Nucleic acid binding proteins

1. A method for preparing 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 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;

iii) synthesizing a polynucleotide encoding the binding protein of (ii);

iv) introducing the polynucleotide of (iii) into a cell; and

v) incubating the cell under conditions in which the encoded nucleic acid binding protein is expressed.

2. A 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 preparing 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 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 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;

iii) synthesizing a polynucleotide encoding the binding protein of (ii);

iv) introducing the polynucleotide of (iii) into a cell; and

v) incubating the cell under conditions in which the encoded nucleic acid binding protein is expressed.

4. A method any one of claims 1 - 3 wherein each zinc finger has the general primary structure

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-linker SEQ ID NO: 3)

−1 1 2 3 4 5 6 7 8 9

wherein X (including X a , X b and X c ) is any amino acid.

5. A method according to claim 4 wherein Xa is Phe/Tyr-X or Pro-Phe/Tyr-X.

6. A 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. A method according to claim 4 wherein X b is Thr or Ile.

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

9. A method according to claim 4 wherein the linker is (SEQ ID NO: 4) Thr-Gly-Glu-Lys or (SEQ ID NO: 5) Thr-Gly-Glu-Lys-Pro.

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

11. A 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. A 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 preparing a nucleic acid binding protein of the Cys2-His2 zinc finger class 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 set in any one of claims 1 to 3 .

14. A method according to claim 13 , 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).

15. A method according to claim 13 , wherein the model zinc finger is a naturally-occurring zinc finger whose structure is selected from one finger of a protein selected from the group consisting of Zif268, GLI, Tramtrack and YY1.

16. A method according to claim 15 wherein the model zinc finger is finger 2 of Zif 268.

17. A method according to claim 13 wherein the binding protein comprises two or more zinc finger binding motifs, placed N-terminus to C-terminus.

18. A method according to claim 14 , 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).

19. A method according to claim 13 wherein the nucleic acid binding protein is obtained by recombinant nucleic acid technology, the method comprising the steps of:

a) preparing a nucleic acid coding sequence encoding two or more model zinc finger domains, placed N-terminus to C-terminus;

b) inserting the nucleic acid sequence into a suitable expression vector; and

c) expressing the nucleic acid sequence in a host organism in order to obtain the nucleic acid binding protein.

20. A method according, to claim 19 comprising the additional steps of subjecting the nucleic acid binding protein to one or more rounds of randomisation and selection.

21. A method according to claim 20 , wherein the randomisation and selection is carried out by phage display technology.

22. A method according to claim 21 , further comprising the steps of:

a) preparing a nucleic acid construct which expresses a fusion protein comprising the nucleic acid binding protein and a minor coat protein of a filamentous bacteriophage;

b) preparing further nucleic acid constructs which express a fusion protein comprising a selectively mutated nucleic acid binding protein and a minor coat protein of a filamentous bacteriophage;

c) causing the fusion proteins defined in steps (a) and (b) to be expressed on the surface of bacteriophage transformed with the nucleic acid constructs; and

d) assaying the ability of the bacteriophage to bind the target nucleic acid sequence and selecting the bacteriophage demonstrating superior binding characteristics.

23. A method according to claim 22 wherein the nucleic acid binding protein is selectively randomised at any one of positions +1, +5, +8, −1, +2, +3 or +6.

24. The method of claim 3 , wherein a plurality of overlapping quadruplets are selected within the target sequence.