The DNA binding properties of semisynthetic zinc finger proteins.

摘要

Engineered DNA-binding domains based on tandem arrays of designed Cys ₂His₂ zinc fingers have proven to be an effective tool for the directed regulation of gene expression. The widespread application of this technology is presently hindered by limitations on the DNA sites that can be targeted by engineered proteins of this type. We present a series of experiments and experimental methods aimed at augmenting our understanding of zinc finger-DNA interactions and further broadening the spectrum of DNA sites amenable to zinc finger design. First, a method is presented for the generation of semisynthetic three-zinc finger proteins in which the N-terminal two fingers are expressed in E. coli and the third is made by chemical peptide synthesis. A fluorescence anisotropy-based DNA-binding assay is then presented which is suitable for the characterization of DNA-binding by these three-finger proteins. This assay was employed in a set of experiments comparing a pair of designed zinc finger proteins to demonstrate that DNA-binding affinity and DNA-binding specificity are not necessarily correlated properties and respond differently to changes in ionic strength. Next, a series of experiments is presented in which unnatural amino acids were incorporated into semisynthetic zinc finger proteins and used to confer novel DNA-binding specificities. Lastly, a pair of additional synthetic peptide chemistry techniques are discussed including the full chemical synthesis of a three finger protein by native chemical ligation and the synthesis of peptides on a glass surface by inkjet microdeposition.