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

Area of Science:

• Molecular Virology
• Structural Biology
• Medicinal Chemistry

Background:

• The SARS-CoV-2 genome contains G-rich segments that form G-quadruplex (GQ) structures.
• These GQ structures regulate viral protein expression essential for replication and pathogenesis.
• The Nsp3 protein's GQ motif is crucial for viral replication but its structural polymorphism and interactions are poorly understood.

Purpose of the Study:

• To investigate the structural polymorphism of the Nsp3 GQ motif.
• To explore the interaction of Nsp3 GQ structures with small molecules.
• To develop a method for probing GQ structures and quantifying ligand binding.

Main Methods:

• Utilized a dual-application environment-responsive nucleoside conjugate, 5-fluorobenzofuran-modified uridine (FBFU).
• Employed fluorescence and 19F nuclear magnetic resonance (NMR) spectroscopy to analyze Nsp3 GQ-forming sequences.
• Incorporated a phosphoramidite substrate probe into the loop region for minimal perturbation and distinct signaling.

Main Results:

• The FBFU probe successfully detected the formation of GQ structures and duplexes.
• A simple and effective 19F NMR approach identified coexisting multiple GQ structures within the Nsp3 gene.
• A fluorescence-based method was developed to quantify the binding affinity of small-molecule ligands to GQs.

Conclusions:

• The study provides valuable insights into the structural diversity of the conserved Nsp3 G-rich region.
• The developed probe aids in understanding GQ structural polymorphism.
• Findings support the development of GQ binders for therapeutic evaluation against SARS-CoV-2.