Development of a tetracycline-inducible programmed ribosomal frameshifting platform for sensitive regulation of mammalian gene expression

Developing engineered genetic switches that can respond to specific stimuli in mammalian systems has great potential to advance next-generation therapies. This study introduces a novel tetracycline-inducible artificial riboswitch that regulates gene expression at the translational level via a modified -1 programmed ribosomal frameshifting (-1 PRF) platform. Our dose-response analysis reveals an EC50 of 0.41 μM, indicating exceptional sensitivity compared to other tetracycline-dependent artificial riboswitches developed for mammalian applications. By varying the precise spacing and composition of the tetracycline aptamer as the stimulatory structure on the -1 PRF platform, a series of switches has been constructed that enable robust induction of gene expression upon ligand addition. To attain a translational gene control platform with broader applicability, we developed a concept that allows it to be integrated upstream of a gene of interest (GOI), enabling precise control without requiring extensive re-engineering and expressing the GOI as a fusion protein. We validate the platform's effectiveness in several genetic contexts, confirming its modularity. As optimization efforts continue to develop artificial riboswitches with optimized properties in mammalian systems, our findings contribute to the development of highly sensitive and efficient regulatory devices with beneficial characteristics, such as reversibility, modularity, and compatibility with RNA-based delivery therapies.