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 analyzing a polypeptide, comprising:
(a) contacting (i) a plurality of fragments of a polypeptide, wherein each fragment is associated directly or indirectly with a recording tag, with (ii) a plurality of binding agents, wherein the recording tags comprise a common barcode, wherein each binding agent comprises a binding moiety and a coding tag comprising identifying information regarding the binding moiety, wherein the binding moiety is capable of binding to one or more amino acids of a fragment of the plurality of fragments, or capable of binding to the one or more amino acids modified by a functionalizing reagent, and wherein each fragment and/or its associated recording tag, or each binding agent, is immobilized directly or indirectly on a substrate;
(b) for each fragment of the plurality of fragments, allowing transfer of information between (i) the recording tag associated with the fragment and (ii) the coding tag of a binding agent of the plurality of binding agents, upon binding between the binding moiety of the binding agent and the one or more amino acids of the fragment, thereby generating an extended recording tag, an extended coding tag, or a di-tag construct for each fragment of the plurality of fragments, wherein the extended recording tag, the extended coding tag, or the di-tag construct comprises nucleic acid sequence information of the common barcode and the identifying information regarding the binding moiety; and
(c) analyzing nucleic acid sequence of the extended recording tag, the extended coding tag, or the di-tag construct to obtain at least partial amino acid sequence information of each fragment of the plurality of fragments and a nucleic acid sequence of the common barcode, and combining the at least partial amino acid sequence information of each fragment of the plurality of fragments using the nucleic acid sequence information of the common barcode to obtain amino acid sequence information regarding the polypeptide.
2 . The method of claim 1 , wherein in (c), the analyzing comprises concatenating the extended recording tags, the extended coding tags, or the di-tag constructs generated for two or more fragments of the plurality of fragments to create a long nucleic acid molecule comprising two or more different extended recording tags, extended coding tags or di-tag constructs, respectively, and wherein the long nucleic acid molecule is sequenced to obtain amino acid sequence information regarding the polypeptide.
3 . The method of claim 1 , wherein the binding moiety of a binding agent of the plurality of binding agents binds to a single amino acid of the fragment.
4 . The method of claim 1 , wherein the binding moiety of a binding agent of the plurality of binding agents binds to a single amino acid of the fragment, which is modified by the functionalizing reagent prior to the binding.
5 . The method of claim 4 , wherein the binding moiety of a binding agent of the plurality of binding agents selectively binds to the modified single amino acid of the fragment.
6 . The method of claim 1 , wherein the binding moiety of a binding agent of the plurality of binding agents binds to a dipeptide or a tripeptide sequence of the fragment.
7 . The method of claim 1 , wherein the binding moiety of a binding agent of the plurality of binding agents comprises an aptamer, a polypeptide, or an antibody or antigen binding fragment thereof.
8 . The method of claim 1 , wherein 1,000,000 or more polypeptide molecules are analyzed in parallel.
9 . The method of claim 1 , wherein the recording tag comprises a barcode for the polypeptide.
10 . The method of claim 1 , wherein in (b), the transfer of information between the recording tag and the coding tag comprises a ligation.
11 . The method of claim 1 , wherein in (b), the transfer of information between the recording tag and the coding tag comprises a primer extension.
12 . The method of claim 1 , wherein the recording tag and the coding tag comprise complementary spacer sequences.
13 . The method of claim 1 , wherein the plurality of fragments of the polypeptide are obtained by a chemical cleavage of the polypeptide.
14 . The method of claim 1 , wherein the plurality of fragments of the polypeptide are obtained by an enzymatic cleavage of the polypeptide.
15 . The method of claim 1 , wherein analyzing the extended recording tag, the extended coding tag, or the di-tag construct comprises obtaining the identifying information regarding the binding moiety bound to each fragment of the plurality of fragments.
16 . The method of claim 1 , wherein in (c), the analyzing comprises determining an amino acid sequence of the polypeptide.
17 . The method of claim 1 , wherein in (c), the analyzing comprises determining an amino acid sequence of each fragment of the plurality of fragments.
18 . A method for analyzing a plurality of polypeptides, comprising:
(a) immobilizing the plurality of polypeptides to a plurality of substrates, wherein each substrate comprises a plurality of recording tags;
(b) fragmenting the polypeptides immobilized on a substrate of the plurality of substrates, thereby generating an immobilized plurality of polypeptide fragments on the substrate;
(c) contacting the immobilized plurality of polypeptide fragments with a plurality of binding agents, wherein each binding agent comprises a binding moiety and a coding tag comprising identifying information regarding the binding moiety, wherein the binding moiety is capable of binding to one or more amino acids of a fragment of the plurality of fragments, or capable of binding to the one or more amino acids modified by a functionalizing reagent;
(d) for each fragment of the immobilized plurality of polypeptide fragments, allowing transfer of information between (i) the recording tag associated with each fragment and (ii) the coding tag, upon binding between the binding moiety and the one or more amino acids of the fragment, thereby generating an extended recording tag, an extended coding tag, or a di-tag construct for each fragment of the immobilized plurality of polypeptide fragments, wherein the extended recording tag, the extended coding tag, or the di-tag construct comprises information from the recording tag and the coding tag; and
(e) concatenating the extended recording tags, the extended coding tags, or the di-tag constructs generated for two or more fragments of the immobilized plurality of polypeptide fragments to create a long nucleic acid molecule comprising two or more different extended recording tags, extended coding tags, or di-tag constructs, or complements thereof, and sequencing the long nucleic acid molecule to obtain amino acid sequence information regarding the plurality of polypeptides.
19 . The method of claim 18 , wherein the binding moiety of a binding agent of the plurality of binding agents binds to a single amino acid of the fragment.
20 . The method of claim 18 , wherein the binding moiety of a binding agent of the plurality of binding agents binds to a single amino acid of the fragment, wherein the single amino acid is modified by the functionalizing reagent prior to the binding.
21 . The method of claim 20 , wherein the binding moiety of a binding agent of the plurality of binding agents selectively binds to the modified single amino acid of the fragment.
22 . The method of claim 18 , wherein the plurality of polypeptides comprises 1,000,000 or more polypeptide molecules.
23 . The method of claim 18 , wherein the recording tag comprises a barcode for the polypeptide.
24 . The method of claim 18 , wherein the recording tag comprises a universal priming site and/or a unique molecular identifier (UMI).
25 . The method of claim 18 , wherein in (d), the transfer of information between the recording tag and the coding tag comprises a primer extension.
26 . The method of claim 18 , wherein the recording tag and the coding tag comprise complementary spacer sequences.
27 . The method of claim 18 , wherein in (e), the analyzing comprises determining an amino acid sequence of at least one polypeptide of the plurality of polypeptides.
28 . The method of claim 18 , wherein in (c), the analyzing comprises determining an amino acid sequence of each fragment of the immobilized plurality of polypeptide fragments.
29 . The method of claim 1 , wherein the common barcode is a unique molecular identifier for the polypeptide.