Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.
Molecules. 2020 Nov 24;25(23):5492. doi: 10.3390/molecules25235492.
RNA 5'-modifications are known to extend the functional spectrum of ribonucleotides. In recent years, numerous non-canonical 5'-modifications, including adenosine-containing cofactors from the group of B vitamins, have been confirmed in all kingdoms of life. The structural component of thiamine adenosine triphosphate (thiamine-ATP), a vitamin B1 derivative found to accumulate in and other organisms in response to metabolic stress conditions, suggests an analogous function as a 5'-modification of RNA. Here, we report the synthesis of thiamine adenosine dinucleotides and the preparation of pure 5'-thiamine-capped RNAs based on phosphorimidazolide chemistry. Furthermore, we present the incorporation of thiamine-ATP and thiamine adenosine diphosphate (thiamine-ADP) as 5'-caps of RNA by T7 RNA polymerase. Transcripts containing the thiamine modification were modified specifically with biotin via a combination of thiazole ring opening, nucleophilic substitution and copper-catalyzed azide-alkyne cycloaddition. The highlighted methods provide easy access to 5'-thiamine RNA, which may be applied in the development of thiamine-specific RNA capture protocols as well as the discovery and confirmation of 5'-thiamine-capped RNAs in various organisms.
RNA 5' - 修饰已知可扩展核糖核苷酸的功能谱。近年来，包括来自 B 族维生素的组的含腺苷辅因子在内的许多非典型 5' - 修饰已在所有生命领域得到证实。硫胺素三磷酸腺苷（硫胺素-ATP）的结构成分是一种维生素 B1 衍生物，在代谢应激条件下发现积累在 和其他生物体中，表明其具有作为 RNA 5' - 修饰的类似功能。在这里，我们报告了硫胺素腺苷二核苷酸的合成以及基于亚磷酰胺化学制备纯 5' - 硫胺帽 RNA。此外，我们提出了通过 T7 RNA 聚合酶将硫胺素-ATP 和硫胺素腺苷二磷酸（硫胺素-ADP）掺入 RNA 的 5' - 帽中。通过噻唑环打开、亲核取代和铜催化的叠氮-炔环加成反应，用生物素特异性修饰含有硫胺修饰的转录本。突出显示的方法提供了易于获得 5' - 硫胺 RNA 的途径，这可应用于开发硫胺特异性 RNA 捕获方案以及在各种生物体中发现和确认 5' - 硫胺帽 RNA。
