Artificial endonuclease

Abstract

The present invention provides artificial endonucleases and methods to prepare and use those endonucleases.

What is claimed is:  
     
         1 . An isolated synthetic peptide or polypeptide comprising a domain which specifically binds a nucleic acid sequence and a domain which specifically binds a metal which is hydrolytic or redox active, wherein the domain which specifically binds the metal is within the domain which specifically binds the nucleic acid sequence.  
     
     
         2 . The peptide or polypeptide of  claim 1  which binds Eu(III) or Ce(IV).  
     
     
         3 . The peptide or polypeptide of  claim 1  which comprises the amino acid sequence TERRRFDKDGNGYISAAELRHVKIWFQNKRAKIK (SEQ ID NO:3), TERRRFRVFDKDGNGYISAAEKIWFQNKRAKIK (SEQ ID NO:4), TRRRRFLSFDKDGDGTITTKEEVWFQNRRMKWK (SEQ ID NO:5) TERRRQQLDKDGDGTIDEREIKIWFQNKRAKIK (SEQ ID NO:7), TERRRQQLDKDGDGTIDEREQIKIWF (SEQ ID NO:10), DEKRPRTAFSGEQLARLKREFNENRYLTERRRLRVFDKDGNGFIS AAEKIWFQNKRAKIKKST (SEQ ID NO:6), or a catalytically active portion thereof.  
     
     
         4 . The peptide or polypeptide of  claim 1  which binds Cu(II).  
     
     
         5 . The peptide or polypeptide of  claim 4  which comprises the amino acid sequence TERRRQQLSSEVGMTCSGCSGQIKIWF (SEQ ID NO:8), or a catalytically active portion thereof.  
     
     
         6 . The peptide or polypeptide of  claim 1  which binds Zn(II).  
     
     
         7 . The peptide or polypeptide of  claim 6  which comprises the amino acid sequence TERRRHELMHAIGFYHEAQIKIWF (SEQ ID NO:9) or a catalytically active portion thereof.  
     
     
         8 . The peptide or polypeptide of  claim 1  which comprises the amino acid sequence TERRRDIDGDGTITAKEKIWF (SEQ ID NO:13), TERRRQQLFDIDGDGTITAKEEAQIKIWF (SEQ ID NO:14), or a catalytically active portion thereof.  
     
     
         9 . The peptide or polypeptide of  claim 1  which comprises at least 20 residues.  
     
     
         10 . The peptide or polypeptide of  claim 1  which comprises a consensus EF-Hand sequence.  
     
     
         11 . The peptide or polypeptide of  claim 1  wherein the domain which specifically binds the nucleic acid sequence is a domain from a transcription factor.  
     
     
         12 . The peptide or polypeptide of  claim 11  wherein the transcription factor is engrailed.  
     
     
         13 . The peptide or polypeptide of  claim 11  wherein the transcription factor comprises a helix-turn-helix domain.  
     
     
         14 . The peptide or polypeptide of  claim 1  wherein the domain which specifically binds the nucleic acid sequence comprises a helix-turn-helix motif, a relaxed helix-turn-helix motif, a winged helix-turn-helix motif, a helix-loop-strand motif, or a hormone receptor motif.  
     
     
         15 . The peptide or polypeptide of  claim 14  which comprises alpha-helices 2 and 3 of a helix-turn-helix motif.  
     
     
         16 . The peptide or polypeptide of  claim 1  which binds dsDNA, dsRNA, ssDNA, ssRNA, A-DNA, B-DNA, or Z-DNA.  
     
     
         17 . The peptide or polypeptide of  claim 1  wherein the domain which specifically binds the nucleic acid sequence is a homeodomain.  
     
     
         18 . The peptide or polypeptide of  claim 1  wherein the hydrolytic metal is a transition metal.  
     
     
         19 . The peptide or polypeptide of  claim 1  which binds Zn(II), Ca(II), Mg(II), Cd(II), Ce(II), Cr(IV), Fe(III), Co(III), Mn(II) or Cu(II).  
     
     
         20 . The peptide or polypeptide of  claim 1  which binds a lanthanide.  
     
     
         21 . The peptide or polypeptide of  claim 1  further comprising a protein transport domain.  
     
     
         22 . An isolated nucleic acid molecule comprising a nucleic acid segment which encodes the peptide or polypeptide of any one of  claims 1  to  21 , or a sequence complementary thereto.  
     
     
         23 . An expression cassette comprising the nucleic acid molecule of  claim 22  which is operably linked to a promoter functional in a host cell.  
     
     
         24 . A host cell comprising the expression cassette of  claim 23  or contacted with the isolated peptide or polypeptide of any one of  claims 1  to  21 .  
     
     
         25 . The host cell of  claim 24  wherein the host cell is a prokaryotic cell.  
     
     
         26 . The host cell of  claim 24  wherein the host cell is a eukaryotic cell.  
     
     
         27 . A vector comprising the expression of cassette of  claim 23 .  
     
     
         28 . A method of using a synthetic endonuclease specific for a particular nucleic acid sequence comprising: contacting a sample comprising nucleic acid with an amount of the peptide or polypeptide of any one of  claims 1  to  21  effective to cleave at least one nucleic acid sequence in the sample.  
     
     
         29 . The method of  claim 28  wherein the sample comprises cells.  
     
     
         30 . A method to detect the presence of a nucleic acid sequence in a sample comprising: 
 a) contacting a sample comprising nucleic acid suspected of containing a nucleic acid sequence recognized by the peptide or polypeptide of  claim 1 , with the peptide or polypeptide of any one of  claims 1  to  21 ; and    b) determining or detecting whether the peptide or polypeptide cleaves the nucleic acid sequence.    
     
     
         31 . The method of  claim 30  wherein the detection or determination is by gel electrophoresis.  
     
     
         32 . A composition comprising the peptide or polypeptide of any one of  claims 1  to  21 .  
     
     
         33 . An isolated synthetic peptide or polypeptide comprising a domain which specifically binds a nucleic acid sequence and a domain which specifically binds a metal which is hydrolytic or redox active, wherein the domain which specifically binds the metal is within the domain which specifically binds the nucleic acid sequence for use in medical therapy