Synthetic hammerhead ribozymes with ligand-responsive tertiary interactions

Abstract

A ligand-responsive hammerhead ribozyme is provided. In some embodiments, the ribozyme comprises: i. a first loop that has been replaced by an RNA aptamer that binds to a ligand; and ii. a second loop comprising a modified sequence, wherein the aptamer and the second loop interact in a ligand-dependent manner and autocatalytic cleavage of the ribozyme is ligand-responsive.

1 . A ligand-responsive hammerhead ribozyme that, relative to a wild-type hammerhead ribozyme, comprises
 i. a first loop that has been replaced by an RNA aptamer that binds to a ligand, and   ii. a second loop comprising a modified sequence, wherein   the first and second loops are loops L1 and L2 respectively, or loops L2 and L1 respectively, and   the aptamer and the loop with the modified sequence interact in a ligand-dependent manner and autocatalytic cleavage of the ribozyme is ligand-responsive.   
     
     
         2 . The ribozyme of  claim 1 , wherein the first loop is loop L1 and the second loop is loop L2. 
     
     
         3 . The ribozyme of  claim 1 , wherein the first loop is loop L2 and the second loop is loop L1. 
     
     
         4 . The ribozyme of  claim 1 , wherein the ribozyme is a Type III hammerhead ribozyme. 
     
     
         5 . The ribozyme of  claim 1 , wherein binding of the ligand to the aptamer causes the first and second loops to disassociate from one another, thereby inhibiting autocatalytic cleavage of the ribozyme. 
     
     
         6 . The ribozyme of  claim 1 , wherein binding of the ligand to the aptamer causes the first and second loops to interact with one another, thereby activating autocatalytic cleavage activity of the ribozyme. 
     
     
         7 . The ribozyme of  claim 1 , wherein the aptamer is at least 20 nucleotides in length. 
     
     
         8 . The ribozyme of  claim 1 , wherein the ligand for the aptamer has a molecular weight of less than 1 kDa. 
     
     
         9 . The ribozyme of  claim 1 , wherein the aptamer is directly connected to a stem region of the ribozyme, thereby eliminating the first or second loop from the ribozyme. 
     
     
         10 . The ribozyme of  claim 1 , wherein the ribozyme comprises:
 a first stem of 4-7 bp;   a second stem of 4-6 bp;   a third stem of 3-6 bp;   wherein: (i) the first and second stems are joined by sequence CUGANGA, (ii) the second and third stems are joined by sequence GAAA, and (iii) the second and third stems are joined by sequence NUH;   the first stem or the second stem is directly joined by an aptamer and the other of the first and second stems terminates in a loop that contains a modified sequence; and   wherein the aptamer and the modified sequence interact in a ligand-dependent manner and autocatalytic cleavage of the ribozyme is ligand-responsive.   
     
     
         11 . The ribozyme of  claim 1 , wherein the ribozyme comprises at least one non-naturally occurring nucleotide. 
     
     
         12 . A construct comprising a nucleic acid encoding the ligand-responsive hammerhead of  claim 1 . 
     
     
         13 . The construct of  claim 12 , wherein the nucleic acid is present in an expression cassette comprising: a promoter, a coding sequence, and a 3′ UTR. 
     
     
         14 . The construct of  claim 12 , wherein the nucleic acid is present in a 3′ UTR of the expression cassette. 
     
     
         15 . A cell comprising the construct of  claim 12 . 
     
     
         16 . The cell of  claim 15 , wherein the cell is a eukaryotic cell. 
     
     
         17 . A method for modulating expression of a gene comprising:
 contacting a cell of  claim 15 , with a ligand for the aptamer, thereby causing the first and second loops to associate or disassociate and modulating expression of the product encoded by the coding sequence.   
     
     
         18 . The method of  claim 17 , wherein the product encoded by the coding sequence is a protein or a non-coding RNA. 
     
     
         19 . The method of  claim 17 , wherein binding of the ligand to the aptamer inhibits autocatalytic cleavage of the ribozyme, thereby increasing expression of the protein encoded by the coding sequence. 
     
     
         20 . The method of  claim 17 , wherein binding of the ligand to the aptamer induces autocatalytic cleavage of the ribozyme, thereby reducing expression of the protein encoded by the coding sequence. 
     
     
         21 - 22 . (canceled)