Triggered RNAi
The present application relates to methods and compositions for triggering RNAi. Triggered RNAi is highly versatile because the silencing targets are independent of the detection targets. In some embodiments, methods of silencing or modulating the expression of a target gene are provided. The methods generally comprise providing an initiator to a cell comprising a detection target and a silencing target gene, wherein the detection target is different from the silencing target gene, wherein binding of the detection target to the initiator initiates formation of an inactivator double-stranded RNA (inactivator dsRNA). The inactivator dsRNA can silence or modulate the expression of the silencing target gene.
1. A method of forming an inactivator dsRNA, the method comprising:
contacting a sample with an initiator, wherein the sample comprises a detection target, wherein the initiator comprises: a) a detection target binding region and b) an initiator region, wherein the detection target binding region can interact with the detection target and wherein the initiator region is not exposed prior to the detection target binding region interacting with the initiator;
contacting the sample with a first substrate monomer that comprises:
i) a silencing target complement region,
ii) an initiator complement region, wherein the initiator complement region hybridizes to the initiator region of the initiator, wherein upon binding of the detection target binding region to the detection target, the initiator region of the initiator is exposed and binds to the initiator complement region of the first substrate monomer, and
iii) a substrate monomer complement region; and
contacting the sample with a second substrate monomer that comprises:
i) a substrate monomer region that hybridizes to the substrate monomer complement region, and
ii) a silencing target region that hybridizes to the silencing target complement region;
wherein upon binding of the initiator complement region of the first monomer to the initiator region of the initiator, the silencing target complement region hybridizes to the silencing target region to form an inactivator dsRNA.
2. The method of claim 1 , wherein the inactivator dsRNA comprises a sequence that can be processed by Dicer, and wherein Dicer processes the inactivator dsRNA to silence a silencing targeting gene.
3. The method of claim 2 , wherein the sample further comprises the silencing target gene.
4. The method of claim 2 , wherein the detection target is different from the silencing target gene.
5. The method of claim 1 , wherein the initiator comprises a nucleic acid hairpin monomer.
6. The method of claim 1 , wherein the first substrate monomer comprises a nucleic acid hairpin monomer that comprises a first hairpin loop region.
7. The method of claim 6 , wherein the first hairpin loop region comprises the second substrate monomer complement region.
8. The method of claim 1 , wherein the second substrate monomer comprises a nucleic acid hairpin monomer that comprises a second hairpin loop region.
9. The method of claim 8 wherein the second hairpin loop region comprises the substrate monomer region.
10. The method of claim 1 , further comprising: contacting the sample with at least one accessory molecule comprising a DNA sequence that binds to a region flanking the initiator binding site of the detection target.
11. The method of claim 1 , wherein the inactivator dsRNA is processed by Dicer.
12. The method of claim 11 , wherein the inactivator dsRNA is processed to produce a 19 bp duplex with 2-nt 3′ overhangs.
13. The method of claim 11 , wherein the inactivator dsRNA comprises a 27-base pair RNA duplex substrate.
14. The method of claim 1 , wherein the inactivator dsRNA is an siRNA.
15. The method of claim 14 , wherein the inactivator dsRNA is a 19 bp duplex with 2-nt 3′ overhangs.
16. The method of claim 1 , wherein the inactivator dsRNA can activate protein kinase R (PKR).
17. The method of claim 1 , wherein the detection target is a nucleic acid.
18. The method of claim 4 , wherein the silencing target gene is an mRNA comprising a sequence different from the sequence of the detection target.
19. The method of claim 1 , wherein the detection target is associated with a disease or disorder.
20. The method of claim 19 , wherein the detection target is an mRNA associated with a cancer.
21. The method of claim 19 , wherein the detection target is a viral nucleic acid.
22. The method of claim 19 , wherein the detection target is an mRNA molecule associated with a disease or disorder.
23. The method of claim 2 , wherein the silencing target gene is an mRNA.
24. The method of claim 23 , wherein the mRNA is fluorescent protein mRNA.
25. The method of claim 23 , wherein the mRNA is a housekeeping gene mRNA.
26. The method of claim 23 , wherein the mRNA is a protein kinase R (PKR) mRNA.
27. The method of claim 1 , wherein the initiator comprises a recognition molecule such that upon binding of the recognition molecule to the detection target, a portion of the initiator is made available to bind to the initiator complement region of the first substrate monomer.
28. The method of claim 27 , wherein the recognition molecule is an aptamer.
29. The method of claim 27 , wherein the detection target is a nucleic acid.
30. The method of claim 27 , wherein the detection target is selected from the group consisting of polypeptides, carbohydrates, lipids and small molecules.
31. The method of claim 1 , wherein the first substrate monomer comprises a sticky end region.
32. The method of claim 1 , wherein the first RNA hairpin monomer comprises an analog RNA nucleotide.
33. The method of claim 1 , wherein the second RNA hairpin monomer comprises an analog RNA nucleotide.