# US 8,772,464 B2 · Aptamer regulated nucleic acids and uses thereof

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- Number: US 8,772,464 B2
- Document ID: US8772464B2
- Status: Granted
- Kind: B2
- Application number: 11/884,110
- Filed: 2006-02-09
- Published: 2012-02-09
- Granted: 2014-07-08
- Earliest claimed: 2006-02-09
- Assignees: California Institute of Technology
- Applicants: Christina D. Smolke; Travis S. Bayer
- Inventors: Christina D. Smolke; Travis S. Bayer
- Web page: https://patentgrep.com/patent/US8772464B2

## CPC

- C12Q 1/686: Polymerase chain reaction [PCR]
- A61P 3/04: Anorexiants; Antiobesity agents
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/04: Antibacterial agents
- A61P 35/00: Antineoplastic agents
- A61P 37/04: Immunostimulants
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/115: Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers}
- C12N 15/635: {Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline}
- C12Q 1/6897: involving reporter genes operably linked to promoters
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C12N 2310/11: Antisense
- C12N 2310/111: spanning the whole gene, or a large part of it
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/16: Aptamers
- 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
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)

## Abstract

The invention relates to aptamer-regulated, ligand-responsive nucleic acids, or “ampliSwitches,” and uses thereof. Particular embodiments include a ligand-responsive nucleic acid that comprises a primer sequence domain and an aptamer domain that is responsive to a ligand.

## Claims

### Claim 1

A nucleic acid comprising:
(i) an aptamer comprising a 5′-end switching aptamer stem sequence; and
(ii) a 3′-end priming sequence;
wherein:
(1) in a first conformation, the 3′-end priming sequence forms a hairpin structure, and the 5′-end switching aptamer stem sequence is single-stranded; and,
(2) in a second conformation, the 5′-end switching aptamer stem sequence hybridizes to a complementary region of the nucleic acid, and the 3′-end priming sequence is available for hybridization to a target template to form a primer:template pair;
wherein binding to a ligand by the aptamer induces a conformational change of the nucleic acid to shift between the first conformation and the second conformation, or between the second conformation and the first conformation.

### Claim 2 (depends on 1)

The nucleic acid of claim 1, wherein said primer:template pair acts as a substrate for an extrinsic enzymatic activity.

### Claim 3 (depends on 2)

The nucleic acid of claim 2, wherein said extrinsic enzymatic activity is a DNA polymerase.

### Claim 4 (depends on 2)

The nucleic acid of claim 2, wherein said extrinsic enzymatic activity is a ligase.

### Claim 5 (depends on 3)

The nucleic acid of claim 3, wherein said polymerase is phi29 or taq polymerase.

### Claim 6 (depends on 1)

The nucleic acid of claim 1, wherein said nucleic acid comprises ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or both.

### Claim 7 (depends on 1)

The nucleic acid of claim 1, wherein said conformational change produces or removes an intramolecular double-stranded feature, including said priming sequence, which double-stranded feature alters the availability of said priming sequence to hybridize to said target template.

### Claim 8 (depends on 1)

The nucleic acid of claim 1, wherein said ligand is selected from the group consisting of small molecules, metal ions, natural products, polypeptides, peptide analogs. nucleic acids, carbohydrates, fatty acids and lipids, a non-peptide hormone (such as steroids) and metabolic precursors or products thereof, and enzyme co-factors, enzyme substrates and products of enzyme-mediated reactions.

### Claim 9 (depends on 1)

An expression construct comprising (i) a coding sequence which, when transcribed, produces the nucleic acid of claim 1, and (ii) one or more transcriptional regulatory sequences that regulate transcription of said nucleic acid in an expression system containing said expression construct.

### Claim 10 (depends on 9)

The expression construct of claim 9, wherein the expression system is a cell.

### Claim 11 (depends on 9)

A recombinant cell engineered with one or more expression constructs of claim 9.

### Claim 12 (depends on 1)

A library comprising two nucleic acids, wherein each nucleic acid comprises the nucleic acid of claim 1, and wherein each aptamer of said two nucleic acids has a different binding affinity for said ligand.

### Claim 13 (depends on 1)

A library comprising two nucleic acids, wherein each nucleic acid comprises the nucleic acid of claim 1, and wherein each nucleic acid has a different thermodynamic stability that affects the likelihood that said conformational change will occur in response to binding to said ligand.

### Claim 14 (depends on 1)

A library comprising two nucleic acids, wherein each nucleic acid comprises the nucleic acid of claim 1, and wherein each aptamer of said two nucleic acids binds to a different ligand.

### Claim 15 (depends on 1)

The nucleic acid of claim 1, which is about 30-500 nucleotides in length.

### Claim 16 (depends on 12)

The library of claim 12, wherein each said nucleic acid is about 30-500 nucleotides in length.

### Claim 17 (depends on 13)

The library of claim 13, wherein each said nucleic acid is about 30-500 nucleotides in length.

### Claim 18 (depends on 14)

The library of claim 14, wherein each said nucleic acid is about 30-500 nucleotides in length.