Zinc finger binding domains for GNN

Abstract

Zinc finger-nucleotide binding polypeptides having binding specificity for target nucleotides containing one or GNN triplets are provided. Compositions containing such polypeptides and the use of such polypeptides and compositions for regulating gene expression are also provided.

1 - 39 . (canceled) 
     
     
         40 . A non-naturally occurring polypeptide which is a zinc finger nucleotide binding polypeptide, which non-naturally occurring polypeptide binds a nucleotide sequence of the form 5′-(GNN)-3′ as a result of the presence of one or more nucleotide sequence binding regions 
       
         
           
                 
                 
               
                     
                   QS/PS/GN/D/T/H/S/ELVR, 
                 
                     
                     
                 
                     
                   DP/SGN/D/E/T/H/K/A/SLVR, 
                 
                     
                     
                 
                     
                   R/KSP/AN/Q/T/D/K/H/V/A/SLVR, 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   TS/PGN/E/T/D/K/H/SLVR. 
                 
             
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         41 . The polypeptide of  claim 40  which has two or more of the nucleotide sequence binding regions. 
     
     
         42 . The polypeptide of  claim 41  which has two to six of the nucleotide sequence binding regions. 
     
     
         43 . The polypeptide of  claim 41  which has two to twelve of the nucleotide sequence binding regions. 
     
     
         44 . The polypeptide of  claim 40  wherein the nucleotide sequence binding region is selected from the group consisting of: 
       
         
           
                 
                 
                 
               
                     
                   QSSNLVR, 
                   (SEQ ID NO: 1) 
                 
                     
                     
                 
                     
                   DPGNLVR, 
                   (SEQ ID NO: 2) 
                 
                     
                     
                 
                     
                   TSGNLVR, 
                   (SEQ ID NO: 4) 
                 
                     
                     
                 
                     
                   TSGELVR, 
                   (SEQ ID NO: 8) 
                 
                     
                     
                 
                     
                   DPGHLVR, 
                   (SEQ ID NO: 10) 
                 
                     
                     
                 
                     
                   TSGHLVR, 
                   (SEQ ID NO: 12) 
                 
                     
                     
                 
                     
                   QSSSLVR, 
                   (SEQ ID NO: 13) 
                 
                     
                     
                 
                     
                   DPGALVR, 
                   (SEQ ID NO: 14) 
                 
                     
                     
                 
                     
                   TSGSLVR, 
                   (SEQ IS NO: 16) 
                 
                     
                     
                 
                     
                   QSGNLVR; 
                   (SEQ ID NO: 18) 
                 
                     
                     
                 
                     
                   QPGNLVR; 
                   (SEQ ID NO: 19) 
                 
                     
                     
                 
                     
                   KSANLVR; 
                   (SEQ ID NO: 22) 
                 
                     
                     
                 
                     
                   QSSTLVR; 
                   (SEQ ID NO: 25) 
                 
                     
                     
                 
                     
                   QPGDLVR; 
                   (SEQ ID NO: 27) 
                 
                     
                     
                 
                     
                   QPGTLVR; 
                   (SEQ ID NO: 30) 
                 
                     
                     
                 
                     
                   TPGELVR; 
                   (SEQ ID NO: 49) 
                 
                     
                     
                 
                     
                   TSGDLVR; 
                   (SEQ ID NO: 50) 
                 
                     
                     
                 
                     
                   QSSDLVR; 
                   (SEQ ID NO: 54) 
                 
                     
                     
                 
                     
                   TPGTLVR; 
                   (SEQ ID NO: 56) 
                 
                     
                     
                 
                     
                   TSGTLVR; 
                   (SEQ ID NO: 58) 
                 
                     
                     
                 
                     
                   QSSHLVR; 
                   (SEQ ID NO: 59) 
                 
                     
                     
                 
                     
                   QSGHLVR; 
                   (SEQ ID NO: 60) 
                 
                     
                     
                 
                     
                   QPGHLVR; 
                   (SEQ ID NO: 61) 
                 
                     
                     
                 
                     
                   TPGHLVR; 
                   (SEQ ID NO: 73) 
                 
                     
                     
                 
                     
                   TSGKLVR; 
                   (SEQ ID NO: 75) 
                 
                     
                     
                 
                     
                   QPGELVR; 
                   (SEQ ID NO: 76) 
                 
                     
                     
                 
                     
                   QSGELVR; 
                   (SEQ ID NO: 77) 
                 
                     
                     
                 
                     
                   DPGSLVR; 
                   (SEQ ID NO: 79) 
                 
                     
                     
                 
                     
                   RSASLVR 
                   (SEQ ID NO: 91) 
                 
                     
                     
                 
                     
                   KSASLVR; 
                   (SEQ ID NO: 97) 
                 
                     
                     
                 
                     
                   KSAALVR; 
                   (SEQ ID NO: 98) 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   TSGELVR. 
                   (SEQ ID NO: 107) 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         45 . The polypeptide of  claim 40  further comprising a transcription regulating polypeptide. 
     
     
         46 . The polypeptide of  claim 45  wherein the transcription regulating polypeptide activates transcription. 
     
     
         47 . The polypeptide of  claim 45  wherein the transcription regulating polypeptide inhibits transcription. 
     
     
         48 . A method to prepare a vector encoding a non-naturally occurring polypeptide which is a zinc finger nucleotide binding polypeptide, comprising:
 a) selecting a heptapeptide that binds 5′-GNA-3′, 5′-GNC-3′, 5′-GNG-3′, or 5′-GNT-3′ in a nucleotide sequence of at least 9 base pairs; and   b) preparing an expression vector that encodes a non-naturally occurring polypeptide having the selected heptapeptide sequence.   
     
     
         49 . The method of  claim 48  wherein the selected heptapeptide is 
       
         
           
                 
                 
               
                     
                   QS/PS/GN/D/T/H/S/ELVR, 
                 
                     
                     
                 
                     
                   DP/SGN/D/E/T/H/K/A/SLVR, 
                 
                     
                     
                 
                     
                   R/KSP/AN/Q/T/D/K/H/V/A/SLVR, 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   TS/PGN/E/T/D/K/H/SLVR 
                 
             
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         50 . A method to detect or determine binding of a non-naturally occurring polypeptide to 5′-GNA-3′, 5′-GNC-3′, 5′-GNG-3′, or 5′-GNT-3′ in the genome of a cell, comprising:
 a) introducing to a host cell an expression vector that encodes a non-naturally occurring polypeptide having a heptapeptide that binds 5′-GNA-3′, 5′-GNC-3′, 5′-GNG-3′, or 5′-GNT-3′ in a nucleotide sequence of at least 9 base pairs; and   b) detecting or determining whether the non-naturally occurring polypeptide binds a nucleotide sequence in the genome of the cell that has 5′-GNA-3′, 5′-GNC-3′, 5′-GNG-3′, or 5′-GNT-3′.   
     
     
         51 . The method of  claim 50  wherein the selected heptapeptide is 
       
         
           
                 
                 
               
                     
                   QS/PS/GN/D/T/H/S/ELVR, 
                 
                     
                     
                 
                     
                   DP/SGN/D/E/T/H/K/A/SLVR, 
                 
                     
                     
                 
                     
                   R/KSP/AN/Q/T/D/K/H/V/A/SLVR, 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   TS/PGN/E/T/D/K/H/SLVR 
                 
             
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         52 . A method to control expression of sequences 3′ to a nucleotide sequence of at least 9 base pairs that includes 5′-GNA-3′, 5′-GNC-3′, 5′-GNG-3′, or 5′-GNT-3′ in the genome of a cell, comprising:
 expressing in a host cell an expression vector that encodes a non-naturally occurring polypeptide having a heptapeptide that binds 5′-GNA-3′, 5′-GNC-3′, 5′-GNG-3′, or 5′-GNT-3′ in a nucleotide sequence of at least 9 base pairs in an amount effective to regulate expression of nucleic acid linked 3′ to the nucleotide sequence and present in the genome of the host cell.