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

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.