Comprehensive Screens and Computational Analysis of Cys2His2 Zinc Fingers Uncovers Complexities of DNA-Binding Specificity
The Cys2His2 Zinc finger DNA-binding domain is the most common domain utilized by metazoan transcription factors and has become a critical domain applied toward synthetic biology and genome engineering applications. Despite decades of research, we are only able to provide a simplistic understanding of how this domain interacts with all target sequences. To increase our understanding of this important domain we created multiple libraries that fully randomize 6 base-recognizing positions of the protein. We screened the 64 million amino acid combinations offered by each library for their ability to bind all possible 3 base pair targets. We applied multiple filters to the recovered data to separate truly enriched proteins from the background. Next, we clustered these enriched proteins and find multiple, distinct strategies this domain may use to bind any 3 base target. We find similar groups of proteins are often enriched to bind similar targets. Conversely, we find that highly similar domains can offer distinct binding specificities. Nonetheless, computational predictions of DNA-binding specificity are in excellent agreement with the specificities offered by a large set of experimentally tested proteins. We are currently applying these exhaustive zinc finger pools to create artificial activators and transcription factor mimics. Ongoing research is investigating a multiple reporter systems to fine-tune specificity and potentially provide specificity and affinity information in a single experiment.