L-DNA-based probes for the detection of radiation-induced DNA strand cleavage

Accurate assessment of DNA strand breaks is essential for evaluating the efficacy of radiation therapy, yet most existing methods rely on indirect detection of reactive oxygen species (ROS), which are unreliable in hypoxic tumor environments and do not consistently correlate with DNA damage. Here, we report a nuclease-resistant, L-DNA-based Förster resonance energy transfer (FRET) probe that directly detects radiation-induced DNA strand cleavage. The probes consist of short single-stranded L-DNA labeled with FAM and TAMRA fluorophores, designed to lose FRET upon strand scission. Both two- and six-thymidine variants (L-T2 and L-T6) were synthesized and shown to resist enzymatic degradation while maintaining fluorescence under irradiation up to 50 Gy. Radiation exposure induces a dose-dependent increase in donor fluorescence, with L-T2 exhibiting greater sensitivity in physiological buffer. The probes are quantitatively responsive, with detectable signal shifts from as little as 1% cleavage and a linear relationship between donor/acceptor emission ratios and DNA breakage. In cells, however, the behavior is more complex. While L-T6 exhibits dose-dependent FRET loss in certain contexts, particularly in radiation-sensitive SKBR3 cells, the extent of cleavage is significantly reduced compared to in vitro conditions. Glutathione depletion failed to enhance intracellular cleavage, suggesting that other mechanisms of protection or sequestration are at play. These findings highlight the challenges of sensing strand scission in the cytoplasm and point to unanticipated barriers to detecting radiation-induced damage in living cells.