In vitro transcription of long RNA containing modified nucleosides

The in vitro synthesis of long RNA can be accomplished using phage RNA polymerase and template DNA. However, the in vitro synthesized RNA, unlike those transcribed in vivo in cells, lacks nucleoside modifications. Introducing modified nucleosides into in vitro transcripts is important because they reduce the potential of RNA to activate RNA sensors and translation of such nucleoside-modified RNA is increased in cell lines, primary cells, and after in vivo delivery. Here, we describe the in vitro synthesis of nucleoside-modified RNA with enhanced translational capacity and reduced ability to activate immune sensors.