Zinc fingers are small DNA-binding modules noted for their occurrence
in a large number of eukaryotic transcription factors, and their use i
n protein engineering. Although it was expected that zinc fingers can
bind to a wide diversity of DNA sequences, previous studies using mode
l zinc finger domains from Zif268 (and Sp1) have revealed a potential
Limitation to the DNA-binding specificity. For example, phage display
selection of individual zinc fingers to recognize trinucleotide DNA su
bsites returned fingers that bound specifically only to triplets of th
e form GNN, i.e., triplets with guanine at the 5' end. Following our r
ecently reported work [Isalan, M., Choo, Y., and Klug, A. (1997) Proc.
Natl. Acad. Sci. U.S.A. 94, 5617-5621], we now show that this limitat
ion can be overcome by the concerted randomization of certain amino ac
id positions in adjacent zinc fingers that specify overlapping DNA sub
sites. This illustrates an important mechanism underlying DNA recognit
ion by arrays of zinc fingers, and points the way to improved strategi
es for the design of highly specific zinc finger proteins that bind an
y given nucleotide sequence.