Depaix Anaïs, Grudzien-Nogalska Ewa, Fedorczyk Bartlomiej, Kiledjian Megerditch, Jemielity Jacek, Kowalska Joanna
Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland.
Centre of New Technologies, University of Warsaw, Warsaw, Poland.
Front Mol Biosci. 2022 Aug 19;9:854170. doi: 10.3389/fmolb.2022.854170. eCollection 2022.
Many eukaryotic and some bacterial RNAs are modified at the 5' end by the addition of cap structures. In addition to the classic 7-methylguanosine 5' cap in eukaryotic mRNA, several non-canonical caps have recently been identified, including NAD-linked, FAD-linked, and UDP-glucose-linked RNAs. However, studies of the biochemical properties of these caps are impaired by the limited access to transcribed RNA probes of high quality, as the typical capping efficiencies with NAD or FAD dinucleotides achieved in the presence of T7 polymerase rarely exceed 50%, and pyrimidine derivatives are not incorporated because of promoter sequence limitations. To address this issue, we developed a series of di- and trinucleotide capping reagents and transcription conditions to provide straightforward access to unconventionally capped RNAs with improved 5'-end homogeneity. We show that because of the transcription start site flexibility of T7 polymerase, RppApG-type structures (where R is either nicotinamide riboside or riboflavin) are efficiently incorporated into RNA during transcription from dsDNA templates containing both φ 6.5 and φ 2.5 promoters and enable high capping efficiencies (∼90%). Moreover, uridine-initiated RNAs are accessible by transcription from templates containing the φ 6.5 promoter performed in the presence of RppUpG-type initiating nucleotides (where R is a sugar or phosphate moiety). We successfully employed this strategy to obtain several nucleotide-sugar-capped and uncapped RNAs. The capping reagents developed herein provide easy access to chemical probes to elucidate the biological roles of non-canonical RNA 5' capping.
许多真核生物和一些细菌的RNA在5'端通过添加帽结构进行修饰。除了真核生物mRNA中的经典7-甲基鸟苷5'帽外，最近还鉴定出了几种非经典帽，包括与NAD、FAD和UDP-葡萄糖连接的RNA。然而，由于难以获得高质量的转录RNA探针，这些帽的生化特性研究受到了阻碍，因为在T7聚合酶存在下，用NAD或FAD二核苷酸实现的典型加帽效率很少超过50%，并且由于启动子序列限制，嘧啶衍生物无法掺入。为了解决这个问题，我们开发了一系列二核苷酸和三核苷酸加帽试剂以及转录条件，以便直接获得具有改善的5'端均一性的非常规加帽RNA。我们表明，由于T7聚合酶的转录起始位点灵活性，RppApG型结构（其中R为烟酰胺核糖或核黄素）在从含有φ 6.5和φ 2.5启动子的双链DNA模板转录过程中有效地掺入RNA中，并实现了高加帽效率（约90%）。此外，通过在RppUpG型起始核苷酸（其中R为糖或磷酸部分）存在下从含有φ 6.5启动子的模板进行转录，可以获得尿苷起始的RNA。我们成功地采用了这种策略来获得几种核苷酸-糖加帽和未加帽的RNA。本文开发的加帽试剂为阐明非经典RNA 5'加帽的生物学作用提供了易于获得的化学探针。
