5' terminal nucleotide determines the immunogenicity of IVT RNAs

Nucleic Acids Res. 2025 Jan 24;53(3):gkae1252. doi: 10.1093/nar/gkae1252.

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.

MeSH terms

  • Adenosine
  • Animals
  • COVID-19 / immunology
  • COVID-19 / prevention & control
  • COVID-19 / virology
  • DEAD Box Protein 58 / genetics
  • DEAD Box Protein 58 / metabolism
  • DNA-Directed RNA Polymerases
  • Humans
  • Interferon-beta / genetics
  • Interferon-beta / immunology
  • Mice
  • Mice, Inbred C57BL
  • RNA, Double-Stranded* / genetics
  • RNA, Double-Stranded* / immunology
  • Receptors, Immunologic
  • SARS-CoV-2 / genetics
  • SARS-CoV-2 / immunology
  • Signal Transduction
  • Transcription, Genetic*
  • Viral Proteins

Substances

  • RNA, Double-Stranded
  • Interferon-beta
  • Adenosine
  • DEAD Box Protein 58
  • RIGI protein, human
  • bacteriophage T7 RNA polymerase
  • Receptors, Immunologic
  • DNA-Directed RNA Polymerases
  • Viral Proteins