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

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.