Probing SARS-CoV-2 G-Quadruplexes in the Nsp3 Gene Segment by Fluorescence and ¹⁹F NMR Using a Functionalized Nucleoside Analog

The SARS-CoV-2 genome is a single-stranded RNA containing highly conserved G-rich segments that can fold into G-quadruplex (GQ) structures. These GQ structures take part in regulating the expression of both structural and nonstructural proteins, which are essential for viral replication and pathogenesis. Particularly, Nsp3 is one of the key nonstructural proteins responsible for the production of the replicase gene and controls the polyprotein expression required for viral replication. However, limited information is available on the structural polymorphism of the Nsp3 GQ motif and its interaction with small molecules, underscoring the need for a further detailed investigation. In this study, we utilized a dual-application environment-responsive nucleoside conjugate, 5-fluorobenzofuran-modified uridine (FBFU), to probe the complex structural features of the Nsp3 GQ-forming sequence using fluorescence and ¹⁹F nuclear magnetic resonance (NMR). The phosphoramidite substrate of the probe incorporated into the loop region is minimally perturbing and reports the formation of GQ structures and duplexes with distinct fluorescence and ¹⁹F NMR signals. Notably, the simple and effective ¹⁹F NMR approach is used to detect coexisting multiple GQ structures in the Nsp3 gene. Further, using the fluorescent component of the nucleoside analog, a method was designed to quantify the binding affinity of small-molecule ligands to the GQs. Taken together, our probe provided valuable insights into the structural diversity of the highly conserved Nsp3 G-rich region, which should aid in developing GQ binders and advance the therapeutic evaluation of this target.