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

在这一应用中，我们打算使用生物物理方法的组合来阐明G-四链体结构的能量折叠情况，特别是实时核磁共振光谱。实验结果将指导J·斯宾纳(布尔诺大学)进行分子动力学模拟。先进的化学和生物化学方法将被开发和应用于制备同位素标记的DNA和笼状DNA。G-四链体的结构非常多态，它们的整体折叠可以根据链的取向、环的几何形状和糖苷类的扭转角而变化。越来越多的证据表明，G-四链结构在许多生物过程中具有调节作用。事实上，G-四链体存在于端粒的3‘端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。事实上，G-四元组的能源格局还远未完全被理解。到目前为止所报道的数据表明，一个非常崎岖的能量图景，具有多个能量最小值，总体上遵循动力学分配机制的缓慢折叠动力学。实时核磁共振最适合于在原子分辨率下获得动力学信息。我们将使用实时核磁共振研究DNA和RNA G-四链的折叠动力学，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-四链折叠。第二种方法依赖于对选定的鸟嘌呤残基进行光笼处理(在Heckel小组中进行的工作)，以阻断特定构象中的寡核苷酸或保持其展开。重折叠是通过将光笼中的DNA/RNA直接照射在核磁共振管中，并将石英光纤连接到激光器上来触发的。特别是，光笼法将被用来研究G-四链形成序列中G-寄存器交换的过程，每个G-链包含3个以上的鸟嘌呤残基。这些数据将与由UV-VIS滞后实验得出的激活能相补充(Mittermaier小组)。这些信息将成为未来分子动力学模拟(斯宾纳小组)的基础，该模拟将提供核磁共振无法检测和表征的快速折叠中间体的细节。此外，我们旨在优化一种基于滚环扩增(RCA)的酶促方法，用于生产mg量的13C，15N标记的单链DNA序列。13C，15N标记DNA的可获得性将使我们能够通过2D核磁共振实验来监测折叠动力学，以及以高分辨率表征选定的G-四链体的结构。

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.