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 of the Cys2-His2 class, wherein the second His in Cys2-His2 is His or Cys, and wherein each zinc finger has an alpha helix and amino acids numbered −1, 1, 2, 3, 4, 5, 6, 7, 8, and 9 with position 1 being the first amino acid of the alpha helix, further wherein adjacent zinc fingers bind synergistically to overlapping quadruplet target subsites, 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 Glu, Asn or Val;

b) if base 4 in the quadruplet is C, then position +6 in the α-helix is Ser, Thr, Val, Ala, Glu or Asn.

2. 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 of the Cys2-His2 class, wherein the second His in Cys2-His2 is His or Cys, and wherein each zinc finger has an alpha helix and amino acids are numbered −1, 1, 2, 3, 4, 5, 6, 7, 8, and 9 with position 1 being the first amino acid of the alpha helix, and adjacent zinc fingers bind synergistically to overlapping quadruplet target subsites, 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 Lys;

b) if base 4 in the quadruplet is A, then position +6 in the α-helix is Glu, Asn or Val;

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

d) if base 4 in the quadruplet is C, then position +6 in the α-helix is Ser, Thr, Val, Ala, Glu or Asn;

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;

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 His or Thr;

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

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

n) if base 1 in the quadruplet is A, then position +2 Arg or Gln;

o) if base 1 in the quadruplet is C, then position +2 is Asn, Gln, Arg, His or Lys;

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

3. The method according to claim 2 , wherein at least one zinc finger has the structure of

SEQ ID NO:115.

4. The method according to claim 2 , wherein the binding protein further comprises a linker that is Thr-Gly-Glu-Lys (SEQ ID NO: 4) or Thr-Gly-Glu-Lys-Pro (SEQ ID NO: 5).

5. The method according to claim 2 , wherein position +9 is Arg or Lys.

6. The method according to claim 3 , wherein positions +1, +5 and +8 are not occupied by any one of the hydrophobic amino acids, Phe, Trp or Tyr.

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

8. A method for designing a nucleic acid binding protein of the Cys2-His2 zinc finger class, wherein the second His in Cys2-His2 is His or Cys, which binds a target nucleic acid sequence, comprising the steps of:

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

b) mutating the finger according to claim 1 .

9. The method according to claim 2 , wherein the binding protein comprises two or more zinc finger binding motifs, placed N-terminus to C-terminus.

10. The method of claim 2 , wherein a plurality of overlapping quadruplets are selected within the target sequence.

11. The method of claim 10 , 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.

12. A method for designing a nucleic acid binding protein of the Cys2-His2 zinc finger class, wherein the second His in Cys2-His2 is His or Cys, which nucleic acid binding protein binds a target nucleic acid sequence, comprising the steps of:

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

b) mutating the finger according to claim 2 .