2'‐ O ‐Acetal Levulinic Ester Ribonucleoside 3'‐Phosphoramidites for the Solid‐Phase Synthesis of Long RNA

Abstract

Solid‐phase RNA synthesis is challenging because the 2'‐hydroxyl requires a protecting group that is stable during chain assembly yet easily removable without degrading the product. Driven by the demand for long RNA therapeutics, we report a synthesis method using 2'‐ O ‐acetal levulinic ester (ALE) ribonucleoside 3'‐ O ‐phosphoramidites. This approach utilizes a rapid, base‐labile on‐column deprotection strategy that preserves the 5'‐ O ‐dimethoxytrityl (DMTr) group, facilitating DMTr‐ON reverse‐phase (RP) purification. The resulting protocols enable the efficient production of long, functional RNAs, such as single‐guide RNAs with superior yield and purity compared to conventional silyl‐based methods, while remaining compatible with diverse chemical modifications. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1 : Automated solid‐phase synthesis of 2'‐ALE RNA

Basic Protocol 2 : Fast on‐column deprotection of 2'‐ALE RNA

Basic Protocol 3 : DMTr‐ON reverse‐phase high‐performance liquid chromatography (RP‐HPLC) purification of long RNA

Support Protocol 1 : Automated deprotection setup of 2'‐ALE RNA

Support Protocol 2 : Denaturing polyacrylamide gel electrophoresis (PAGE) purification of RNA