Combining simulations and single-molecule fluorescence spectroscopy to understand SARS-CoV-2 nucleocapsid protein RNA interaction

SARS-CoV-2 causes the respiratory disease COVID-19 that has resulted in a worldwide pandemic. An outstanding question revolves around how SARS-CoV-2 and related coronaviruses package their relatively long 30 kb genomes into a relatively small (<100 nm) virion. Genome packaging is driven by the nucleocapsid protein (N), a five-domain protein that consists of an intrinsically disordered N-terminal domain (NTD), folded RNA binding domain (RBD), disordered linker domain, folded dimerization domain, and a C-terminal disordered domain. This domain architecture is conserved in coronaviruses, hinting at the importance of interspersed disordered and folded domains in mediating N-protein biological function. We sought to characterize if and how the first two domains (NTD-RBD) cooperate to bind RNA. To provide molecular-level insight into NTD-RBD RNA recognition we paired single-molecule fluorescence spectroscopy with a coarse-grained computational model of the NTD-RBD and single-stranded RNA (ssRNA), applying a finite size correction method that allows us to quantify binding affinities. Our models suggest that the RBD weakly binds ssRNA through nonspecific electrostatic interactions. Furthermore, the disordered NTD increases binding affinity through additional nonspecific electrostatic interactions and remains disordered in the bound state. We also assessed how the SARS-CoV-2 Omicron variant NTD-RBD affects binding affinity. The computational models accurately recapitulate results from our single molecule experiments. Lastly, we investigated how the NTD-RBD interacts with double-stranded RNA hairpins (dsRNA) using single-molecule fluorescence spectroscopy. Our results show that the NTD-RBD favors ssRNA regions over dsRNA. We provide mechanistic insight into how the NTD-RBD recognizes RNA and uncover positively charged regions on both the NTD and RBD that drive RNA binding. Taken together, our results provide new information on the mechanism of interactions between the N-protein and its RNA genome, setting the basis for new therapeutic strategies to target these interactions.