KITS FOR ANALYSIS USING NUCLEIC ACID ENCODING AND/OR LABEL
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.
1 . A method for analyzing a plurality of polypeptides in a sample, comprising:
(a) attaching each polypeptide of the plurality of polypeptides to a nucleic acid tag to generate a nucleic acid-polypeptide chimera, wherein at least 1,000,000 polypeptides are attached to the nucleic acid tags;
(b) hybridizing the nucleic acid tag in the nucleic acid-polypeptide chimera to a capture nucleic acid attached to a solid support;
(c) covalently attaching the nucleic acid-polypeptide chimera to the solid support; and
(d) contacting the covalently attached nucleic acid-polypeptide chimera with a plurality of binding agents, and wherein each binding agent of the plurality of binding agents binds to a component of one or more polypeptides of the plurality of polypeptides and comprises (i) a nucleic acid coding tag with identifying information regarding the binding agent or (ii) a detectable label, thereby analyzing the one or more polypeptides in the covalently attached nucleic acid-polypeptide chimera.
2 . The method of claim 1 , wherein each polypeptide of the plurality of polypeptides is attached to the nucleic acid tag via an N-terminal amino acid (NTAA) residue or C-terminal amino acid (CTAA) residue.
3 . The method of claim 1 , wherein each polypeptide of the plurality of polypeptides is attached to the nucleic acid tag via a lysine residue.
4 . The method of claim 1 , wherein the plurality of binding agents comprises 3 different binding agents each selective for a different component of one or more polypeptides of the plurality of polypeptides.
5 . The method of claim 4 , wherein each of the at least 3 different binding agents is configured to bind to an NTAA residue or CTAA residue of one or more polypeptides of the plurality of polypeptides.
6 . The method of claim 1 , wherein each binding agent of the plurality of binding agents comprises the detectable label which comprises a fluorescently moiety, a color-coded nanoparticle, a quantum dot, or any combination thereof.
7 . The method of claim 1 , wherein each binding agent of the plurality of binding agents comprises the nucleic acid coding tag with identifying information regarding the binding agent.
8 . The method of claim 1 , wherein each polypeptide of the plurality of polypeptides is chemically conjugated to an internal nucleotide of the nucleic acid tag.
9 . The method of claim 1 , wherein the capture nucleic acid comprises a hairpin region.
10 . The method of claim 1 , wherein at least some polypeptides of the plurality of polypeptides are each attached to multiple nucleic acid tags and are each fragmented or digested into smaller polypeptides, with each smaller polypeptide attached on average to one nucleic acid tag.
11 . The method of claim 1 , wherein the plurality of polypeptides is a proteome or subset thereof.
12 . The method of claim 1 , wherein the nucleic acid tag comprises a barcode or a unique molecular identifier (UMI).
13 . The composition of claim 12 , wherein the barcode comprises an analyte barcode, a compartment barcode, a partition barcode, a sample barcode, a fraction barcode, or any combination thereof.
14 . The method of claim 1 , wherein the solid support is a bead or a flow cell.
15 . The method of claim 1 , wherein the capture nucleic acid is attached to the solid support via a click chemistry reaction.
16 . The method of claim 1 , wherein the nucleic acid tags attached to the at least 1,000,000 polypeptides share a common sequence.
17 . The method of claim 16 , wherein the common sequence comprises a universal primer sequence.
18 . The method of claim 1 , wherein the nucleic acid-polypeptide chimeras are randomly attached to the solid support.