Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80/82, 6020, Innsbruck, Austria.
Angew Chem Int Ed Engl. 2020 Sep 21;59(39):17062-17069. doi: 10.1002/anie.202006577. Epub 2020 Jul 29.
We present the access to [5- F, 5- C]-uridine and -cytidine phosphoramidites for the production of site-specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5- F, 5- C]-pyrimidine labels into five RNAs-the 30 nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61 nt human hepatitis B virus ϵ (hHBV ϵ) RNA, the 49 nt SAM VI riboswitch aptamer domain from B. angulatum, the 29 nt apical stem loop of the pre-microRNA (miRNA) 21 and the 59 nt full length pre-miRNA 21. The main stimulus to introduce the aromatic F- C-spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole-dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic C- F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5- F, 5- C]/[5- F] pyrimidine labeling. For the 61 nt hHBV ϵ we find a beneficial F- C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [ F, C]-labeling of the SAM VI aptamer domain and the pre-miRNA 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting.
我们介绍了[5-F，5-C]-尿苷和胞苷磷酰胺的合成方法，用于制备多达 65 个核苷酸（nt）的定点修饰 RNA。这些磷酰胺被用于将[5-F，5-C]-嘧啶标签引入五个 RNA 中，分别是 30nt 的人类免疫缺陷病毒反式激活反应（HIV TAR）2 RNA、61nt 的乙型肝炎病毒ϵ（hHBV ϵ）RNA、来自 B. angulatum 的 49nt SAM VI 核糖开关适体结构域、29nt miRNA 21 的前体 miRNA 21 的顶端茎环结构和 59nt 全长前 miRNA 21。将芳香族 F-C-自旋拓扑结构引入 RNA 的主要动机来自于 Boeszoermenyi 等人的一项工作，其中偶极-偶极相互作用和化学位移各向异性弛豫机制相互抵消，从而表现出有利于芳香族蛋白质侧链的 TROSY 特性。这种芳香族 C-F 标记方案现在被转移到 RNA 中。我们提供了一种基于稀释[5-F，5-C]/[5-F]嘧啶标记的固相合成的共振分配方案。对于 61nt 的 hHBV ϵ，我们发现 F-C TROSY 增强有益，在更大的 RNA 中应该更加明显，这将有助于更大 RNA 的 NMR 研究。SAM VI 适体结构域和前 miRNA 21 的[F，C]标记进一步为使用生物正交稳定同位素报告核在体内 NMR 中观察配体结合和 miRNA 处理提供了可能性，这是在生物学相关环境中进行的。
