Modular fluorescent sensor motifs are needed to design fluorescent sensors for detecting various cellular processes and functional molecules. Here, we took advantage of the versatility of a new sensor motif to design a series of sensors called SPOTon. SPOTon sensors integrate the signal from either opioids, protein−protein interactions, or protease activities to generate persistent green fluorescence. We demonstrate that SPOTon can be engineered with temporal gating to allow detection of these cellular events during a user-defined time window, providing temporal information about cellular processes and functional molecule release. These SPOTon sensors all show a high signal-to-noise ratio, up to 38 for chemical gated opioid detection, 147 for chemical gated protein−protein interaction detection, and 85 for protease activity detection.

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A circularly permuted AsLOV2 domain has been designed to photocontrol peptides by caging their C-terminus. This photoswitch provides a new module for engineering optogenetic tools and a general approach to lower the background of LOV-based tools.