Abstract
Fluorescence resonance energy transfer (FRET), especially with fluorescent proteins of donor and acceptor, has been widely used to measure biomolecular interactions. To overcome limitations of existing approaches for quantitative FRET, we here put forward a novel platform of dual-switching FRET (dsFRET), with a photoswitchable donor as well as a photoswitchable acceptor. With the photoswitchable capability from the FRET pair we constructed, neither donor-only nor acceptor-only samples would be required as control reference for calculation. Experiments of dsFRET and traditional 33-FRET were conducted and compared in both dimer and two-hybrid forms. Our data demonstrate that dsFRET has higher accuracy and stability than 33 -FRET, mainly benefited from in-situ references. Further development of dsFRET has been pursued, to enhance the performance of this new methodology, and also to extend its applications, such as in subcellular FRET and in-vivo FRET.
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