Nucleic acid binding proteins

1. A method for designing a nucleic acid binding protein that binds to a target nucleotide sequence, wherein the binding protein comprises a plurality of zinc fingers, and adjacent zinc fingers bind synergistically to overlapping quadruplet target subsites, and wherein the method comprises:

i) selecting a quadruplet within the target nucleotide sequence;

ii) designing the binding protein such that binding of a zinc finger to the quadruplet is obtained by choosing the sequence of particular residues of the zinc finger depending on the nucleotide sequence of the quadruplet, as follows:

a) if base 4 in the quadruplet is A, then position +6 in the α-helix is Gln and position ++2 is not Asp;

b) if base 4 in the quadruplet is C, then position +6 in the α-helix may be any residue, as long as position ++2 in the α-helix is not Asp.

2. The method according to claim 1 , wherein binding to base 4 of the quadruplet by a zinc finger is additionally determined as follows:

c) if base 4 in the quadruplet is G, then position +6 in the α-helix is Arg, or position +6 is Ser or Thr and position ++2 is Asp;

d) if base 4 in the quadruplet is T, then position +6 in the α-helix is Ser or Thr and position ++2 is Asp.

3. A method for designing a nucleic acid binding protein that binds to a target nucleotide sequence, wherein the binding protein comprises a plurality of zinc fingers, and adjacent zinc fingers bind synergistically to overlapping quadruplet target subsites, and wherein the method comprises:

i) selecting a quadruplet within the target nucleotide sequence;

ii) designing the binding protein such that binding of a zinc finger to the quadruplet is obtained by choosing the sequence of particular residues of the zinc finger depending on the nucleotide sequence of the quadruplet, as follows:

a) if base 4 in the quadruplet is G, then position +6 in the α-helix is Arg; or position +6 is Ser or Thr and position ++2 is Asp;

b) if base 4 in the quadruplet is A, then position +6 in the α-helix is Gln and position ++2 is not Asp;

c) if base 4 in the quadruplet is T, then position +6 in the α-helix is Ser or Thr and position ++2 is Asp;

d) if base 4 in the quadruplet is C, then position +6 in the α-helix may be any amino acid, provided that position ++2 in the α-helix is not Asp;

e) if base 3 in the quadruplet is G, then position +3 in the α-helix is His;

f) if base 3 in the quadruplet is A, then position +3 in the α-helix is Asn;

g) if base 3 in the quadruplet is T, then position +3 in the α-helix is Ala, Ser or Val; provided that if it is Ala, then the residues at −1 or +6 are small residues;

h) if base 3 in the quadruplet is C, then position +3 in the α-helix is Ser, Asp, Glu, Leu, Thr or Val;

i) if base 2 in the quadruplet is G, then position −1 in the α-helix is Arg;

j) if base 2 in the quadruplet is A, then position −1 in the α-helix is Gln;

k) if base 2 in the quadruplet is T, then position −1 in the α-helix is Asn or Gln;

l) if base 2 in the quadruplet is C, then position −1 in the α-helix is Asp;

m) if base 1 in the quadruplet is G, then position +2 is Asp;

n) if base 1 in the quadruplet is A, then position +2 is not Asp;

o) if base 1 in the quadruplet is C, then position +2 is not Asp;

p) if base 1 in the quadruplet is T, then position +2 is Ser or Thr.

4. The method of claims 3 , wherein each zinc finger has the general primary structure with positions numbered such that position 1 is the first position of an alpha helix

−1 1 2 3 4 5 6 7 8 9

X a Cys X 2-4 Cys-X 2-3 -Phe-X c -X-X-X-X-Leu-X-X-His-X-X-X b His/Cys, SEQ ID NO:25) wherein X (including X a , X b and X c ) is any amino acid.

5. The method according to claim 4 , wherein X a is Phe/Tyr-X or Pro-Phe/Tyr-X.

6. The method according to claim 5 , wherein X 2-4 is selected from any one of:

Ser-X, Glu-X, Lys-X, Thr-X, Pro-X and Arg-X.

7. The method according to claim 4 , wherein X b is Thr or Ile.

8. The method according to claim 4 , wherein X 2-3 is Gly-Lys-Ala, Gly-Lys-Cys, Gly-Lys-Ser, Gly-Lys-Gly, Met-Arg-Asn or Met-Arg.

9. The method according to claim 4 , wherein at least one zinc finger is linked to a linker that is Thr-Gly-Glu-Lys (SEQ ID NO: 4) or Thr-Gly-Glu-Lys-Pro (SEQ ID NO: 5).

10. The method according to claim 4 , wherein position +9 is Arg or Lys.

11. The method according to claim 4 , wherein positions +1, +5 and +8 are not occupied by any one of the hydrophobic amino acids Phe, Trp or Tyr.

12. The method according to claim 11 , wherein positions +1, +5 and +8 are occupied by the residues Lys, Thr and Gln respectively.

13. A method for designing a nucleic acid binding protein which binds a target nucleic acid sequence, comprising the steps of:

a) selecting a model zinc finger domain from the group consisting of naturally occurring zinc fingers and consensus zinc fingers; and

b) mutating the finger according to the rules of claim 2 .

14. A method for designing a nucleic acid binding protein which binds a target nucleic acid sequence, comprising the steps of:

a) selecting a model zinc finger domain from the group consisting of naturally occurring zinc fingers and consensus zinc fingers; and

b) mutating the finger according to the rules of claim 4 .

15. The method according to claim 14 , wherein the model zinc finger is a consensus zinc finger whose structure is selected from the group consisting of the consensus structure Pro-Tyr-Lys-Cys-Pro-Glu-Cys-Gly-Lys-Ser-Phe-Ser-Gln-Lys-Ser-Asp-Leu-Val-Lys-His-Gln-Arg-Thr-His-Thr-Gly (SEQ ID NO: 6), and the consensus structure Pro-Tyr-Lys-Cys-Ser-Glu-Cys-Gly-Lys-Ala-Phe-Ser-Gln-Lys-Ser-Asn; Leu-Thr-Arg-His-Gln-Arg-Ile-His-Thr-Gly-Glu-Lys-Pro (SEQ ID NO: 7).

16. The method according to claim 14 , wherein the model zinc finger is a naturally-occurring zinc finger of Zif268.

17. The method according to claim 16 , wherein the model zinc finger is finger 2 of Zif 268.

18. The method according to claim 14 , wherein the binding protein comprises two or more zinc finger binding motifs, placed N-terminus to C-terminus.

19. The method according to claim 15 , wherein the N-terminal zinc finger is preceded by a leader peptide having the sequence Met-Ala-Glu-Glu-Lys-Pro (SEQ ID NO: 8).

20. The method of any one of claims 1 , 2 and 3 , wherein a plurality of overlapping quadruplets are selected within the target sequence.

21. The method of claim 20 , wherein step (ii) comprises designing a first zinc finger that binds to a first of the overlapping quadruplets and a second zinc finger that binds to a second of the overlapping quadruplets.