Probing structure and druggable space of viral G-quadruplexes using dual-functional nucleoside analogs

Abstract

Nucleic acids, beyond their canonical double-stranded DNA or single-stranded RNA forms, can adopt diverse secondary structures such as G-quadruplexes (GQs), i-motifs, and pseudoknots. GQs, in particular, regulate critical biological processes like replication, transcription, and translation. These structures are conserved across a range of organisms, including viruses, where putative GQ-forming sequences often remain unchanged within species, making them attractive targets for antiviral strategies. However, the structural polymorphism and dynamic nature of GQs pose challenges in identifying biologically relevant topologies. To address this, we developed nucleoside-based probe systems to study viral GQs and their interactions with small-molecule ligands using fluorescence and ¹⁹F NMR spectroscopy.In our study, we employed two sensitive nucleoside analogs-5-fluoro-2′-deoxyuridine (FdU) and 5-fluorobenzofuran-2′- deoxyuridine (FBFdU)-as structure-responsive probes to investigate the conformational landscape of the HIV-1 long terminal repeat (LTR). These probes differentiated GQ and hairpin structures in both cell-free and cellular environments. We used them to explore the druggable regions of individual LTR segments, determining ligand binding constants via fluorescence assays and identifying binding sites using ¹⁹F NMR. The stabilization of GQs by ligands also showed inhibitory effects on enzymatic activity, suggesting a regulatory role in viral replication. Taking this forward, the FBFU probe was employed to probe the GQs of SARS-CoV- 2, which belongs to the family of RNA viruses. The probe detected the existence of multimeric structures. 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 viral G-rich regions, which should aid in developing GQ binders and advance the therapeutic evaluation of this target.