A Single Dimer of the SARS-CoV‑2 N Protein Can Associate with Multiple Fragments of Single-Stranded and Stem-Loop...

The nucleocapsid (N) protein of SARS-CoV-2 binds to the viral genomic RNA (gRNA), forming ribonucleoprotein (RNP) granules. However, the detailed molecular structures of the RNP and how they form are largely unknown. To understand the interaction between the N protein and different structural units of RNA, we used circular dichroism (CD) spectroscopy, fluorescence correlation spectroscopy (FCS), and single-molecule Förster resonance energy transfer (sm-FRET) spectroscopy. We selected polyadenylate chains of 40, 30, and 20 bases with a single-stranded structure and three stem loops of 50, 41, and 29 bases; the latter three were taken from the gRNA. We labeled their 5' and 3' ends by Alexa488 and Alexa647, respectively, for the FCS and sm-FRET measurements. We found that the N protein began binding to the single-stranded RNA at concentrations between 10 and 100 nM. Binding of the N protein to one of the stem loops occurred at concentrations below 10 nM without melting the stem loop. For all samples, the binding of multiple molecules of the RNA fragments to a single dimer of the N protein was observed. These results demonstrate that the N protein acts as a nonspecific binder to both single-stranded and stem-loop RNA structures and that it might be able to contract a long RNA chain by bridging its multiple segments. We propose that RNP granules may fold due to the association of the numerous stem loops of gRNA triggered by the N protein assembly.