Mild, biocompatible deprotection method for synthetic RNAs, including RNAs containing sensitive modifications such as 5'-diphosphates

The synthesis of high-quality RNA oligonucleotides, including those with modifications such as 5'diphosphate or triphosphate groups, requires mild deprotection methods that maintain chemical integrity while enabling efficient downstream processing. Conventional approaches using tbutylammonium fluoride (TBAF) are effective for unmodified RNA molecules, but they pose challenges for purification, scale-up, and compatibility for RNA containing thermolabile modifications. In this study, we present an alternative deprotection protocol employing triethylamine trihydrofluoride (TEA•3HF) under mild conditions. This method preserves RNA and 5'-terminal phosphate integrity, yielding a product profile comparable to the TBAF control. The reduced temperature and streamlined workflow minimize degradation of the RNA backbone and chemical modifications, improving suitability for in vivo applications where thermal sensitivity and innate immune activation are concerns. Furthermore, the approach avoids toxic reagents and leverages readily available materials, enhancing reproducibility and supply chain resilience. Overall, this TEA•3HF-based method offers a robust, efficient, and biologically compatible strategy for preparing diphosphorylated RNA oligonucleotides suitable for therapeutic and research applications.