Abstract
In vitro transcription (IVT) is a technology of vital importance that facilitated the production of mRNA therapeutics and drove numerous breakthroughs in RNA biology. T7 polymerase-produced RNAs can begin with either 5'-triphosphate guanosine (5'-pppG) or 5'-triphosphate adenosine (5'-pppA), generating potential agonists for the RIG-I/type I interferon response. While it is established that IVT can yield highly immunogenic double-stranded RNA (dsRNA) via promoterless transcription, the specific contribution of initiating nucleosides to this process has not been previously reported. Our study shows that IVT-derived RNAs containing 5'-pppA are significantly more immunogenic compared with their 5'-pppG counterparts. We observed heightened levels of dsRNAs triggered by IVT with 5'-pppA RNA, activating the RIG-I signaling pathway in cultured cells, as well as in ex vivo and in vivo mouse models, where the IFN-β gene was substituted with the mKate2 fluorescent reporter. Elevated levels of dsRNA were found in both short and long 5'-pppA RNAs, including those of COVID-19 vaccines. These findings reveal the unexpected source of IVT RNA immunogenicity, offering valuable insights for both academic research and future medical applications of this technology.