The development of RNA-based genetic circuits has recently gained attention due to their capability to perform computation in biological systems with minimal risk of genome integration, making them attractive as a basis for developing potential therapeutics. This progress is driven by the development of modified ribonucleosides and their incorporation into synthetic mRNA, yielding modified RNA (modRNA). This technology allows the suppression of cellular innate immune response normally associated with introducing synthetic mRNA into the body, opening a path to leverage synthetic RNA in medicine, as demonstrated by the success of mRNA vaccines during the COVID-19 pandemic.

Due to limited access to the transcriptional control tools commonly used in synthetic biology, modRNA-based systems are primarily based on post-transcriptional regulation. One such method utilizes the binding specificity of certain pairings of RNA-binding proteins (RBP) with specific mRNA sequences (termed "switches") to control exogenous gene expression. However, most available systems suppress gene expression in response to binding by the specific RBP. By contrast, a system prioritizing target gene expression while shutting down all others would be tremendously powerful, but such systems have not been available until now.

To this end, the research team recently constructed one such "exclusive selector" system, which ramps up the expression of target genes over all others, by exploiting the unique biology of SARS-CoV-2. Non-structural protein 1 (Nsp1), encoded in the SARS-CoV-2 genome, is a translational suppressor that selectively suppresses the expression of host mRNA to favor viral RNA for translation by recognizing specific features in their 5' regions. To test if modified nucleosides affect Nsp1's negative influence on modRNAs, the team evaluated combinations of modified nucleosides to synthesize viral sub-genomic RNA 5'-UTR (sgUTR)-containing modRNAs. The team found pseudouridine (Ψ) to be poorly tolerated by Nsp1, while the combination of 5-methylcytosine (m5C) and N1-methylpseudouridine (m1Ψ) performs best across two different cell lines, allowing the synthesis of Nsp1-resistant modRNAs.

To demonstrate the utility of Nsp1-resistant modRNAs, the team created an Nsp1-responsive toxin-antitoxin system consisting of Nsp1-resistant Barnase toxin-expressing modRNA and an excess of Nsp1-sensitive Barstar antitoxin-expressing modRNA. While excessive Barstar counteracts the toxicity of Barnase under normal conditions, the presence of Nsp1 suppresses Barstar expression, thus unleashing the cytotoxic RNA-degrading activity of Barnase and effectively halting all translation, including viral RNA. The team demonstrated the high sensitivity of this system, which is activated by even minuscule Nsp1 expression, and its versatility across three different cell lines, suggesting a robust system.

Altogether, Nsp1 exhibits differential sensitivity to modRNAs containing various modified nucleosides in their viral 5'-leader features, with the combination of m5C and m1Ψ being the most well-tolerated. The modified nucleoside combination was used to synthesize Nsp1-resistant modRNA, allowing the construction of a Nsp1-responsive post-transcriptional "exclusive selector" genetic circuit. The new system introduces a novel mechanism for constructing post-transcriptional genetic circuits, broadening its potential applications in therapeutics and biomedical research.

The results of this research were published online in ACS Synthetic Biology on June 6, 2025.

• doi: 10.1021/acssynbio.5c00075