Position-Specific Nucleoside Sugar Modifications in mRNA ORF: Enhancing Translational Function through Complete Chemical Synthesis of mRNA

In this study, we systematically evaluated the efficacy of chemical modifications in enhancing the translational activity and stability of mRNA. We employed a primarily chemistry-based synthetic approach for the modified mRNA, which is crucial for the position-specific introduction of chemical modifications, enabling an intricate and detailed structure-activity relationship (SAR) study, hitherto unattainable with conventional methods. A pivotal innovation herein was the introduction of 2´-F modification for the nucleosugar in the open reading framework (ORF) at a site-specific position within the codon unit (the first nucleoside in the codon unit, 1st NC), which significantly bolstered the biological stability of mRNA without compromising its translational activity. This precise control of chemical modification patterns has emerged as a key to achieving higher peptide expression levels than conventional in vitro-transcribed mRNA. Further evaluations of the modifications at the 5´-UTR and poly(A) tail with other types of nucleoside and phosphate analogs also exemplified the importance of terminal modifications on mRNA for improved translational activity. These findings offer a novel framework for molecular design and development of effective mRNA-based therapeutics.