Design, synthesis, and evaluation of fluorescent probes targeting DNA polymerase theta (Polθ) for inhibitor screening and cellular imaging.

DNA polymerase theta (Polθ) is an emerging therapeutic target in homologous recombination-deficient cancer, yet fluorescent probes for Polθ inhibitor screening and cellular imaging remain largely unexplored. To address this gap, we report the rational design, synthesis, and biological evaluation of small-molecule fluorescent probes targeting the polymerase domain of Polθ (Polθ-pol). These probes were synthesized by conjugating a Polθ-pol recognition scaffold to fluorescein- and 4-chloro-7-nitrobenzofurazan-derived fluorophores through flexible linkers. Spectroscopic characterization revealed solvent-dependent absorption and emission profiles. Fluorescence polarization assay demonstrated concentration-dependent and saturable binding of all probes to Polθ-pol, with Kd values in nanomolar range. Among the series, Fluorescein-derived Probe-2 exhibited the strongest binding affinity and enabled a robust FP competition assay for successful evaluation of Polθ inhibitors, establishing it as a promising fluorescent tracer for inhibitor screening. Furthermore, live-cell fluorescence imaging showed that NBD-derived probes displayed comparatively stronger cellular fluorescence. Probe-5 showed co-localization with Polθ and increased γ-H2AX foci formation in HCT-116 cells. Collectively, this study provides the first set of small-molecule fluorescent probes for both Polθ-pol inhibitor screening and investigation of Polθ-associated processes in living cells.