Our laboratory employs modern synthetic organic chemistry, continuous directed molecular evolution, and protein design to develop innovative molecular technologies that report on or modulate key (bio)chemical regulators in living systems. Our approach is to build functional molecules, both small molecules, peptides, and entire enzymes, to solve problems in biomedical science. Currently, the group is focused on three primary project areas:
Chemical biology approaches to interrogate signaling by protein chemical modifications
Although hundreds of proteins in the human proteome are subjected to regulation by lysine and cysteine acylation, the regulation of these modifications is largely unknown. Therefore, we are developing tools to uncover how cells regulate deacylation and other “eraser” activities, and how these modifications regulate signaling. Our approach is to develop small molecule fluorescent imaging reagents that report on the “erasers” of lysine and cysteine PTMs. For example, hundreds of human proteins are modified by reversible palmitoylation of cysteine residues (S-palmitoylation), but the regulation and dynamics of depalmitoylation in cells is poorly understood. We recently developed DPPs, small molecule fluorescent probes to monitor the endogenous activity levels of “erasers” of S-palmitoylation, acyl-protein thioesterases (APTs). Live-cell analysis with DPPs revealed rapid growth factor-mediated inhibition of the depalmitoylation activity of APTs, exposing a novel regulatory mechanism of dynamic lipid signaling. Ultimately, we are driven to uncover how chemical modifications to the proteome are regulated, with a particular focus on dynamic subcellular trafficking.
Activity-responsive RNA polymerases to analyze and control cell fate
We are pioneering a new approach for both bioanalysis the cellular engineering that utilizes activity-responsive RNA polymerases (ARs) as a new family of easily programmable biosensors. We recently unveiled protease-responsive RNAPs (PRs) as a strategy to respond to protease activities by production of defined RNA outputs, and showed that PRs can encode multidimensional protease activities in defined sequences of RNA in both prokaryotic and mammalian cells. RNAPs in principle provide a new platform for biosensor creation, but engineering such complex enzymes is challenging. Therefore, we used evolution to create a proximity-dependent “split” RNAP as a versatile biosensor platform. We have now generated split RNAP-based AR sensors for small molecules, protein-protein interactions, light, catalytic processes, and more, with robust sensitivity and dynamic range (>300-fold). We are deploying ARs as new synthetic biology tools to control genetic therapies, to create biosensing platforms to analyze endogenous (bio)chemical processes using high-throughput sequencing, and to develop new rapid evolution platforms to reprogram biomolecules and small molecule therapies.
Synthetic biology approaches to study and control the epitranscriptome
All genetic information contained within the DNA of living systems is carried through an intermediate RNA biopolymer prior to conversion to protein. Recently, a variety of RNA chemical modifications have been discovered, opening up the new field of epitranscriptomics. However, an inability to accurately measure and manipulate specific chemical marks on specific RNAs has impeded the elucidation of the biological roles of these clearly important modifications. Therefore, we are developing new evolution and protein-engineering approaches that will allow us to develop tools to site-specifically chemically modify RNA regulatory mechanisms on single target RNAs in live cells. Our versatile technologies will lay down the framework for the comprehensive analysis of RNA post-transcriptional regulation, shifting the paradigm for how genetic information flow is regulated in mammalian systems.
