Methods and kits using nucleic acid encoding and/or label
Inventors
Chee, Mark S. • Gunderson, Kevin L. • Weiner, Michael Phillip • Shi, Lei • MURANAKA, Norihito
Assignees
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Publication Number
US-12129463-B2
Publication Date
2024-10-29
Expiration Date
Abstract
Methods and Kits for analyzing macromolecules, including peptides, polypeptides, and proteins, employing nucleic acid encoding are disclosed. The sample analysis kits employ nucleic acid encoding and/or nucleic acid recording of a molecular interaction and/or reaction, such as recognition events (e.g., between an antigen and an antibody, between a modified terminal amino acid residue, or between a small molecule or peptide therapeutic and a target, etc.). Assays that do not require the cyclic transfer of information between a coding tag and a recording tag are also disclosed, including single cycle assays.
Core Innovation
The invention provides a method for assaying interactions between a set of polypeptides from different biological samples and a library of binding agents by using nucleic-acid recording tags and nucleic-acid coding tags. Polypeptides from different biological samples are labeled with recording tags comprising a polypeptide-specific barcode and a sample-specific barcode, pooled, and immobilized on a plurality of solid supports, with multiple molecules of different polypeptides on each solid support spatially separated by at least 50 nm.
The immobilized polypeptides are contacted with a library of binding agents, each comprising a polypeptide or an aptamer and associated with a nucleic acid coding tag providing identifying information regarding the binding agent. Following binding, identifying information is transferred between nucleic acid coding tag(s) associated with the binding agent and nucleic acid recording tag(s) associated with the immobilized polypeptides by primer extension and/or ligation, generating extended nucleic acid recording tag(s) and/or extended nucleic acid coding tag(s).
The extended nucleic acid recording tag(s) and/or extended nucleic acid coding tag(s) are analyzed by nucleic acid sequencing to obtain identifying information regarding the binding agents and the polypeptides, including their sample origin. The method thereby assaying the interactions and creates a polypeptide-agent binding matrix having size of at least 10^6, with dependent refinements describing non-covalent or covalent binding, a larger matrix size of at least 10^9, density constraints, and polypeptide-specific barcodes as unique molecular identifiers.
Claims Coverage
The independent claim covers immobilized, sample-barcoded polypeptides, a nucleic-acid-tagged binding-agent library, transfer of identifying information by primer extension and/or ligation, and sequencing-based readout to form a polypeptide-agent binding matrix of at least 10^6. The claim set includes 8 inventive features, with dependent refinements addressing binding mode, matrix scale, density constraints, and barcode function.
Barcoded recording-tag immobilization of sample polypeptides
Polypeptides from different biological samples are labeled with recording tags each comprising a polypeptide-specific barcode and a sample-specific barcode, pooled, and immobilized on a plurality of solid supports, wherein multiple molecules of different polypeptides on each solid support are spatially separated by at least 50 nm.
Coding-tag-associated binding agents for identifying information
Contacting the immobilized polypeptides with a library of binding agents, wherein each binding agent comprises a polypeptide or an aptamer and is associated with a nucleic acid coding tag providing identifying information regarding the binding agent.
Primer extension and/or ligation transfer to extended tags
Following binding, identifying information is transferred between nucleic acid coding tag(s) associated with the binding agent and nucleic acid recording tag(s) associated with the immobilized polypeptides by primer extension and/or ligation, generating extended nucleic acid recording tag(s) and/or extended nucleic acid coding tag(s).
Sequencing readout to identify both partners and sample origin
Analyzing the extended nucleic acid recording tag(s) and/or extended nucleic acid coding tag(s) by nucleic acid sequencing to obtain identifying information regarding the binding agents and the polypeptides including their sample origin.
Polypeptide-agent binding matrix creation
Assaying the interactions between the set of polypeptides and the library of binding agents and creating a polypeptide-agent binding matrix having size of at least 10^6.
Non-covalent versus covalent binding refinement
The binding agents bind non-covalently to the immobilized polypeptides and/or bind covalently to the immobilized polypeptides.
Increased polypeptide-agent binding matrix scale
The method is performed using a polypeptide-agent binding matrix having a size of at least 10^9.
Polypeptide-specific barcode as a unique molecular identifier
Each polypeptide-specific barcode of the recording tags is a unique molecular identifier.
The coverage centers on immobilizing barcoded polypeptides, contacting them with coding-tagged binding agents, transferring identifying information by primer extension and/or ligation, and reading out both partners by nucleic acid sequencing to form a large polypeptide-agent binding matrix. Dependent refinements specify binding mode, matrix scale, barcode role as a unique molecular identifier, and density-related constraints on solid supports.
Stated Advantages
Allows assaying interactions between polypeptides from different biological samples and a library of binding agents.
Enables identification of binding agents and polypeptides including sample origin by nucleic acid sequencing.
Creates a polypeptide-agent binding matrix having size of at least 10^6.
Optionally reaches larger polypeptide-agent binding matrix sizes, including at least 10^9 as stated in a dependent claim.
Documented Applications
Assaying interactions between a set of polypeptides from different biological samples and a library of binding agents to produce a polypeptide-agent binding matrix.
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