Structure, dynamics and kinetics of folding of G-quadruplex nucleic acids

G-四链体核酸折叠的结构、动力学和动力学

• 批准号: 392117191
• 负责人: Professor Dr. Harald Schwalbe
• 金额: --
• 依托单位: Institut für Organische Chemie und Chemische Biologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/392117191?language=en
• 关键词: Structure; dynamics; kinetics; folding; quadruplex

In this application, we intend to elucidate the energy folding landscape of G-quadruplex structures using a combination of biophysical methods, including in particular real time NMR spectroscopy. Experimental findings will guide molecular dynamics simulations by J. Sponer (Brno University). Sophisticated chemical and biochemical approaches will be developed and applied for the preparation of isotope labeled DNA and caged DNAs.The structures of G-quadruplexes are very polymorphic and their overall folding can vary in terms of strands orientation, geometry of the loops and of the glycosidic torsion angles. There is increasing evidence for a regulatory role of G-quadruplex structures in many biological processes. In fact, G-quadruplex are found at the 3'-overhang of telomeres and in the promoter regions of many oncogenes and are nowadays considered a novel target in anticancer therapy. However, more information is needed to fully understand the structure and the folding dynamics of G-quadruplexes and to unravel the details of its interaction with ligands and protein binding partners. In fact, the G-quadruplex energy landscape is far from being completely understood. The data reported until now suggest a very rugged energy landscape with multiple energy minima and an overall slow folding kinetics that follows a kinetic partition mechanism.Real-time NMR is optimally suited to obtain kinetic information at atomic resolution.We will use real-time NMR to investigate the folding kinetics of DNA and RNA G-quadruplexes, employing two different approaches. The first approach allows us to trigger the G-quadruplex refolding by injection of KCl directly in the NMR tube using a rapid mixing device. The second approach relies on the photocaging of selected guanine residues (work performed in the Heckel group) to block the oligonucleotide in a specific conformation or to maintain it unfolded. The refolding is triggered by illumination of the photocaged DNA/RNA directly in the NMR tube with a quartz fiber connected to a laser. In particular, the photocaging approach will be used to investigate the process of G-register exchange in G-quadruplex forming sequences containing more than 3 guanine residues per G-tract. These data will be complemented with activation energies derived by UV-vis hysteresis experiments (Mittermaier group). The information will be then the base for future MD simulations (Sponer group) that will provide details on the fast folding intermediates that cannot be detected and characterized by NMR. Furthermore, we aim at optimizing an enzymatic method based on the rolling circle amplification (RCA) for the production of 13C,15N labeled single stranded DNA sequences in mg amount. The availability of 13C,15N labeled DNA will allow us to monitor the folding kinetics with 2D NMR experiments as well as to characterize at high resolution the structure of selected G-quadruplexes.

在这个应用中，我们打算利用生物物理方法的组合来阐明g -四联体结构的能量折叠景观，特别是实时核磁共振波谱。实验结果将指导J. Sponer（布尔诺大学）的分子动力学模拟。复杂的化学和生化方法将被开发和应用于同位素标记DNA和笼化DNA的制备。g -四重复合物的结构是非常多形性的，它们的整体折叠可以在链的方向、环的几何形状和糖苷扭转角方面发生变化。越来越多的证据表明g -四重结构在许多生物过程中起调节作用。事实上，g -四重体存在于端粒的3'悬垂和许多癌基因的启动子区域，目前被认为是抗癌治疗的新靶点。然而，需要更多的信息来充分了解g -四联体的结构和折叠动力学，并揭示其与配体和蛋白质结合伙伴相互作用的细节。事实上，g -四重体的能量格局还远未被完全理解。迄今为止报告的数据表明，存在一个非常崎岖的能量景观，具有多个能量极小值和遵循动力学分配机制的整体缓慢折叠动力学。实时核磁共振最适合获得原子分辨率的动力学信息。我们将使用实时核磁共振来研究DNA和RNA g -四联体的折叠动力学，采用两种不同的方法。第一种方法允许我们通过使用快速混合装置直接在核磁共振管中注入KCl来触发g -四重体再折叠。第二种方法依赖于选定的鸟嘌呤残基的光笼（在Heckel组中进行的工作），以阻断特定构象的寡核苷酸或保持其展开。再折叠是通过直接在核磁共振管中用石英纤维连接到激光照射光笼子中的DNA/RNA来触发的。特别是，光笼方法将用于研究每个g -基团含有超过3个鸟嘌呤残基的g -四重体形成序列中g -寄存器交换的过程。这些数据将与由UV-vis迟滞实验得出的活化能相补充（Mittermaier组）。这些信息将成为未来MD模拟（Sponer组）的基础，该模拟将提供无法通过NMR检测和表征的快速折叠中间体的详细信息。此外，我们旨在优化一种基于滚动环扩增（RCA）的酶促方法，以生产mg量的13C，15N标记单链DNA序列。13C，15N标记DNA的可用性将使我们能够通过二维核磁共振实验监测折叠动力学，并以高分辨率表征选定的g -四联体的结构。