Kits for analysis using nucleic acid encoding and/or label
Inventors
Chee, Mark S. • Beierle, John M. • MURANAKA, Norihito • Gunderson, Kevin L. • Weiner, Michael Phillip • Shi, Lei • James, Robert C. • Monfregola, Luca
Assignees
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Publication Number
US-12292446-B2
Publication Date
2025-05-06
Expiration Date
Abstract
Kits and methods of using the 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.). Additional barcoding reagents, such as those for cycle-specific barcoding (e.g., “clocking”), compartment barcoding, combinatorial barcoding, spatial barcoding, or any combination thereof, may be included in the kits. The sample may comprise macromolecules, including peptides, polypeptides, and proteins, and the recording may generate molecular interaction and/or reaction information, and/or polypeptide sequence information. The kits may be used in high-throughput, multiplexed, and/or automated analysis, and are suitable for analysis of a proteome or subset thereof.
Core Innovation
The invention relates to a kit for analyzing a polypeptide by functionalizing an N-terminal amino acid (NTAA) of the polypeptide to generate a functionalized NTAA. The kit includes one or more nucleic acid recording tags configured to associate directly or indirectly with the polypeptide or with a component obtained by cleavage of the functionalized NTAA, and each nucleic acid recording tag comprises a unique molecular identifier (UMI).
The kit further includes one or more binding agents, each comprising a binding moiety that binds to the functionalized NTAA or the component obtained by cleavage and a nucleic acid coding tag attached to the binding moiety. The nucleic acid coding tag comprises a barcode sequence that comprises identifying information regarding the binding moiety, and the nucleic acid recording tags and nucleic acid coding tags are configured to allow transfer of sequence information of the barcode sequence and/or the UMI by ligation or primer extension upon binding.
By ligation or primer extension, the kit generates one or more extended nucleic acid constructs each comprising the barcode sequence or a complement thereof and the UMI or a complement thereof. The disclosure also describes cyclic transfer of nucleic-acid coding tag information to nucleic-acid recording tags or di-tags using repeated binding cycles, and includes multiplexing features such as compartment/spatial/cycle barcoding, universal priming sites, spacers, and error-correcting codes.
Claims Coverage
The consolidated claim coverage reflects one independent claim and includes four core inventive features: NTAA functionalization, UMI-containing nucleic acid recording tags, binding agents carrying nucleic acid coding tags with barcode sequences, and transfer of sequence information by ligation or primer extension.
Functionalizing reagent for n-terminal amino acid labeling
A functionalizing reagent that reacts with an N-terminal amino acid (NTAA) of the polypeptide to generate a functionalized NTAA of the polypeptide.
Nucleic acid recording tags with unique molecular identifiers
One or more nucleic acid recording tags configured to associate directly or indirectly with the polypeptide or with a component obtained by cleavage of the functionalized NTAA, wherein each nucleic acid recording tag comprises a unique molecular identifier (UMI).
Binding agents with nucleic acid coding tags and barcode sequences
One or more binding agents each comprising a binding moiety that binds to the functionalized NTAA or the component obtained by cleavage of the functionalized NTAA, and a nucleic acid coding tag attached to the binding moiety comprising a barcode sequence that comprises identifying information regarding the binding moiety.
Ligation or primer extension transfer of barcode and UMI information
The nucleic acid recording tags and the nucleic acid coding tags are configured to allow transfer of sequence information of the barcode sequence and/or the UMI by ligation or primer extension upon binding, thereby generating one or more extended nucleic acid constructs each comprising the barcode sequence or a complement thereof and the UMI or a complement thereof.
The consolidated claim coverage centers on a kit that combines NTAA functionalization with UMI-bearing nucleic acid recording tags and barcode-bearing binding agents, with barcode and/or UMI sequence transfer by ligation or primer extension to form extended nucleic acid constructs.
Stated Advantages
Enables analyzing a polypeptide using NTAA functionalization with nucleic acid recording tags carrying UMIs and binding agents carrying barcode coding tags, where binding followed by ligation or primer extension generates extended nucleic acid constructs containing barcode and UMI information.
Allows transfer of sequence information of the barcode sequence and/or the UMI by ligation or primer extension to generate extended nucleic acid constructs.
Supports repeated binding cycles and cyclic transfer of nucleic-acid coding tag information to nucleic-acid recording tags or di-tags.
Documented Applications
Analyzing a polypeptide, including peptide/protein analysis with analysis of resulting first, second, and third-order tags.
Processing and analysis of extended recording/coding/di-tag libraries using sequencing and UMI-based mapping.
Multi-cycle NTAA/proteome analysis for proteome partitioning and mapping/phasing of proteoforms [procedural detail omitted for safety].
Array-based spatial proteomics, including tissue slice contexts, for recording tag coupling/transfer [procedural detail omitted for safety].
Single cell proteomics workflows using droplet/compartment barcoded beads and droplet barcoding [procedural detail omitted for safety].
Target enrichment by hybrid capture for proteomics workflows [procedural detail omitted for safety].
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