Use of ribozymes for functionating genes

Abstract

Methods and compositions for identifying a gene or genes associated with the generation of a specific cellular phenotype or a specific cellular response using combinatorial libraries of catalytic RNA directed against RNA sequences encoding structural or functional polypeptide motifs. The invention is exemplified by use of a combinatorial ribozyme library to target sequence in mRNAs encoding zinc finger, protein kinase and integrin motifs.

What is claimed:  
     
         1 . A method for identifying one or more members of a combinatorial ribozyme library that alters a phenotype of a host cell, said method comprising the steps of: 
 growing a cell culture comprising host cells, wherein said host cells comprise a transcription product of a mammalian target nucleic acid encoding a motif of interest, wherein the nucleotide sequence of said mammalian target nucleic acid is unknown;    contacting said cell culture with a plurality of members of a combinatorial ribozyme library, which bind to said transcription product, whereby expression of said transcription product is disrupted, wherein: 
 each member of library comprises a catalytic domain that cleaves a sequence within the transcription product of the target nucleic acid that encodes a motif of interest or variant thereof; and  
 the library comprises a plurality of such members;  
   identifying host cells that exhibit one or more phenotypic changes, whereby said host cells exhibiting one or more phenotypic changes are isolated, whereby said one or more members of said combinatorial library are identified.    
     
     
         2 . The method of  claim 1 , further comprising the step of; 
 utilizing the nucleotide sequence of said one or more members of said library that disrupt expression of said transcription product as a probe to identify nucleotide sequences of said transcription product and said mammalian target nucleic acid, whereby said nucleotide sequences of said transcription product and said mammalian target nucleic acid are obtained.    
     
     
         3 . The method of  claim 1 , wherein said host cell culture comprises a plurality of mammalian cells, bacterial cells, invertebrate cells or plant cells.  
     
     
         4 . The method of  claim 1 , wherein said motif is a zinc finger motif, a receptor protein kinase motif, or an integrin motif.  
     
     
         5 . The method of  claim 1 , wherein said transcription product is mRNA.  
     
     
         6 . A double stranded DNA molecule, comprising: 
 a sense strand and an antisense strand, wherein said sense strand codes for a catalytic domain, which when expressed as RNA, disrupts expression of an mRNA transcribed from a target nucleic acid encoding a motif of interest and binding regions flanking said catalytic domain for binding said RNA to said mRNA, and wherein a means for determining directionality of expression is included in said double stranded DNA.    
     
     
         7 . A vector comprising a double-stranded DNA of  claim 6 .  
     
     
         8 . The vector of  claim 7 , wherein said double-stranded DNA further comprises regulatory elements for expression.  
     
     
         9 . The double stranded DNA of  claim 6 , wherein said means for determining directionality of expression comprises a different non blunt-ended restriction enzyme site at each end of said double stranded DNA.  
     
     
         10 . The double stranded DNA of  claim 6 , wherein said RNA is a ribozyme.  
     
     
         11 . A retrovirus expression vector comprising: 
 a retrovirus plasmid vector comprising a double stranded DNA of  claim 6 .    
     
     
         12 . The retrovirus expression vector of  claim 11 , wherein said vector comprises supercoiled DNA.  
     
     
         13 . A retrovirus packaging cell line comprising: 
 a retrovirus expression vector of  claim 11 .    
     
     
         14 . An adeno-associated virus expression vector comprising: 
 an adeno-associated virus plasmid vector comprising a double stranded DNA of  claim 6 .    
     
     
         15 . An adeno-associated virus packaging cell line comprising: 
 an adeno-associated virus expression vector of  claim 14  and an adeno-associated virus helper plasmid.    
     
     
         16 . A plasmid expression vector comprising: 
 a double stranded DNA of  claim 6 .    
     
     
         17 . A combinatorial library comprising: 
 a plurality of double-stranded DNA molecules of  claim 6 , wherein said binding regions are degenerate.    
     
     
         18 . The combinatorial library of  claim 17 , wherein said target nucleic acid encodes a protein.  
     
     
         19 . The combinatorial library of  claim 18 , wherein said protein is an enzyme.  
     
     
         20 . The combinatorial library of  claim 28 , wherein said enzyme is a protein kinase or a protease.  
     
     
         21 . The combinatorial library of  claim 18 , wherein said protein contains a zinc-finger domain.  
     
     
         22 . The combinatorial library of  claim 18 , wherein said protein contains an integrin domain.  
     
     
         23 . The combinatorial library of  claim 18  wherein said protein is a signaling molecule or a structural protein.  
     
     
         24 . The double stranded DNA of  claim 6 , wherein said motif is a zinc finger motif, a protein kinase motif or an integrin motif.  
     
     
         25 . A retrovirus particle, comprising: 
 a genome encoding an RNA comprising a catalytic domain that cleaves mRNA transcribed from a target nucleic acid encoding a motif of interest, and binding sequences flanking said catalytic domain for binding said RNA to said mRNA.    
     
     
         26 . A mammalian cell, comprising: 
 one or more double stranded DNA(s) comprising a sense strand and an antisense strand, wherein said sense strand codes for a catalytic domain, which when expressed as RNA, cleaves a mRNA sequence transcribed from a target nucleic acid encoding a motif of interest, and binding regions flanking said catalytic domain for binding said RNA to said mRNA, and wherein a means for determining directionality of expression is included in said one or more double stranded DNA(s).    
     
     
         27 . A adeno-associated virus, comprising: 
 a genome encoding of an RNA comprising a catalytic domain for cleavage of an mRNA transcribed from a target nucleic acid comprising a sequence encoding a structural motif of interest, and binding regions flanking catalytic domain for binding said RNA to said mRNA.    
     
     
         28 . A ribozyme, comprising: 
 a catalytic domain and binding domains complementary to a nucleotide sequence encoding a motif of interest, that disrupts expression of said nucleotide sequence.    
     
     
         29 . The ribozyme of  claim 28 , wherein said nucleotide sequence is RNA.