Aptamer regulated nucleic acids and uses thereof
The invention relates to trans-acting ligand-responsive nucleic acids and uses thereof. In particular, a ligand responsive nucleic acid comprises an effector domain and an aptamer domain that is responsive to a ligand.
1. A nucleic acid comprising (i) a substrate sequence that can form a hairpin RNA stem as a substrate for an RNaseIII family nuclease, and (ii) an aptamer that binds to a ligand, wherein said aptamer comprises a sequence that can form an aptamer stem, the formation of said aptamer stem competes with the formation of said hairpin RNA stem,
wherein binding of the ligand to said aptamer causes a conformational change to render the substrate sequence non-functional by destabilizing the hairpin RNA stem, through stabilizing the aptamer stem, and,
wherein said RNaseIII family nuclease processes said hairpin RNA through an RNA interference (RNAi) mechanism in attenuating expression of a target gene.
2. The nucleic acid of claim 1 , wherein said nucleic acid is a ribonucleic acid (RNA).
3. The nucleic acid of claim 1 , wherein said RNaseIII family nuclease processes said hairpin RNA to produce siRNA.
4. The nucleic acid of claim 1 , wherein the ligand is a small molecule having a molecular weight less than 2500 amu.
5. The nucleic acid of claim 1 , wherein the ligand is a metal ion.
6. The nucleic acid of claim 1 , wherein the ligand is a polypeptide, a peptide, a nucleic acid, a carbohydrate, a fatty acid, a lipid, a non-peptide hormone, or a metabolic precursor or product thereof.
7. The nucleic acid of claim 1 , wherein the ligand is selected from enzyme co-factors, enzyme substrates and products of enzyme-mediated reactions.
8. An expression construct comprising (i) a coding sequence which, when transcribed, produces the RNA of claim 2 , and (ii) one or more transcriptional regulatory sequences that regulate transcription of said RNA in a cell containing said expression construct.
9. A library of aptamer-regulated nucleic acids, each independently comprising the nucleic acid of claim 1 , or an expression construction encoding said nucleic acid.
10. A recombinant cell engineered with the expression construct of claim 9 .
11. A cell comprising the nucleic acid of claim 1 .
12. The nucleic acid of claim 1 , wherein said RNaseIII family nuclease is Dicer.
13. The nucleic acid of claim 3 , wherein said siRNA is between about 19 and about 30 nucleotides in length.
14. The nucleic acid of claim 3 , wherein said siRNA is between about 21 and about 23 nucleotides in length.
15. The nucleic acid of claim 1 , wherein said nucleic acid further comprises an intercalator or an alkylating agent.
16. The nucleic acid of claim 1 , wherein said aptamer is responsive to pH, temperature, or salt concentration.
17. The nucleic acid of claim 1 , wherein said aptamer of said nucleic acid is altered so that the K d for binding of a ligand to the aptamer is higher or lower than the K d of an unaltered aptamer.
18. The nucleic acid of claim 1 , wherein said nucleic acid comprises one or more said aptamers or one or more said substrate sequences.
19. The nucleic acid of claim 1 , wherein said nucleic acid interacts with and responds to multiple ligands.
20. The nucleic acid of claim 1 , wherein said nucleic acid is a cooperative ligand controlled nucleic acid wherein multiple ligands sequentially bind to multiple aptamer domains to allosterically regulate an effector domain.