Building an RNA-Based Toggle Switch Using Inhibitory RNA Aptamers

Abstract

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.

Autoinhibited transient, gated, and cascaded dynamic transcription of RNAs

“…While recent efforts addressed the triggered dynamic operation of transcriptional circuits ( 51 , 52 ), the out-of-equilibrium operation of transcription machineries and particularly the design of transient gated and cascaded transcriptional networks, as a means to modulate gene expression circuits ( 8 ), are unprecedented. In the present study, we introduce a versatile mechanism to generate transient dissipative machineries by applying a transcription machinery that transcribes the T7 RNA polymerase (RNAP) aptamer ( 53 , 54 ) to inhibit the RNAP. The time-dependent inhibition of RNAP leads to the transient blockage of the target transcription machinery.…”

Harmonizing the growing fluorogenic RNA aptamer toolbox for RNA detection and imaging

“…When combined with two aptamers demonstrating high affinity and inhibitory activity towards T7 and SSP6 RNA polymerase each, a circuit can be produced that ‘lock’ at a high concentration of RNA 1 and a low concentration of RNA 2 or the inverse as reported by the fluorogenic aptamers. 125 While many other examples exist, these two examples serve to demonstrate the versatility in experimental design that is possible by exploiting the synthesis of fluorogenic aptamers as reporter constructs.…”

Evaluating DFHBI-Responsive RNA Light-Up Aptamers as Fluorescent Reporters for Gene Expression

“…10−13 RNA reporters have potential advantages compared to protein-based reporters: the dynamics of activation and degradation are potentially faster, their expression may impose a lower metabolic burden, as it only involves the transcription process, and their structure and function are predictable and versatile due to Watson−Crick base pairing. 14,15 Therefore, some attention has been devoted to RNA reporters for certain applications such as implementing RNA-based circuits with fluorescent reporters, 16,17 synthetic circuits requiring fast dynamics, transcription-based studies, or sequence-specific RNA−RNA and RNA−protein interactions. 13 RNA aptamers are transcripts that have been optimized for high-affinity binding to specific ligands by exponential enrichment (SELEX).…”

“…Recent advances in nucleic acid engineering and RNA biology , have enabled the emergence and development of alternative reporters based on RNA molecules. − RNA reporters have potential advantages compared to protein-based reporters: the dynamics of activation and degradation are potentially faster, their expression may impose a lower metabolic burden, as it only involves the transcription process, and their structure and function are predictable and versatile due to Watson–Crick base pairing. , Therefore, some attention has been devoted to RNA reporters for certain applications such as implementing RNA-based circuits with fluorescent reporters, , synthetic circuits requiring fast dynamics, transcription-based studies, or sequence-specific RNA–RNA and RNA–protein interactions …”

“…Over the past few years, these RNA light-up aptamers have been mainly implemented as tags to track and localize mRNA molecules in bacterial and mammalian cells using fluorescence microscopy, , as biosensors for therapy and diagnostic applications, − and as reporters in synthetic biology for in vitro and in vivo applications. ,, However, it remains a challenge to express fluorescent aptamers in living cells due to several factors affecting their performance, such as transport of the fluorophore across the membrane, toxicity to the cell, or stability of the aptamer. Regarding the latter, several strategies have been proposed to improve RNA stability in living cells and prevent degradation of the RNA aptamer.…”