Regulation of gene expression by aptamer-modulated rnase p cleavage

Abstract

The present disclosure provides polynucleotide constructs for the modulation of target gene expression by aptamer-mediated ribonuclease cleavage of the target gene RNA and methods of using the constructs to modulate gene expression in response to the presence or absence of a ligand that binds the aptamer. The polynucleotide constructs contains a ribonuclease substrate sequence (e.g., an RNase P substrate) and a riboswitch comprising an effector region and an aptamer such that when the aptamer binds a ligand, target gene expression occurs.

We claim: 
     
         1 . A polynucleotide cassette for the regulation of the expression of a target gene comprising an RNase P substrate sequence linked to a riboswitch, wherein the riboswitch comprises an effector region and an aptamer sequence, wherein the effector region comprises sequence complimentary to a portion of the RNase P substrate sequence. 
     
     
         2 . The polynucleotide cassette of  claim 1 , wherein the aptamer binds a small molecule ligand. 
     
     
         3 . The polynucleotide cassette of  claim 1 , wherein the RNase P substrate sequence comprises a sequence encoding a tRNA, mascRNA, MEN beta tRNA-like structure, viral tRNA-like structure, RNase P model substrate, and homologous sequences that can initiate RNase P cleavage. 
     
     
         4 . The polynucleotide cassette of  claim 1 , wherein the effector region comprises a sequence capable of forming a stem structure upon ligand binding the aptamer, optionally wherein the effector region stem is 6 to 12 base pairs. 
     
     
         5 . The polynucleotide cassette according to any one of  claims 1  to  4 , wherein the aptamer sequence is located 5′ to the RNase P substrate sequence and the effector region comprises sequence complimentary to the leader sequence of the RNase P substrate. 
     
     
         6 . The polynucleotide cassette of  claim 5 , wherein the acceptor stem of the RNase P substrate and the riboswitch effector region are separated by 0, 1, 2, 3, or 4 nucleotides. 
     
     
         7 . The polynucleotide cassette of  claim 5 , wherein the effector region additionally comprises sequence complementary to the acceptor stem sequence of the RNase P substrate. 
     
     
         8 . The polynucleotide cassette according to any one of  claims 1  to  4 , wherein the aptamer sequence is located 3′ to the RNase P substrate sequence and the effector region comprises sequence complimentary to the 3′ acceptor stem of the RNase P substrate sequence. 
     
     
         9 . The polynucleotide cassette of  claim 8 , wherein the effector region sequence complimentary to the 3′ acceptor stem of the RNase P substrate is 1 to 7 nucleotides. 
     
     
         10 . A method of modulating the expression of a target gene comprising:
 a. inserting the polynucleotide cassette of any one of  claims 1  to  4  into a an untranslated region (UTR) of the target gene,   b. introducing the target gene comprising the polynucleotide cassette into a cell, and   c. exposing the cell to a small molecule ligand that specifically binds the aptamer in an amount effective to increase expression of the target gene.   
     
     
         11 . The method of  claim 10 , wherein the aptamer sequence of the polynucleotide cassette is located 5′ to the RNase P substrate sequence and the effector region comprises sequence complimentary to the leader sequence of the RNase P substrate. 
     
     
         12 . The method of  claim 11 , wherein the acceptor stem of the RNase P substrate and the riboswitch effector region are separated by 0, 1, 2, 3, or 4 nucleotides. 
     
     
         13 . The method of  claim 10 , wherein the aptamer sequence of the polynucleotide cassette is located 3′ to the RNase P substrate sequence and the effector region comprises sequence complimentary to the 3′ acceptor stem of the RNase P substrate sequence. 
     
     
         14 . The method of  claim 13 , wherein the effector region sequence that is complimentary to the 3′ acceptor stem of the RNase P substrate is 1 to 7 nucleotides. 
     
     
         15 . The method of  claim 10 , wherein the polynucleotide cassette is inserted into the 5′ untranslated region of the target gene. 
     
     
         16 . The method of  claim 10 , wherein the polynucleotide cassette is inserted into the 3′ untranslated region of the target gene. 
     
     
         17 . The method of  claim 10 , wherein two or more of the polynucleotide cassettes are inserted into the target gene. 
     
     
         18 . The method of  claim 17 , wherein the two or more polynucleotide cassettes comprise different aptamers that specifically bind to different small molecule ligands. 
     
     
         19 . The method of  claim 17 , wherein the two or more polynucleotide cassettes comprise the same aptamer. 
     
     
         20 . The method of  claim 10 , wherein the target gene further comprises a gene regulation cassette that modulates target gene expression by aptamer-mediated regulation of alternative splicing. 
     
     
         21 . The method of  claim 10 , wherein the target gene comprising the polynucleotide cassette is incorporated in a vector for the expression of the target gene. 
     
     
         22 . The method of  claim 21 , wherein the vector is a viral vector. 
     
     
         23 . The method of  claim 22 , wherein the viral vector is selected from the group consisting of adenoviral vector, adeno-associated virus vector, and lentiviral vector. 
     
     
         24 . A vector comprising a target gene that contains a polynucleotide cassette according to any one of  claims 1  to  4 . 
     
     
         25 . The vector of  claim 24 , wherein the vector is a viral vector. 
     
     
         26 . The vector of  claim 25 , wherein the viral vector is selected from the group consisting of adenoviral vector, adeno-associated virus vector, and lentiviral vector. 
     
     
         27 . The vector of  claim 24 , wherein the target gene further comprises a gene regulation cassette that modulates target gene expression by aptamer-mediated regulation of alternative splicing.