Department of Chemistry, University of Zurich, Zurich, Switzerland.
Methods Mol Biol. 2022;2439:191-204. doi: 10.1007/978-1-0716-2047-2_13.
Fast and efficient site-specific labeling of long RNAs is one of the main bottlenecks limiting distance measurements by means of Förster resonance energy transfer (FRET) or electron paramagnetic resonance (EPR) spectroscopy. Here, we present an optimized protocol for dual end-labeling with different fluorophores at the same time meeting the restrictions of highly labile and degradation-sensitive RNAs. We describe in detail the dual-labeling of a catalytically active wild-type group II intron as a typical representative of long functional RNAs. The modular procedure chemically activates the 5'-phosphate and the 3'-ribose for bioconjugation with a pair of fluorophores, as shown herein, or with spin labels. The mild reaction conditions preserve the structural and functional integrity of the biomacromolecule and results in covalent, dual-labeled RNA in its pre-catalytic state in yields suitable for both ensemble and single-molecule FRET experiments.
快速且高效的长 RNA 位点特异性标记是通过Förster 共振能量转移（FRET）或电子顺磁共振（EPR）光谱法进行距离测量的主要瓶颈之一。在此，我们提出了一种优化的双端标记方案，可同时满足高度不稳定和易降解的 RNA 的限制条件，使用不同的荧光染料进行双重标记。我们详细描述了催化活性野生型 II 类内含子的双标记，作为长功能 RNA 的典型代表。该模块化程序通过化学方法激活 5'-磷酸和 3'-核糖，以便与一对荧光染料（如本文所示）或自旋标记物进行生物偶联。温和的反应条件可保持生物大分子的结构和功能完整性，并产生预催化状态的共价、双重标记 RNA，其产率适合于ensemble 和单分子 FRET 实验。
