Biosensor Optimization Using a Fo?rster Resonance Energy Transfer Pair Based on mScarlet Red Fluorescent Protein and an mScarlet-Derived Green Fluorescent Protein

Genetically encoded biosensors based on Fo''rster resonance energy transfer (FRET) are indispensable tools for monitoring biochemical changes in cells. Green and red fluorescent protein-based FRET pairs offer advantages over the classically employed cyan and yellow fluorescent protein pairs, such as better spectral separation, lower phototoxicity, and less autofluorescence. Here, we describe the development of an mScarlet-derived green fluorescent protein (designated as mWatermelon) and its use as a FRET donor to the red fluorescent protein mScarlet-I as a FRET acceptor. We tested the functionality of this FRET pair by engineering biosensors for the detection of protease activity, Ca2+, and K+. Furthermore, we described a strategy to enhance the FRET efficiency of these biosensors by modulating the intramolecular association between mWatermelon and mScarlet-I.

Automated summary

Genetically encoded biosensors based on Forster resonance energy transfer (FRET) are essential tools for monitoring biochemical changes in cells. Green and red fluorescent protein-based FRET pairs offer advantages over cyan and yellow fluorescent protein pairs, including better spectral separation, lower phototoxicity, and less autofluorescence. In this study, a green fluorescent protein called mWatermelon, derived from mScarlet, was developed and used as a FRET donor to mScarlet-I, a red fluorescent protein, as the FRET acceptor. The functionality of this FRET pair was tested by constructing biosensors for the detection of protease activity, Ca2+, and K+. Furthermore, a strategy to enhance the FRET efficiency of these biosensors by modulating the intramolecular association between mWatermelon and mScarlet-I was described.