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 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.

Figure 1. Schematic overview of the constructs used in this study.

Donor and acceptor fluorophore are connected by a peptide stretch (Linker A: SGLRSRYPFASEL) or by the Epac1(ΔDEP, CD) domain . Within this stretch, the amino acids PF were replaced by the Epac domain itself, leaving linkers B: SGLRSRY and C: ASEL. For truncated donor constructs (CFPΔ and GFPΔ) GITLGMDELYK was deleted from the donor FPs and SGLRS from the linker. In tandem acceptor constructs the acceptors were separated by a supplementary linker (Linker D: PNFVFLIGAAGILFVSGEL) except for tdHcRed and tdTomato which have distinctive linkers, namely NG(GA)₆PVAT) and (GHGTGSTGSGSSGTASSEDNNMA), respectively.

Figure 2. FRET in donor-linker-acceptor constructs as detected by frequency-domain FLIM.

The indicated constructs were expressed in HEK293 cells and FRET efficiency E was determined as detailed in Material and Methods. Shown are mean (bars), standard deviation (SD) and standard error of the mean (SEM) of 20–400 cells. For further detail, see text.

Figure 3. FRET span in cAMP sensors.

The indicated constructs were expressed in HEK-293 cells and assayed for cAMP-induced changes in donor to acceptor ratio on a fluorescence microscope equipped with dual photometers. Donor and acceptor emission were read out simultaneously, and the baseline ratio was set to 1.0 at the onset. FRET span ΔR was determined by calculating the ratio change following addition of IBMX and Forskolin. This raises intracellular cAMP levels maximally and saturates the sensor. For further detail, see the text and Methods.

Figure 4. Typical FRET responses in HEK293 cell expressing CFP^nd-Epac-cp¹⁷³Venus to stimulation with IBMX/Forskolin.

(A) CFP and YFP emission from a single HEK293 cell expressing the improved cAMP sensor were detected at 4 samples per second, following addition of IBMX and Forskolin. The YFP/CFP ratio dropped by almost 35% within minutes. Shown is a typical recording. (B) A single cell spectral fingerprint, obtained before (black) and after (grey) IBMX and Forskolin using a spectrometer. For further detail see Methods.

Figure 5. Speckle formation.

HEK293 cells were transfected with indicated constructs and further cultured for 24–36 hours. Shown are confocal sections chosen to maximally visualize any speckles. Note that most speckles go unnoticed by wide-field fluorescence microscopy. Also indicated are estimated percentages of cells showing at least some speckles. Scale bar, 11 µm.

Figure 6. UV-induced photochromism.

Change in ratio of YFP to CFP emission in CFP^nd-linker-YFP^nd (squares) and CFP^nd-linker-Venus^d (triangles) following exposure to UV light for the indicated times. CFP^nd-linker-YFP^nd as well as free YFP (data not shown) display a dose-dependent increase in emission that maximizes at about 10%, whereas Venus and cp¹⁷³Venus (not shown) are insensitive to UV exposure. See Methods for further detail.

Figure 7. Slow green-to-red maturation of tdTomato and its effects on FRET.

(A) Cells expressing CFP^nd-EPAC-tdTomato for 24 hr display a spectrum of colors when viewed by eye using an Omega X154 triple-color (CFP-YFP-RFP) cube. For reproduction reasons, the confocal picture shows a mix of green (470–530 nm) and red (570–670) emission to closely match the image visible by eye. In contrast, CFP^nd-EPAC-mRFP and CFP^nd-EPAC-mCherry show a more homogeneous red color. (B) Cell-to-cell variability in maturation of CFP^nd-linker-tdTomato causes significant deviations in the fluorescence decay times detected in the CFP channel, as measured by frequency-domain FLIM. Scale bar, 12 µm.