Synthetic RNA systems offer unique advantages such as faster response, increased specificity, and programmability compared to conventional protein-based networks. Here, we demonstrate an in vitro RNA-based toggle switch using RNA aptamers capable of inhibiting the transcriptional activity of T7 or SP6 RNA polymerases. The activities of both polymerases are monitored simultaneously by using Broccoli and malachite green light-up aptamer systems. In our toggle switch, a T7 promoter drives the expression of SP6 inhibitory aptamers, and an SP6 promoter expresses T7 inhibitory aptamers. We show that the two distinct states originating from the mutual inhibition of aptamers can be toggled by adding DNA sequences to sequester the RNA inhibitory aptamers. Finally, we assessed our RNA-based toggle switch in degrading conditions by introducing controlled degradation of RNAs using a mix of RNases. Our results demonstrate that the RNA-based toggle switch could be used as a control element for nucleic acid networks in synthetic biology applications.

Synthetic RNA systems offer unique advantages such as faster response, increased specificity, and programmability compared to conventional protein-based networks. Here, we demonstrate an in-vitro RNA-based toggle switch using RNA aptamers capable of inhibiting the transcriptional activity of T7 or SP6 RNA polymerases. The activities of both polymerases are monitored simultaneously by using Broccoli and Malachite green light-up aptamer systems. In our toggle switch, a T7 promoter drives the expression of SP6 inhibitory aptamers, and an SP6 promoter expresses T7 inhibitory aptamers. We show that the two distinct states originating from the mutual inhibition of aptamers can be toggled by adding DNA sequences to sequester the RNA inhibitory aptamers. Finally, we assessed our RNA-based toggle switch in cell-like conditions by introducing controlled degradation of RNAs using a mix of RNases. Our results demonstrate that the RNA-based toggle switch could be used as a control element for nucleic acid networks in synthetic biology applications.Graphical TOC Entry