Co-transcriptional modifications of 2′-hydroxyls on synthetic mRNA enhance ribonuclease resistance and lipid nanoparticle thermostability

The innate fragility of messenger RNA (mRNA) stems from 2'-hydroxyls on the ribose that are responsible for making mRNA sensitive to enzymatic and thermal degradation, posing formidable challenges in efficacy, <i>in vivo</i> stability, global-scale distribution, and long-term storage of mRNA nanomedicines. However, 2'-OH modifications are difficult to introduce, both enzymatically and synthetically, and their potential impact on mRNA translation remains poorly understood. Here, we synthesized 2'-O-methyl (2'-O-Me)- or 2'-fluoro (2'-F)-substituted mRNA through <i>in vitro</i> transcription processes by employing T7 RNA polymerase mutants, aiming to reveal how these 2'-modifications dictate the stability and activity of modified mRNAs. We found that 2'-O-Me and 2'-F modifications protected mRNA from enzymatic degradation, thereby extending the lifetime of intact mRNA in RNase-abundant environments. We observed that higher stability was conferred mostly in exchange for a decrease in translatability, but 2'-modified mRNA still showed detectable activity both <i>in vitro</i> and <i>in vivo</i>. Furthermore, when formulated into lipid nanoparticles (LNPs) and stored at ambient temperature, 2'-modified mRNA led to significantly slower decay in activity. Our findings demonstrate how co-transcriptional 2'-modifications can enhance the stability of synthetic mRNA with application in mRNA LNP formulations.