Sequential encoding methods and related kits
The present disclosure relates to methods and kits for analyzing a macromolecule. In some embodiments, the present disclosure relates to macromolecule analysis methods which employ barcoding and nucleic acid encoding of molecular recognition events. Also provided herein is a method and related kits for transferring information using a plurality of enzymes, including for performing a ligation, extension, and cleavage reaction with nucleic acid molecules associated with the macromolecule for analysis. In some embodiments, the macromolecule for analysis comprises a peptide, a polypeptide, or a protein.
1 . A method for analyzing a macromolecule, comprising the steps of:
(a) providing a macromolecule and an associated recording tag joined to a support, wherein the recording tag comprises a double stranded region;
(b) contacting the macromolecule with a binding agent, wherein the binding agent comprises a coding tag with identifying information regarding the binding agent, wherein the binding agent binds to the macromolecule;
(c) covalently joining the 5′ end of the recording tag to the 3′end of the coding tag by a nucleic acid joining reagent;
(d) following (c), extending the recording tag using the coding tag as a template for a polymerase, thereby generating a double stranded extended recording tag; and
(e) cleaving both strands of the double stranded extended recording tag with a double strand nucleic acid cleaving reagent to generate a 3′ overhang in the extended recording tag, wherein a length of the 3′ overhang in the extended recording tag is less than 6 base pairs;
whereby information is transferred from the coding tag to the recording tag to generate the extended recording tag.
2 . The method of claim 1 , wherein in step (d), the double strand nucleic acid cleaving reagent recognizes a recognition sequence of the double stranded extended recording tag.
3 . The method of claim 1 , wherein the cleavage in step (e) releases the binding agent from the macromolecule.
4 . The method of claim 1 , wherein steps (b), (c), (d) and (e) are repeated sequentially one or more times in a cyclic manner.
5 . The method of claim 1 , wherein the macromolecule is a polypeptide.
6 . The method of claim 5 , wherein the polypeptide is obtained by fragmenting a protein from a biological sample.
7 . The method of claim 4 , further comprising removing a portion of the macromolecule prior to repeating step (b).
8 . The method of claim 5 , further comprising removing an N-terminal amino acid (NTAA) of the polypeptide to expose a new NTAA of the polypeptide prior to repeating step (b).
9 . The method of claim 4 , wherein the 3′ overhang of the extended recording tag generated by the double strand nucleic acid cleaving reagent in step (e) is available to hybridize with a second coding tag when step (b) is repeated.
10 . The method of claim 1 , wherein in step (b), the macromolecule is contacted with a plurality of binding agents, wherein the plurality of binding agents comprises the binding agent, and each binding agent of the plurality of binding agents comprises a coding tag with identifying information regarding the corresponding binding agent.
11 . The method of claim 5 , further comprising treating the polypeptide with a reagent for modifying a terminal amino acid of the polypeptide prior to step (b).
12 . The method of claim 1 , wherein the recording tag associated with the macromolecule comprises a nucleic acid hairpin.
13 . The method of claim 1 , wherein the nucleic acid joining reagent of step (c), the polymerase of step (d) and the double strand nucleic acid cleaving reagent of step (e) are provided simultaneously.
14 . The method of claim 1 , wherein the double strand nucleic acid cleaving reagent is a type IIS restriction enzyme.
15 . The method of claim 1 , further comprising analyzing one or more of the extended recording tags, wherein analyzing the extended recording tag(s) comprises a nucleic acid sequencing method.