Abstract
Light-activated fluorescence represents a potent tool for investigating subcellular structures and dynamics, offering enhanced control over the temporal and spatial aspects of the fluorescence signal. While alkyl-substituted tetrazine has previously been reported as a photo-trigger for various fluorophore scaffolds, its limited photochemical efficiency and high activation energy have constrained its widespread application at the biomolecular level. In this study, we demonstrate that a single sulfur atom substitution of tetrazine greatly enhances the photochemical properties of tetrazine conjugates and significantly improves their photocleavage efficiency. Notably, the resulting sulfur-tetrazine can be activated using a lower-energy light source, thus transforming it into a valuable visible-light photo-trigger. To introduce this photo-trigger into biological systems, we have developed a series of visible-light activatable small molecular dyes, along with a photoactivatable noncanonical amino acid containing sulfur-tetrazine. Using the Genetic Code Expansion technology, this novel amino acid is genetically incorporated into fluorescent protein molecules, serving as a phototrigger to create an innovative photoactivatable protein. These advancements in tetrazine-scaffold photo-trigger design open up new avenues for generating photoactivatable biomolecules, promising to greatly facilitate the exploration of biological functions and structures.