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 composition comprising:
(i) one or more solid supports, wherein each solid support comprises at least 100,000 polypeptide molecules covalently attached to the solid support, wherein each of the at least 100,000 polypeptide molecules is associated with a nucleic acid tag, and wherein adjacent polypeptide molecules on the same solid support are spaced apart from each other on a surface or within a volume of the solid support at an average distance of about 50 nm or greater; and
(ii) a plurality of binding agents each bound to one of the at least 100,000 polypeptide molecules, wherein each of the plurality of binding agents comprises a coding tag that comprises an encoder sequence comprising identifying information regarding the binding agent, and wherein the coding tag of at least one binding agent that is bound to a polypeptide molecule of the at least 100,000 polypeptide molecules attaches to the nucleic acid tag associated with the polypeptide molecule by either hybridizing to a complementary sequence on the nucleic acid tag or forming a covalent bond with the nucleic acid tag,
wherein the composition comprises at least 3 different binding agents, wherein each of the at least 3 different binding agents is selective for a different polypeptide component, and wherein each of the at least 3 different binding agents is bound to (i) a N-terminal amino acid (NTAA) residue of a polypeptide molecule, and the different polypeptide component is a different NTAA residue of the polypeptide molecules, or (ii) a C-terminal amino acid (CTAA) residue of a polypeptide molecule, and the different polypeptide component is a different CTAA residue of the polypeptide molecules.
2. The composition of claim 1 , wherein each polypeptide molecule is covalently attached to the nucleic acid tag associated therewith.
3. The composition of claim 1 , wherein the polypeptide molecules are randomly attached to the support(s).
4. The composition of claim 1 , wherein each polypeptide molecule is covalently attached to one of the support(s) through an oligonucleotide.
5. The composition of claim 4 , wherein the oligonucleotide is covalently attached to the C-terminal amino acid residue of the polypeptide molecule.
6. The composition of claim 4 , wherein the oligonucleotide comprises the nucleic acid tag associated with the polypeptide molecule.
7. The composition of claim 6 , wherein the oligonucleotide comprises a nucleic acid hairpin.
8. The composition of claim 1 , wherein each polypeptide molecule comprises an N-terminal or C-terminal amino acid residue modified by a functionalizing reagent.
9. The composition of claim 1 , wherein each polypeptide molecule comprises an N-terminal amino acid (NTAA) residue modified by a functionalizing reagent.
10. The composition of claim 1 , wherein the nucleic acid tag associated with each polypeptide molecule comprises a barcode or a unique molecular identifier (UMI).
11. The composition of claim 10 , wherein the barcode comprises an analyte barcode, a compartment barcode, a partition barcode, a sample barcode, a fraction barcode, or any combination thereof.
12. The composition of claim 10 , wherein the nucleic acid tags associated with the polypeptide molecules share a common sequence.
13. The composition of claim 10 , wherein the common sequence comprises a universal primer sequence and a common spacer sequence flanking the barcode or UMI.
14. The composition of claim 1 , wherein the one or more solid supports are one or more beads or one or more flow cells.
15. The composition of claim 14 , comprising at least 500,000 polypeptides covalently attached to the same bead.
16. The composition of claim 1 , wherein the plurality of binding agents comprises binders bound to modified N-terminal or C-terminal amino acid residues of at least some of the polypeptide molecules.
17. The composition of claim 1 , wherein the plurality of binding agents comprises an aminopeptidase or a variant, mutant, or modified molecule thereof; an aminoacyl-tRNA synthetase or a variant, mutant, or modified molecule thereof; an anticalin or a variant, mutant, or modified molecule thereof; a ClpS or a variant, mutant, or modified molecule thereof; a UBR box protein or a variant, mutant, or modified molecule thereof; a vancomycin or a variant, mutant, or modified molecule thereof; an antibody or an antigen-binding fragment thereof; or an aptamer; or any combination thereof.