Analysis of the Effects of Sugar Modifications on RNA Chemical Ligation Reactions

Chemical ligation of RNA fragments is an effective method for synthesizing long RNAs. It is particularly useful for incorporating site-specific modifications into long RNAs; however, its low reaction efficiency remains a major challenge. Herein, 5'-amino-2'-substituted uridine or cytidine nucleotides are synthesized, which are attached to the 5' end of a synthetic RNA and conjugated with a phosphate group at the 3' end of an alternative RNA fragment, to perform a head-to-tail-type RNA ligation. It is examined how the 2' position of nucleotides with 5'-amino or 3'-phosphate groups in the ligated site affects the ligation reaction. The 2'-fluoro-5'-amino-nucleotide shows enhanced reactivity compared with the 2'-ribo- or 2'-deoxy-5'-amino-nucleotide. In contrast, the 2'-O-methyl modification demonstrates optimal efficacy with the 3'-phosphate nucleotide. Interestingly, the 2'-fluoro-5'-amino nucleotide maintains remarkable reactivity, even under acidic conditions. A pre-miRNA comprising 84 nucleotides is synthesized through ligation, and the intracellular functionality of the ligated RNA is confirmed. This study elucidates the effect of 2' substituents on chemical ligation reactions, providing insights into the chemical synthesis of long RNAs.