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

## TL;DR

This study explores how sugar modifications at the RNA ligation site affect the efficiency of RNA synthesis, revealing that specific substitutions improve reaction performance.

## Contribution

The study identifies optimal 2′-substituent combinations for RNA ligation, enhancing synthesis efficiency and functionality.

## Key findings

- 2′-fluoro-5′-amino nucleotides show higher reactivity compared to ribo- or deoxy-variants.
- 2′-O-methyl modification with 3′-phosphate nucleotides achieves the highest ligation efficiency.
- 2′-fluoro-5′-amino nucleotides maintain reactivity under acidic conditions.

## Abstract

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.

The effect of substitutions at the 2′ position of the nucleotides at the ligation site on phosphoramidate‐type chemical ligation reaction efficiency is examined. The efficiency varies depending on the substituents, with the combination of a fluorine group on 5′NRNA and an O‐methyl group on RNA3′P showing the highest efficiency.© 2025 WILEY‐VCH GmbH