Förster resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) are two major biophysical techniques for studying nanometer-scale motion dynamics within living cells. Both techniques read photoemission from the transient RET-excited acceptor, which makes RET and detection processes inseparable. We here report a novel hybrid strategy, bioluminescence assisted switching and fluorescence imaging (BASFI) using a bioluminescent Renilla luciferase RLuc8 as the donor and a photochromic fluorescent protein Dronpa as the acceptor. When in close proximity, RET from RLuc8 switches Dronpa from its original dark state to a stable bright state, whose fluorescence is imaged subsequently with... More
Förster resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) are two major biophysical techniques for studying nanometer-scale motion dynamics within living cells. Both techniques read photoemission from the transient RET-excited acceptor, which makes RET and detection processes inseparable. We here report a novel hybrid strategy, bioluminescence assisted switching and fluorescence imaging (BASFI) using a bioluminescent Renilla luciferase RLuc8 as the donor and a photochromic fluorescent protein Dronpa as the acceptor. When in close proximity, RET from RLuc8 switches Dronpa from its original dark state to a stable bright state, whose fluorescence is imaged subsequently with an external laser. Such decoupling between RET and imaging processes in BASFI promises high photon flux as in FRET and minimal bleedthroughs as in BRET. We demonstrated BASFI with Dronpa-RLuc8 fusion constructs and drug-inducible intermolecular FKBP-FRB protein-protein interactions in live cells with high sensitivity, resolution, and specificity. Integrating the advantages of FRET and BRET, BASFI will be a valuable tool for various biophysical studies.
