Characterization and design of C2H2 zinc finger proteins as custom DNA binding domains

As the storage medium for the source code of life, DNA is fundamentally linked to all cellular processes. Nature employs hundreds of sequence-specific DNA binding proteins as transcription factors and repressors to regulate the flow of genetic expression and replication. By adapting these DNA-binding domains to target desired genome locations, they can be harnessed to treat diseases by regulating genes and repairing diseased gene sequences.\nThe C2H2 zinc finger motif is perhaps the most promising and versatile DNA binding framework. Each C2H2 zinc finger domain (module) is capable of recognizing approximately three adjacent nucleotide bases in standard B form DNA. Through directed mutagenesis, novel zinc finger modules (ZFMs) can be selected for most of the 64 possible DNA triplets. By assembling multiple ZFMs with the appropriate linkers, zinc finger proteins (ZFPs) can be generated to specifically bind extended DNA sequence motifs.\nSeveral methods of varying complexity are currently available for ZFP engineering. ZFPs generated from the relatively simple modular design method often fail to function in vivo. Those generated using the most reliable module subsets, those recognizing triplets with a 5' guanine (GNN), only function successfully only an estimated 50% of the time, while modularly assembled ZFPs comprising primarily non-GNN modules rarely function in vivo. These low success rates are extremely problematic for applications requiring multiple ZFPs that target adjacent sequence motifs. More complex ZFP engineering approaches provide enhanced success rates, as compared to modular design, with the drawback that they are also more labor intensive and require additional biological expertise.\nIn this research we developed and engineered novel ZFPs, analyzed characteristics of functional custom zinc finger proteins and their targets, formulated algorithms predictive of ZFP success for both modular assembly and OPEN (Oligomerized Pool Engineering) selection methods, and generated a web-based server and software tools to aid others in the successful application of this technology.