Question

You have 3 Donor/ Acceptor FRET pairs. Venus/ mplum, mTurquoise2/ EYFP and EBFP/ EGFP. Which pair is more ideal, less ideal and give two reasons to support conclusion.

Answer 1

Ans) FP-based FRET sensors have succeeded in exploring the molecular mechanisms underlying cancer, immunological and neurological diseases and play a key role in drug discovery. Nonetheless, there remain FRET sensors that suffer from low dynamic range when detecting subtle or transient biochemical responses in living cells.

- For example, RhoA activation in neuronal growth cones during ephrinA-stimulated retraction exhibits only a 5% FRET change with Clover-mRuby2. Optimizing existing FRET pairs, whether in brightness and folding or maturation, could increase FRET dynamic range.

- Currently, mNeonGreen-mRuby3 or mClover3-mRuby3 has the largest r0s among all monomeric FP-based FRET pairs and exhibits the largest FRET dynamic ranges in kinase FRET sensors. Compared to mClover3, mNeonGreen is slightly brighter than mClover3 (6%) and more photostable and thus may be a probable starting point for evolving a single bright pair with even larger FRET dynamic range for live cell imaging.

- The QY of LanYFP, the parental tetrameric FP of mNeonGreen, is 0.95 [46]. Moreover, the highest reported QY of engineered avGFP derivatives is 0.93, from mTurquoise2, while the QY of mNeonGreen is 0.8. This may suggest that variants of mNeonGreen can be developed with even higher QY. Unlike avGFP-derived FPs, many engineered RFPs, even for oligomeric ones, fold poorly or mature slowly in cells.

- Although mRuby3 is better than mRuby2 in maturation or folding, its maturation and folding is still worse than mCherry (unpublished data). In turn, the folding and maturation of mRuby3 can be further improved, which would enable more fluorescent mRuby3 molecules in the FRET complex and result in increased dynamic range. This is especially important when performing long-term imaging, since a FP’s maturation during imaging could induce FRET.

- With the development of two-photon FLIM (2pFLIM), FRET imaging has been extended from cultured cells to living animals [118]. Due to the limited number of FLIM-FRET pairs, however, it has been difficult to simultaneously image two molecular events with 2pFLIM in vivo. Developing new FLIM-FRET pairs compatible with existing pairs is required. Many 2pFLIM sensors use EGFP or mEGFP as donors due to efficient FRET with RFPs and high photostability under 2P excitation, and it would be useful to develop a new FRET pair compatible with GFP-based 2pFLIM-FRET pairs that would enable imaging of multiple biochemical events in the same cell in vivo. For this purpose, a cyan-excitable orange or red FP like CyOFP1 would be a good starting point as a donor, as a single 2P excitation wavelength, for example, 940 nm, could excite both donors [71]. CyOFP1 has an unusually high QY of 0.76 and decays as a single exponential with a long lifetime of 3.66 ns, which makes it attractive as a 2pFLIM-FRET donor, but also has some bleed-through in the emission channel for EGFP and is not fully monomeric. A red-shifted monomeric CyOFP1 paired with a far-red acceptor with high EC, for example mCardinal, could function with a GFP-based 2pFLIM-FRET pair to monitor two molecular activities simultaneously in vivo.

- Overall, the development of a variety of FPs has made FRET viable in a larger number of contexts. Brighter and more photostable FPs have increased the FRET imaging window, large stokes shift FPs have enabled the monitoring of multiple signals simultaneously, and photoswitchable and photoactivatable FPs have provided greater control over imaging without the use of controls. The choice of FP FRET pairs in biosensors is strongly dependent on the system in which the sensor will be used. Factors such as pH and folding or maturation time can significantly affect the photophysical performance of a given pair , such that a FRET pair optimized for a particular sensor or particular system may not perform as well in other contexts. Therefore, validation of FP performance in a variety of contexts (e.g., in vivo and in vitro) combined with the optimization of photophysical properties, is crucial to the development of generalizable FRET pairs for single cell or in vivo imaging.