Global incorporation of synthetic adenosine analogs reveals poly(A)-dependent translation differences in mRNA

Chemical modifications of nucleosides are essential for enhancing the efficacy of therapeutic mRNAs. While uridine analogs like N1-methylpseudouridine (m1Ψ) are well studied, adenine modifications remain underexplored, despite adenine's abundance and exclusive role in the poly(A) tail of eukaryotic mRNA. Inspired by the translational benefits of N4-acetylcytidine (ac4C), we designed and synthesized a series of novel N6-acylated adenosine analogs. These modified nucleotides were globally incorporated into mRNAs via in vitro transcription and systematically evaluated against established modifications, including N6-methyl (m6A), 2-amino (am2A), and 7-deaza (c7A) derivatives. Among these, N6-acetyl modification (ac6A) exhibited the best overall performance, supporting efficient transcription, high translational output, sustained intracellular abundance, and comparable to native innate immunogenicity. Our study also reveals that the poly(A) tail is highly sensitive to chemical modifications. We found that incorporating modified adenines into the poly(A) consistently impairs translation efficiency. This context-dependent effect was most evident with am2A, which enhanced translation 3-fold in tail-less mRNAs but reduced it to near-background levels upon polyadenylation. Structural simulations suggest that am2A group may sterically and electrostatically interfere with poly(A) binding protein (PABP) recognition, providing a plausible structural basis for the observed translation suppression. Overall, this work expands the chemical space of adenine modifications with effective N6-acylated analogs and shows that all modifications can interfere with poly(A) tail function. These findings highlight the potential value of region-specific modification as a design consideration for future mRNA therapeutics.