Construction of the FRET Pairs for the Visualization of Mitochondria Membrane Potential in Dual Emission Colors

Mitochondria membrane potential (MMP) play significant roles during metabolism, signaling, and other important bioevents. Visualization of MMP levels is essential for many biological researches. However, fluorescent probes for monitoring MMP levels in dual emission colors are still deficient, which greatly limited the development of relative research areas. In this work, a pair of fluorescent probes have been designed and synthesized to monitor the MMP levels in dual emission colors based on Forster resonance energy transfer (FRET) mechanism. The FRET donor (FixD) is constructed by linking a benzyl chloride group to a fluorophore with bright-green emission. The FixD could target mitochondria and be immobilized in mitochondria by linking to the thiol group of mitochondrial proteins. The FRET acceptor (LA) is designed with green absorption and deep-red emission. In live cells with high MMP levels, FixD and LA both ta rget mitochondria, and deep-red (DR) emission could be detected with the excitation of 405 nm. Particularly, the spectral shift of fluorescence upon the decrease of MMP is up to 110 nm, which is greatly favorable for the clear observation of MMP levels. With the decrease of MMP, LA would be released from mitochondria while FixD would still be immobilized in mitochondria, and decreased DR emission and increased green fluorescence could be detected due to the absence of FRET. In this manner, the MMP levels could be monitored in dual emission colors.