Fluorescent Probes for Plasma Membrane Proteins Based on Nile Red

Fluorescent probes are very important in basic research and in medicine, because of their sensitivity, versatility and quantitative capabilities. They allow the imaging of tissues or cellular compartments and the monitoring of biological events in vivo. An ideal probe is specific and turns fluorescent only upon binding to its target. In recent years, efforts have been made for the development of such fluorogenic probes. These probes allow specific labeling without the requirement of extensive washing steps for removal of the excess of the dye before imaging. They allow the real-time monitoring of molecular events and they can be used in cases where a washing step is not possible, like in in vivo imaging. With the work described in this thesis, we demonstrate the design of a fluorogenic probe based on the molecule Nile Red and SNAP-tag. It takes advantage of Nile Red, a solvatochromic dye highly fluorescent in an apolar environment, like the plasma membrane and almost non- fluorescent in an aqueous environment. We present our idea to tune the affinity of Nile Red for the plasma membrane by chemically derivatizing it, so that it would insert into the membrane only upon binding to its target receptor. Then, Nile Red - being in an apolar environment - would fluoresce, whereas the excess of the dye - being in a polar aqueous environment - would remain in its dark state. Moreover, we chose to direct our Nile Red derivatives to SNAP-tag, a self- labeling tag, in order to demonstrate the generality of our approach. We describe the design of a large repertoire of Nile Red derivatives with different linkers and charges and we apply the probe for the visualisation of a pharmacologically interesting receptor, the SNAP-tagged human insulin receptor. We then present the idea of using our Nile Red derivatives as potential voltage sensors targeted to SNAP-tag. We show the voltage sensitivity of NR12S, a molecule based on Nile Red, and describe our hypotheses on the lack of voltage sensitivity of our Nile Red derivatives and on the position of the latter after the reaction with SNAP-tag. With this work, we introduce a turn-on probe for SNAP-tagged plasma membrane receptors that would become a valuable tool in bioimaging. Moreover, we suggest Nile Red as a new fluorophore for the generation of voltage sensors.