A comparison of donor-acceptor pairs for genetically encoded FRET sensors: Application to the Epac cAMP sensor as an example

Abstract

We recently reported on CEP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP-levels in living cells. In this Study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in blosensors such as CFP-Epac-YFP, Our strategy was to prepare a wide range of constructs of consisting of different donor and acceptor fluorescent proteins separated by a short linker, Constructs were expressed in HEk293 cells and tested for FRET and other relevant properties. The most promising pairs were subsequently used in an attempt to improve the FRETT spati of the Epac-based cAMP sensor. The resuits show significant albeit not perfect correlation between performance in the sparer construct and in the Epac sensor. Finally, this strategy enabled us to identify improved sensors both for detection by sensitized emission and by fluorescent lifetime imaging. The present overview should be helpful in guiding development of future FRET sensors. © 2008 van der Krogt et al.