Department of Chemistry, Konstanz Research School of Chemical Biology and Zukunftskolleg, University of Konstanz, Konstanz, Germany.
Nat Protoc. 2013 Jan;8(1):131-47. doi: 10.1038/nprot.2012.136. Epub 2012 Dec 20.
Double electron-electron resonance (DEER) is an electron paramagnetic resonance (EPR) technique used to determine distance distributions in the nanometer range between spin labels by measuring their dipole-dipole interactions. Here we describe how in-cell DEER can be applied to spin-labeled DNA sequences to unravel their conformations in living cells by long-range distance measurements in cellula. As EPR detects unpaired electron spins only, diamagnetic molecules provide no background and do not reduce detection sensitivity of the specific signal. Compared with in-cell NMR spectroscopy, low concentrations of spin-labeled molecules can be used owing to the higher sensitivity of EPR per spin. This protocol describes the synthesis of the spin labels, their introduction in DNA strands, the injection of labeled DNA solutions in cells and the performance of in-cell EPR measurements. Completion of the entire protocol takes ~20 d.
双电子电子共振（DEER）是一种电子顺磁共振（EPR）技术，用于通过测量其偶极-偶极相互作用来确定纳米范围内自旋标记物之间的距离分布。在这里，我们描述了如何将细胞内 DEER 应用于自旋标记的 DNA 序列，通过在细胞内进行长程距离测量来揭示其在活细胞中的构象。由于 EPR 仅检测不成对的电子自旋，抗磁性分子不会提供背景并且不会降低特定信号的检测灵敏度。与细胞内 NMR 光谱学相比，由于每个自旋的 EPR 灵敏度更高，可以使用低浓度的自旋标记分子。该方案描述了自旋标记物的合成，它们在 DNA 链中的引入，标记 DNA 溶液在细胞中的注入以及细胞内 EPR 测量的性能。整个方案的完成大约需要 20 天。
