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

Nov 24, 2025Molecular therapy. Nucleic acids

Chemical changes during mRNA production improve its resistance to breakdown and the stability of its delivery particles

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Abstract

2'-O-methyl and 2'-fluoro modifications significantly enhance the stability of mRNA.

2'-O-methyl and 2'-fluoro modifications protect mRNA from enzymatic degradation.
These modifications extend the lifetime of intact mRNA in environments rich in RNase.
Higher stability of modified mRNA is generally associated with decreased translatability.
Despite reduced translatability, 2'-modified mRNA retains detectable activity.
When combined with lipid nanoparticles and stored at ambient temperature, 2'-modified mRNA shows significantly slower decay in activity.

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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,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 throughtranscription 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 bothand. 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. in vivo in vitro in vitro in vivo

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

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