European Field Application Scientist Molecular Devices Wokingham, England, United Kingdom
Abstract: POLQ is a multifunctional enzyme with template-dependent DNA polymerase, ATP-dependent helicase, and dNTP-dependent endonuclease activities that plays important roles in DSB repair. POLQ is synthetic lethal with BRCA-1 and ATM mutations, and both the polymerase and ATPase activities are being targeted with small molecules for anti-cancer drug discovery. Here, we used the Transcreener ADP2 Assay combined with Molecular Devices SpectraMax® i3x Multi-Mode Microplate reader to detect POLQ ssDNA-dependent ATPase activity, a component of its helicase function. Transcreener assays use highly selective antibodies to detect nucleotides in a homogenous format with far-red fluorescent readouts, including fluorescence polarization (FP), fluorescence intensity (FI), and time-resolved Förster energy transfer (TR-FRET). Their single addition mix-&-read format and outstanding reagent stability makes them well-suited for automated workflows. The SpectraMax i3x reader measures absorbance, fluorescence, and luminescence with user-installable detection modules that expand its capabilities to include FP, TR-FRET, dual injection, western blot detection, and more.
We demonstrated robust detection of ssDNA-dependent PolQ ATPase initial velocity activity with FP, TR-FRET, or FI readouts, each yielding a Z’ > 0.7. The FP assay was used for a pilot screen of 1280 bioactives, resulting in a hit rate of 1.8% and an interference rate of 0.1%. Dose-response assays in FP, TR-FRET, and FI detection modes yielded similar IC50 values for one of the hits and the probe inhibitor, suramin. This study clearly demonstrates the utility of the Transcreener ADP2 Assay combined with the Molecular Devices SpectraMax i3x reader for the discovery of POLQ helicase inhibitors.
