Design of orthogonal and portable RNA devices for post-transcriptional regulation by reverse engineering type I toxin-antitoxin systems

Type I toxin-antitoxin (TA) systems are widely distributed in the prokaryotic world, known for their antitoxin RNA interfering with the cognate toxin mRNA translation. Beyond their canonical role in plasmid maintenance, these systems represent evolutionarily optimized regulatory modules with rapid kinetics and modular architectures. Here, we repurposed them as post-transcriptional regulatory RNA devices via reverse engineering. After isolating the core of type I TA pairs and demonstrating the independence between their structure and repression function, we reconstructed artificial TA pairs termed SRTS-OPRTS. A platform for generating orthogonal SRTS-OPRTS pairs with cross-species application (Bacillus subtilis, Escherichia coli, and Corynebacterium glutamicum) was developed by introducing structure and energy constraints. As an individual expressing element or a co-expressing 3' UTR tag within specific mRNA, SRTS achieved quantitative regulation of the gene with 3' UTR cognate OPRTS. Such portability enabled convenient construction of dynamic mutually inhibitory switches, where genes tagged by SRTS and OPRTS could regulate each other. Leveraging this approach, a selective lethal system was further constructed to enrich high-fluorescent mutants, resulting in up to 11.32-fold enhancement in mean fluorescence intensity. Overall, these synthetic RNA devices provide portable tools for gene regulation and offer a robust foundation for constructing dynamic genetic circuits.