Enhancing mRNA stability and translational potential through tailored modifications at the 3' end

Poly(A) tails regulate mRNA turnover and translation through multiple RNA-protein interactions, and deadenylation is the initiating step of the major cytoplasmic decay pathways. Here, we report a simple post-transcriptional strategy to protect the 3' end of synthetic mRNA by enzymatically ligating short, chemically modified 5'-phosphorylated dinucleotides. Using T4 RNA ligase 1, we attached 2'-O-methyl and/or phosphorothioate containing A- or G-dinucleotides to the 3' end of model oligoadenylates and to an IVT Gaussia luciferase (GLuc) mRNA bearing an ∼150-nt poly(A) tail. All ligated products showed strong resistance to human CNOT7-mediated deadenylation in vitro, whereas the unmodified control mRNA underwent poly(A) removal. In rabbit reticulocyte lysate, 3'-modified GLuc mRNAs translated comparably to the control, indicating minimal interference with the translational machinery. In mammalian cells (A549, JAWSII and HEK293), the protein output depended on the dinucleotide structure and cell type; two adenosine donors-pApA_m and the D1 phosphorothioate stereoisomer pAp_sA_m-consistently performed best, increasing the cumulative GLuc production up to 163% in HEK293 and up to 79% in A549. These results establish dinucleotide ligation as a minimal and modular 3'-end engineering approach that enhances resistance to deadenylation and can improve the translational performance of therapeutic mRNA candidates.