SARS-CoV-2 remains a leading cause of death in Australia, responsible for around 5000 deaths in 2023 alone [1]. While vaccination programs and public health campaigns have undoubtedly aided the fight against this pathogen, we still lack effective antivirals against SARS-CoV-2. Antivirals that are useful against all SARS-CoV-2 variants as well as future SARS-CoV viruses are particularly sought after. The highly conserved and essential RNA-binding protein Nsp9 is therefore an attractive target. However, the RNA-binding specificity and exact functions of Nsp9 are not yet well understood.
Guided by the proposed interaction between Nsp9 and 7SL RNA [2], a highly dynamic RNA involved in protein trafficking, we used nuclear magnetic resonance spectroscopy to characterise the binding interaction between Nsp9 and a variety of RNA constructs. By testing the binding of single-stranded, hairpin RNAs, and their modified counterparts, we found that Nsp9 showed relatively stronger interactions with flexible, dynamic RNA loops. We therefore explored whether Nsp9 could be targeted by modified hairpin-forming single-stranded oligonucleotides (SSOs). Here, we demonstrated that a previously designed hairpin-forming SSO, containing both phosphorothioate bonds and 2’-O-methyl modifications bound Nsp9 in a similar manner to 7SL S domain. In contrast, an unmodified SSO with the same sequence interacted much more weakly with Nsp9 suggesting that the modifications likely help mimic the dynamics of 7SL to improve binding affinity. With modified RNA offering other favourable drug properties like increased stability and improved cellular uptake, our results highlight the potential for using modified SSOs to target RNA-binding proteins that prefer binding dynamic RNAs.