N1-Methylpseudouridine

N1-Methylpseudouridine is a methylpseudouridine and enhances translation through eIF2α-dependent and independent mechanisms by increasing ribosome density in mRNA. (In Vitro) :In HEK293T cells, Incorporation of N1-Methylpseudouridine into mRNA modifies mRNAs produced higher amounts of luc than the standard Luc mRNA . Incorporation of N1-Methylpseudouridine nucleoside modification in both Luc and GFP mRNA enhances the initiation step of translation, in part by suppressing eIF2α phosphorylation. In addition, polysome formation and growth on the N1-Methylpseudouridine-containing Luc mRNA is enhanced due to the reduction of elongation rate. (In Vivo) :mRNAs containing the N1-Methylpseudouridine modification alone and/or in combination with 5-methylcytidine (m5C) outperformed the current state-of-the-art pseudouridine (Ψ) and/or m5C/Ψ-modified mRNA platform by providing up to ~44-fold (when comparing double modified mRNAs) or ~13-fold (when comparing single modified mRNAs) higher reporter gene expression upon transfection into cell lines or mice, respectively. We show that (m5C/) N1-Methylpseudouridine-modified mRNA resulted in reduced intracellular innate immunogenicity and improved cellular viability compared to (m5C/) Ψ-modified mRNA upon in vitro transfection.