IISER MOHALI

Abstract:
N is a structural protein of SARS-CoV2 and forms the ribonucleoprotein granules having a diameter of ~16 nm upon the association of ~12 molecules of N and ~800-base regions of viral genomic RNA (gRNA). N also participates in the selective packaging of gRNA inside of virus. In addition, many in vitro experiments demonstrated the formation of liquid-like droplets of N upon the association with short RNA fragments. However, the structural properties and the folding mechanism of the ribonucleoprotein granules are largely unknown. We used single molecule FRET and FCS spectroscopies to understand the interaction between N and different secondary structures of RNA. We used poly-adenylate bases with different lengths and labeled their 3’ and 5’ termini with donor and acceptor fluorophores. We also labeled the two termini of 50-base stem loop with the donor and acceptor pair. We found that the N protein started to bind to the single-stranded RNAs at concentrations between 10 and 100 nM. The binding of the N protein to the stem loop occurred at the concentration less than 10 nM without melting the stem loop. For all the 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 non-specific binder to both single-stranded and stem-loop units of RNA, and that the N protein might contract a long RNA chain by bridging its multiple segments. Our recent trials to observe the compaction process of long RNA samples having ~800 bases will also be presented.