Dimerization of zinc fingers mediated by peptides evolved in vitro from random sequences

Peptides that mediate dimerization of attached zinc finger DNA-binding domains have been evolved in vitro starting from random sequences. We first used phage display to select dimerization elements from libraries of random 15-residue polypeptides that were fused to the N terminus of the zinc finger domains. We then reoptimized these peptides by sequentially randomizing five-residue blocks (proceeding across the peptide in three steps) and selecting variant peptides that further stabilized the protein-DNA complex. Biochemical experiments confirmed that the selected peptides promote dimerization of the zinc fingers on an appropriate DNA target site. These results demonstrate that dimerization units can be obtained readily from random polypeptide libraries of moderate complexity. Our success reemphasizes the utility of searching random peptide libraries in protein design projects, and the sequences presented here may be useful when designing novel transcription factors.