Synthesis of mRNAs with a Varied Chemically Modified Poly(A) Tail and Study of Their Translation Efficiencies ^†

Comprehensive Summary RNA modifications have revealed essential regulatory roles in messenger RNA (mRNA) metabolism to affect cellular gene regulation, and have also received widespread applications in RNA therapeutics such as mRNA vaccine and protein replacement. Most efforts focus on mRNA internal base and ribose modifications, however, chemical modification within mRNA poly(A) tail remains unexplored. In this work, we synthesized luciferase and GFP (Green Fluorescent Protein) mRNAs with a fully chemically modified poly(A) tail, in which adenosine is replaced by either base‐modified N 6 ‐methyladenosine (m 6 A)/ N 6 ‐ethyladenosine (Et 6 A) or ribose‐modified 2’‐ O ‐methyladenosine (Am), and investigated the effect of these tail modifications on mRNA stability and translation efficiency upon transfection into cells using fluorescent and chemiluminescent reporter assays. The results showed that all these modifications impaired translation without affecting mRNA stability. Further study demonstrated that modified poly(A) tail weakens its binding to PABPC1 (Polyadenylate‐Binding Protein 1), which reduces the formation of mRNA head‐to‐tail loop and thus decreases the translation efficiency. Our finding reveals the translation‐regulatory role of the adenosine modifications within poly(A) tail, offering a new way for manipulating mRNA translation inside cells.