“Homotransfer FRET Sensors for Biological Exploration”

Mark A. Rizzo, University of Maryland School of Medicine

Genetically-encoded biosensors can reveal the dynamics of molecular regulation within living cells and tissues with unprecedented detail. Conventionally, these reagents incorporate Forster transfer pairs of different colors into their design to create a ratiometric readout. While this approach ought to be inherently quantitative, the signal-to-noise of intensity-based measurements can limit the utility of the approach. To overcome these limitations for biological applications, two strategies have been developed. First, existing FRET biosensors were converted to homotransfer “Fluorescence Anisotropy Reporters (FLAREs).” Measuring FRET via fluorescence polarizations improves the signal-to-noise of the sensors and enables multiplexing of different colors. Second, an improved red fluorescent protein was developed entirely in silico using statistics-guided engineering. The new red fluorescent protein, mVermilion, is twice as bright as mCherry and shows improved fluorescence polarization properties for FLARE-type sensors.