Cytosolic RNA:DNA hybrids activate the cGAS-STING axis

Intracellular recognition of non‐self and also self‐nucleic acids can result in the initiation of potent pro‐inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2′–5′), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP‐1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS–STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA. Synopsis Next to dsDNA, cGAS can bind to cytoplasmic RNA:DNA hybrids. This results in the formation of cGAMP, which subsequently binds to and activates STING, leading to antiviral gene expression. Cytosolic delivery of RNA:DNA hybrids triggers antiviral gene expression in myeloid cells. RNA:DNA hybrid recognition depends on the cGAS–STING axis. Hybrids directly bind to cGAS, resulting in its enzymatic activity. Graphical Abstract RNA:DNA hybrids are a novel class of intracellular PAMP molecules that can directly stimulate cGAS to produce cGAMP leading to the induction of antiviral response genes.