SANS investigations of the condensates between SARS-CoV-2 nucleocapsid protein and model viral RNA

This proposal aims to study the internal structure of the aggregates formed by the SARS-CoV-2 Nucleocapsid (N) protein and RNA, a relevant information for the development of possible therapies. Indeed, N-protein is positively charged at neutral pH, so it shows a strong binding affinity with negatively charged RNAs. Moreover, previous gel filtration and DLS results suggested N-protein oligomerization [1]. Since these features are similar to those of other proteins that have been reported to undergo liquid–liquid phase separation (LLPS) with nucleic acids, we propose to study the in-solution structural and aggregational features of the N-protein in the presence of a model RNA by fully exploiting the contrast variation SANS technique.
For the proposed study, we have already produced the SARS-CoV-2 N-protein through the recombinant DNA technology and we will take advantage of recent fluorescence results that evidence how, at fixed N-protein concentration, liquid-like droplets form at high RNA concentrations. GENFIT software will be used to obtain the form factor of the N-protein in solution, the form factor of the protein inside the condensates, and, finally, the structure factor that describes the correlation between proteins within the condensates.