim 1 , wherein the macromolecule comprises a polypeptide and the binding agent or a binding agent from the plurality of binding agents is capable of binding to a N-terminal amino acid (NTAA) of the polypeptide or to a modified NTAA of the polypeptide.

10. The method of claim 9 , wherein analyzing the macromolecule comprises identifying at least one amino acid residue of the polypeptide.

11. The method of claim 10 , wherein providing a macromolecule comprises providing the polypeptide associated with a recording tag; the binding agent or each binding agent from the plurality of binding agents is associated with a coding tag with identifying information regarding the binding agent; obtaining an information about the binding agent comprises transferring an information from the coding tag to the recording tag after binding of the binding agent to the macromolecule to generate an extended recording tag; and identifying at least one amino acid residue of the polypeptide comprises analyzing the extended recording tag.

12. The method of claim 11 , further comprising:
providing an adaptor molecule comprising a first hybridization sequence and a secondary tag, wherein the first hybridization sequence is substantially complementary to at least a portion of the coding tag, to allow hybridization between the first hybridization sequence and the coding tag; and
transferring information of the secondary tag to the recording tag to generate an extended recording tag, wherein the information of the secondary tag is transferred from the adaptor molecule to the recording tag after the coding tag associated with the binding agent hybridizes with the first hybridization sequence on the adaptor molecule.

13. The method of claim 11 , wherein transferring information of the coding tag to the recording tag is performed after the stabilizing components are linked together.

14. The method of claim 13 , wherein transferring information comprises contacting the coding tag with a reagent for transferring the identifying information, the reagent comprising a reagent for primer extension reaction, a chemical ligation reagent or a biological ligation reagent.

15. The method of claim 14 , wherein the stable complex is disrupted after the transfer of information from the coding tag to the recording tag by removing the linking agent from the stable complex or by introducing a destabilizing agent.

16. The method of claim 10 , further comprising contacting the polypeptide with a N-terminal modifier agent prior to binding of the binding agent to the polypeptide to form the modified NTAA of the polypeptide.

17. The method of claim 16 , further comprising removing the modified NTAA of the polypeptide after transferring the information from the coding tag to the recording tag to expose a new NTAA of the polypeptide.

18. The method of claim 17 , further comprising repeating at least one more time prior to analyzing the extended recording tag the steps of:
contacting the polypeptide with a N-terminal modifier agent to form the modified NTAA of the polypeptide;
contacting the polypeptide with a binding agent capable of binding to the modified NTAA of the polypeptide or with a plurality of binding agents wherein at least one binding agent of the plurality of binding agents is capable of binding to the modified NTAA of the polypeptide, wherein each binding agent of the plurality of binding agents is associated with the second stabilizing component;
linking the first and second stabilizing components together to form a stable complex comprising the binding agent, the macromolecule and the stabilizing components; and/or
optionally, removing the modified NTAA of the polypeptide.

19. The method of claim 10 , wherein the extended recording tag is analyzed using a nucleic acid sequencing method.

20. The method of claim 1 , wherein the macromolecule comprises a first stabilizing component, and the binding agent comprises a second stabilizing component.

21. The method of claim 1 , wherein the stable complex stabilizes interaction between the binding agent and the macromolecule during obtaining information about the binding agent bound to the macromolecule.

Assignees

• Encodia Inc

Inventors

• Gunderson Kevin L
• Muranaka Norihito

Classifications

• G01N33/54353

  with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393) · CPC title

• C40B70/00

  Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes · CPC title

• C40B20/04

  Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes · CPC title

• G01N33/54306 Primary

  Solid-phase reaction mechanisms · CPC title

• G01N33/582 Primary

  with fluorescent label · CPC title