Dye-mediated FRET strategy for constructing semi-synthetic large Stokes shift far-red fluorescent protein

Abstract

Red fluorescent proteins with large Stokes shift (LSS-RFPs) are advantageous for multicolor imaging applications that allow simultaneous visualizations of multiple biological events. But it is difficult to develop LSS-RFPs by extending the emission wavelength of RFPs to far-red region. Here, we employed Förster resonance energy transfer (FRET) strategy to engineer the far-red fluorescent proteins with large Stokes shift. LSS-mApple and LSS-mCherry were constructed by fusing HaloTag to mApple and mCherry, allowing the fluorophore TMSiR to be connected to these RFPs. FRET between RFPs and TMSiR enabled them to apply the excitation of donor RFPs to emit far-red fluorescence of acceptor TMSiR. The Stokes shifts of LSS-mApple and LSS-mCherry were 97 nm and 75 nm, respectively. The high FRET efficiency of LSS-mCherry (E_FRET = 83.7 %) can greatly reduce the fluorescence from the donor channel, which did not affect co-imaging with mCherry. In addition, LSS-mCherry also showed excellent photostability (t_1/2 = 449.3 s), enabling stable confocal fluorescence imaging for 15 min under continuous strong excitation. Furthermore, LSS-mCherry was applied for fluorescence labeling and imaging of the nucleus, mitochondria, lysosomes, and endoplasmic reticulum in living cells. Finally, we applied LSS-mCherry to perform multi-color bioimaging of 2–4 channels, and there was no obvious crosstalk between these channels.