Global Incorporation of Synthetic ATP Analogs Reveals Poly(A)-Dependent Translation Differences in mRNA
Abstract
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 ATP analogs. we systematically evaluated these analogs against established modifications, including N6-methyl (m6A), 2-amino (am2A) and 7-deaza (c7A) derivatives, by incorporating them into mRNAs via global substitution. Our investigation highlights the efficacy of the newly developed N6-acetyl (ac6A) modification. In non-polyadenylated mRNAs, ac6A substitution significantly enhanced translation, achieving a threefold increase over unmodified mRNA. Importantly, in polyadenylated mRNAs, ac6A mRNA maintained translation efficiency comparable to natural mRNA, demonstrating its high biocompatibility. Surprisingly, am2A modification displayed striking poly(A)-dependent translational behavior. While am2A modified mRNA showed a threefold translation increase in the absence of a poly(A) tail, its efficiency dropped to 6% of natural levels upon poly(A) addition. Structural simulations revealed that am2A group introduces steric clashed and electrostatic repulsion with poly(A) binding protein (PABP), hindering closed-loop formation and reducing translation. Overall, this work expands the chemical space of adenine modifications with effective N6-acylated analogs and highlights that region-specific modifications can be strategically exploited to optimize translation efficiency for mRNA therapeutic design.
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