Towards Dynamic and Complex RNA Circuits: A Novel Approach to Address Retroactivity with Positive Feedback Insulation

The creation of de novo designed RNA transcriptional regulatory systems with high dynamic range and orthogonality is allowing for the creation of RNA-based circuits that process signals solely at the transcriptional level. While much progress has been made, challenge remains to construct more complex and dynamic circuitry analogous to the ones achieved using protein-based regulators. One of the major challenges is high retroactivity seen in RNA regulatory systems. For example, in small RNA activating systems called STARs, a regulator RNA is irreversibly consumed for each regulatory event. To address this, we propose a novel approach to overcome retroactivity by implementing a positive-feedback insulation strategy that in essence compensates for the loss of regulatory RNAs. We present here a theoretical analysis of our strategy, called RNA compensator, and demonstrate that it can effectively mitigate the retroactivity not only in multi-state RNA circuits, e.g., self-activator and toggle switches, but also in dynamic RNA circuits like oscillators. In addition, we improve the connectivity between RNA modules by reshaping their input-output responses using tandem STAR and RNA sequestration. Finally, I will present our recent work creating broad-host RNA transcriptional regulators that use host-agnostic phage-derived RNA polymerases. Combining the works above, a toolset is developed to create modular, robust, and broad-host RNA circuits, providing a new platform for future synthetic RNA research.