Nucleic acid binding proteins

1. A method for binding a nucleic acid binding protein to a target nucleotide sequence, wherein the binding protein comprises a plurality of zinc fingers, and further wherein at least two 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 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;

(iii) preparing a nucleic acid encoding the binding protein of (ii); and

(iv) introducing the nucleic acid of (iii) into a cell,

whereby the encoded nucleic acid binding protein is expressed and binds to the target sequence.

2. A method for binding a nucleic acid binding protein to a target sequence, wherein the binding protein comprises a plurality of zinc fingers and further wherein at least two adjacent zinc fingers bind synergistically to overlapping quadruplet target subsites,

wherein the protein was designed by:

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;

wherein the method comprises:

(A) preparing a nucleic acid encoding the binding protein; and

(B) introducing the nucleic acid into a cell, whereby the encoded nucleic acid binding protein is expressed and binds to the target sequence.

3. A method according to claim 1 , 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/Cys-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.

4. A method according to claim 1 comprising the additional steps of subjecting the nucleic acid binding protein to one or more rounds of randomization and selection in order to improve the characteristics thereof.

5. A method according to claim 4 , wherein the randomization and selection is carried out by phage display technology.

6. A method according to claim 5 , comprising the steps of:

a) preparing a nucleic acid construct which express 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;

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

7. A method according to claim 4 , wherein the nucleic acid binding protein is selectively randomized at any one of positions +1, +5, +8, −1, +2, +3 or +6.

8. A method according to claim 7 , wherein, in the nucleic acid binding protein, position +6 of a zinc finger and positions −1, +1, +2 and +3 of an adjacent zinc finger are randomized.

9. A method according to claim 1 , wherein the binding protein further comprises a nucleic acid cleaving domain.

10. A method according to claim 2 , wherein the binding protein further comprises a nucleic acid cleaving domain.