DNA intercalation assay

To test whether the Polθ inhibitors were causing signal interference by intercalating DNA, ART558 was dispensed into 384-well black low volume non-binding plates (Greiner). For each compound, 100 nL of a 1.5-fold dilution series was dispensed starting from a 12 mM top concentration of compound in DMSO using the D300e digital dispenser (Tecan). A DMSO control was also included. Wells were normalised and backfilled with DMSO such that all wells contained 1% (v/v) after the addition of 10 μL of Polθ DNA product solution. 10 μL of a solution containing 200 nM Polθ product DNA in assay buffer (25 mM Tris-HCl pH 7.5, 12.5 mM NaCl, 0.5 mM MgCl₂, 5% (v/v) glycerol, 0.01% (v/v) Triton x-100, 0.01% (w/v) Bovine γ-Globulin, 1 mM dithiothreitol) and 5 μL of detection reagent (25 mM Tris-HCl pH 7.5, 10 mM EDTA, 2.5% (v/v) PicoGreen) were dispensed separately using a Tempest liquid handler (Formulatrix). Plates were then read on the CLARIOstar Plus (BMG Labtech) using the settings described above. Two known DNA intercalators: mitoxantrone and doxorubicin were used as positive controls. IC₅₀ data were analysed using GraphPad Prism V.8.4.2 (GraphPad Software Inc, San Diego, CA).