Site-directed spin-labeling of nucleotides and the use of in-cell EPR to determine long-range distances in a biologically relevant environment
Site-directed spin-labeling of nucleotides and the use of in-cell EPR to determine long-range distances in a biologically relevant environment
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DOI：
10.1038/nprot.2012.136
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发表时间：
2013-01-01
期刊：
影响因子：
14.8
通讯作者：
Drescher, Malte
中科院分区：
文献类型：
--
作者：
Azarkh, Mykhailo;Singh, Vijay;Drescher, Malte
双电子-电子共振（DEER）是一种电子顺磁共振（EPR）技术，用于通过测量它们的偶极-偶极相互作用来确定自旋标记之间在纳米范围内的距离分布。在这里，我们描述了如何在细胞内的DEER可以应用到自旋标记的DNA序列解开它们的构象在活细胞中的细胞内的长距离测量。由于EPR仅检测未成对电子自旋，因此抗磁性分子不提供背景，并且不会降低特定信号的检测灵敏度。与细胞内NMR光谱相比，由于EPR每次自旋的灵敏度较高，因此可以使用低浓度的自旋标记分子。该方案描述了自旋标记的合成，它们在DNA链中的引入，在细胞中注射标记的DNA溶液和在细胞中EPR测量的性能。整个方案的完成需要大约20天。
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 similar to 20 d.