Investigation of FLASH Proton Radiation Induced DNA Double Strand Breaks in Plasmid DNA Using Atomic Force Microscopy

This study investigates how free radical scavenger concentration, radiation dose and linear energy transfer (LET) influence FLASH proton radiation-induced DNA double-strand breaks (DSBs) in a cell-free plasmid DNA model, using atomic force microscopy (AFM).

pUC-19 plasmid DNA was prepared in HEPES buffer at concentrations of 2 and 10 mM and irradiated with FLASH or conventional dose rate (CDR) proton beams to doses of 1 or 3 kGy in both plateau and Bragg peak regions. AFM was used to image the irradiated DNA samples, enabling measurement of individual DNA fragments for quantification of fragment size distributions and the number of double strand breaks (DSB) per DNA.

At 2-mM HEPES concentration, FLASH consistently induced fewer DSBs relative to CDR proton radiation in both plateau and Bragg peak regions. This pattern persisted at 10 mM HEPES concentration at the 1 kGy dose; however, at 3 kGy dose, FLASH generated more DSBs than CDR radiation. Furthermore, the number of DSBs per plasmid was larger at the Bragg peak than at plateau for both FLASH and CDR radiation at 2 mM HEPES, whereas at 10 mM HEPES concentration, this effect is dose-rate dependent.

AFM-measured DNA fragment distributions and DSB metrics provide nanometer-scale benchmarks for comparing FLASH and CDR proton radiation under controlled chemical conditions. At low scavenger concentrations, FLASH proton radiation consistently yields fewer DSBs than CDR, regardless of dose or beam position. However, this protective effect diminishes at higher scavenger concentrations and reverses at 3 kGy in both the plateau and Bragg peak regions. These results suggest that the initial DNA damage from FLASH radiation is governed by a synergistic interplay between radical chemistry, total dose, and LET.