DESIGN OF POLYDACTYL ZINC-FINGER PROTEINS FOR UNIQUE ADDRESSING WITHIN COMPLEX GENOMES

Zinc-finger proteins of the Cys(2)-His(2) type represent a class of ma lleable DNA-binding proteins that may be selected to bind diverse sequ ences, Typically, zinc-finger proteins containing three zinc-finger do mains, like the murine transcription factor Zif268 and the human trans cription factor Sp1, bind nine contiguous base pairs, To create a clas s of proteins that would be generally applicable to target unique site s within complex genomes, we have utilized structure-based modeling to design a polypeptide linker that fuses two three-finger proteins, Two six-fingered proteins were created and demonstrated to bind 18 contig uous bp of DNA in a sequence-specific fashion, Expression of these pro teins as fusions to activation or repression domains allows transcript ion to be specifically up- or down-modulated within human cells, Polyd actyl zinc-finger proteins should be broadly applicable as genome-spec ific transcriptional switches in gene therapy strategies and the devel opment of novel transgenic plants and animals.