# Synthesis of long and functionally active RNAs facilitated by acetal levulinic ester chemistry

## TL;DR

Researchers developed a new method using acetal levulinic ester chemistry to synthesize long, functional RNAs up to 215 nucleotides in length.

## Contribution

The study introduces 2′-acetal levulinic ester (2′-ALE) chemistry for efficient synthesis of long RNAs with high functional activity.

## Key findings

- 2′-ALE phosphoramidites enabled synthesis of RNAs up to 215 nucleotides with >99% coupling efficiency.
- Synthetic RNAs including sgRNAs and mRNAs showed robust functional activity in cell-free and cellular systems.
- Incorporating 2′-O-methyl-adenosine in the poly(A) tail significantly enhanced protein expression.

## Abstract

Recent advances in RNA-based therapeutics have created a demand for synthetic RNAs that are 100 nucleotides (nts) or longer. In this study, we present the use of 2′-acetal levulinic ester (2′-ALE) phosphoramidites for the synthesis of long RNAs that are at least 215 nts in length. We have developed protocols for rapid (2–4 min) and efficient coupling (>99%) of 2′-ALE monomers and established a rapid, on-column deprotection of RNA strands requiring short alkylamine treatments at room temperature. The results of these studies enabled the successful syntheses of sgRNAs (99 nt), sgRNA tagged with fluorogenic Mango II and Broccoli aptamers (130–170 nt), and 5′-capped minimal mRNAs (200–215 nt), each exhibiting robust functional activity in both cell-free and cellular systems. We also found that the incorporation of 2′-O-methyl-adenosine in the poly(A) tail of synthetic mRNAs markedly enhanced protein expression, highlighting the ALE platform’s compatibility for systematic exploration of RNA chemical diversity. Collectively, these results establish 2′-ALE chemistry as a promising platform for the synthesis of long and functionally active RNAs.

Graphical Abstract