Ni-NTA Bead-Based Real-Time Monitoring of PARPs Inhibitor Binding

This study establishes a real-time fluorescence microscopy platform for visualizing ligand binding dynamics to His-tagged proteins bound to Ni-NTA agarose beads. By preserving solution-phase kinetics while enabling sub-minute temporal resolution in physiological buffers, the methodology overcomes critical limitations of surface-based techniques and gel electrophoretic methods. We applied this platform to investigate inhibitor action within a nucleosomal system, a more physiologically relevant context than free DNA. Through studies of PARP2-nucleosome interactions modulated by clinical inhibitors (talazoparib, veliparib) and by reaction of poly(ADP-ribosyl)ation in the presence of NAD+, we demonstrate direct spatial and temporal resolution of chromatin-protein dynamics. The virtually unlimited compatibility of the platform with buffers, real-time monitoring capabilities, and elimination of covalent immobilization artifacts provide an advanced understanding of the mechanisms of drug-chromatin interaction.