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

在本申请中，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景观，特别是包括真实的时间NMR光谱。实验结果将指导J. Sponer（布尔诺大学）的分子动力学模拟。先进的化学和生物化学方法将被开发和应用于同位素标记的DNA和笼状DNA的制备。G-四链体的结构是非常多态的，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变化。越来越多的证据表明G-四链体结构在许多生物过程中起着调节作用。事实上，G-四链体存在于端粒的3 '-突出端和许多癌基因的启动子区域，并且现在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，并解开其与配体和蛋白质结合伙伴的相互作用的细节。事实上，G-quadruplex能量景观远未被完全理解。到目前为止，已有的数据显示了一个非常崎岖的能量景观，有多个能量最小值，总体折叠动力学缓慢，遵循动力学分配机制。实时NMR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NMR研究DNA和RNA G-四链体的折叠动力学，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在NMR管中直接注射KCl来触发G-四链体重折叠。第二种方法依赖于选定的鸟嘌呤残基的光笼化（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未折叠。通过用连接到激光器的石英纤维直接在NMR管中照射光笼化的DNA/RNA来触发重折叠。特别是，光笼的方法将被用来调查的过程中的G-寄存器交换G-四链体形成的序列含有超过3个鸟嘌呤残基每个G-道。这些数据将与紫外-可见滞后实验（Mittermaier组）得出的活化能进行补充。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，我们的目标是优化基于滚环扩增（RCA）的酶促方法，用于生产mg量的13 C，15 N标记的单链DNA序列。13 C，15 N标记的DNA的可用性将使我们能够监测的折叠动力学与2D NMR实验，以及在高分辨率的选定G-四链体的结构进行表征。

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.