BPS2026 – Single-stranded RNA binding to SARS-CoV-2 nucleocapsid protein: A fluorescence investigation

The nucleocapsid protein of SARS-CoV-2 binds to its genomic RNA to package it into the virus. Among the five domains of the protein, three domains, the N-terminal domain (NTD), the RNA binding domain (RBD), and linker together bind single-stranded RNA. We incorporate the fluorescent nucleobase 2-aminopurine at different sites in a 21 nucleotide RNA derived from the viral genome and measure its fluorescence free and bound to NTD-RBD-linker and NTD-RBD. Steady-state fluorescence monitors binding, and we probe the dynamics of the bound RNA using time-correlated single photon counting fluorescence experiments. We find that the bound RNA nucleobases are flexible, suggesting that the protein is interacting with the RNA backbone. RNA binding also quenches the fluorescence of one of the tryptophan residues in the RNA binding domain, located on one of its flexible loops. We model the path of the RNA on the protein surface.