Design and Evaluation of Synthetic RNA-Based Incoherent Feed-forward Loop Circuits

De-novo-designed synthetic RNA regulators [1-3] have been utilized to construct complex RNA-based synthetic gene circuits in living cells. We constructed an RNA-only type 1 incoherent feedforward loop (IFFL) circuit in vivo using these regulators and explored their dynamic behaviors [4,5]. However, there were no significant timescale differences between direct activation and indirect inhibition, and no pulses were observed in the experiment. These results were confirmed by mathematical modeling and simulation results for a wide range of conditions. To increase the delay of the inhibitory pathway, we introduced a protein synthesis process into the circuit and designed an RNA-protein hybrid IFFL circuit using toehold switch (THS) and Tet repressor. Simulation results indicated that pulse generation could be achieved using this RNA-protein hybrid model, which was further validated by experimental realization in E. coli. This research demonstrates that RNA-based circuits can serve as modular and composable parts for synthetic biological circuits to achieve desired dynamics [6], and further suggests that previously unattainable transcription-only and translation-only RNA-based IFFL circuits could be implemented.2