Nucleic acid binding polypeptides characterized by flexible linkers connected nucleic acid binding modules

A method according to any preceding claim, in which the binding affinity and/or specificity of the modified polypeptide to a nucleic acid sequence is increased compared to the binding affinity and/or specificity of an unmodified polypeptide.

A method according to any preceding claim, in which the nucleic acid sequence comprises a sequence which is bound by the unmodified polypeptide.

A method according to any preceding claim in which the nucleic acid sequence comprises a sequence bound by the unmodified nucleic acid binding polypeptide, into which one or more nucleic acid residues has been inserted.

A method according to any preceding claim, in which the nucleic acid residue(s) are inserted between target subsites bound by the first and second binding domains of the unmodified polypeptide.

A method of making a nucleic acid binding polypeptide, the method comprising the steps of:

• (a) providing a first binding domain and a second binding domain, at least one of the first and second binding domains consisting of one or two nucleic acid binding module(s);
• and
• (b) linking the first and second binding domains with a linker sequence comprising five or more amino acid residues.

A nucleic acid binding polypeptide comprising a first binding domain and a second binding domain linked by a linker sequence comprising five or more amino acid residues, in which at least one of the first and second binding domains consists of one or two nucleic acid binding module(s).

A non-naturally occurring nucleic acid binding polypeptide comprising a first binding domain comprising a nucleic acid binding module and a second binding domain comprising a nucleic acid binding module, the first and second binding domains being linked by a linker sequence comprising a structured linker.

A nucleic acid binding polypeptide comprising a first binding domain comprising a nucleic acid binding module and a second binding domain comprising a nucleic acid binding module, the first and second binding domains being linked by a linker sequence comprising a structured linker;

• a third binding domain consisting of one or two contiguous nucleic acid binding modules and a fourth binding domain consisting of one or two contiguous nucleic acid binding modules, the third and fourth binding domains being linked by a second linker sequence comprising five or more amino acid residues.

A method or polypeptide according to any preceding claim, in which the nucleic acid binding module is a zinc finger of the Cys2-His2 type.

A method or polypeptide according to any preceding claim, in which the nucleic acid binding module is selected from the group consisting of naturally occurring zinc fingers and consensus zinc fingers.

A method or polypeptide according to any preceding claim, in which the first linker sequence comprises between 5 and 8 amino acid residues.

A method or polypeptide according to any preceding claim, in which the first linker sequence is provided by insertion of one or more amino acid residues into a canonical linker sequence.

A method or polypeptide according to any preceding claim, in which the first linker sequence comprises a sequence selected from:

• GGEKP, GGQKP, GGSGEKP, GGSGQKP, GGSGGSGEKP, and GGSGGSGQKP.

A method or polypeptide according to any preceding claim, in which the nucleic acid binding polypeptide comprises a nucleic acid sequence selected from SEQ ID Nos:

• 22, 23, 24, 25, 26 and 27.

A method or polypeptide according to any preceding claim, in which the structured linker comprises an amino acid sequence which is not capable of specifically binding nucleic acid.

A method or polypeptide according to any preceding claim, in which the structured linker is derived from a zinc finger by mutation of one or more of its base contacting residues to reduce or abolish nucleic acid binding activity of the zinc finger.

A method or polypeptide according to any preceding claim, in which the structured linker comprises the amino acid sequence of TFIIIA finger IV.

A method or polypeptide according to any preceding claim, in which the zinc finger is finger 2 of wild type Zif268 mutated at positions −1, 2, 3 and 6.

A method or polypeptide according to any preceding claim, in which the nucleic acid binding polypeptide comprises substantially the sequence of Zif-ZnF-GAC (SEQ ID NO:

• 55), GAC-F4-Zif (SEQ ID NO:
• 54) or TF(1-4)-ZIF (SEQ ID NO:
• 53).

A method or polypeptide according to any preceding claim, in which the or each linker sequence comprises one or more further sequence(s), each further sequence comprising a canonical linker sequence, preferably GEKP, GERP, GQKP or GQRP, optionally comprising one or more amino acid sequences inserted into the canonical sequence.

Use of a structured linker in a method of making a nucleic acid binding polypeptide.

A nucleic acid binding polypeptide comprising a repressor domain and a plurality of nucleic acid binding domains, the nucleic acid binding domains being linked by at least one non-canonical linker.

Use of a nucleic acid binding domain comprising two zinc finger modules as a basic unit in the construction of a nucleic acid binding polypeptide.

A method of producing a nucleic acid binding polypeptide, the method comprising providing a first and a second nucleic acid binding domain each comprising two zinc finger modules, and linking the first and second nucleic acid binding domains with a a structured linker sequence or a flexible linker sequence.

Use of a amino acid sequence comprising five or more amino acid residues as a flexible linker to join two or more nucleic acid binding domains comprising two zinc finger modules.

Use of an amino acid sequence comprising a zinc finger which is not capable of specifically binding nucleic acid, as a structured linker to join two or more nucleic acid binding domains comprising two zinc finger modules.

A nucleic acid binding polypeptide comprising units of zinc finger binding domains linked by flexible and/or structured linkers, each zinc finger binding domain comprising two zinc finger modules.