Poster: An HTS Assay for Detection of XRN1 Exoribonuclease Activity with Unmodified RNA

XRN1 is a 5’ to 3’ exoribonuclease that degrades both single- and double-stranded RNA to nucleotide 5’ monophosphate products. It plays an important role in mRNA turnover and in innate immunity, by damping activation of cytosolic RNA sensors, e.g., PKR and MDA5.  Loss of XRN1 has been linked to high interferon signaling in cancer cell culture models, making it an attractive target for cancer immunotherapy. Here we describe development of HTS-compatible assays for screening and profiling inhibitors using the Transcreener AMP²/GMP² Assay.  Existing assays for RNAses rely on fluorescently labeled RNAs, which impose limitations on the substrates that can be used and may affect enzyme binding or catalysis.   WIth the Transcreener AMP²/GMP² Assay, purine monophosphates produced by XRN1 are directly detected by a competitive immunoassay with a far-red, fluorescence polarization (FP) or time-resolved Förster-resonance-energy-transfer (TR-FRET) readout.