Structural elucidation of HIV-1 G-quadruplexes in a cellular environment and their ligand binding using responsive 19F-labeled nucleoside probes.

Understanding the structure and recognition of highly conserved regulatory segments of the integrated viral DNA genome that forms unique topologies can greatly aid in devising novel therapeutic strategies to counter chronic infections. In this study, we configured a probe system using highly environment-sensitive nucleoside analogs, 5-fluoro-2'-deoxyuridine (FdU) and 5-fluorobenzofuran-2'-deoxyuridine (FBFdU), to investigate the structural polymorphism of HIV-1 long terminal repeat (LTR) G-quadruplexes (GQs) by fluorescence and 19F NMR. FdU and FBFdU, serving as hairpin and GQ sensors, produced distinct spectral signatures for different GQ topologies adopted by LTR G-rich oligonucleotides. Importantly, systematic 19F NMR analysis in Xenopus laevis oocytes gave unprecedented information on the structure adopted by the LTR G-rich region in the cellular environment. The results indicate that it forms a unique GQ-hairpin hybrid architecture, a potent hotspot for selective targeting. Furthermore, structural models generated using MD simulations provided insights on how the probe system senses different GQs. Using the responsiveness of the probes and Taq DNA polymerase stop assay, we monitored GQ- and hairpin-specific ligand interactions and their synergistic inhibitory effect on the replication process. Our findings suggest that targeting GQ and hairpin motifs simultaneously using bimodal ligands could be a new strategy to selectively block the viral replication.

Synthesis of N2-trans-isosafrole-dG-adduct Bearing DNAs and the Bypass Studies with Human TLS Polymerases κ and η.

Safrole is a natural product present in many plants and plant products, including spices and essential oils. During cellular metabolism, it converts to a highly reactive trans-isosafrole (SF) intermediate that reacts with genomic DNA and forms N2-SF-dG and N6-SF-dA DNA adducts, which are detected in the oral tissue of cancer patients with betel quid chewing history. To study the SF-induced carcinogenesis and to probe the role of low fidelity translesion synthesis (TLS) polymerases in bypassing SF adducts, herein, we report the synthesis of N2-SF-dG modified DNAs using phosphoramidite chemistry. The N2-SF-dG modification in the duplex DNA does not affect the thermal stability and retains the B-form of helical conformation, indicating that this adduct may escape the radar of common DNA repair mechanisms. Primer extension studies showed that the N2-SF-dG adduct is bypassed by human TLS polymerases hpolκ and hpolη, which perform error-free replication across this adduct. Furthermore, molecular modeling and dynamics studies revealed that the adduct reorients to pair with the incoming nucleotide, thus allowing the effective bypass. Overall, the results indicate that hpolκ and hpolη do not distinguish the N2-SF-dG adduct, suggesting that they may not be involved in the safrole-induced carcinogenicity.