Macromolecule analysis employing nucleic acid encoding

Patent metadata

Field	Value
Publication number	US-12320813-B2
Application number	US-202418659334-A
Country	US
Kind code	B2
Filing date	May 9, 2024
Priority date	May 2, 2016
Publication date	Jun 3, 2025
Grant date	Jun 3, 2025

Abstract

A method for analyzing macromolecules, including peptides, polypeptides, and proteins, employing nucleic acid encoding is disclosed.

First claim

The invention claimed is:

1. A method for analyzing peptides of a single cell from a sample comprising a population of cells, the method comprising:
(a) partitioning cells of the population of cells into a plurality of compartments, wherein
i) a single cell comprising peptides and at least one bead from a plurality of beads are comprised within a single compartment; and
ii) each bead of the plurality of beads comprises a plurality of nucleic acid recording tags attached thereto, wherein each nucleic acid recording tag of the plurality of nucleic acid recording tags comprises a functional moiety configured to covalently link the nucleic acid recording tag to a peptide of the peptides of the single cell;
(b) lysing cells in the compartments and allowing immobilization of the peptides released from the single cell on the at least one bead comprised within the single compartment, thereby generating at least one bead comprising immobilized peptides of the single cell, wherein each of the immobilized peptides is associated with a nucleic acid recording tag of the plurality of nucleic acid recording tags;
(c) releasing the beads comprising immobilized peptides from the plurality of compartments and pooling the released beads;
(d) combinatorically indexing the immobilized peptides on the released beads with barcodes through a series of split-and-pool steps, thereby generating at least one bead comprising immobilized barcoded peptides of the single cell; and
(e) analyzing the immobilized barcoded peptides on the released beads, thereby analyzing peptides of the single cell.

2. The method of claim 1 , wherein prior to immobilization of the peptides released from the single cell on the at least one bead, the peptides are digested with a protease, and or peptides produced by the protease digestion are immobilized on the at least one bead.

3. The method of claim 1 , wherein, on each bead of the plurality of beads, the nucleic acid recording tags are spaced apart from each other on a surface or within a volume of the bead by at least 50 nm.

4. The method of claim 1 , wherein analyzing the immobilized barcoded peptides on the released beads comprises: (i) contacting the released beads with a plurality of binding agents capable of binding to the immobilized barcoded peptides; (ii) following binding of a binding agent from the plurality of binding agents to an immobilized barcoded peptide, obtaining information regarding the binding agent.

5. A method for analyzing peptides of a single cell from a sample comprising a population of cells, the method comprising:
(a) partitioning cells of the population of cells into a plurality of compartments such that a single cell comprising peptides is comprised within a single compartment, wherein the compartment comprises polymer-forming subunits;
(b) lysing cells in the compartments, polymerizing the polymer-forming subunits to form single cell gel beads and allowing immobilization of the peptides from the single cell inside a single cell gel bead within the compartment, thereby generating at least one single cell gel bead comprising immobilized peptides of the single cell;
(c) releasing the single cell gel beads comprising the immobilized peptides from the plurality of compartments and pooling the released beads;
(d) barcoding the immobilized peptides on the released single cell gel beads via combinatorial indexing through a series of split-and-pool steps by adding a plurality of recording tags, wherein each recording tag of the plurality of recording tags comprises a barcode and a functional moiety configured to covalently link the recording tag to an immobilized peptide of the immobilized peptides, thereby generating at least one single cell gel bead comprising immobilized barcoded peptides of the single cell; and
(e) analyzing the immobilized barcoded peptides on the released single cell gel beads, thereby analyzing peptides of the single cell.

6. The method of claim 5 , wherein the peptides are digested with a protease inside the single cell gel bead.

7. The method of claim 5 , wherein, on each single cell gel bead, the recording tags are spaced apart from each other within a volume of the single cell gel bead by at least 50 nm.

8. The method of claim 5 , wherein analyzing the immobilized barcoded peptides on the released single cell gel beads comprises: (i) contacting the released single cell gel beads with a plurality of binding agents capable of binding to the immobilized barcoded peptides; (ii) following binding of a binding agent from the plurality of binding agents to an immobilized barcoded peptide, obtaining information regarding the binding agent.

9. The method of claim 5 , wherein prior to barcoding the immobilized peptides on the released single cell gel beads, the peptides are contacted with a labeling reagent that is configured to provide the peptides with a reactive coupling moiety capable of reacting with the functional moiety of the recording tags.

Assignees

• Encodia Inc

Inventors

• Chee Mark S
• Gunderson Kevin L
• Weiner Michael Phillip

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C40B70/00

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

• C40B40/04

  Libraries containing only organic compounds · 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

• G01N33/6818 Primary

  Sequencing of polypeptides · CPC title