TRIGGERED RNAi

Abstract

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.

What is claimed is: 
     
         1 . A method of silencing a target gene, the method comprising:
 contacting a sample comprising a detection target and a silencing target gene with an initiator comprising a detection target binding region and an initiator region, wherein the detection target binding region can interact with the detection target, and wherein the detection target is different from the silencing target gene;   contacting the sample with a first substrate complex and a second substrate complex:   wherein the first substrate complex comprises a second substrate complex complement region and the second substrate complex comprises a first substrate monomer complement region, wherein the second substrate complex complement region is complementary to the first substrate complex complement region;   wherein the first substrate complex comprises a silencing target region and an initiator complement region that is complementary to the initiator region of the initiator, wherein upon binding of the detection target binding region of the initiator to the detection target, the initiator region of the initiator is made available to bind to the initiator complement region of the first substrate complex; and   wherein the second substrate complex comprises a silencing target complement region, wherein upon binding of the initiator complement region to the initiator region, the silencing target region of the first substrate complex is made available to bind to the silencing target complement region of the second substrate complex;   wherein an inactivator double-stranded RNA (inactivator dsRNA) is formed by hybridization of the silencing target complement region to the silencing target region, and wherein the inactivator dsRNA is processed to silence the silencing target gene.   
     
     
         2 . The method of  claim 1 , wherein the first substrate complex comprises a nucleic acid duplex. 
     
     
         3 . The method of  claim 1 , wherein the second substrate complex comprises a nucleic acid duplex. 
     
     
         4 . The method of  claim 3  wherein the first substrate complex comprises a region complementary to a region of the second substrate complex. 
     
     
         5 . The method of  claim 1 , wherein the first substrate complex comprises a first bulge loop region. 
     
     
         6 . The method of  claim 5 , wherein the second substrate complex comprises a second bulge loop region, wherein a portion of the first bulge loop region is complementary to the second bulge loop region. 
     
     
         7 . The method of  claim 1 , wherein the initiator comprises a nucleic acid hairpin monomer. 
     
     
         8 . The method of  claim 1 , wherein the inactivator dsRNA is processed by Dicer. 
     
     
         9 . The method of  claim 8 , wherein the inactivator dsRNA is processed to produce a 19 bp duplex with 2-nt 3′ overhangs. 
     
     
         10 . The method of  claim 8 , wherein the inactivator dsRNA comprises a 27-base pair RNA duplex substrate. 
     
     
         11 . The method of  claim 1 , wherein the inactivator dsRNA is an siRNA. 
     
     
         12 . The method of  claim 11 , wherein the inactivator dsRNA is a 19 bp duplex with 2-nt 3′ overhangs. 
     
     
         13 . The method of  claim 1 , wherein the inactivator dsRNA activates protein kinase R (PKR).