General composition framework for ligand-controlled RNA regulatory systems
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.
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.
2. 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.
3. The method of claim 1 , wherein the polynucleotide is an RNA.
4. The method of claim 1 , wherein said substrate is an siRNA precursor, an miRNA precursor, or an shRNA precursor.
5. 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.
6. The method of claim 1 , wherein the switching strand and the competing strand do not have an overlapping region.
7. The method of claim 1 , wherein the switching strand and the competing strand have substantially the same sequence.
8. The method of claim 1 , wherein the switching strand and the competing strand are separated by one or more nucleotides.
9. 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.
10. The method of claim 9 , wherein the extent of the conformation change is amenable to adjustment or tuning.
11. 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.
12. 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.
13. The method of claim 11 , wherein said modifying is effectuated by changing base-pairing complementarity.
14. 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.
15. The method of claim 10 , wherein said adjustment or tuning is effectuated by changing the size of the modular sensor domain.