NMR研究多重共存的端粒G-四链体折叠结构之间相互动态转换及调控

G-quadruplexes folded at ends of telomeric DNA are able to inhibit the telomerase activity, and thus have been regarded as an anti-cancer drug target. However, the vast majority of drug designs have focused solely on targeting the most thermodynamically stable telomeric G-quadruplexes. Our preliminary work and other recent publications have found that, under both kinetic and thermodynamic controls simultaneously, there was a dynamic equilibrium and spontaneously mutual conversion among different coexisting G-quadruplex structures adopted by a single fragment of telomeric DNA. For most of biological processes, the biological macromolecules with proper functions are obligated to fold into appropriate structures within the demanded period of time. Especially, telomeric DNA which was highly protected and regulated by the sheltering proteins, was only liberated during specific physiological processes, such as DNA replication and transcription. Potentially forming a G-quadruplex during these processes might be dominated by kinetic rather than thermodynamic control. Accordingly, the kinetically-favored metastable G-quadruplex structure, however with faster formation and adequate lifetime, would have biological impacts more relevantly. Taken advantages of its real-time detection with atomic resolution, liquid NMR is applied mainly in this project to characterize any factors, simulated in vivo environment, which may impact on the dynamic equilibrium between kinetically and thermodynamically-favored G-quadruplex structures of telomeric DNA fragments; secondly to explore the intermediates involved in the folding/unfolding process during mutual conversions between these coexisting G-quadruplexes, thirdly to determine thermodynamic and especially the kinetic G-quadruplex structures of telomeric DNA. As a result, the off-target risks in the anti-cancer drug design targeting telomeric G-quadruplexes are expected to be further improved.

端粒DNA形成的G-四链体结构能抑制端粒酶活性，因而成为抗癌药物靶点，但目前多数药物设计还仅是针对该G-四链体靶点在最终平衡态下的单一热力学稳定结构。我们前期工作及新近他人报道均发现，某些单一的端粒DNA片段序列形成G-四链体时，同时受到热力学控制和动力学控制的综合影响，呈现出不同折叠结构共存并自发相互动态转化的现象。生物大分子要在生物体系中产生功能，则要求其在相应时间内形成恰当的结构；鉴于端粒在体内受到庇护蛋白等严格保护调控，这就使得那些具备动力学速度优势、能够首先形成并具备足够寿命的亚稳态G-四链体结构更应得到关注。本项目尝试利用液体核磁共振原子水平高分辨率和实时在线观测的优势，表征端粒G-四链体多重结构共存体系及动态相互转换过程中所涉及的中间体；从模拟细胞环境出发，探索能够影响动力学和热力学结构之间动态平衡的调控因素；解析动力学优势结构的三维模型。完善药物设计策略，降低药物脱靶风险。

端粒DNA形成的G-四链体结构能抑制端粒酶活性，通常作为抗癌药物靶点。但目前几乎所有的相关药物设计还仅是针对该类G-四链体靶点在最终平衡态下的单一热力学稳定结构，完全忽视了那些具备动力学速度优势、能够最先形成并且具备足够寿命的亚稳态G-四链体结构。而本项目发现了某些单一的端粒DNA片段序列折叠形成G-四链体时，同时受到热力学控制和动力学控制的综合影响，造成不同G-四链体结构多重共存且自发相互动态转化。.通过液体NMR技术，本项目深入研究了端粒3-最末端侧翼碱基在发生变更后是如何影响由人类端粒三个重复单元片段靶标与单重复单元片段探针之间组装而成的异分子间G-四链体拓扑结构以及形成过程：钠离子溶液中组装成动力学有利的分子间LLP型G-四链体（left loop progression，连接环为左走向），以及热力学有利的分子间RLP型G-四链体（right loop progression，连接环为右走向）。NMR表征了动态相互转换过程中所涉及的中间体，探索了能够影响动力学和热力学结构之间动态平衡的调控因素。尽管呈假镜面对称的LLP型与RLP型G-四链体在空间结构上的差异非常小，但我们还是找到了能够区分以上两种G-四链体的荧光有机小分子“硫黄素T”：处于低当量的硫黄素T可以优先识别RLP型G-四链体结构并通过芳香环平面堆叠的方式与之结合，这对于理解端粒G-四链体构型的调控以及其靶向药物的设计等领域都具有重要意义。.另外还发现：人源端粒DNA序列d(GTTAGG)更偏好在Na+溶液中自聚集形成非对称的三聚G-四链体，能耐受等量的K+离子。该三聚G-四链体位于缺口两侧的沟槽在上下G-四分体层之间宽度不一致，这是该结构的重要特征之一，可以潜在地作为特异的结构识别位点。此外，该三聚G-四链体不是一个完全静止的静态组装结构，时刻处于动态的链交换平衡之中，首次揭示了动态自组装形成的G-四链体有序结构。.以上相关研究成果已在高水平国际期刊Nucleic Acids Research上发表了两篇论文。