Alpha-helix substitution: Novel approach for the design of a new zinc finger peptides for AT-rich sequence

A novel strategy for the design of a zinc finger peptide on the basis of α-helix substitution has been demonstrated. SplHM is a helix-substituted mutant for the wild-type Sp1(zf123) and its α-helix of each finger is replaced by that of fingers 4–6 of CF2-II. The circular dichroism spectrum of Spi HM suggests that Sp1 HM has an ordered secondary structure similar to Sp1(zf123). From the analyses of the DNA binding affinity and specificity by gel mobility shift assay, it is clearly indicated that SplHM specifically binds to the AT-rich sequence (5′-GTA TAT ATA-3′) with 3 nM dissociation constants. Moreover, the zinc finger peptides for the sequence alternating between the AT- and GC-rich subsites can also be created by the α-helix substitution. This strategy is evidently effective and is also more convenient than the phage display method. Consequently, our design method is widely applicable to creating zinc finger peptides with novel binding specificities.