Macromolecule analysis employing nucleic acid encoding
Inventors
Chee, Mark S. • Gunderson, Kevin L. • Weiner, Michael Phillip
Assignees
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Publication Number
US-12320813-B2
Publication Date
2025-06-03
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 by partitioning a sample comprising a population of cells into a plurality of compartments, such that a single cell comprising peptides and at least one bead are comprised together within a single compartment. Each bead comprises nucleic acid recording tags attached thereto, and each nucleic acid recording tag comprises a functional moiety configured to covalently link the nucleic acid recording tag to a peptide of the single cell. After lysing cells in the compartments, peptides released from the single cell are immobilized on the bead and associated with a nucleic acid recording tag.
The immobilized peptides are then released with the beads and pooled, and the peptides are combinatorially indexed with barcodes through a series of split-and-pool steps, thereby generating at least one bead comprising immobilized barcoded peptides of the single cell. The immobilized barcoded peptides on the released beads are analyzed to analyze peptides of the single cell.
A further embodiment uses polymer-forming subunits in the compartments to form single cell gel beads. After lysing, polymerization forms single cell gel beads and immobilizes peptides inside a single cell gel bead within the compartment, followed by release and pooling of the single cell gel beads and barcoding by combinatorial split-and-pool steps using recording tags with barcodes and functional moieties configured to covalently link the recording tags to immobilized peptides.
Claims Coverage
Two independent claims are present. Together they cover compartmentalized single-cell peptide immobilization on tagged beads or in single cell gel beads, covalent linkage of recording tags to peptides, combinatorial split-and-pool barcode indexing, and analysis of immobilized barcoded peptides.
Compartmentalized single-cell peptide immobilization on beads with nucleic-acid recording tags
Partitioning cells into a plurality of compartments wherein a single cell comprising peptides and at least one bead are comprised within a single compartment; each bead comprises a plurality of nucleic acid recording tags attached thereto, and each nucleic acid recording tag comprises a functional moiety configured to covalently link the nucleic acid recording tag to a peptide of the peptides of the single cell; lysing cells and allowing immobilization of released peptides on the bead so each immobilized peptide is associated with a nucleic acid recording tag.
Combinatorial split-and-pool barcode indexing and analysis of immobilized barcoded peptides
Releasing the beads comprising immobilized peptides and pooling; combinatorially indexing the immobilized peptides on the released beads with barcodes through a series of split-and-pool steps to generate immobilized barcoded peptides of the single cell; analyzing the immobilized barcoded peptides on the released beads to analyze peptides of the single cell.
Single cell gel bead formation from polymer-forming subunits
Partitioning cells into compartments so that a single cell comprising peptides is within a single compartment, where the compartment comprises polymer-forming subunits; lysing cells and polymerizing the polymer-forming subunits to form single cell gel beads and allowing immobilization of the peptides inside a single cell gel bead within the compartment.
Covalent recording-tag barcoding of immobilized peptides on single cell gel beads
Releasing the single cell gel beads and pooling; 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 comprises a barcode and a functional moiety configured to covalently link the recording tag to an immobilized peptide.
The inventive features center on compartment-based association of single cells with tagged beads or single cell gel beads, covalent linkage of recording tags to immobilized peptides, combinatorial split-and-pool barcode indexing, and analysis of the immobilized barcoded peptides.
Stated Advantages
Documented Applications
Analyzing peptides of a single cell from a sample comprising a population of cells using compartmentalization, bead- or gel-bead-based immobilization of peptides with nucleic acid recording tags, combinatorial barcoding via split-and-pool steps, and analysis of the immobilized barcoded peptides.
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