Macromolecule analysis employing nucleic acid encoding
Inventors
Chee, Mark S. • Gunderson, Kevin L. • Weiner, Michael Phillip
Assignees
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Publication Number
US-12019078-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 provides a plurality of labeled polypeptides in a plurality of compartments, where each polypeptide is labeled with a nucleic acid tag or a nucleic acid barcode or UMI tag. Each compartment comprises nucleic acid compartment tags with barcodes that are the same within an individual compartment and different across compartments, and the compartment tags or barcode/UMI information are transferred to the nucleic acid tags to create recording tag-labeled polypeptides.
The recording tag-labeled polypeptides are generated by transferring nucleic acid compartment tag information or coding-tag information to recording tags via primer extension and/or ligation. Each recording tag-labeled polypeptide includes one or more recording tags, including two or more separate recording tags, that comprise the barcode, the UMI tag, or a complement thereof.
The invention then fragments the recording tag-labeled polypeptides to obtain recording tag-labeled peptides, where each recording tag-labeled peptide comprises a recording tag with the barcode, the UMI tag, or a complement thereof. The framework includes compartment occupancy with fewer than a single polypeptide per compartment and supports sequence-based readout of peptide or protein binding events using sequencing methods including NGS and/or single-molecule sequencing.
Claims Coverage
Independent claims are directed to compartmented labeled polypeptides, transfer of compartment barcode or barcode/UMI information to nucleic acid tags via primer extension and/or ligation to form recording tag-labeled polypeptides, and fragmentation to obtain recording tag-labeled peptides that retain the barcode, UMI tag, or complement. Across the independent claims, the inventive features include compartment barcode partitioning, barcode/UMI-to-recording tag transfer, multi-recording-tag construction, and compartment occupancy constraints.
Compartmented barcode-labeled polypeptides for recording-tag peptide generation
Providing a plurality of labeled polypeptides in a plurality of compartments, wherein each polypeptide is labeled with a nucleic acid tag or a nucleic acid barcode or UMI tag, and each compartment comprises nucleic acid compartment tags with barcodes that are the same within an individual compartment and different across compartments.
Primer extension and/or ligation transfer from compartment tags to nucleic-acid tags
Transferring a nucleic acid compartment tag, coding-tag information, or a complement thereof to each nucleic acid tag of the polypeptide via primer extension and/or ligation, thereby generating recording tag-labeled polypeptides comprising one or more recording tags that include the barcode, UMI tag, or a complement thereof.
Fragmentation to obtain recording tag-labeled peptides
Fragmenting the recording tag-labeled polypeptides to obtain recording tag-labeled peptides, wherein each recording tag-labeled peptide comprises a recording tag including the barcode, the UMI tag, or the complement thereof.
Barcode/UMI-labeled polypeptide with distinct nucleic acid tags
Providing a labeled polypeptide in a compartment, where the polypeptide is labeled with a nucleic acid barcode or a UMI tag and one or more nucleic acid tags distinct from the nucleic acid barcode or UMI tag.
Transferring barcode/UMI to distinct nucleic acid tags via primer extension and/or ligation
Transferring the barcode or UMI tag, or a complement thereof, to each of the one or more distinct nucleic acid tags attached to the polypeptide via primer extension and/or ligation, thereby generating a recording tag-labeled polypeptide comprising two or more separate recording tags each including the barcode or UMI tag, or the complement thereof.
Compartment occupancy fewer than a single polypeptide per compartment
Providing a plurality of labeled polypeptides in a plurality of compartments, where each compartment comprises on average fewer than a single polypeptide, and each polypeptide is labeled with a nucleic acid barcode or a UMI tag and one or more nucleic acid tags.
Generating multi-recording-tag polypeptides by barcode/UMI transfer and fragmenting
For each polypeptide, transferring the barcode or UMI tag, or a complement thereof, to each of the one or more nucleic acid tags via primer extension and/or ligation to generate recording tag-labeled polypeptides comprising two or more separate recording tags, and fragmenting the recording tag-labeled polypeptides to obtain recording tag-labeled peptides that each comprise the barcode or UMI tag, or the complement thereof.
Across the independent claims, the inventive coverage centers on compartmentalizing labeled polypeptides, transferring compartment barcode and/or barcode/UMI information to nucleic acid tags via primer extension and/or ligation to form recording tag-labeled polypeptides with one or more recording tags, including two or more separate recording tags, and fragmenting to obtain recording tag-labeled peptides that retain the barcode, UMI, or complements. The independent claim set also includes compartment occupancy constraints.
Stated Advantages
Highly parallel, digital readout of peptide/protein binding events.
Identification and quantitation of the peptide/protein binding events by sequencing.
Documented Applications
Highly parallel, digital readout of peptide/protein binding events using nucleic-acid-encoded macromolecule analysis with extended recording tags that are sequenced for identification and quantitation.
Next-generation protein assay (NGPA) based on sequencing-encoded tag representations of peptide/protein binding events.
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