Method for target site selection and discovery

Abstract

Nucleic acid catalysts, method of screening/selection for nucleic acid catalysts, synthesis of ribozyme libraries and discovery of gene sequences involved in a biological process are described.

1 . A method comprising: 
 a) providing a random binding arm hairpin ribozyme library to mammalian cells in culture under conditions suitable for a hairpin ribozyme from the library to down-regulate the expression of a gene in the mammalian cells;    b) determining the cells in which the expression of a gene has been down-regulated under step (a);    c) determining the nucleotide sequence of at least one portion of the binding arm of the hairpin ribozyme in the cell of step (b); and    d) identifying the gene, the expression of which is down-regulated in step (a), using the nucleotide sequence from step (c).    
     
     
         2 . The method of  claim 1 , wherein said library of hairpin ribozyme is encoded by an expression vector in a manner which allows expression of said hairpin ribozymes.  
     
     
         3 . The method of  claim 2 , wherein said expression vector comprises: 
 a) a transcription initiation region;    b) a transcription termination region; and    c) a sequence encoding at least one said hairpin ribozyme, wherein said sequence is operably linked to said initiation region and said termination region, in a manner which allows expression, delivery, or expression and delivery of said hairpin ribozyme.    
     
     
         4 . The method of  claim 2 , wherein said expression vector comprises: 
 a) a transcription initiation region;    b) a transcription termination region;    c) an open reading frame for a polypeptide; and    d) a sequence encoding at least one said hairpin ribozyme, wherein said sequence is operably linked to the 3′-end of said open reading frame; wherein said sequence is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression, delivery, or expression and delivery of said hairpin ribozyme.    
     
     
         5 . The method of  claim 2 , wherein said expression vector comprises: 
 a) a transcription initiation region;    b) a transcription termination region;    c) an intron; and    d) a sequence encoding at least one said hairpin ribozyme, wherein said sequence is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression, delivery, or expression and delivery of said hairpin ribozyme.    
     
     
         6 . The method of  claim 2 , wherein said expression vector comprises: 
 a) a transcription initiation region;    b) a transcription termination region;    c) an intron;    d) an open reading frame for a polypeptide; and    e) a sequence encoding at least one said hairpin ribozyme, wherein said sequence is operably linked to the 3′-end of said open reading frame; wherein said sequence is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression,delivery, or expression and delivery of said hairpin ribozyme.    
     
     
         7 . The method of  claim 2 , wherein said expression vector is derived from an expression vector selected from the group consisting of retrovirus, adenovirus, adenoassociated virus, and alphavirus expression vectors.  
     
     
         8 . The method of  claim 2 , wherein said expression vector is operably linked to an RNA polymerase II promoter element.  
     
     
         9 . The method of  claim 2 , wherein said expression vector is operably linked to an RNA polymerase III promoter element.  
     
     
         10 . The method of  claim 9 , wherein said RNA polymerase III promoter element is derived from a transfer RNA gene.  
     
     
         11 . The method of  claim 9 , wherein said RNA polymerase III promoter element is derived from a U6 small nuclear RNA gene.  
     
     
         12 . The method of  claim 9 , wherein the hairpin ribozyme comprises a sequence at its 5′-end homologous to the terminal 27 nucleotides encoded by said U6 small nuclear RNA gene.  
     
     
         13 . The method of  claim 9 , wherein said RNA polymerase III promoter is derived from a TRZ RNA gene.  
     
     
         14 . The method of  claim 1 , wherein said substrate binding arm is of a length between 12 and 100 nucleotides.  
     
     
         15 . The method  claim 1 , wherein said hairpin ribozyme binding arm is of a length between 14 and 24 nucleotides.  
     
     
         16 . The method of  claim 1 , wherein said hairpin ribzoyme comprises two binding arms.  
     
     
         17 . The method of  claim 16 , wherein said hairpin ribozyme binding arms are of similar length.  
     
     
         18 . The method of  claim 16 , wherein said hairpin ribozyme binding arms are of different length.  
     
     
         19 . The method of  claim 1 , wherein said library of hairpin ribozyme is a multimer library.