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.

在此应用中，我们打算使用生物物理方法的组合，包括特别是实时NMR光谱法，阐明G四链体结构的能量折叠景观。实验发现将指导J. Sponer（Brno University）的分子动力学模拟。将开发并应用复杂的化学和生化方法，用于制备标有DNA和笼式DNA的同位素。G-四链体的结构非常多晶型，其总体折叠可能会因链的定向，glamcosidicic的几何学和glycosidicic的几何学而有所不同扭转角。有越来越多的证据表明，G四链体结构在许多生物过程中的调节作用。实际上，在端粒的3'-遍布和许多肿瘤基因的启动子区域中发现了g-四链体，如今被认为是抗癌治疗中的新靶标。但是，需要更多信息来充分了解G四链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。实际上，G-四链体能量景观远非完全理解。到目前为止，报道的数据表明，具有多个能量最小的非常坚固的能量景观以及遵循动力学分区机制的总体缓慢折叠动力学。真实时代NMR最适合在原子分辨率下获得动力学信息。我们将使用实时NMR使用两种不同的方法研究DNA和RNA G-四链体的折叠动力学。第一种方法使我们能够使用快速混合装置直接在NMR管中注入KCL来触发G Quadruplex。第二种方法依赖于选定的鸟嘌呤残基（在Heckel组执行的工作）的光定位以在特定构型中阻断寡核苷酸或保持其展开。重新折叠是通过直接在NMR管中使用的石英纤维连接到激光器的NMR管中的光塑料DNA/RNA触发的。特别地，光定位方法将用于研究G-Qu-四链体形成序列中含有3种以上鸟嘌呤残基的G-Qu-Qu-Quendister交换过程。这些数据将与UV-VIS磁滞实验（Mittermaier组）得出的激活能相互补充。该信息将成为未来MD模拟（Sponer Group）的基础，该基础将提供有关无法检测和表征NMR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圆扩增（RCA）的酶促方法，用于生产13C，15N的15N标记为Mg量的单链DNA序列。 13C，标记为DNA的13C的可用性将使我们能够通过2D NMR实验监测折叠动力学，并在高分辨率上表征所选G Quadruplexes的结构。