Chemogenetic modulation of luciferase emission color for imaging and sensing

Bioluminescent luciferases have emerged as powerful tools for bioimaging, enabling to image biological systems without external excitation light, reducing thus phototoxicity and eliminating background autofluorescence. Advanced imaging requires luciferases that deliver high photon output for enhanced sensitivity, tunable emission colors for multicolor imaging, and red-shifted emission for effective deep tissue imaging. Here, we introduce LumiFAST, a small tunable luciferase engineered by fusing the bright blue-light emitting NanoLuc with the tunable chemogenetic fluorescent reporter pFAST. pFAST binds and stabilizes the fluorescent state of a variety of synthetic fluorogenic chromophores (also called fluorogens). Its proximity with NanoLuc leads to efficient bioluminescence resonance energy transfer (BRET), enabling customizable red-shifted emission. Thanks to the small size of pFAST, LumiFAST maintains a compact structure, while its modular design allows emission color to be tuned from cyan to green, yellow, orange and red simply by changing the fluorogen. Systematic optimization of the fusion topology and linker length yielded an optimal variant with apparent BRET efficiencies reaching up to 90 %. The red-shifted emission of LumiFAST enables dual-color microscopy imaging when used alongside NanoLuc and allows imaging through thick scattering media. Beyond imaging, our insights into the structural factors governing efficient BRET allowed us to engineer biosensors based on NanoLuc and pFAST for the visualization of protease activity and protein-protein interactions in live cells.