Parkinson Gary N, Collie Gavin W
UCL School of Pharmacy, University College London, London, UK.
Discovery Sciences, R&D, AstraZeneca, Cambridge, UK.
Methods Mol Biol. 2019;2035:131-155. doi: 10.1007/978-1-4939-9666-7_8.
The application of X-ray crystallographic methods toward a structural understanding of G-quadruplex (G4) motifs at atomic level resolution can provide researchers with exciting opportunities to explore new structural arrangements of putative G4 forming sequences and investigate their recognition by small molecule compounds. The crowded and ordered crystalline environment requires the self-assembly of stable G4 motifs, allowing for an understanding of their inter- and intramolecular interactions in a packed environment, revealing thermodynamically stable topologies. Additionally, crystallographic data derived from these experiments in the form of electron density provides valuable opportunities to visualize various solvent molecules associated with G4s along with the geometries of the metal ions associated within the central channel-elements critical to the understanding G4 stability and topology. Now, with the advent of affordable, commercially sourced and purified synthetic DNA and RNA molecules suitable for immediate crystallization trials, and combined with the availability of specialized and validated crystallization screens, researchers can now undertake in-house crystallization trials without the need for local expertise. When this is combined with access to modern synchrotron platforms that offer complete automation of the data collection process-from the receipt of crystals to delivery of merged and scaled data for the visualization of electron density-the application of X-ray crystallographic techniques is made open to nonspecialist researchers. In this chapter we aim to provide a simple how-to guide to enable the reader to undertake crystallographic experiments involving G4s, encompassing the design of oligonucleotide sequences, fundamentals of the crystallization process and modern strategies used in setting up successful crystallization trials. We will also describe data collection strategies, phasing, electron density visualization, and model building. We will draw on our own experiences in the laboratory and hopefully build an appreciation of the utility of the X-ray crystallographic approaches to investigating G4s.
运用X射线晶体学方法在原子水平分辨率上对G-四链体（G4）基序进行结构解析，可为研究人员提供令人兴奋的机会，以探索假定的G4形成序列的新结构排列，并研究小分子化合物对它们的识别。拥挤且有序的晶体环境要求稳定的G4基序进行自组装，从而有助于理解它们在堆积环境中的分子间和分子内相互作用，揭示热力学稳定的拓扑结构。此外，这些实验以电子密度形式获得的晶体学数据提供了宝贵的机会，可用于可视化与G4相关的各种溶剂分子以及与中央通道元素相关的金属离子的几何结构，而这些对于理解G4的稳定性和拓扑结构至关重要。如今，随着价格合理、可从商业渠道获取和纯化的适合立即进行结晶试验的合成DNA和RNA分子的出现，再结合专门且经过验证的结晶筛选方法，研究人员现在无需当地专业知识就能进行内部结晶试验。当这与使用现代同步加速器平台相结合时，该平台可提供从晶体接收至合并和缩放数据以进行电子密度可视化的数据收集过程的完全自动化，X射线晶体学技术的应用便向非专业研究人员开放。在本章中，我们旨在提供一个简单的操作指南，使读者能够进行涉及G4的晶体学实验，包括寡核苷酸序列的设计、结晶过程的基本原理以及成功进行结晶试验所采用的现代策略。我们还将描述数据收集策略、相位确定、电子密度可视化和模型构建。我们将借鉴我们在实验室中的经验，希望能让读者认识到X射线晶体学方法在研究G4方面的实用性。
