Binding proteins for recognition of DNA

Abstract

Disclosed are libraries of DNA sequences encoding zinc finger binding motifs for display on a particle, together with methods of designing zinc finger binding polypeptides for binding to a particular target sequence and, inter alia, use of designed zinc finger polypeptides for various in vitro or in vivo applications.

We claim: 
     
       1. A library of DNA sequences, each sequence encoding a zinc finger polypeptide for display, the zinc finger polypeptide comprising at least one zinc finger having partially randomised allocation of amino acids, the partially randomised zinc finger having a random allocation of amino acids at positions −1, +2, +3 and +6 and at least one of positions +1, +5 or +8, position +1 being the first amino acid in the α-helix of the zinc finger. 
     
     
       2. A library according to  claim 1 , wherein the partially randomised zinc finger has random allocation of amino acids at each of positions +1, +5 and +8. 
     
     
       3. A library according to  claim 1 , wherein the encoded partially randomised zinc finger comprises the zinc finger of the Zif 268 polypeptide. 
     
     
       4. A library according to  claim 1  as a fusion with a DNA sequence encoding the minor coat protein of bacteriophage fd. 
     
     
       5. A method of designing a zinc finger polypeptide for binding to a particular target DNA sequence, comprising the steps of:
 comparing the binding to one or more DNA triplets of each of a plurality of zinc finger polypeptides having a partially randomized zinc finger, the zinc finger polypeptides being encoded by a library in accordance with  claim 1 ; and   selecting those nucleic acid sequences encoding randomised zinc fingers which bind to the target DNA sequence.   
     
     
       6. A method of designing a zinc finger polypeptide for binding to a particular target DNA sequence, comprising the steps of:
 screening against at least a portion of the target DNA sequence, a plurality of zinc finger polypeptides having a partially randomised zinc finger, the portion of the target DNA sequence being sufficient to allow binding of some of the zinc finger polypeptides, the plurality of zinc finger polypeptides being encoded by a library in accordance with  claim 1 ;   comparing the binding to one or more DNA triplets of each of said plurality of zinc finger polypeptides having a partially randomised zinc finger positioned between two or more zinc fingers having defined amino acid sequence; and selecting those nucleic acid sequences encoding randomised zinc fingers which bind to the target DNA sequence.   
     
     
       7. A method of designing a zinc finger polypeptide for binding to a particular target DNA sequence, the method comprising the steps of:
 screening against at least a portion of the target DNA sequence, zinc finger polypeptides having a partially randomised zinc finger, the portion of the target DNA sequence being sufficient to allow binding of some of the zinc finger polypeptides, the zinc finger polypeptides being encoded by a library in accordance with  claim 1 ;   comparing the binding to one or more DNA triplets of each of said zinc finger polypeptides having a partially randomised zinc finger;   selecting certain of the screened randomised zinc fingers for analysis of binding characteristics; and combining those sequences encoding desired zinc fingers to form a sequence encoding a single zinc finger polypeptide.   
     
     
       8. A method for producing a zinc finger polypeptide for binding to a particular target DNA sequence, comprising the steps of:
 screening against at least a portion of the target DNA sequence, zinc finger polypeptides having a partially randomised zinc finger, the portion of the target DNA sequence being sufficient to allow binding of some of the zinc finger polypeptides, the zinc finger polypeptides being coded by a library in accordance with  claim 1 ;   selecting those nucleic acid sequences encoding randomised zinc fingers which bind to the target DNA sequence; and expressing the selected nucleic acid sequences to produce zinc finger polypeptides which bind to the target DNA sequence.   
     
     
       9. A library according to  claim 1 , wherein the zinc finger polypeptide is displayed on a viral particle. 
     
     
       10. A library according to  claim 1 , wherein the partially randomised zinc finger is positioned between two or more zinc fingers. 
     
     
       11. A method of designing a zinc finger polypeptide for binding to a particular target DNA sequence, comprising the steps of:
 screening against at least a portion of the target DNA sequence, zinc finger polypeptides having a partially randomised zinc finger, the portion of the target DNA sequence being sufficient to allow binding of some of the zinc finger polypeptides, the zinc finger polypeptides being encoded by a library in accordance with  claim 1 ; and   selecting those nucleic acid sequences encoding randomised zinc fingers which bind to the target DNA sequence.   
     
     
       12. A method according to  claim 11 , wherein two or more rounds of screening are performed. 
     
     
       13. A method of designing a zinc finger polypeptide for binding to a particular target DNA sequence, wherein sequences encoding individual zinc fingers selected by the method of  claim 11  are randomly combined in the appropriate order to encode zinc finger polypeptides, the zinc finger polypeptides being screened against the target sequence, that combination of zinc finger sequences encoding a zinc finger polypeptide which binds to the target DNA sequence. 
     
     
       14. A method of modifying a nucleic acid sequence of interest present in a sample mixture by binding thereto a zinc finger polypeptide, wherein the zinc finger polypeptide is designed in accordance with  claim 11 , comprising contacting the sample mixture with a zinc finger polypeptide having affinity for at least a portion of the sequence of interest, so as to allow the zinc finger polypeptide to bind specifically to the sequence of interest. 
     
     
       15. A method according to  claim 14 , further comprising the step of separating the zinc finger polypeptide and the sequence of interest specifically bound thereto, from the rest of the sample. 
     
     
       16. A method according to  claim 14 , wherein the zinc finger polypeptide is bound to a solid phase support. 
     
     
       17. A method according to  claim 14 , wherein the presence of the zinc finger polypeptide bound to the sequence of interest is detected by the addition of one or more detection reagents. 
     
     
       18. A method according to  claim 14 , wherein the DNA sequence of interest is present in an acrylamide or agarose gel matrix, or is present on the surface of a membrane. 
     
     
       19. A DNA library according to  claim 1 , consisting of 64 sequences, each sequence comprising a different one of the 64 possible permutations of a DNA triplet, the library being arranged in twelve sublibraries, wherein for any one sub-library one base in the triplet is defined and the other two bases are randomised. 
     
     
       20. A library according to  claim 19  wherein the sequences are biotinylated. 
     
     
       21. A library according to  claim 19 , wherein the sequences are associated with separation means. 
     
     
       22. A library according to  claim 21 , wherein the separation means is selected from the group consisting of microtitre plate, magnetic bead, non-magnetic bead, sedimentation particle, and affinity chromatography column. 
     
     
       23. A kit for making a zinc finger polypeptide for binding to a nucleic acid sequence of interest, comprising: a library of DNA sequences according to  claim 1  encoding zinc finger polypeptides into a vector; a vector molecule that accepts one or more sequences from the library; and instructions for use. 
     
     
       24. A kit according to  claim 23 , wherein the vector directs the expression of the cloned sequences as a single zinc finger polypeptide. 
     
     
       25. A kit according to  claim 23 , wherein the vector directs the expression of the cloned sequences as a single zinc finger polypeptide displayed on the surface of a viral particle. 
     
     
       26. A kit for making a zinc finger polypeptide for binding to a nucleic acid sequence of interest, comprising: a library of DNA sequences in accordance with  claim 1 ; and instructions for use. 
     
     
       27. A kit according to  claim 26 , further comprising a DNA library consisting of 64 sequences, each sequence comprising a different one of the 64 possible permutations of a DNA triplet, the library being arranged in twelve sub-libraries, wherein for any one sub-library one base in the triplet is defined and the other two bases are randomized. 
     
     
       28. A kit according to  claim 27  further comprising appropriate buffer solutions and/or reagents for detection of bound zinc fingers. 
     
     
       29. A kit according to  claim 28  further comprising a vector suitable for accepting one or more sequences selected from the library of DNA sequences encoding zinc fingers. 
     
     
       30. A library of DNA sequences, each sequence encoding a zinc finger polypeptide for display, the zinc finger polypeptide comprising at least one zinc finger having partially randomised allocation of amino acids, the partially randomised zinc finger having a random allocation of amino acids at positions −1, +1, +2, +3 and +6, position +1 being the first amino acid in the α-helix of the zinc finger.  
     
     
       31. A library according to  claim 30 , wherein the partially randomised zinc finger further has a random allocation of amino acids at position +5. 
     
     
       32. A library according to  claim 31 , wherein the zinc finger polypeptide is displayed on a viral particle..  
     
     
       33. A library according to  claim 31 , wherein the partially randomised zinc finger is positioned between two or more zinc fingers. 
     
     
       34. A library according to  claim 30 , wherein the partially randomised zinc finger further has a random allocation of amino acids at position +8. 
     
     
       35. A library according to  claim 34 , wherein the zinc finger polypeptide is displayed on a viral particle. 
     
     
       36. A library according to  claim 34 , wherein the partially randomised zinc finger is positioned between two or more zinc fingers. 
     
     
       37. A library of DNA sequences, each sequence encoding a zinc finger polypeptide for display, the zinc finger polypeptide comprising at least one zinc finger having partially randomised allocation of amino acids, the partially randomised zinc finger having a random allocation of amino acids at positions −1, +2, +3, +5 and +6, position +1 being the first amino acid in the α-helix of the zinc finger. 
     
     
       38. A library according to  claim 37 , wherein the partially randomised zinc finger further has a random allocation of amino acids at position +8. 
     
     
       39. A library according to  claim 38 , wherein the zinc finger polypeptide is displayed on a viral particle. 
     
     
       40. A library according to  claims 38 , wherein the partially randomised zinc finger is positioned between two or more zinc fingers. 
     
     
       41. A library of DNA sequences, each sequence encoding a zinc finger polypeptide for display, the zinc finger polypeptide comprising at least one zinc finger having partially randomised allocation of amino acids, the partially randomised zinc finger having a random allocation of amino acids at positions −1, +2, +3, +6 and +8, position +1 being the first amino acid in the α-helix of the zinc finger. 
     
     
       42. A method for preparing a nucleic acid binding protein, comprising:
 synthesizing a polynucleotide encoding the nucleic acid binding protein of claim 50;   introducing the polynucleotide into a cell; and   expressing the nucleic acid binding protein from the introduced polynucleotide.   
     
     
       43. The method of claim 42, wherein the functional domain comprises an activation domain. 
     
     
       44. The method of claim 43, wherein the activation domain is VP16. 
     
     
       45. The method of claim 42, wherein the functional domain comprises a repression domain. 
     
     
       46. The method of claim 42, wherein the functional domain comprises a nuclear localization signal. 
     
     
       47. The method of claim 46, wherein the nuclear localization signal is a large T antigen of SV40. 
     
     
       48. The method of claim 42, wherein the functional domain comprises a catalytic domain of a restriction enzyme. 
     
     
       49. The method of claim 42, wherein the cell is a mammalian cell. 
     
     
       50. A nucleic acid binding protein that binds to a target nucleotide sequence, wherein the protein comprises
 (a) three or more zinc fingers selected based on their ability to bind a target DNA sequence, each finger comprising an antiparallel beta sheet packed against an alpha helix, the alpha helix including 10 amino acid residues numbered −1 to +9, wherein the region numbered −1 to +6 is a non-naturally occurring amino acid sequence and further wherein the alpha helix region of at least two non-naturally occurring zinc fingers comprise a seven amino acid residue sequence as shown in residues 1 to 7 of any of SEQ ID NOs:20-37, 39-51, 54, 59, 68, 72-74, 76, 77, 81, 85, 95, 100, 103, 116 and 119; and   (b) a heterologous functional domain selected from the group consisting of an activation domain, a repression domain, a nuclear localization signal, and a catalytic domain of a restriction enzyme.   
     
     
       51. The nucleic acid binding protein of claim 50, wherein the target nucleotide sequence comprises a plurality of contiguous triplets. 
     
     
       52. The nucleic acid binding protein of claim 50, wherein the functional domain comprises an activation domain. 
     
     
       53. The nucleic acid binding protein of claim 52, wherein the activation domain is VP16.