Synthetic biology-based methods are promising strategies for the manipulation of biological systems to both interrogate and control biological regulatory systems. Elegant methods have been developed to sense and respond to RNA patterns and to create RNA and DNA-based computation systems. RNA is an attractive biomolecule for engineering because it can easily be programmed to interact with other nucleic acids based on specific nucleotide binding. However, RNA parts are not well suited for integration with protein-based chemical modifications that regulate cellular systems. Therefore, a general strategy is needed to link protein-based chemical and biochemical events to programmable RNA output signals. The broad interests of my group are to develop and exploit novel continuous directed systems to create such next-generation synthetic biology-based diagnostics and therapies, in order to combat cancer.

To accomplish this, we are developing Activity-Responsive RNA Polymerases (ARs), which “record” specific biochemical events in defined sequences of RNA, as an enabling technology to simultaneously monitor and regulate multiple enzyme activities in living cells. By combining the capacity of ARs to amplify conversion of specific DNAs to RNAs via catalysis with our laboratory’s ability to rapidly generate orthogonal RNA Polymerases by directed evolution, many sensors can be simultaneously produced within the same cell, each encoding different activities in separate RNAs. The RNA signals can be “read” by high-throughput sequencing (HTS) for multidimensional analyses and identification of disease-specific molecular signatures, or can be integrated into gene circuits for tailored therapeutics. The long-term goal is to use ARs for analysis of and response to endogenous biochemical activities in order to both study the underlying mechanisms of complex cancer signaling networks and to treat the disease using a new class of synthetic biology-based reagents.

Progress:

Thanks to the generous support of the Cancer Research Foundation, we recently developed the first RNA polymerase-based “smart” reagents that respond to specific biochemical signals in live cancerous cells. In work recently reported in the Journal of the American Chemical Society (J. Am. Chem. Soc. 137, 15996, 2015) we disclosed our new protease-responsive RNA polymerases (PRs), molecular sensors that “record” specific protease activities in defined sequences of RNA, as an enabling technology to simultaneously monitor and respond to biochemical events in living cells. To accomplish this, we deployed continuous directed evolution to create a panel of three T7 RNAP variants with orthogonal DNA promoter specificity. We then engineered protease-responsiveness into each RNAP variant using an approach we recently developed that involves tethering catalytically inactive T7 lysozyme, which inhibits T7 RNAP, through a flexible linker containing a target protease substrate. The effective concentration forces the complex into the lysozyme-bound, RNAP-inactive state. Proteolysis of the target sequence releases an active RNAP that transcribes from a specific DNA promoter. We demonstrated that PRs function in live mammalian cells and respond to specific protease activities by driving programmed gene expression outputs. Our results establish RNAP-based molecular recording devices as a new strategy for the detection of or response to endogenous signaling events for both interrogating and engineering biological systems.

Current focus:

Now that we have validated the approach of using evolved RNA polymerases proteins as “smart” reagents that drive cellular outputs, we are now expanding the technology toward a more general platform. Our current focus is on creating reagents that respond to biomolecular interactions (protein-protein interactions, protein-small molecule interactions, protein-RNA interactions) as well as other biochemical signaling events.

Goal of funded work:

The primary goal of our funded work was to leverage advances in chemical and synthetic biology to develop new approaches to diagnose and treat cancer. Specifically, we developed an entirely new type of genetic biosensor: Activity-Responsive RNA Polymerases (ARs), which “record” specific biochemical events in defined sequences of RNA. This enabling technology opens up new opportunities for cell analysis and therapeutic development. The long-term goal is to use ARs for analysis of and response to endogenous biochemical activities in order to both study the underlying mechanisms of complex cancer signaling networks and to treat the disease using a new class of synthetic biology-based reagents.

Progress:

Thanks to the generous support of the CRF, we recently developed the first RNA polymerase-based “smart” reagents that respond to specific biochemical signals in live cancerous cells, which we reported in the Journal of the American Chemical Society (J. Am. Chem. Soc. 137, 15996, 2015). Since our last progress report, we dramatically this technology through the development of a proximity-dependent split RNAP biosensor system, which we recently reported in Nature Chemical Biology (Nat. Chem. Biol. 13, 432-438, 2017). Biosensors that transduce target chemical and biochemical inputs into genetic outputs are essential for bioengineering and synthetic biology. Current biosensor design strategies are often limited by a low signal-to-noise ratio, the extensive optimization required for each new input, and poor performance in mammalian cells. After discovering that interactions between fused proteins modulate the assembly of a split T7 RNAP, we optimized the split RNAP components for protein-protein interaction detection by phage-assisted continuous evolution (PACE). We then applied the resulting activity-responsive RNAP (AR) system to create biosensors that can be activated by light and small molecules, demonstrating the ‘plug-and-play’ nature of the platform. Finally, we validated that ARs can interrogate multidimensional protein-protein interactions and trigger RNA nanostructure production, protein synthesis, and gene knockdown in mammalian systems, illustrating the versatility of ARs in synthetic biology applications.

Moving Forward:

Now, with our new technologies in hand, we are deploying these tools in a variety of ways to better understand and treat cancer. For example, we are monitoring pharmacological engagement of therapeutic compounds in live cells to better understand target engagement and poly-pharmacology. We are developing new rapid evolution systems to attempt to “evolve” therapeutic leads, dramatically accelerating the drug discovery process. Finally, we are deploying our biosensors in synthetic biology-based “smart” therapeutic approaches