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-12130291-B2
Publication Date
2024-10-29
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 immobilizing one or more polypeptide molecules on one or more solid supports so that each polypeptide molecule is associated with a nucleic acid tag and adjacent polypeptide molecules are spaced apart by an average distance of about 50 nm or greater. In the composition, at least 100,000 polypeptide molecules are covalently attached to each solid support, and coding-tagged binding agents enable attachment and recording of identifying information by hybridizing to a complementary sequence on the nucleic acid tag or forming a covalent bond with the nucleic acid tag.
A key aspect is controlling binding selectivity by providing at least 3 different binding agents that are selective for different polypeptide components defined by N-terminal amino acid (NTAA) residues or C-terminal amino acid (CTAA) residues. Each of the at least 3 different binding agents is bound to a distinct NTAA residue or distinct CTAA residue of the polypeptide molecules, thereby associating different encoder information with different terminal polypeptide components on the same solid support.
The document also describes nucleic-acid extended recording tag, coding tag, and di-tag library concatenation strategies for single-molecule nanopore sequencing with a direct solid-support readout. Recording tags, barcode elements, spacer sequences, universal priming site elements, and protected ssDNA embodiments are used to transfer or assemble coding-tag information across binding cycles while maintaining error-correcting or coding-based robustness.
Claims Coverage
The consolidated claim coverage identifies one independent composition claim centered on polypeptide-loaded solid supports, nucleic acid tags, and coding-tagged binding agents. Across the provided claim content, there are 3 inventive features: high-density covalently attached polypeptides with spacing, coding tags with encoder sequence identifying information that attach to nucleic acid tags, and at least 3 selective binding agents for different NTAA or CTAA residue components.
Solid support with covalently attached polypeptides and nucleic acid tags
One or more solid supports wherein each solid support comprises at least 100,000 polypeptide molecules covalently attached to the solid support, each polypeptide molecule associated with a nucleic acid tag, and adjacent polypeptide molecules spaced apart at an average distance of about 50 nm or greater.
Coding-tagged binding agents with encoder sequence identifying information
A plurality of binding agents each bound to one of the at least 100,000 polypeptide molecules, each binding agent comprising a coding tag with an encoder sequence identifying information regarding the binding agent, wherein the coding tag attaches to the nucleic acid tag by hybridizing to a complementary sequence on the nucleic acid tag or by forming a covalent bond with the nucleic acid tag.
At least three terminal-residue-selective binding agents
At least 3 different binding agents, each selective for a different polypeptide component, wherein each different binding agent is bound to a different N-terminal amino acid (NTAA) residue or a different C-terminal amino acid (CTAA) residue of the polypeptide molecules.
The claim coverage centers on a solid-support composition that displays densely loaded, spatially spaced polypeptides associated with nucleic acid tags, and uses coding-tagged binding agents to associate identifying information with terminal-residue-selective binding events. The same core structure is reinforced across the independent claim content.
Stated Advantages
The approach is described as reducing non-specific binding and intermolecular transfer that can generate false positives.
It is described as supporting error-correcting or coding-based robustness.
It enables digital protein (peptide/protein) identification and quantitation from sequencing-derived recording tags.
It enables multiplexing using sample barcodes as a digital alternative to RPPA.
It is described as improving electrical distinguishability in duplex interrupted nanopore sequencing.
It reduces proteome dynamic range through depletion, fractionation, and compartmentalization.
Incorrect pairing events decrease or disappear at extreme dilution.
Documented Applications
Single-molecule nanopore sequencing with a direct solid-support readout.
Digital protein (peptide/protein) identification and quantitation.
Multiplexing using sample barcodes.
Compartmentalized proteome mapping using compartment tags such as barcodes, spacers, and UMIs.
Sequencing-derived recording tags associated to a compartment or compartmental proteome.
Reference protein sequence database comparison.
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