Understanding structure and recognition of highly conserved regulatory segments of the integrated viral DNA genome that forms unique topologies can greatly aid devise 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. Further, 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.

中文翻译：

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使用响应性 19F 标记核苷探针对细胞环境中的 HIV-1 G 四链体及其配体结合进行结构阐明

了解形成独特拓扑结构的整合病毒 DNA 基因组中高度保守的调控片段的结构和识别，可以极大地帮助设计对抗慢性感染的新治疗策略。在本研究中，我们使用高度环境敏感的核苷类似物5-氟-2'-脱氧尿苷（FdU）和5-氟苯并呋喃-2'-脱氧尿苷（FBFdU）配置了探针系统，以研究HIV-1的结构多态性通过荧光和¹⁹ F NMR 检测长末端重复 (LTR) G-四链体 (GQ) 。 FdU 和 FBFdU 作为发夹和 GQ 传感器，为 LTR 富含 G 的寡核苷酸采用的不同 GQ 拓扑产生不同的光谱特征。重要的是，对非洲爪蟾卵母细胞的系统¹⁹ F NMR 分析提供了关于细胞环境中富含 LTR G 区域的结构的前所未有的信息。结果表明，它形成了独特的 GQ-发夹混合结构，是选择性靶向的有效热点。此外，使用 MD 模拟生成的结构模型提供了有关探针系统如何感知不同 GQ 的见解。利用探针的响应性和Taq DNA 聚合酶终止测定，我们监测了 GQ 和发夹特异性配体相互作用及其对复制过程的协同抑制作用。我们的研究结果表明，使用双峰配体同时靶向 GQ 和发夹基序可能是选择性阻断病毒复制的新策略。