Institute of Physical and Theoretical Chemistry, Rheinische Friedrich Wilhelms University, Bonn, Germany.
Life & Medical Sciences Institute (LIMES), Chemische Biologie, c/o Kekulé-Institut für organische Chemie, Bonn, Germany.
Methods Mol Biol. 2022;2439:205-221. doi: 10.1007/978-1-0716-2047-2_14.
Electron paramagnetic resonance (EPR) is a spectroscopic method for investigating structures, conformational changes, and dynamics of biomacromolecules, for example, oligonucleotides. In order to be applicable, the oligonucleotide has to be labeled site-specifically with paramagnetic tags, the so-called spin labels. Here, we provide a protocol for spin labeling of long oligonucleotides with nitroxides. In the first step, a short and commercially available RNA strand is labeled with a nitroxide via a copper-(I)-catalyzed azide-alkyne cycloaddition (CuAAC), also referred to as "click" reaction. In the second step, the labeled RNA strand is fused to another RNA sequence by means of enzymatic ligation to obtain the labeled full-length construct. The protocol is robust and has been shown experimentally to deliver high yields for RNA sequences up to 81 nucleotides, but longer strands are in principle also feasible. Moreover, it sets the path to label, for example, long riboswitches, ribozymes, and DNAzymes for coarse-grained structure determination and enables to investigate mechanistical features of these systems.
电子顺磁共振（EPR）是一种用于研究生物大分子（例如寡核苷酸）结构、构象变化和动力学的光谱方法。为了使其适用，寡核苷酸必须经过特异性标记的顺磁标记物，即所谓的自旋标记物。在这里，我们提供了一种用氮氧自由基对长寡核苷酸进行自旋标记的方案。在第一步中，通过铜（I）催化的叠氮化物-炔烃环加成（CuAAC），也称为“点击”反应，将短的、商业可得的 RNA 链用氮氧自由基标记。在第二步中，通过酶连接将标记的 RNA 链与另一个 RNA 序列融合，以获得标记的全长构建体。该方案稳健，实验表明，它可用于高达 81 个核苷酸的 RNA 序列，获得高产量，但原则上也可以标记更长的链。此外，它为粗粒结构测定标记例如长的核糖开关、核酶和 DNA 酶铺平了道路，并能够研究这些系统的机械特征。
