Simple Enzymatic\nIncorporation of 2′OMeU Nucleotide\nat the End of the Poly‑A Tail for Enhancement of the mRNA Stability\nand Protein Expression

This study focused on the efficient post-transcriptional\nincorporation\nof a modified nucleoside at the end of the poly-A tail of mRNA. The\nmodified mRNA was obtained in high yield and served to enhance protein\nexpression. Utilizing poly-U polymerase, our method successfully enabled\na single 2′OMeU residue to be incorporated into mRNA, which\nunexpectedly provided significant stabilization, even with only a\nsingle incorporation, to enhance the resistance of mRNA to degradation\nby cellular exonuclease. This stabilization effect allowed the mRNA\nto remain viable within the cell for an extended period to ultimately\nincrease the translation efficiency at least 3-fold. This approach\nto mRNA modification at the 3′ end with a single 2′OMeU\nresidue, by utilizing a straightforward tailing method, surpasses\nother ligation methods in terms of mRNA modification efficiency. Collectively,\nour results highlight the potential of this method to significantly\nadvance the development of highly effective mRNA-based therapies in\nthe future.