Access to High‐Purity 7m G‐cap RNA in Substantial Quantities by a Convenient All‐Chemical Solid‐Phase Method
Résumé
Abstract Given the importance of mRNA with 5’‐cap, easy access to RNA substrates with different 7m G caps, of high quality and in large quantities is essential to elucidate the roles of RNA and the regulation of underlying processes. In addition to existing synthetic routes to 5’‐cap RNA based on enzymatic, chemical or chemo‐enzymatic methods, we present here an all‐chemical method for synthetic RNA capping. The novelty of this study lies in the fact that the capping reaction is performed on solid‐support after automated RNA assembly using commercial 2’‐ O ‐propionyloxymethyl ribonucleoside phosphoramidites, which enable final RNA deprotection under mild conditions while preserving both 7m G‐cap and RNA integrity. The capping reaction is efficiently carried out between a 5’‐phosphoroimidazolide RNA anchored on the support and 7m GDP in DMF in the presence of zinc chloride. Substantial amounts of 7m G‐cap RNA (from 1 to 28 nucleotides in length and of any sequence with or without internal methylations) containing various cap structures ( 7m GpppA, 7m GpppA m , 7m Gppp m6 A, 7m Gppp m6 A m , 7m GpppG, 7m GpppG m ) were obtained with high purity after IEX‐HPLC purification. This capping method using solid‐phase chemistry is convenient to perform and provides access to valuable RNA substrates as useful research tools to unravel specific issues regarding cap‐related processes.
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