Structural Design and Validation of Linkers for Zinc Finger Proteins

Abstract

The possibility of using engineered DNA binding proteins to either control gene expression (artificial transcription factors) or to alter the sequence or structure of specific genes (Zinc Finger Nucleases) is a highly appealing idea with a wide range of applications in biological research and molecular therapeutics. Zinc finger proteins (ZFPs), belonging to Cys2His2 family constitute the most common DNA binding motifs found in eukaryotes. ZFP normally occur in repeats of two to three zinc finger motifs (ZFMs) to bind 6-9 contiguous DNA base pairs in a sequence specific manner. Several methods of varying complexity are available to engineer ZFPs that can target all the 64 codons in the genome. Although ZFPs are becoming a powerful tool for site specific modification in the genome, several challenges remain before the full potential of ZFPs can be realized. The engineered ZFPs generated using the present design platforms target mostly base triplets with 5' Guanine (GNN, where N is any nucleotide) and the non-GNN or AT rich modules are difficult to target. In the present project we attempt to address this challenge by designing linker regions between the ZFP motifs to target non-contiguous base pairs in the DNA. This will increase the number of targetable DNA sequences by an order of magnitude and will help to realize the full potential of ZFPs. Using structure based methods, we provide an extensive library of possible linker molecules that can be introduced between the individual zinc finger motifs to skip up to 10 base pairs between adjacent zinc finger protein recognition sites in the DNA sequences. We also performed a proof of principle experiment to validate the binding affinity and specificity of one of the computationally designed ZFP to its target DNA sequence.