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

Abstract

RNA-based regulators are promising tools for building synthetic biological systems that provide a powerful platform for achieving a complex regulation of transcription and translation. Recently, de novo-designed synthetic RNA regulators, such as the small transcriptional activating RNA (STAR), toehold switch (THS), and three-way junction (3WJ) repressor, have been utilized to construct RNA-based synthetic gene circuits in living cells. In this work, we utilized these regulators to construct type 1 incoherent feed-forward loop (IFFL) circuits in vivo and explored their dynamic behaviors. A combination of a STAR and 3WJ repressor was used to construct an RNA-only IFFL circuit. However, due to the fast kinetics of RNA–RNA interactions, there was no significant timescale difference between the direct activation and the indirect inhibition, that no pulse was observed in the experiments. These findings were confirmed with mechanistic modeling and simulation results for a wider range of conditions. To increase delay in the inhibition pathway, we introduced a protein synthesis process to the circuit and designed an RNA–protein hybrid IFFL circuit using THS and TetR protein. Simulation results indicated that pulse generation could be achieved with this RNA–protein hybrid model, and this was further verified with experimental realization in E. coli. Our findings demonstrate that while RNA-based regulators excel in speed as compared to protein-based regulators, the fast reaction kinetics of RNA-based regulators could also undermine the functionality of a circuit (e.g., lack of significant timescale difference). The agreement between experiments and simulations suggests that the mechanistic modeling can help debug issues and validate the hypothesis in designing a new circuit. Moreover, the applicability of the kinetic parameters extracted from the RNA-only circuit to the RNA–protein hybrid circuit also indicates the modularity of RNA-based regulators when used in a different context. We anticipate the findings of this work to guide the future design of gene circuits that rely heavily on the dynamics of RNA-based regulators, in terms of both modeling and experimental realization.

Model-Based Investigation of the Relationship between Regulation Level and Pulse Property of I1-FFL Gene Circuits

“…Experimental Construction of the TX Circuit. In our previous work, Hong et al, 30 we obtained a pulse generation with an HY-2 Circuit but not the HY-1 Circuit; this finding agrees with the simulation analysis presented here, which suggests that the TX and HY-2 Circuits have the highest chance of generating a pulse. Therefore, we then sought to perform a quick experimental validation on whether the TX Circuit can generate a pulse or not, with moderate efforts.…”

“…This observation agrees with our experimental results on the TX Circuit presented in this study and the experimental results on HY-1 and HY-2 Circuits in the previous study. 30 The mechanistic models developed in this study have represented the detailed dynamics and interactions in the system in lumped kinetic parameters, with the parameter values inferred from previous experiments. While the model is able to predict the circuit dynamics in experiments, as exemplified by the E. coli experiments presented here, we anticipate a detailed model that accounts for the specific experimental designs that would further improve the prediction accuracy, as demonstrated in our previous work on the HY-1 and HY-2 Circuits.…”

Detection of pks Island mRNAs Using Toehold Sensors in Escherichia coli

“…Synthetic biology is a burgeoning field that aims to design novel biological components, networks, and organisms by combining biological knowledge and technology with engineering principles [1,2]. Over the past decades, continued progress in the ability to redesign biological systems has succeeded in the construction of synthetic biological devices such as toggle switches [3,4], oscillators [4,5], counters [6], memory systems [7], pulse generators [8,9], and majority sensors [10]. The growing repertoire of sophisticated genetic circuitry in synthetic biological systems could find applications in medical and industrial fields, paving the way for precision medicine [11], cancer therapy [12,13], vaccine developments [14], and biosensors [15].…”

“…The toehold switch design is mostly free from sequence constraints compared to earlier synthetic riboregulators [20], and consequently, could achieve a wide dynamic range and high programmability [19]. Several recent works employed toehold switches for synthetic biological circuitry, including cellular logic computation [21], translational repressing riboregulators [22], incoherent feed-forward loop circuits [9], synthetic transcription terminators [23], protein quality control system [24], and modulators of riboswitch circuits [25]. The versatility of toehold switches can be further showcased by recent developments of paper-based toehold switch systems as in vitro RNA detection platforms for Zika virus detection [26], Coronavirus detection [27], and gut microbiota analysis [28] in combination with well-known isothermal RNA amplification techniques (e.g., nucleic acid sequencebased amplification (NASBA) [29,30] or reverse transcription loop-mediated isothermal amplification (RT-LAMP) [31]).…”

Reimagining the Power of Nucleic Acids as Therapeutic and Diagnostic Agents

“…The research article by Hong et al explores how RNA molecules can be applied in synthetic biological systems to create artificial platforms that can regulate complex cellular processes such as transcription and translation [ 5 ]. These authors utilized three specific types of de novo-designed synthetic RNA regulators to construct RNA-based synthetic gene circuits.…”