HPLC purification of in vitro transcribed long RNA

In vitro transcription of DNA with phage RNA polymerases is currently the most efficient method to produce long sequence-specific RNA. While the reaction can yield large quantities of RNA, it contains impurities due to various unwanted activities of the polymerases. Here, we described an easily performed HPLC purification that removes multiple contaminants from in vitro transcribed RNA and is scalable. The purified RNA is translated at much greater levels, especially in primary cells and in vivo. HPLC purification of RNA containing modified nucleosides that suppress RNA-mediated activation of innate immune sensors leads to a non-immunogenic RNA with superior translational capacity.