Life & Medical Sciences Institute, Chemical Biology & Medicinal Chemistry Unit, University of Bonn, Gerhard-Domagk-Str. 1, 53121, Bonn, Germany.
Institute for Physical and Theoretical Chemistry, University of Bonn, Wegelerstr. 12, 53115, Bonn, Germany.
Angew Chem Int Ed Engl. 2020 May 11;59(20):7891-7896. doi: 10.1002/anie.201916447. Epub 2020 Mar 13.
We present herein a novel nitroxide spin label-containing RNA triphosphate TPT3 and its application for site-specific spin-labeling of RNA through in vitro transcription using an expanded genetic alphabet. Our strategy allows the facile preparation of spin-labeled RNAs with sizes ranging from short RNA oligonucleotides to large, complex RNA molecules with over 370 nucleotides by standard in vitro transcription. As a proof of concept, inter-spin distance distributions are measured by pulsed electron paramagnetic resonance (EPR) spectroscopy in short self-complementary RNA sequences and in a well-studied 185 nucleotide non-coding RNA, the B. subtilis glmS ribozyme. The approach is then applied to probe for the first time the folding of the 377 nucleotide A-region of the long non-coding RNA Xist, by PELDOR.
我们在此提出了一种新型的含硝氧自由基的 RNA 三磷酸 TPT3，并将其应用于通过扩展遗传密码子的体外转录实现 RNA 的定点自旋标记。我们的策略允许通过标准的体外转录轻松制备大小从短 RNA 寡核苷酸到超过 370 个核苷酸的大型复杂 RNA 分子的自旋标记 RNA。作为概念验证，通过脉冲电子顺磁共振（EPR）光谱测量了短的自互补 RNA 序列和经过充分研究的 185 个核苷酸非编码 RNA，枯草芽孢杆菌 glmS 核酶中的自旋间距分布。然后，该方法首次应用于 PELDOR 探测长非编码 RNA Xist 的 377 个核苷酸 A 区的折叠。
