Systems and methods for biomolecule retention

Patent metadata

Field	Value
Publication number	US-12071618-B2
Application number	US-202418416639-A
Country	US
Kind code	B2
Filing date	Jan 18, 2024
Priority date	Mar 11, 2021
Publication date	Aug 27, 2024
Grant date	Aug 27, 2024

Abstract

Compositions, systems, and methods for the display of analytes such as biomolecules are described. Display of analytes is achieved by coupling of the analytes to displaying molecules that are configured to associate with surfaces or interfaces. Arrays of analytes may be formed from the described systems for utilization in assays and other methods.

First claim

The invention claimed is:

1. A method, comprising:
a) providing a solid support comprising a plurality of sites, wherein each individual site of the plurality of sites comprises:
i) one and only one nucleic acid nanostructure coupled to the individual site; and
ii) one and only one polypeptide of interest attached to the one and only one nucleic acid nanostructure;

b) delivering binding reagents to the solid support, thereby coupling binding reagents to polypeptides of interest at sites of the plurality of sites;
c) repeating step b) using second binding reagents instead of the binding reagents, wherein the second binding reagents are different from the binding reagents;
d) for each individual site, detecting presence or absence of coupling of each individual binding reagent and second binding reagent to the polypeptide of interest; and
e) based upon presence or absence of coupling of each individual binding reagent and second binding reagent to the polypeptide of interest, identifying the polypeptide of interest at each individual site of the plurality of sites.

2. The method of claim 1 , wherein providing the solid support comprises providing the solid support contacted with a fluidic medium comprising a denaturing species.

3. The method of claim 1 , wherein delivering binding reagents to the solid support comprises delivering a fluidic medium comprising the binding reagents and a denaturing species.

4. The method of claim 1 , wherein the binding reagents comprise affinity reagents.

5. The method of claim 4 , wherein an affinity reagent of the affinity reagents comprises an aptamer.

6. The method of claim 4 , wherein an affinity reagent of the affinity reagents comprises an antibody.

7. The method of claim 4 , wherein coupling binding reagents to polypeptides of interest comprises coupling an affinity reagent to an epitope of the polypeptide of interest.

8. The method of claim 4 , wherein coupling binding reagents to polypeptides of interest comprises coupling an affinity reagent to a modified N-terminal amino acid of the polypeptide of interest.

9. The method of claim 8 , further comprising cleaving the modified N-terminal amino acid of the polypeptide of interest after coupling the affinity reagent to the modified N-terminal amino acid.

10. The method of claim 1 , wherein delivering the binding reagents to the solid support comprises delivering an N-terminal modifying agent.

11. The method of claim 1 , wherein a polypeptide of interest has a full-length primary amino acid structure.

12. The method of claim 1 , wherein a polypeptide of interest is a fragment of a full-length primary amino acid structure.

13. The method of claim 1 , wherein providing the solid support comprises providing the solid support comprising a first species of polypeptide and a second species of polypeptide.

14. The method of claim 13 , wherein the first species of polypeptide and the second species of polypeptide have a dynamic range of at least 10 4 .

15. The method of claim 1 , wherein providing the solid support comprises providing the solid support comprising at least 1000 unique polypeptide species of interest.

16. The method of claim 1 , wherein providing the solid support comprises providing the solid support comprising at least 25% of polypeptide species of a proteome.

17. The method of claim 1 , wherein detecting presence or absence of coupling of each individual binding reagent to the polypeptide of interest comprises detecting presence or absence of fluorescent signals at an address of the site comprising the polypeptide of interest.

18. The method of claim 17 , wherein detecting presence or absence of fluorescent signals at an address of the site comprising the polypeptide of interest occurs for each instance of delivering binding reagents to the solid support.

19. The method of claim 1 , wherein detecting presence or absence of coupling of each individual binding reagent to the polypeptide of interest comprises detecting presence or absence of a nucleic acid tag.

20. The method of claim 1 , wherein identifying the polypeptide of interest comprises identifying a species of the polypeptide of interest.

21. The method of claim 1 , wherein identifying the polypeptide of interest comprises identifying an amino acid sequence of the polypeptide of interest.

22. The method of claim 1 , wherein identifying the polypeptide of interest comprises identifying a proteoform of the polypeptide of interest.

23. The method of claim 1 , wherein the polypeptide of interest is attached to the nucleic acid nanostructure by a linking moiety.

24. The method of claim 23 , wherein the linking moiety provides a separation gap of at least 10 nanometers (nm) between the polypeptide of interest and the solid support.

25. The method of claim 1 , further comprising removing the binding reagents from the polypeptides of interest.

26. The method of claim 25 , wherein removing the binding reagents occurs between step b) and step c).

27. The method of claim 25 , wherein removing the binding reagents occurs after step c).

28. The method of claim 14 , wherein the first species of polypeptide and the second species of polypeptide have a dynamic range of at least 106.

29. The method of claim 14 , wherein identifying the polypeptide of interest at each individual site of the plurality of sites comprises identifying a polypeptide of the first species of polypeptide and a polypeptide of the second species of polypeptide.

30. The method of claim 15 , wherein identifying the polypeptide of interest at each individual site of the plurality of sites comprises identifying the 1000 unique polypeptide species of interest.

31. The method of claim 16 , wherein identifying the polypeptide of interest at each individual site of the plurality of sites comprises identifying the at least 25% of polypeptide species of the proteome.

32. The method of claim 24 , wherein the linking moiety provides a separation gap of at least 20 nanometers (nm) between the polypeptide of interest and the solid support.

Assignees

• Nautilus Subsidiary Inc

Inventors

• Aksel Tural
• Hendricks Stephen
• Ikwa Elvis
• Indermuhle Pierre
• Ingle Sadie
• Inman Christina
• Mallick Parag
• Rinker Torri Elise
• Tan Steven

Classifications

• G01N33/5436

  with ligand physically entrapped within the solid phase (liposomes G01N33/5432; immunological test elements G01N33/54386) · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6837

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• B82Y5/00

  Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery · CPC title

• B01J2219/0061

  The surface being organic · CPC title