# US 9,040,495 B2 · General composition framework for ligand-controlled RNA regulatory systems

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- Number: US 9,040,495 B2
- Document ID: US9040495B2
- Status: Granted
- Kind: B2
- Application number: 13/585,919
- Filed: 2012-08-15
- Published: 2013-04-25
- Granted: 2015-05-26
- Earliest claimed: 2007-08-28
- Assignees: California Institute of Technology
- Applicants: Christina D. Smolke; Maung Nyan Win; Chase Beisel
- Inventors: Christina D. Smolke; Maung Nyan Win; Chase Beisel
- Web page: https://patentgrep.com/patent/US9040495B2

## CPC

- 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)}
- A61K 31/7088: Compounds having three or more nucleosides or nucleotides
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- 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 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- G01N 33/537: with separation of immune complex from unbound antigen or antibody
- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/16: Aptamers
- C12N 2310/531: Stem-loop; Hairpin
- C12N 2320/00: Applications; Uses
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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 invention provides an improved design for the construction of extensible nucleic acid-based, ligand-controlled regulatory systems, and the nucleic acid regulatory systems resulting therefrom. The invention contemplates improving the design of the switches (ligand-controlled regulatory systems) through the design of an information transmission domain (ITD). The improved ITD eliminates free-floating ends of the switching and the competing strands, and localizes competitive hybridization events to a contiguous strand of competing and switching strands in a strand-displacement mechanism-based switch, thereby improving the kinetics of strand-displacement. The improved regulatory systems have many uses in various biological systems, including gene expression control or ligand-concentration sensing.

## Claims

### Claim 1

A method for rendering expression of a target gene in a cell dependent on the presence or absence of a molecule, comprising introducing into the cell a polynucleotide comprising:
(1) a modular actuator domain comprising a substrate for RNase III, wherein said substrate, when processed by RNase III, produces an siRNA or miRNA that targets a transcript of said target gene,
(2) a modular sensor domain that detects concentration or status change of the molecule, wherein the modular sensor domain comprises an aptamer,
(3) an information transmission domain (ITD) between the modular actuator domain and the modular sensor domain, said information transmission domain comprising:
(a) a general transmission region,
(b) a switching strand, and,
(c) a competing strand,
wherein the switching strand and the competing strand are in a continuous sequence and compete to bind to the general transmission region through hybridization interactions,
(i) wherein detection of the concentration or status change by the modular sensor domain, through binding of said molecule to said modular sensor domain, favors a conformation change in the modular actuator domain;
(ii) wherein said conformation change is mediated by a strand-displacement mechanism in the ITD to favor the binding of the general transmission region to one of said switching strand and said competing strand; and wherein said hybridization interactions are sufficient to allow said conformation change to occur by the strand-displacement mechanism; and,
(iii) wherein said conformation change modulates the ability of said substrate to be processed by RNase III to produce the siRNA or miRNA for targeting said transcript, at a rate dependent upon the presence or absence of said molecule.

### Claim 2 (depends on 1)

The method of claim 1, wherein the target gene is a reporter gene, the method further comprising:
(A) measuring the amount of expression of said reporter gene; and
(B) correlating the amount of expression of said reporter gene with the amount of the molecule, thereby determining the amount of the molecule in the cell.

### Claim 3 (depends on 1)

The method of claim 1, wherein the polynucleotide is an RNA.

### Claim 4 (depends on 1)

The method of claim 1, wherein said substrate is an siRNA precursor, an miRNA precursor, or an shRNA precursor.

### Claim 5 (depends on 1)

The method of claim 1, wherein said ability of said substrate to be processed by RNase III to produce said siRNA or miRNA comprises: an ability to be incorporated into a RISC complex to serve as an siRNA or miRNA guide sequence, or an ability to be an RNase III substrate.

### Claim 6 (depends on 1)

The method of claim 1, wherein the switching strand and the competing strand do not have an overlapping region.

### Claim 7 (depends on 1)

The method of claim 1, wherein the switching strand and the competing strand have substantially the same sequence.

### Claim 8 (depends on 1)

The method of claim 1, wherein the switching strand and the competing strand are separated by one or more nucleotides.

### Claim 9 (depends on 1)

The method of claim 1, wherein said conformation change enhances said ability of said substrate to be processed by RNase III to produce said siRNA or miRNA.

### Claim 10 (depends on 9)

The method of claim 9, wherein the extent of the conformation change is amenable to adjustment or tuning.

### Claim 11 (depends on 10)

The method of claim 10, wherein said adjustment or tuning is effectuated by modifying base-pairing interactions formed between the general transmission region and the switching strand, and/or base-pairing interactions formed between the general transmission region and the competing strand.

### Claim 12 (depends on 11)

The method of claim 11, wherein said modifying is effectuated by changing the length of base pairs at one or both ends of the duplex formed between the general transmission region and the switching strand, and/or the duplex formed between the general transmission region and the competing strand.

### Claim 13 (depends on 11)

The method of claim 11, wherein said modifying is effectuated by changing base-pairing complementarity.

### Claim 14 (depends on 10)

The method of claim 10, wherein said adjustment or tuning is effectuated by changing the binding affinity between the modular sensor domain and the molecule without changing the size of the modular sensor domain.

### Claim 15 (depends on 10)

The method of claim 10, wherein said adjustment or tuning is effectuated by changing the size of the modular sensor domain.