# US 2023/0056532 A1 · Methods for information transfer and related kits

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- Number: US 2023/0056532 A1
- Document ID: US20230056532A1
- Status: Published application
- Kind: A1
- Application number: 17/790,876
- Filed: 2021-01-05
- Published: 2023-02-23
- Earliest claimed: 2020-01-07
- Assignees: Encodia, Inc.
- Applicants: Encodia, Inc.
- Inventors: Kevin L. GUNDERSON; Norihito MURANAKA
- Web page: https://patentgrep.com/patent/US20230056532A1

## CPC

- G01N 33/54353: {with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393)}
- C12Q 1/6813: Hybridisation assays
- C40B 20/04: Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
- C40B 70/00: Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
- G01N 33/54306: {Solid-phase reaction mechanisms}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

The present disclosure relates to methods and kits for analyzing a macromolecule including information transfer between molecules, such as transfer of identifying information between nucleic acid molecules. In some embodiments, the macromolecule for analysis comprises a peptide, a polypeptide, or a protein. In some embodiments, the present disclosure relates to macromolecule analysis methods which employ barcoding and nucleic acid encoding of molecular recognition events, and/or detectable labels. Provided herein is a programmable system for information transfer comprising one or more adaptor molecules.

## Claims

### Claim 1

A method for analyzing a macromolecule, comprising the steps of:
(a) providing a macromolecule and an associated recording tag joined to a support;
(b) contacting the macromolecule with a binding agent capable of binding to the macromolecule, wherein the binding agent comprises a coding tag with identifying information regarding the binding agent, to allow binding between the macromolecule and the binding agent;
(c) providing an adaptor molecule comprising a first hybridization sequence and a secondary tag, wherein the first hybridization sequence is substantially complementary to at least a portion of the coding tag, to allow hybridization between the first hybridization sequence and the coding tag, wherein step (c) is performed before, after or simultaneously with step (b);
(d) transferring information of the secondary tag to the recording tag to generate an extended recording tag, wherein the information of the secondary tag is transferred from the adaptor molecule to the recording tag after the coding tag associated with the binding agent hybridizes with the first hybridization sequence on the adaptor molecule; and
(e) analyzing the extended recording tag.

### Claim 2 (depends on 1)

The method of claim 1, wherein step (b) comprises contacting a plurality of macromolecules with a plurality of binding agents and step (c) comprises providing a plurality of adaptor molecules, wherein the plurality of adaptor molecules comprises at least one adaptor molecule capable of hybridizing to at least one coding tag associated with the binding agent.

### Claim 3 (depends on 1)

The method of claim 1, wherein multiple coding tags associated with the binding agent are configured to hybridize to adaptor molecules comprising the same secondary tag.

### Claim 4 (depends on 1)

The method of claim 1, wherein the adaptor molecule further comprises a second hybridization sequence substantially complementary to a sequence at the 3′ terminus of the recording tag or substantially complementary to a region on the recording tag generated from a previous information transfer of the secondary tag from the adaptor molecule to the recording tag, and wherein information transfer of the secondary tag from the adaptor molecule to the recording tag occurs after:
the first hybridization sequence on the adaptor molecule hybridizes to the coding tag of the binding agent; and
the second hybridization sequence of the adaptor molecule hybridizes to a portion of the recording tag.

### Claim 5 (depends on 1)

The method of claim 1, wherein the macromolecule is a polypeptide, analyzing the macromolecule comprises determining at least a portion of an amino acid sequence of the polypeptide, and recording tag, the coding tag and the adaptor molecule comprise a DNA molecule, an RNA molecule, a PNA molecule, a BNA molecule, an XNA, molecule, an LNA molecule, a γPNA molecule, or a combination thereof.

### Claim 6 (depends on 4)

The method of claim 4, wherein the second hybridization sequence on the adaptor molecule comprises the secondary tag information or portion thereof on a different adaptor molecule.

### Claim 7 (depends on 1)

The method of claim 1, wherein information transfer from the secondary tag to the recording tag is mediated by a DNA ligase or DNA polymerase.

### Claim 8

(canceled)

### Claim 9 (depends on 7)

The method of claim 7, wherein the adaptor molecule comprises a spacer to stop extension after transfer of information from the secondary tag to the recording tag.

### Claim 10 (depends on 1)

The method of claim 1, wherein the secondary tag comprises a binding cycle specific sequence.

### Claim 11 (depends on 10)

The method of claim 10, wherein the adaptor molecule comprises from 5′ to 3′ direction: the first hybridization sequence, the secondary tag, and the second hybridization sequence, and the secondary tag comprises an identifying information regarding the binding agent, a binding cycle-specific barcode, a unique molecular identifier, or a combination thereof.

### Claim 12 (depends on 5)

The method of claim 5, wherein the binding agent is configured to bind to an N-terminal amino acid (NTAA) residue of the polypeptide.

### Claim 13 (depends on 5)

The method of claim 5, wherein the method further comprises the following step: (a′) modifying an N-terminal amino acid (NTAA) residue of the polypeptide, thereby producing a modified NTAA residue, and the binding agent is configured to bind to the modified NTAA residue of the polypeptide.

### Claim 14 (depends on 13)

The method of claim 13, further comprising the following steps after step (d) and before step (e): (i) removing the modified NTAA residue of the polypeptide to expose immediately adjacent amino acid residue of the polypeptide as a new NTAA residue; (ii) modifying the new NTAA residue, thereby producing a new modified NTAA residue; (iii) contacting the polypeptide with a second binding agent capable of binding to the new modified NTAA residue of the polypeptide, wherein the second binding agent comprises a second coding tag with identifying information regarding the second binding agent, to allow binding between the polypeptide and the binding agent; (iv) providing a second adaptor molecule comprising a second first hybridization sequence substantially complementary to at least a portion of the second coding tag, and a second secondary tag, to allow hybridization between the second first hybridization sequence and the second coding tag; (v) transferring an information of the second secondary tag to the extended recording tag, wherein the information of the second secondary tag is transferred from the second adaptor molecule to the extended recording tag after the second coding tag associated with the second binding agent hybridizes with the second first hybridization sequence on the second adaptor molecule.

### Claim at position 15

15-16. (canceled)

### Claim 17

A kit for analyzing a macromolecule, comprising:
a binding agent comprising a coding tag, which comprises identifying information regarding the binding agent, wherein the binding agent is configured to bind to the macromolecule associated with a recording tag joined to a support;
an adaptor molecule comprising a first hybridization sequence substantially complementary to at least a portion of the coding tag, and a secondary tag,
wherein an information of the secondary tag is configured for transferring from the adaptor molecule to the recording tag to generate an extended recording tag after the coding tag associated with the binding agent hybridizes with the first hybridization sequence on the adaptor molecule;
optionally, a means for transferring the information of the secondary tag or a means for analyzing the extended recording tag.

### Claim 18 (depends on 17)

The kit of claim 17, wherein the kit comprises a plurality of binding agents or a plurality of adaptor molecules, wherein the plurality of adaptor molecules comprises at least one adaptor molecule capable of hybridizing to at least one coding tag associated with the binding agent.

### Claim 19 (depends on 17)

The kit of claim 17, wherein the adaptor molecule further comprises a second hybridization sequence substantially complementary to a sequence at the 3′ terminus of the recording tag or substantially complementary to a region on the recording tag generated from a previous information transfer of the secondary tag from the adaptor molecule to the recording tag, and wherein information transfer of the secondary tag from the adaptor molecule to the recording tag is configured to occur after:
the first hybridization sequence on the adaptor molecule hybridizes to the coding tag of the binding agent; and
the second hybridization sequence of the adaptor molecule hybridizes to a portion of the recording tag.

### Claim 20 (depends on 17)

The kit of claim 17, wherein the macromolecule is a polypeptide, analyzing the macromolecule comprises determining at least a portion of an amino acid sequence of the polypeptide, and recording tag, the coding tag and the adaptor molecule comprise a DNA molecule, an RNA molecule, a PNA molecule, a BNA molecule, an XNA, molecule, an LNA molecule, a γPNA molecule, or a combination thereof.

### Claim 21 (depends on 20)

The kit of claim 20, wherein the means for transfer the information of the secondary tag comprises DNA ligase or DNA polymerase, and the means for analyzing the extended recording tag comprises a sequencing primer.

### Claim 22 (depends on 19)

The kit of claim 19, wherein the adaptor molecule comprises from 5′ to 3′ direction: the first hybridization sequence, the secondary tag, and the second hybridization sequence, and the secondary tag comprises an identifying information regarding the binding agent, a binding cycle-specific barcode, a unique molecular identifier, or a combination thereof.

### Claim 23 (depends on 20)

The kit of claim 20, further comprising a support for immobilizing the polypeptide and/or the recording tag.

### Claim 24

(canceled)