A General Strategy to Endow Dyes with Genic Fluorescence for Wash-Free Imaging of Proteins in Living Cells

Abstract

Fluorescent dyes are the main fluorophore for protein labeling and fluorescent imaging in living cells. However, due to the inevitable nonspecific binding of the dye to non-targets within the cell, background signals are generated, which severely affect the imaging quality. Here, we endow the dye with genic fluorescence by introducing a fluorescent proteins FRET pair, such that only the dye bound to the target protein emits fluorescence (λex= FPs, λem= Dyes), while nonspecifically bound dye remains non-fluorescent. This significantly improves the signal-to-noise ratio (SNR) in wash-free imaging. This strategy is achieved by fusing the Halo-tag to fluorescent proteins (sfGFP, mCherry) that can generate FRET with Halo dyesO-RhoandSi-Rho. The FPs serve as the donor, and the FRET mechanism imparts genic fluorescence property toO-RhoandSi-Rho. Since the excitation wavelength of the donor (λex= FPs) cannot excite the unspecifically dye fluorescence, background fluorescence interference is reduced. We further improved the SNR by increasing the FRET efficiency between the FPs and dyes. The system was used for four-color super-resolution imaging of target proteins and dynamic SIM tracking of mitochondria. In addition, the genic FRET fluorophore constructed with the ultra-stable mStayGold showed significant photobleaching resistance and can be used for long-term dynamic super-resolution imaging. Theoretically, by selecting appropriate FRET pairs, this method can improve the SNR of any fluorophore, providing a new labeling strategy for live-cell wash-free imaging.