A ZINC-FINGER DIRECTORY FOR HIGH-AFFINITY DNA RECOGNITION

We have used two manovalent phage display libraries containing variant s of the Zif268 DNA-binding domain to obtain families of zinc fingers that bind to alterations in the last 4 bp of the DNB sequence of the Z if268 consensus operator, GCG TGGGCG. Affinity selection was performed by altering the Zif268 operator three base pairs at a time, and simul taneously selecting for sets of 16 related DNA sequences. In this way, only four experiments were required to select far ail possible 64 com binations of DNA triplet sequences. The results show that (i) for high -affinity DNA binding in the range observed for the Zif268 wild-type c omplex (K-d=0.5-5 nM), finger 1 specifically requires the arginine at the carboxy terminus of its recognition helix that forms a bidentate h ydrogen-bond with the guanine base (G) under bar in the crystal struct ure of Zif268 complexed to its DNA operator sequence GCG TGG <(G)under bar CG>; (ii) when the guanine base (G) is replaced by A, C, or T, a lower-affinity family (K-d greater than or equal to 50 nM) can be dete cted that shows an overall tendency to bind G-rich DNA; (iii) the resi dues at position 2 on the finger 2 recognition helix do not appear to interact strongly with the complementary 5' base in the finger 1 bindi ng site; and (iv) unexpected substitutions at the amino terminus of fi nger 1 can occasionally result in specificity for the 3' base in the f inger I binding site, A DNA recognition directory was constructed for high-affinity zinc fingers that recognize all three bases in a DNA tri plet for seven sequences of the type GNN. Similar approaches may be ap plied to other zinc fingers to broaden the scope of the directory.