One-pot ligation of multiple mRNA fragments on dsDNA splint advancing regional modification and translation.

Region-specific RNA modifications are crucial for advancing RNA research and therapeutics, including messenger RNA (mRNA)-based vaccines and immunotherapy. However, the predominant method, synthesizing regionally modified mRNAs with short single-stranded DNA (ssDNA) splints, encounters challenges in ligating long mRNA fragments due to the formation of RNA self-folded complex structures. To address this issue, we developed an efficient strategy using an easily obtained long double-stranded DNA (dsDNA) as a ligation splint after in situ denaturing, while parts of this dsDNA are the templates for transcribing mRNA fragments.