Methods for preparing analytes and related kits

Patent metadata

Field	Value
Publication number	US-11634709-B2
Application number	US-202117458199-A
Country	US
Kind code	B2
Filing date	Aug 26, 2021
Priority date	Apr 30, 2019
Publication date	Apr 25, 2023
Grant date	Apr 25, 2023

Abstract

Provided herein are methods for preparing and treating an analyte (e.g., a macromolecule or a plurality of macromolecules, peptides, polypeptides, and proteins) for analysis. In some embodiments, the analyte is prepared and treated in a method that includes the use of bait and capture nucleic acids, solid supports, and reaction mixtures including the bait and capture nucleic acids. In some embodiments, the analyte is coupled to a solid support. Also provided are kits containing components for performing the provided methods for preparing the analytes. In some embodiments, the methods are for preparing an analyte for sequencing. Provided herein are methods for preparing and treating an analyte (e.g., a macromolecule or a plurality of macromolecules, peptides, polypeptides, and proteins) for analysis. In some embodiments, the analyte is prepared and treated in a method that includes the use of bait and capture nucleic acids, solid supports, and reaction mixtures including the bait and capture nucleic acids. In some embodiments, the analyte is coupled to a solid support. Also provided are kits containing components for performing the provided methods for preparing the analytes. In some embodiments, the methods are for preparing an analyte for sequencing.

First claim

What is claimed is:

1. A method for treating a polypeptide analyte, comprising:
attaching the polypeptide analyte to a bait nucleic acid to generate a nucleic acid-analyte chimera, wherein the bait nucleic acid is covalently attached to the polypeptide analyte to generate the nucleic acid-analyte chimera;
bringing the nucleic acid-analyte chimera into proximity with a solid support by hybridizing the bait nucleic acid in the nucleic acid-analyte chimera to a capture nucleic acid attached to the solid support;
covalently coupling the nucleic acid-analyte chimera to the solid support, wherein a plurality of the nucleic acid-analyte chimeras is coupled on the solid support and adjacent nucleic acid-analyte chimeras coupled on the solid support are spaced apart from each other at an average distance of about 50 nm or greater;
contacting the polypeptide analyte covalently coupled to the solid support with a binding agent capable of binding to the polypeptide analyte, wherein the binding agent comprises a coding tag that comprises identifying information regarding the binding agent; and
transferring the identifying information regarding the binding agent from the coding tag to the bait nucleic acid or capture nucleic acid attached to the polypeptide analyte.

2. The method of claim 1 , wherein the polypeptide analyte is attached to the 3′ end of the bait nucleic acid or to the 5′ end of the bait nucleic acid.

3. The method of claim 1 , wherein the polypeptide analyte is attached to an internal position of the bait nucleic acid.

4. The method of claim 1 , further comprising attaching a barcode to the coupled nucleic acid-analyte chimera, wherein the barcode comprises a compartment barcode, a partition barcode, a sample barcode, a fraction barcode, or any combination thereof.

5. The method of claim 1 , wherein the polypeptide analyte is obtained by fragmenting proteins from a biological sample.

6. The method of claim 1 , wherein the bait nucleic acid and/or capture nucleic acid further comprises a universal priming site, wherein the universal priming site comprises a priming site for amplification, sequencing, or both.

7. The method of claim 1 , further comprising repeating one or more times:
contacting the polypeptide analyte with an additional binding agent capable of binding to the polypeptide analyte, wherein the additional binding agent comprises a coding tag that comprises identifying information regarding the additional binding agent; and
transferring the identifying information regarding the additional binding agent from the coding tag to the bait nucleic acid or capture nucleic acid attached to the polypeptide analyte.

8. The method of claim 1 , wherein adjacent nucleic acid-analyte chimeras coupled on the solid support are spaced apart from each other at an average distance of about 100 nm or greater.

9. The method of claim 1 , wherein adjacent nucleic acid-analyte chimeras coupled on the solid support are spaced apart from each other at an average distance of about 250 nm or greater.

10. The method of claim 1 , comprising covalently coupling the bait nucleic acid in the nucleic acid-analyte chimera to the capture nucleic acid attached to the solid support, thereby covalently coupling the nucleic acid-analyte chimera to the solid support.

11. The method of claim 10 , wherein the covalent coupling comprises covalently cross-linking the bait nucleic acid and the capture nucleic acid.

12. The method of claim 10 , wherein the covalent coupling comprises enzymatic ligation or chemical ligation of the bait nucleic acid and the capture nucleic acid.

13. The method of claim 1 , wherein the bait nucleic acid or the capture nucleic acid comprises a splint nucleic acid strand that bridges the bait nucleic acid and the capture nucleic acid via hybridization for covalent coupling of the bait nucleic acid and the capture nucleic acid.

14. The method of claim 13 , wherein the capture nucleic acid comprises the splint nucleic acid strand which comprises a sequence complementary to the bait nucleic acid.

15. The method of claim 1 , wherein the capture nucleic acid comprises an intramolecular duplex formed between mutually complementary nucleic acid regions, and the capture nucleic acid comprises a loop region connecting the mutually complementary nucleic acid regions.

16. The method of claim 1 , wherein the polypeptide analyte is indirectly attached to the bait nucleic acid via a linker and the nucleic acid-analyte chimera is indirectly coupled to the solid support.

17. The method of claim 1 , wherein the solid support is a porous bead.

18. The method of claim 1 , wherein the capture nucleic acid comprises a nucleic acid hairpin.

19. The method of claim 1 , wherein the transfer of the identifying information regarding the binding agent from the coding tag to the bait nucleic acid or capture nucleic acid occurs through a primer extension reaction or ligation.

20. A nucleic acid-analyte composition comprising a plurality of nucleic acid-analyte chimeras and a plurality of capture nucleic acids attached to a solid support, the nucleic acid-analyte composition generated by the steps of:
attaching a polypeptide analyte to a bait nucleic acid to generate a nucleic acid-analyte chimera, wherein the bait nucleic acid is covalently attached to the polypeptide analyte to generate the nucleic acid-analyte chimera;
bringing the nucleic acid-analyte chimera into proximity with the solid support by hybridizing the bait nucleic acid in the nucleic acid-analyte chimera to a capture nucleic acid attached to the solid support;
covalently coupling the nucleic acid-analyte chimera to the solid support, wherein the plurality of nucleic acid-analyte chimeras is coupled on the solid support and adjacent nucleic acid-analyte chimeras coupled on the solid support are spaced apart at an average distance of about 50 nm or greater;
contacting the polypeptide analyte covalently coupled to the solid support with a binding agent capable of binding to the polypeptide analyte, wherein the binding agent comprises a coding tag that comprises identifying information regarding the binding agent; and
transferring the identifying information regarding the binding agent from the coding tag to the bait nucleic acid or capture nucleic acid attached to the polypeptide analyte.

21. The nucleic acid-analyte composition of claim 20 , wherein the polypeptide analyte is attached to the 3′ end of the bait nucleic acid or to the 5′ end of the bait nucleic acid.

22. The nucleic acid-analyte composition of claim 20 , wherein the capture nucleic acid, the nucleic acid-analyte chimera, and/or the bait nucleic acid further comprises a barcode.

23. The nucleic acid-analyte composition of claim 20 , wherein the capture nucleic acid comprises a nucleic acid hairpin.

24. The nucleic acid-analyte composition of claim 20 , wherein the bait nucleic acid and/or capture nucleic acid further comprises a universal priming site, wherein the universal priming site comprises a priming site for amplification, sequencing or both.

25. The nucleic acid-analyte composition of claim 20 , wherein the solid support is a porous bead.

26. A kit for polypeptide analysis, comprising:
(a) a plurality of bait nucleic acids, each of the bait nucleic acids is configured to be covalently attached to a polypeptide analyte;
(b) a solid support comprising a plurality of capture nucleic acids att

Assignees

• Encodia Inc

Inventors

• Chee Mark S
• Gunderson Kevin L
• Muranaka Norihito
• Weinert Brian Tate

Classifications

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C07K19/00

  Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes} · CPC title