Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
Slovenian NMR Center, National Institute of Chemistry, Ljubljana, Slovenia.
PLoS One. 2022 Jul 8;17(7):e0264662. doi: 10.1371/journal.pone.0264662. eCollection 2022.
Solution NMR spectroscopy is a well-established tool with unique advantages for structural studies of RNA molecules. However, for large RNA sequences, the NMR resonances often overlap severely. A reliable way to perform resonance assignment and allow further analysis despite spectral crowding is the use of site-specific isotope labeling in sample preparation. While solid-phase oligonucleotide synthesis has several advantages, RNA length and availability of isotope-labeled building blocks are persistent issues. Purely enzymatic methods represent an alternative and have been presented in the literature. In this study, we report on a method in which we exploit the preference of T7 RNA polymerase for nucleotide monophosphates over triphosphates for the 5' position, which allows 5'-labeling of RNA. Successive ligation to an unlabeled RNA strand generates a site-specifically labeled RNA. We show the successful production of such an RNA sample for NMR studies, report on experimental details and expected yields, and present the surprising finding of a previously hidden set of peaks which reveals conformational exchange in the RNA structure. This study highlights the feasibility of site-specific isotope-labeling of RNA with enzymatic methods.
溶液核磁共振波谱学是一种成熟的工具，在 RNA 分子结构研究方面具有独特的优势。然而，对于较大的 RNA 序列，NMR 共振往往严重重叠。一种可靠的方法是在样品制备中使用位点特异性同位素标记，以进行共振分配并允许进一步分析，尽管光谱拥挤。虽然固相寡核苷酸合成具有几个优点，但 RNA 长度和同位素标记构建块的可用性仍然是存在的问题。纯粹的酶方法是另一种选择，并已在文献中提出。在这项研究中，我们报告了一种方法，我们利用 T7 RNA 聚合酶对 5' 位置的核苷酸单磷酸比对三磷酸的偏好，这允许 RNA 的 5' 标记。连续连接到未标记的 RNA 链上会产生特异性标记的 RNA。我们展示了这种用于 NMR 研究的 RNA 样品的成功生产，报告了实验细节和预期产量，并提出了一个令人惊讶的发现，即一组先前隐藏的峰揭示了 RNA 结构中的构象交换。这项研究强调了酶法对 RNA 进行位点特异性同位素标记的可行性。
