Bidirectional photoriboswitch for translational regulation in mammalian cells

Photoriboswitches are invaluable tools for synthetic biology because they provide direct, precise, and on-demand control of translation. While most existing photoriboswitches are predominantly one-directional, this study presents a bidirectional photoriboswitch platform capable of mimicking the orthogonal RNA-binding protein-mediated up-/downregulation of multiple mRNAs. Inspired by intein-based split protein reconstitution and light-induced protein dimerization, we first engineered a Light-OFF system that uses photodimerization to block intein reconstitution. We then validated that this Light-OFF system can function orthogonally with a Light-ON system that reconstitutes a different split intein pair under light. Together, a bidirectional photoriboswitch is generated, which senses light to orthogonally reconstitute RNA-binding proteins to tune the translation of their respective target mRNAs. We have demonstrated that the photoriboswitch produces bidirectional translation regulation. Importantly, the functional modules of the photoriboswitch can be easily altered to adapt for different purposes, allowing the bidirectional photoriboswitch to serve as a versatile tool for translation regulation.