Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.
Chemistry Branch, Department of Science and Mathematics, Faculty of Petroleum and Mining Engineering, Suez University, Suez 43721, Egypt.
Nucleic Acids Res. 2020 Apr 6;48(6):2830-2840. doi: 10.1093/nar/gkaa086.
The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level. The ability to measure distances of 2-10 nm is particularly important: deformations arising from protein binding commonly fall within this range, but the reliable measurement of such distances for a conformational ensemble remains a significant challenge. Using several techniques, we show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2' position offers a robust and minimally perturbing tool for obtaining such measurements. For two nitroxides, we present results from EPR spectroscopy, X-ray crystal structures of B-form spin-labelled DNA duplexes, molecular dynamics simulations and nuclear magnetic resonance spectroscopy. These four methods are mutually supportive, and pinpoint the locations of the spin labels on the duplexes. In doing so, this work establishes 2'-alkynyl nitroxide spin-labelling as a minimally perturbing method for probing DNA conformation.
确定核酸中特定点之间的距离对于理解其在分子水平上的行为至关重要。能够测量 2-10nm 的距离尤为重要：蛋白质结合引起的变形通常在此范围内，但对于构象整体可靠地测量此类距离仍然是一个重大挑战。我们使用多种技术表明，用三唑取代的 2'位氮氧自由基标记的寡核苷酸的电子顺磁共振（EPR）光谱为获得此类测量提供了一种稳健且最小干扰的工具。对于两个氮氧自由基，我们展示了来自 EPR 光谱、B 型自旋标记 DNA 双链体的 X 射线晶体结构、分子动力学模拟和核磁共振光谱的结果。这四种方法相互支持，并确定了双链体上自旋标记的位置。通过这样做，这项工作确立了 2'-炔基氮氧自由基自旋标记作为探测 DNA 构象的最小干扰方法。
