Precise control of gene expression in response to cellular RNA signatures represents a major goal in synthetic biology and RNA therapeutics. Synthetic RNA devices that sense and respond to specific transcripts can, in principle, distinguish and reprogram target cells based on their unique molecular profiles. As part of the SmaRT (Self-activating mRNA Transcripts) project, we are developing a modular platform based on engineered Tetrahymena Group I ribozymes to achieve programmable, RNA-dependent activation of mRNA translation in mammalian cells.
To establish an efficient splicing-based control system, we first screened five intron insertion sites within the NanoLuc luciferase coding sequence to identify the optimal cis-splicing configuration in HEK293T cells. Building on this foundation, we designed and tested multiple ribozyme split sites to determine the most effective trans-splicing configuration, which served as the base construct for programmable RNA sensors. We then developed a conditionally activated ribozyme design that operates with only two RNA species: a single sensor RNA and a cognate activator RNA. In this configuration, splicing and translation occur selectively in the presence of target transcripts, providing a sequence-specific control mechanism for mRNA activity.
These studies demonstrate the feasibility of constructing RNA-sensing translational switches capable of distinguishing target-cell transcripts through programmable intron architectures. This work establishes a compact and tuneable framework for cell-type–specific mRNA translation control, with broad implications for diagnostic RNA sensors, conditional mRNA therapeutics, and programmable synthetic-biology circuits.