# US 2019/0314397 A1 · Methods For Directed Folding Assembly Or Dimerization Of Proteins By Templated Assembly Reactions

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- Number: US 2019/0314397 A1
- Document ID: US20190314397A1
- Status: Published application
- Kind: A1
- Application number: 16/462,332
- Filed: 2017-11-17
- Published: 2019-10-17
- Earliest claimed: 2016-11-21
- Assignees: TriBiotica LLC
- Applicants: TriBiotica LLC
- Inventors: Ian Dunn; Matthew Lawler
- Web page: https://patentgrep.com/patent/US20190314397A1

## CPC

- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- C07D 401/12: linked by a chain containing hetero atoms as chain links
- C07D 401/14: containing three or more hetero rings
- C12N 15/1068: {Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- G01N 33/50: Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing (measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q)
- C12N 2310/3513: Protein; Peptide
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- 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)

## Abstract

The present disclosure provides nucleic acid molecules, compositions, and kits comprising the same, and methods for producing templated assembly products.

## Claims

### Claim 1

A haplomer-ligand complex comprising:
a haplomer, wherein the haplomer comprises a polynucleotide that is substantially complementary to a target nucleic acid molecule; and
a ligand linked to the 5′ or 3′ terminus of the haplomer, wherein the ligand comprises a ligand partner binding site.

### Claim 2 (depends on 1)

A composition or kit comprising a first haplomer-ligand complex of claim 1 and a second haplomer-ligand complex of claim 1, wherein:
the ligand of the first haplomer-ligand complex is linked to the 5′ terminus of the polynucleotide of the first haplomer-ligand complex; and
the ligand of the second haplomer-ligand complex is linked to the 3′ terminus of the polynucleotide of the second haplomer-ligand complex.

### Claim 3

A bottle haplomer-ligand complex comprising:
a) a bottle haplomer, wherein the bottle haplomer comprises a polynucleotide, wherein the polynucleotide comprises:
i) a first stem portion comprising from about 10 to about 20 nucleotide bases;
ii) an anti-target loop portion comprising from about 16 to about 40 nucleotide bases and having a first end to which the first stem portion is linked, wherein the anti-target loop portion is substantially complementary to a target nucleic acid molecule; and
iii) a second stem portion comprising from about 10 to about 20 nucleotide bases linked to a second end of the anti-target loop portion, wherein the first stem portion is substantially complementary to the second stem portion; and
b) a ligand linked to the terminal end of either the first stem portion or the second stem portion, wherein the ligand comprises a ligand partner binding site;
wherein the Tm of the anti-target loop portion:target nucleic acid molecule is greater than the Tm of the first stem portion:second stem portion.

### Claim 4 (depends on 3)

A composition or kit comprising:
a bottle haplomer-ligand complex of claim 3; and
a second haplomer-ligand complex comprising:
a nucleotide portion comprising from about 6 to about 20 nucleotide bases that is substantially complementary to the stem portion of the bottle haplomer-ligand complex that is linked to the ligand of the bottle haplomer-ligand complex; and
a ligand linked to the 5′ or 3′ terminus of the nucleotide portion of the second haplomer-ligand complex, wherein the ligand comprises a ligand partner binding site;
wherein the Tm of the second haplomer-ligand complex:first or second stem portion linked to the ligand of the bottle haplomer-ligand complex is less than or equal to the Tm of the first stem portion:second stem portion of the bottle haplomer-ligand complex.

### Claim 5

A compound having the formula:
where m is from 3 to 6; having the formula:
where m is from 3 to 6; having the formula;
where n is from 1 to 6; having the formula:
where x is from 1 to 6; having the formula:
where x is from 1 to 6; or having the formula:

### Claim 6

A fusion protein comprising a fragment of a protein of interest fused to a ligand binding domain, wherein the ligand binding domain is an FKBP domain or an FRB domain.

### Claim 7 (depends on 6)

A composition or kit comprising a first fusion protein of claim 6 and a second fusion protein of claim 6, wherein the protein of interest of the first fusion protein and the protein of interest of the second fusion protein can dimerize or fold together.

### Claim 8 (depends on 1, 6)

A method for the directed assembly of a protein comprising:
contacting a target nucleic acid molecule with a first haplomer-ligand complex of claim 1;
contacting the target nucleic acid with a second haplomer-ligand complex of claim 1;
contacting the first haplomer-ligand complex with a first fusion protein of claim 6; and
contacting the second haplomer-ligand complex with a second fusion protein of claim 6;
wherein the ligand of the first haplomer-ligand complex is linked to the 5′ terminus of the polynucleotide of the first haplomer-ligand complex;
wherein the ligand of the second haplomer-ligand complex is linked to the 3′ terminus of the polynucleotide of the second haplomer-ligand complex;
wherein the polynucleotide of the first haplomer-ligand complex is substantially complementary to a target nucleic acid molecule;
wherein the polynucleotide of the second haplomer-ligand complex is substantially complementary to the target nucleic acid molecule at a site in spatial proximity to the polynucleotide of the first haplomer-ligand complex;
wherein the ligand of the first haplomer-ligand complex and the ligand binding domain of the first fusion protein can interact; and
wherein the ligand of the second haplomer-ligand complex and the ligand binding domain of the second fusion protein can interact;
thereby resulting in the folding or dimerization of the fragment of the protein of interest of the first fusion protein with the fragment of the protein of interest of the second fusion protein.

### Claim 9 (depends on 1, 6)

A method for the directed assembly of a protein comprising:
contacting a target nucleic acid molecule with a first haplomer-ligand complex of claim 1;
contacting the target nucleic acid with a second haplomer-ligand complex of claim 1;
contacting the first haplomer-ligand complex with a first fusion protein of claim 6; and
contacting the second haplomer-ligand complex with a second fusion protein of claim 6;
wherein the ligand of the first haplomer-ligand complex is linked to the 5′ terminus of the polynucleotide of the first haplomer-ligand complex;
wherein the ligand of the second haplomer-ligand complex is linked to the 3′ terminus of the polynucleotide of the second haplomer-ligand complex;
wherein the polynucleotide of the first haplomer-ligand complex is substantially complementary to a target nucleic acid molecule;
wherein the polynucleotide of the second haplomer-ligand complex is substantially complementary to the target nucleic acid molecule at a site in spatial proximity to the polynucleotide of the first haplomer-ligand complex;
wherein the ligand of the first haplomer-ligand complex and the ligand binding domain of the first fusion protein can interact; and
wherein the ligand of the second haplomer-ligand complex and the ligand binding domain of the second fusion protein can interact;
thereby resulting in the folding or dimerization of the fragment of the protein of interest of the first fusion protein with the fragment of the protein of interest of the second fusion protein.

### Claim 10 (depends on 1, 6)

A method for the directed assembly of a protein comprising:
contacting a target nucleic acid molecule with a complex formed by the interaction of a first haplomer-ligand complex of claim 1 with a first fusion protein of claim 6, wherein the ligand of the first haplomer-ligand complex is linked to the 5′ terminus of the polynucleotide of the first haplomer-ligand complex, and wherein the ligand of the first haplomer-ligand complex interacts with the ligand binding domain of the first fusion protein; and
contacting the target nucleic acid molecule with a complex formed by the interaction of a second haplomer-ligand complex of claim 1 with a second fusion protein of claim 6, wherein the ligand of the second haplomer-ligand complex is linked to the 5′ terminus of the polynucleotide of the second haplomer-ligand complex, and wherein the ligand of the second haplomer-ligand complex interacts with the ligand binding domain of the second fusion protein;
thereby resulting in the folding or dimerization of the fragment of the protein of interest of the first fusion protein with the fragment of the protein of interest of the second fusion protein.

### Claim 11 (depends on 1, 6)

A method for the directed assembly of a protein comprising:
contacting a target nucleic acid molecule with a complex formed by the interaction of a first haplomer-ligand complex of claim 1 with a first fusion protein of claim 6, wherein the ligand of the first haplomer-ligand complex is linked to the 5′ terminus of the polynucleotide of the first haplomer-ligand complex, and wherein the ligand of the first haplomer-ligand complex interacts with the ligand binding domain of the first fusion protein; and
contacting the target nucleic acid molecule with a complex formed by the interaction of a second haplomer-ligand complex of claim 1 with a second fusion protein of claim 6, wherein the ligand of the second haplomer-ligand complex is linked to the 5′ terminus of the polynucleotide of the second haplomer-ligand complex, and wherein the ligand of the second haplomer-ligand complex interacts with the ligand binding domain of the second fusion protein;
thereby resulting in the folding or dimerization of the fragment of the protein of interest of the first fusion protein with the fragment of the protein of interest of the second fusion protein.

### Claim 12 (depends on 1, 3, 6)

A method for the directed assembly of a protein comprising:
contacting a target nucleic acid molecule with a bottle haplomer-ligand complex of claim 3,
contacting the target nucleic acid with a second haplomer-ligand complex of claim 1, wherein the second haplomer-ligand complex comprises a nucleotide portion that is substantially complementary to the stem portion of the bottle haplomer-ligand complex that is linked to the ligand of the bottle haplomer-ligand complex;
contacting the bottle haplomer-ligand complex with a first fusion protein of claim 6, wherein the ligand of the bottle haplomer-ligand complex and the ligand binding domain of the first fusion protein can interact; and
contacting the second haplomer-ligand complex with a second fusion protein of claim 6, wherein the ligand of the second haplomer-ligand complex and the ligand binding domain of the second fusion protein can interact;
thereby resulting in the folding or dimerization of the fragment of the protein of interest of the first fusion protein with the fragment of the protein of interest of the second fusion protein.

### Claim 13 (depends on 1, 3, 6)

A method for the directed assembly of a protein comprising:
contacting a target nucleic acid molecule with a bottle haplomer-ligand complex of claim 3;
contacting the target nucleic acid with a second haplomer-ligand complex of claim 1, wherein the second haplomer-ligand complex comprises a nucleotide portion that is substantially complementary to the stem portion of the bottle haplomer-ligand complex that is linked to the ligand of the bottle haplomer-ligand complex;
contacting the bottle haplomer-ligand complex with a first fusion protein of claim 6, wherein the ligand of the bottle haplomer-ligand complex and the ligand binding domain of the first fusion protein can interact; and
contacting the second haplomer-ligand complex with a second fusion protein of claim 6, wherein the ligand of the second haplomer-ligand complex and the ligand binding domain of the second fusion protein can interact;
thereby resulting in the folding or dimerization of the fragment of the protein of interest of the first fusion protein with the fragment of the protein of interest of the second fusion protein.

### Claim 14 (depends on 1, 3, 6)

A method for the directed assembly of a protein comprising:
contacting a target nucleic acid molecule with a bottle haplomer-ligand complex of claim 3;
contacting the target nucleic acid molecule with a second haplomer-ligand complex of claim 1, wherein the second haplomer-ligand complex comprises a nucleotide portion that is substantially complementary to the stem portion of the bottle haplomer-ligand complex that is linked to the ligand of the bottle haplomer-ligand complex;
contacting the bottle haplomer-ligand complex with a first fusion protein of claim 6, wherein the ligand of the bottle haplomer-ligand complex and the ligand binding domain of the first fusion protein can interact; and
contacting the second haplomer-ligand complex with a second fusion protein of claim 6, wherein the ligand of the second haplomer-ligand complex and the ligand binding domain of the second fusion protein can interact;
thereby resulting in the folding or dimerization of the fragment of the protein of interest of the first fusion protein with the fragment of the protein of interest of the second fusion protein.

### Claim 15 (depends on 1, 3, 6)

A method for the directed assembly of a protein comprising:
contacting a target nucleic acid molecule with a bottle haplomer-ligand complex of claim 3;
contacting the target nucleic acid molecule with a second haplomer-ligand complex of claim 1, wherein the second haplomer-ligand complex comprises a nucleotide portion that is substantially complementary to the stem portion of the bottle haplomer-ligand complex that is linked to the ligand of the bottle haplomer-ligand complex,
contacting the bottle haplomer-ligand complex with a first fusion protein of claim 6, wherein the ligand of the bottle haplomer-ligand complex and the ligand binding domain of the first fusion protein can interact; and
contacting the second haplomer-ligand complex with a second fusion protein of claim 6, wherein the ligand of the second haplomer-ligand complex and the ligand binding domain of the second fusion protein can interact;
thereby resulting in the folding or dimerization of the fragment of the protein of interest of the first fusion protein with the fragment of the protein of interest of the second fusion protein.