Functionalizing water-in-oil droplet inner surface to characterize and modulate RNA/protein interactions – SURF

Ribonucleoprotein particles (RNPs) are made of RNA associated with proteins and play a central role in biological systems (maintenance of cellular homeostasis, establishment of infectious or pathological processes). The identification of the components of these RNPs has exploded over the past decade thanks to the use of high-throughput analytical approaches. The fine characterization of the interaction of the components of these RNPs then requires the analysis of a large number of mutants. While several high-throughput methodologies have been developed for the analysis of RNA mutant libraries, progress has been scarcer on the protein side. To fill this gap, we propose "SURF", a highly multidisciplinary project aiming at developing a new chemistry for the efficient capture of target RNAs on the surface of water-in-oil droplets produced, manipulated, and analyzed at rates of several million per hour in microfluidic devices. Gene libraries encoding mutants of the studied protein fused to a fluorescent domain will be expressed in vitro at a rate of one mutant (produced in large numbers of copies) per droplet. Thus, a mutant able to interact with the target RNA sequence will lead to a relocation of the fluorescent protein on the surface of the droplet, making it easily discriminable from a drop containing a mutant unable to recognize its target (fluorescence remaining diffuse in the droplet). Applied and validated with various biological models, this technology will not only allow to finely characterize the formation of RNPs, but also to reprogram their specificity, and even to identify molecules able to modulate this interaction. Finally, this project will also be an opportunity to explore surfactants made of alternative chemistries having a lower impact on the environment.