Split RNA switch: Programmable and precise control of gene expression by ensemble of pre- and post-translational regulation

Regulating gene expression in response to biomolecules is a powerful strategy for monitoring intracellular environments and controlling cellular programs. RNA switch is a synthetic mRNA-based technology that controls gene expression at the translational level in response to cellular RNA and protein molecules, thus enabling cell type-specific gene regulation and showing promise for gene therapy, regenerative medicine, and cell therapy. However, single RNA switches often lack the specificity required for practical applications due to low ON/OFF ratios and difficulty in finding distinct and single biomolecule targets. To address these issues, we developed "split RNA switches" that integrate outputs from multiple RNA switches by exploiting protein splicing, a post-translational modification mechanism. We demonstrated that split RNA switches significantly improve the ON/OFF ratio of microRNA (miRNA)-responsive ON switch systems by canceling undesirable leaky OFF level. We achieved efficient and robust target cell purification based on endogenous miRNA profiles, which was impossible with an ON switch alone. Additionally, we constructed multi-output and multi-input RNA-based synthetic circuits using split RNA switches to enable the detection of multiple miRNAs for precise gene control with logical operations. Split RNA switches illustrate a novel application of protein splicing and demonstrate the potential of post-translational processing as a comprehensive solution for advancing translational control technologies toward widespread mRNA therapies.