Methods and kits using nucleic acid encoding and/or label
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.
1. A method for assaying interactions between a set of polypeptides from different biological samples and a library of binding agents, comprising:
(a) generating a set of immobilized polypeptides by
(i) labeling polypeptides from different biological samples with recording tags each comprising a polypeptide-specific barcode and a sample-specific barcode, thereby generating polypeptide-recording tag conjugates from different biological samples,
(ii) pooling the polypeptide-recording tag conjugates from different biological samples together; and
(iii) immobilizing the polypeptide-recording tag conjugates from different biological samples on a plurality of solid supports,
wherein for each solid support, multiple molecules of different polypeptides immobilized on the solid support are spatially separated by at least 50 nm;
(b) contacting:
the set of immobilized polypeptides with
the library of binding agents, wherein each binding agent from the library of binding agents comprises a polypeptide or an aptamer, and is associated with a nucleic acid coding tag that provides identifying information regarding the binding agent;
(c) following binding of one or more binding agents to one or more immobilized polypeptides, allowing transfer of identifying information regarding the one or more binding agent by primer extension and/or ligation between
nucleic acid coding tag(s) associated with the one or more binding agents and
nucleic acid recording tag(s) associated with the one or more immobilized polypeptides,
wherein the transfer of identifying information generates extended nucleic acid recording tag(s) associated with the one or more immobilized polypeptides and/or extended nucleic acid coding tag(s) associated with the one or more binding agents; and
(d) analyzing the extended nucleic acid recording tag(s) and/or extended nucleic acid coding tag(s) by nucleic acid sequencing to obtain the identifying information regarding the one or more binding agents and the identifying information regarding the one or more polypeptides including their sample origin,
thereby 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 .
2. The method of claim 1 , wherein the transfer of identifying information in (c) generates the extended nucleic acid recording tag(s) associated with the one or more immobilized polypeptides, and the method further comprises the following steps after (c) and before (d): (i) contacting the one or more immobilized polypeptides with a second library of binding agents, wherein each binding agent from the second library of binding agents comprises a polypeptide or an aptamer, and is attached to a second nucleic acid coding tag that provides identifying information regarding the binding agent; and (ii) following binding of one or more binding agents of the second library of binding agents to the one or more immobilized polypeptides, allowing transfer of identifying information regarding the one or more binding agent by primer extension and/or ligation from the second nucleic acid coding tag(s) associated with the one or more binding agents to the extended nucleic acid recording tag(s) associated with the one or more immobilized polypeptides, thereby generating further extended nucleic acid recording tag(s) associated with the one or more immobilized polypeptides, and wherein the further extended nucleic acid recording tag(s) are analyzed in (d) by nucleic acid sequencing.
3. The method of claim 1 , wherein each binding agent from the library of binding agents is associated with nucleic acid coding tag by using a mRNA/cDNA polynucleotide, which encodes the binding agent.
4. The method of claim 1 , wherein the set of polypeptides comprises immunoglobulin molecules from a biological sample, and the library of binding agents comprises polypeptide antigens or peptide antigens.
5. The method of claim 1 , wherein for each solid support, a density of immobilized nucleic acid recording tags is at least about 2-fold greater than a density of immobilized polypeptides.
6. The method of claim 1 , wherein during step (a) (iii) the polypeptide-recording tag conjugates are hybridized to capture nucleic acid tags immobilized on the plurality of solid supports.
7. The method of claim 1 , wherein each of the plurality of solid supports is a bead.
8. The method of claim 1 , wherein in (a), each immobilized polypeptide is covalently attached to the solid support.
9. The method of claim 1 , wherein in (a), multiple molecules of different polypeptides immobilized on the solid support are spatially separated by at least 100 nm.
10. The method of claim 1 , wherein in (c), the one or more binding agents bind(s) non-covalently to the one or more immobilized polypeptides.
11. The method of claim 1 , wherein in (c), the one or more binding agents bind(s) covalently to the one or more immobilized polypeptides.
12. The method of claim 1 , wherein each binding agent from the library of binding agents is covalently attached to the nucleic acid coding tag.
13. The method of claim 1 , wherein the polypeptide-agent binding matrix has size of at least 10 9 .
14. The method of claim 1 , wherein the step (a) (ii) is performed before the step (a) (iii).
15. The method of claim 1 , wherein each polypeptide-specific barcode of recording tags is a unique molecular identifier.