Rna-based logic circuits with rna binding proteins, aptamers and small molecules

Abstract

Engineered synthetic RNA-based genetic circuits are provided that are regulated exclusively at the post-transcriptional level.

1 . A synthetic RNA circuit comprising
 a first RNA molecule comprising at least one sequence recognized by at least one first microRNA that is/are specifically expressed in a first cell type, and a sequence encoding a protein that specifically binds to a RNA motif and inhibits protein production; and   a second RNA molecule comprising at least one sequence recognized by at least one second microRNA that is/are not expressed in the first cell type or is expressed at a low level relative to a second cell type, at least one RNA motif and a sequence encoding an output molecule.   
     
     
         2 . The synthetic RNA circuit of  claim 1 , wherein the output molecule is a protein. 
     
     
         3 .- 21 . (canceled) 
     
     
         22 . The synthetic RNA circuit of  claim 1 , wherein in a cell that expresses the at least one first microRNA but does not express the at least one second microRNA, the at least one first microRNA represses translation of or degrades the sequence encoding the protein that specifically binds to a RNA motif and inhibits protein production, thereby allowing expression of the output molecule. 
     
     
         23 .- 39 . (canceled) 
     
     
         40 . A method of treating cancer in a mammal comprising administering to a mammal the synthetic RNA circuit of  claim 1 . 
     
     
         41 . A method of inducing an immune response in a mammal comprising administering to a mammal the synthetic RNA circuit of  claim 1 . 
     
     
         42 . (canceled) 
     
     
         43 . A synthetic RNA circuit comprising:
 (1) a first RNA molecule comprising at least one sequence recognized by a first protein that specifically binds to a RNA motif and inhibits protein production, and a sequence encoding an output molecule; and/or   a second RNA molecule comprising at least one sequence recognized by a second protein that specifically binds to a RNA motif and inhibits protein production or a second RNA molecule comprising at least one sequence recognized by an siRNA molecule or a microRNA molecule, and a sequence encoding the first protein that specifically binds to a RNA motif and inhibits protein production; and/or   a third RNA molecule comprising at least one sequence recognized by an siRNA molecule or a microRNA molecule, and a sequence encoding the second protein that specifically binds to a RNA motif and inhibits protein production; or   (2) an RNA molecule comprising a sequence encoding a destabilization domain fused to an output protein,   wherein the destabilization domain facilitates degradation of the output protein in the absence of a small molecule that binds to the destabilization domain; or   (3) an RNA molecule comprising a sequence encoding a TetR protein and a sequence encoding an output protein, and an aptamer sequence that is bound by the TetR protein in the absence of tetracycline;   wherein the aptamer sequence is positioned relative to the sequence encoding the output protein so that it suppresses translation of the output protein in the absence of tetracycline.   
     
     
         44 . The synthetic RNA circuit of  claim 43 , further comprising the siRNA molecule or microRNA molecule that binds to the third RNA molecule. 
     
     
         45 . (canceled) 
     
     
         46 . The synthetic RNA circuit of  claim 43 , wherein the output molecule is a protein. 
     
     
         47 . The synthetic RNA circuit of  claim 43 , wherein the output molecule or output protein is a therapeutic protein, a cell death protein, a fluorescent protein, an antigen, a selection protein, or an immunomodulator 
     
     
         48 .- 79 . (canceled) 
     
     
         80 . A method of treating cancer in a mammal comprising administering to a mammal the synthetic RNA circuit of  claim 43 . 
     
     
         81 . A method of inducing an immune response in a mammal comprising administering to a mammal the synthetic RNA circuit of  claim 43 . 
     
     
         82 .- 122 . (canceled) 
     
     
         123 . A synthetic RNA circuit comprising:
 (1) a first RNA molecule comprising at least one sequence recognized by a first protein that specifically binds to a RNA motif and inhibits protein production, a sequence encoding a second protein that specifically binds to a RNA motif and inhibits protein production, and at least one sequence recognized by a first siRNA molecule or microRNA molecule; and   a second RNA molecule comprising at least one sequence recognized by the second protein that specifically binds to a RNA motif and inhibits protein production, a sequence encoding the first protein that specifically binds to a RNA motif and inhibits protein production, and at least one sequence recognized by a second siRNA molecule or microRNA molecule, or   (2) a first RNA molecule comprising a sequence encoding a destabilization domain fused to a protein that specifically binds to a RNA motif and inhibits protein production; and a second RNA molecule comprising at least one sequence recognized by the protein that specifically binds to a RNA motif and inhibits protein production, and a sequence encoding an output molecule;   wherein the destabilization domain facilitates degradation of the protein that specifically binds to a RNA motif and inhibits protein production in the absence of a small molecule that binds to the destabilization domain.   
     
     
         124 .- 204 . (canceled) 
     
     
         205 . The synthetic RNA circuit of  claim 123 , wherein the output molecule is a protein. 
     
     
         206 . The synthetic RNA circuit of  claim 205 , wherein the protein is a therapeutic protein, a cell death protein, a fluorescent protein, an antigen, a selection protein, or an immunomodulator. 
     
     
         207 .- 240 . (canceled) 
     
     
         241 . A method of treating cancer in a mammal comprising administering to a mammal the synthetic RNA circuit of  claim 123 . 
     
     
         242 . A method of inducing an immune response in a mammal comprising administering to a mammal the synthetic RNA circuit of  claim 123 . 
     
     
         243 .- 279 . (canceled) 
     
     
         280 . A synthetic RNA circuit comprising:
 (a) a first RNA molecule comprising a sequence encoding a destabilization domain fused to an RNA binding protein that is capable of inhibiting protein production; and   (b) a second RNA molecule comprising at least one RNA motif that is capable of binding to the RNA binding protein, and a sequence encoding an output molecule; wherein the destabilization domain is capable of facilitating degradation of the RNA binding protein in the absence of a small molecule that binds to the destabilization domain.   
     
     
         281 . The synthetic RNA circuit of  claim 280 , wherein the output molecule is a protein. 
     
     
         282 . The synthetic RNA circuit of  claim 281 , wherein the protein comprises a therapeutic protein, a cell death protein, a fluorescent protein, an antigen, a selection protein, or an immunomodulator. 
     
     
         283 . The synthetic RNA circuit of  claim 280 , further comprising a subgenomic promoter.