RNA-Based Fluorescent Sensor with RhoBAST

Cellular targets can be imaged using biosensors composed of RNA. However, current RNA-based sensors encounter several challenges, including low brightness, poor photostability, slow response kinetics, and green background noise. In addition, these sensors are often limited to detecting cellular single rather than multiple targets, or are unable to image targets within subcellular compartments. To address these issues, we develop a novel RNA-based sensor by using RhoBAST fluorogenic RNA aptamer to detect various small molecules and Ca²⁺ in living cells. Importantly, RhoBAST-based sensors exhibit high red fluorescence intensity, robust photostability, and fast activation kinetics compared to previous fluorescent sensors. With these advantageous performances, we also create an orthogonal sensor for the simultaneous subcellular imaging of multiple small molecules. Together, we provide a highly bright, stable, and rapidly responsive red fluorescent sensor composed of RNA for sensing subcellular multiple targets in living mammalian cells.