Engineering & Evolution of Proteins that Target Specific DNA Sites

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My program focuses on how nature uses proteins to recognize specific DNA sequences. We adopt a protein design and engineering approach to understand how the protein scaffold can be used to target a specific DNA site with high affinity and specificity. Protein engineering is a growing field that has increasingly attracted researchers, and new multidisciplinary tools and methods are being developed that enable our design approach. Such tools can come from research far afield, for example, computational methods developed by computer scientists and mathematicians.We have developed minimalist hybrid proteins (MHP) as a platform to study how larger native proteins recognize specific DNA sequences, especially sites of 6-12 base pairs in length. Our MHPs are small (25-75 amino acids), and they mimic very well the DNA-binding function of the native transcription factors that they are based on, for they bind their targets with high DNA sequence specificity and binding affinity.Our lab also develops new tools that facilitate making unnatural proteins with desired properties. We target the E-box DNA site (5'-CACGTG), which is targeted by proteins Myc and Max and is involved in >50% of all cancers. The E-box network is ideal for us to tackle, as it is a well-studied system with abundant literature. We design specific mutations to generate new proteins that bind a desired DNA target. Mutations can be rationally designed, and we also use nonrational methods including directed evolution in the yeast one-hybrid (Y1H) and bacterial one-hybrid (B1H) systems, and the new phage-assisted continuous evolution (PACE) system we just started to implement in the lab. For in vivo detection of protein:DNA complexation, we have used the classic Y1H, which is one of the earliest systems developed for detecting a protein:DNA interaction in live cells. Because yeast are highly prone to giving false signals, we sought to develop a simpler in vivo tool for assaying protein:DNA interactions. We therefore developed the FRep (FRET Reporter) assay that is performed in E. coli. Preliminary FRep data (a screen of just 15 mutants from a much larger library) has allowed us to isolate a protein showing strong and specific binding to E-box. FRep, B1H and PACE are bacterial and are more straightforward to use than yeast.This proposal is therefore focused on two methodological aspects of my program: 1) biophysically understanding FRep, our method for detection of protein:DNA interactions, and 2) developing the B1H and particularly PACE evolution systems as methods that will enable us to more efficiently and successfuly generate MHPs with desired properties.Our MHP approach may someday impact the field of drug design. For example, organic synthetic chemists have been targeting the Myc/Max transcription factor by generating small-molecule inhibitors of the Myc/Max protein/protein interaction. However, no useful small molecule has been developed despite over two decades of work. Our research may complement these efforts by providing new strategies for drug development.We also collaborate with Linda Penn, a cancer researcher at Princess Margaret Hospital, and Warren Chan, a nano-engineer in UofT Engineering. All of my trainees are involved in this diverse collaboration, and they come from diverse background in chemistry and biology. They are exposed to a broad range of research skills and languages including molecular biology, health-related research, chemistry, bioengineering, and nanotechnology. My trainees learn to think in an interdisciplinary manner and to communicate in various scientific and engineering disciplines. The diversity and synergistic value of this collaboration is a great strength of the training my program provides.