Synthesis of Precisely Modified Ribonucleic Acid to Reveal the Site-Specific Effect of Modification on Message Ribonucleic Acid Translation

In the past few decades, RNA modifications have significantly promoted the development of mRNA therapy. However, limited knowledge has been revealed on the site-specific effect of the modification, which raised biosafety concerns on mis-modified or overmodified mRNA drugs. Here in this study, we proposed a template-directed strategy for the synthesis of site-specifically modified long ssRNA. Long single-strand RNAs (over 300 nt) with single-base resolution modifications have been successfully prepared, including m⁶A, m⁵C, m¹Ψ, Ψ, I, Br-dU, Cy3, Cy5, FAM, 2'-F, 2'-OMe, 2'-MOE, 2'-Propargyl, LNA, cET, and PS modifications. Based on this method, the impact of the precisely modified patterns of mRNA on translation has been investigated. We have demonstrated that the modification of m¹Ψ on specific sites could increase translation fidelity while retaining low immunogenicity. It can be anticipated that our study will provide a guideline for mRNA modification to precisely adjust their functions, which will further promote next-generation mRNA drugs and RNA epigenetics research.