Development of a universal fluorescence sybody-based biosensor for the in-vivo study of protein dynamics

The development of genetically-encoded fluorescence sensors revolutionized the monitoring of biochemical processes in living cell by reporting a change in their fluorescence properties in response to a specific aspect of their environment. However, the design of such biosensors is not trivial and available tools only target a limited set of phenomena. Current limitations prevent the monitoring of crucial processes such as the activity of membrane associated receptors which are a rich target for drug development. Simple binders are much easier to develop than biosensors, for instance nanobodies can bind a plethora of targets with high affinity and specificity, sometimes for a particular conformation. The host research group recently developed a molecular design to convert nanobodies into fluorescent biosensors. To elicite a fluorescent signal in presence of the target, this NanoBlock sensors require the development of an intracellular blocking peptide that will be displaced upon binding to the target. This limiting step prevent the application of the NanoBlock design to a large number of targets.This objective of this proposal is to develop an entirely new class of fluorescent biosensors by engineering the NanoBlock technology so that nanobodies can be converted into fluorescent biosensors without the need for a distinct blocking peptide creating a plug and play system. This UniverSy technology will open the possibilities to visualize many more cellular processes to gain fundamental insight on protein activity and the development of therapeutics targeting these processes. The development of the UniverSy sensor will consist in the generation of dual affinity nanobodies binding their original target and a specific peptide sequence; the demonstration of the versatility of the UniverSy framework to target various proteins in vitro and in living cell and the use of UniverSy in a fluorescence assay to quantify the activation of GPCR for drug screening applications.