Toehold Switch-Based Genetic Modulator for Tuning of Riboswitch Sensor

Abstract BackgroundSynthetic biological circuits are widely utilized to control microbial cell functions. Natural and synthetic riboswitches are attractive classes of sensor modules for use in synthetic biological applications. However, tuning the dose-response parameters of riboswitch circuits is challenging because considerable understanding of riboswitch mechanism and screening of mutant libraries are generally required. Therefore, novel molecular parts and strategies for controlling the dose-response parameters of riboswitch circuits are needed.ResultsHere, we developed a toehold switch-based genetic modulator that combines a previously reported hybrid input construct, which consists of riboswitch and transcriptional repressor, and de-novo-designed riboregulators named as toehold switches. First, the introduction of a pair of toehold switch and trigger as a downstream signal-processing module resulted in a functional riboswitch circuit. Next, several optimization strategies that focused on the stoichiometric ratio of RNA components greatly improved the fold-change. Finally, further characterizations confirmed low leakiness and high orthogonality for multiple toehold switches, indicating its applicability in riboswitch circuits in a seamless manner.ConclusionsThe toehold switch-based genetic modulator improved the dynamic range and dramatically shifted the operational range compared to the previous sensors only with hybrid input construct. The programmable RNA-RNA interactions amenable to in silico design and optimization can facilitate further development of RNA-based genetic modulators for flexible tuning of riboswitch circuitry and synthetic biosensors.