Fluorescence-based biosensors are widely used in the analysis of a variety of cellular signaling processes, including visualization of secondary messenger molecules such as cyclic adenosine monophosphate (cAMP). For highly quantitative measurements, a phenomenon known as Förster resonance energy transfer (FRET) is often exploited in sensors. These sensors undergo conformational changes in the protein structure upon analyte binding, which translates into a change in the fluorescence levels of the fluorophores on the sensor. The ratio between these fluorescence levels can be directly mapped to analyte concentration. Many FRET-based biosensors use blue and yellow fluorophores connected <i>via</i> an analyte-specific binding domain, but this FRET pair is not without drawbacks. Long-term excitation with blue light is known to be cytotoxic, presenting temporal limitations on analyte visualization. The wide emission peak of most cyan-fluorescent proteins (CFPs) also causes bleed through between emission channels when imaging. Furthermore, there is a need for FRET-based biosensors that can be coupled with blue-light activated optogenetic tools. When directly exciting the blue fluorophore in these sensors, the optogenetic tool is also excited, making it impossible to uncouple visualization and stimulation. To address these needs, we have developed the first series of yellow-red FRET-based biosensors for the quantification of cellular cAMP. We present a number of novel constructs using combinations of several yellow donor fluorophores, two variations on the Epac cAMP-binding domain, and the Scarlet-I acceptor fluorophore. We also present a new tandem mScarlet-I FRET acceptor, which we have termed tdScarlet-I. We assessed the dynamic ranges of these constructs in mammalian cells and the best sensors were expressed in <i>E. coli</i> and purified for further optical characterization, including excitation/emission spectra and photostability. These results provide a framework from which other yellow-red FRET sensors may be developed and utilized for the study of cellular signaling dynamics.

Novel genetically encoded yellow-red FRET biosensors for visualizing cAMP dynamics

256th National Meeting (2018)

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