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 GpppAm , 7m Gpppm6 A, 7m Gpppm6 Am , 7m GpppG, 7m GpppGm ) 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.
© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.

Salmonella infections require the delivery of bacterial effectors into the host cell that alter the regulation of host defense mechanisms. The secreted cysteine protease GtgE from S. Typhimurium manipulates vesicular trafficking by modifying the Rab32 subfamily via cleaving the regulatory switch I region. Here we present a comprehensive biochemical, structural, and computational characterization of GtgE in complex with Rab32. Interestingly, GtgE solely processes the inactive GDP-bound GTPase. The crystal structure of the Rab32:GDP substrate in complex with the inactive mutant GtgEC45A reveals the molecular basis of substrate recognition. In combination with atomistic molecular dynamics simulations, the structural determinants for protein and activity-state specificity are identified. Mutations in a central interaction hub lead to loss of the strict GDP specificity. Our findings shed light on the sequence of host cell manipulation events during Salmonella infection and provide an explanation for the dependence on the co-secreted GTPase activating protein SopD2.