A comparison of donor-acceptor pairs for genetically encoded FRET sensors: application to the Epac cAMP sensor as an example.
We recently reported on CFP-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 biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs 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 FRET span of the Epac-based cAMP sensor. The results show significant albeit not perfect correlation between performance in the spacer 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.
van der Krogt
Gerard N M
GN
Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Ogink
Janneke
J
Ponsioen
Bas
B
Jalink
Kees
K
D001426:Bacterial Proteins / Q000737:chemistry; D015374:Biosensing Techniques; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods*; D017930:Genes, Reporter; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry; D008954:Models, Biological; D012984:Software
D001426:Bacterial Proteins; D020662:Guanine Nucleotide Exchange Factors; D008164:Luminescent Proteins; C116122:RAPGEF3 protein, human; C054562:yellow fluorescent protein, Bacteria; D000242:Cyclic AMP