General composition framework for ligand-controlled regulatory systems

• US 20090143327A1
• Filed: 09/18/2008
• Published: 06/04/2009
• Est. Priority Date: 08/28/2007
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Beisel, Chase L., Win, Maung Nyan, Smolke, Christina D.
• Granted Patent: US 8,367,815 B2
• Time in Patent Office: Days
• US Class Current: 514/44.R
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/12 catalytic nucleic acids, e.g. ribozymes
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2320/50 Methods for regulating/modulating their activity
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

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

1. A polynucleotide comprising:

(1) a modular actuator domain having one or more functional activities, wherein said modular actuator domain is not a ribozyme,

(2) a modular sensor domain that detects concentration change of a molecule, or status change of an environmental condition (pH, ion concentration, temperature), and,

(3) an information transmission domain between the modular actuator domain and the modular sensor domain, said information transmission domain comprising;

(a) a general transmission region,

(b) a switching strand,

(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;

wherein detection of the concentration or status change by the modular sensor domain favors a conformation change in the modular actuator domain;

wherein said conformation change is mediated by a strand-displacement mechanism in the information transmission domain to favor the binding of the general transmission region to one of said switching strand and said competing strand; and

,

wherein said conformation change modulates said functional activities.

2. The polynucleotide of claim 1, which is an RNA, a DNA, or a combination thereof.

3. The polynucleotide of claim 1, wherein said modular actuator domain comprises an antisense sequence, an siRNA or precursor thereof, an miRNA or precursor thereof, an shRNA or precursor thereof, an RNase III substrate, an alternative splicing element, or an RNAi targeting sequence.

4. The polynucleotide of claim 1, wherein said functional activities comprise an ability to hybridize with a target polynucleotide, 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.

5. The polynucleotide of claim 1, wherein said modular sensor domain is an aptamer.

6. The polynucleotide of claim 1, wherein said modular sensor domain binds the molecule, preferably specifically.

7. The polynucleotide of claim 1, wherein the switching strand and the competing strand substantially do not overlap.

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

9. The polynucleotide of claim 1, wherein the switching strand and the competing strand are in tandem.

10. The polynucleotide of claim 1, wherein said conformation change increases said functional activities.

11. The polynucleotide of claim 1, wherein the extent of the conformation change is amenable to (rational) adjustment/tuning.

12. The polynucleotide of claim 11, wherein said adjustment/tuning is effectuated by modifying base-pairing interactions among the general transmission region, the switching strand, and/or the competing strand.

13. The polynucleotide of claim 12, wherein said adjustment/tuning is effectuated by changing the length of the paring 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.

14. The polynucleotide of claim 12, wherein said adjustment/tuning is effectuated by changing base-pairing complementarity.

15. The polynucleotide of claim 11, wherein said adjustment/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.

16. The polynucleotide of claim 11, wherein said adjustment/tuning is effectuated by changing the size of the modular sensor domain.

19. A vector or expression construct encoding the polynucleotide of claim 1.

20. The vector or expression construct of claim 19, further comprising one or more transcriptional regulatory sequences that regulate transcription from said vector or expression construct in a cell containing said vector or expression construct.

21. A cell engineered to include the polynucleotide of claim 1, or the vector or expression construct of claim 19.

22. A method for regulating expression of a recombinant gene, comprising:

(i) providing a cell of claim 21,

(ii) contacting the cell with said molecule in an amount that alters the activity of said modular actuator domain.

23. A cell comprising:

a metabolic pathway of one or more reactions that are regulated at least in part by a target gene; and

one or more polynucleotide of claim 1 that act as control elements on said metabolic pathway by regulating expression of said target gene through said modular actuator domain, wherein binding of said molecule to said modular sensor domain (e.g., aptamer) causes a change in the intramolecular interaction of said information transmission domain, such that there is a change in the regulation of said target gene by said modular actuator domain, at a rate dependent upon the presence or absence of said molecule.

24. The cell of claim 23, wherein the metabolic pathway includes at least one reaction mediated by an enzyme, and at least one of said polynucleotide regulates expression of said enzyme.

25. The cell of claim 23, wherein said 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.

26. 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 of claim 1 comprising 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, wherein, binding of said molecule to said modular sensor domain (e.g., aptamer) causes a change in the intramolecular interaction of said information transmission domain, such that said substrate is processed by RNase III to produce the siRNA or miRNA to target said transcript, at a rate dependent upon the presence or absence of said molecule.

27. A method of determining the amount of an analyte in a cell which expresses a reporter gene, comprising:

(1) introducing into the cell a polynucleotide of claim 1 comprising 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 reporter gene, wherein binding of said analyte to said modular sensor domain (e.g., aptamer) causes a change in the intramolecular interaction of said information transmission domain, such that said substrate is processed by RNase III to produce said siRNA or miRNA to inhibit expression of said reporter gene, at a rate dependent upon the presence or absence of said analyte;

(2) measuring the amount of expression of said reporter gene; and

(3) correlating the amount of expression of said reporter gene with the amount of analyte, thereby determining the amount of the analyte in the cell.

28. A method for treating or preventing infection by a pathogenic agent, comprising administering to a patient a sufficient amount of a polynucleotide of claim 1, wherein said molecule is produced as a consequence of infection by said pathogenic agent, and wherein said modular actuator domain inhibits the function of one or more genes essential for successful infection by said pathogenic agent.

29. A method for causing phenotypic regulation of cell growth, differentiation or viability in cells of a patient, comprising introducing into cells in said patient a polynucleotide of claim 1, where said modular sensor domain (e.g., aptamer) binds to the molecule, the concentration of which is dependent on cellular phenotype, wherein binding of said molecule to said modular sensor domain favors a conformational change that increases (or decreases) said functional activities of said modular actuator domain, and said increased or decreased functional activities of said modular actuator domain modulates expression of a target gene essential for altering the regulation of cell growth, differentiation or viability in said cells.

30. A pharmaceutical preparation comprising a polynucleotide of claim 1, or an expression construct which, when transcribed, produces an RNA including said polynucleotide, and a pharmaceutically acceptable carrier suitable for use administration to a human or non-human patient.

17. A method for rational design of a modular polynucleotide, the method comprising:

(1) providing a modular actuator domain having one or more functional activities, wherein said modular actuator domain is not a ribozyme,

(2) providing a modular sensor domain that detects concentration change of a molecule, or status change of an environmental condition (pH, ion concentration, temperature), and,

(3) providing an information transmission domain between the modular actuator domain and the modular sensor domain, said information transmission domain comprising;

(a) a general transmission region,

(b) a switching strand,

(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,

wherein detection of the concentration or status change by the modular sensor domain favors a conformation change in the modular actuator domain;

wherein said conformation change is mediated by a strand-displacement mechanism in the information transmission domain to favor the binding of the general transmission region to one of said switching strand and said competing strand; and

,

wherein said conformation change modulates said functional activities.

18. A method for improving the design of a sensor-regulated polynucleotide, said polynucleotide comprising:

(1) an actuator domain having one or more functional activities, wherein said actuator domain is not a ribozyme, and,

(2) a sensor domain that detects concentration change of a molecule, or status change of an environmental condition (pH, ion concentration, temperature),

the method comprising;

providing an information transmission domain between the actuator domain and the sensor domain, said information transmission domain comprising;

(a) a general transmission region,

(b) a switching strand,

(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;

wherein detection of the concentration or status change by the sensor domain favors a conformation change in the actuator domain;

wherein said conformation change is mediated by a strand-displacement mechanism in the information transmission domain to favor the binding of the general transmission region to one of said switching strand and said competing strand; and

,

wherein said conformation change modulates said functional activities.