Institute of Physical Biology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Institute of Biological Information Processing, IBI-7: Structural Biochemistry, Forschungszentrum Jülich, Jülich, Germany.
Methods Mol Biol. 2022;2439:131-151. doi: 10.1007/978-1-0716-2047-2_10.
Catalytically active DNA oligomers (or DNAzymes) offer a broad spectrum of functions as well as applications. Although known for over two decades, the DNAzyme's mode-of-actions are still poorly understood, mainly due to lack of high-resolution structural insights. Due to their molecular size, structural flexibility, and dynamic interactions with metal-ion cofactors, solution nuclear magnetic resonance spectroscopy (NMR) can serve as optimal tool to obtain mechanistic insights of DNAzymes. In this respect, nearly all states of the DNAzyme and its substrate during the catalytic cycle are accessible. The instructions and protocols provided in the following may assist the initial steps of an NMR-based characterization of DNAzymes. To reduce the initial setup requirements and foster exciting new research projects, the discussed approaches focus on experiments that do not require cost-intensive isotope labeling strategies.
催化活性 DNA 寡聚物(或 DNA 酶)具有广泛的功能和应用。尽管已经有二十多年的历史,但 DNA 酶的作用模式仍未被充分理解,主要是由于缺乏高分辨率的结构见解。由于其分子大小、结构灵活性以及与金属离子辅因子的动态相互作用,溶液核磁共振波谱(NMR)可以作为获得 DNA 酶机制见解的最佳工具。在这方面,可以获得催化循环过程中 DNA 酶及其底物的几乎所有状态。以下提供的说明和方案可能有助于对 DNA 酶进行基于 NMR 的表征的初始步骤。为了降低初始设置要求并促进令人兴奋的新研究项目,所讨论的方法侧重于不需要昂贵的同位素标记策略的实验。