Macromolecule analysis employing nucleic acid encoding
Inventors
Chee, Mark S. • Gunderson, Kevin L. • Weiner, Michael Phillip
Assignees
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Publication Number
US-12019077-B2
Publication Date
2024-06-25
Expiration Date
Abstract
A method for analyzing macromolecules, including peptides, polypeptides, and proteins, employing nucleic acid encoding is disclosed.
Core Innovation
The invention relates to analyzing peptides of a single cell from a sample comprising a population of cells by using nucleic acid recording tags on beads to immobilize peptides in a compartmentalized workflow. Cells are partitioned into a plurality of compartments, and each single cell containing peptides together with at least one bead is held in the same compartment. Each nucleic acid recording tag includes a compartment-specific barcode and a functional moiety configured to covalently link the nucleic acid recording tag to a peptide, thereby immobilizing the peptide on the bead via the nucleic acid recording tag attached thereto.
After lysis in the compartments, the released peptides from the single cell become immobilized on the at least one bead, generating immobilized barcoded peptides associated with the nucleic acid recording tag. The beads are released from the compartments and pooled. The pooled beads are then analyzed simultaneously by contacting the released beads with binding agents capable of binding to the immobilized barcoded peptides and obtaining information regarding the binding agent after binding.
The description includes nucleic-acid encoding for extended tag readout for macromolecules and peptides, including extended recording tags produced through coding-tag transfer to immobilized recording tags across one or multiple binding cycles. Peptide-specific cyclic NTAA labeling and alternative transfer schemes are described, including transfer from recording-to-coding tags and di-tag constructs, with tag components that can include UMIs, barcodes, universal priming sites, and spacers.
Claims Coverage
The claims cover 3 inventive features.
Compartmentalized peptide immobilization on barcoded nucleic acid recording tags
Partitioning cells of the population of cells into a plurality of compartments, wherein a single cell comprising peptides and at least one bead from a plurality of beads are comprised within a single compartment, and each bead comprises a plurality of nucleic acid recording tags attached thereto, wherein each nucleic acid recording tag comprises a barcode specific for each compartment and a functional moiety configured to covalently link the nucleic acid recording tag to a peptide of the peptides of the single cell, thereby immobilizing the peptide on the bead via the nucleic acid recording tag attached thereto.
Lysis to generate immobilized barcoded peptides associated with recording tags
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 barcoded peptides of the single cell, wherein each of the immobilized barcoded peptides is associated with a nucleic acid recording tag of the plurality of nucleic acid recording tags.
Pooling and simultaneous analysis using binding agents that provide binding-agent information
Releasing the beads comprising the immobilized barcoded peptides from the plurality of compartments and pooling the released beads, and analyzing the immobilized barcoded peptides on the released beads, wherein 10,000 or more immobilized barcoded peptides are analyzed simultaneously, and the analyzing comprises contacting the released beads with a plurality of binding agents capable of binding to the immobilized barcoded peptides and following binding of a binding agent to an immobilized barcoded peptide, obtaining information regarding the binding agent, thereby analyzing the immobilized barcoded peptides of the single cell.
The claims focus on compartmentalized immobilization of single-cell peptides on barcoded nucleic acid recording tags, lysis-generated immobilized barcoded peptides associated with the tags, and pooled simultaneous analysis with binding agents.
Stated Advantages
Allows simultaneous analysis of 10,000 or more immobilized barcoded peptides.
Documented Applications
Analyzing peptides of a single cell from a sample comprising a population of cells by compartmentalization, nucleic-acid-tag immobilization, bead pooling, and binding-agent-based analysis.
Obtaining information regarding binding agents after binding to immobilized barcoded peptides for analyzing peptides of the single cell.
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