Quantification of Multiple DNA Damage Types in Additive-Free Aqueous Plasmid DNA under Helium Atmospheric-Pressure Plasma Jet Irradiation

Abstract Low-temperature plasma (LTP) has attracted increasing attention due to its potential applications in plasma medicine. LTP can modulate the generation of reactive species through plasma–liquid interaction, resulting in modifications of the biomolecular properties of irradiated systems such as DNA. Nevertheless, the quantitative characterization of multiple types of DNA damage and their kinetic responses in additive-free aqueous systems under LTP irradiation remains insufficiently understood. This study systematically examined the effects of a helium atmospheric-pressure plasma jet (APPJ) on multiple types of damage in plasmid DNA. Single-strand breaks (SSBs), double-strand breaks (DSBs), and oxidative base lesions assessed by enzymatic treatment were quantified using gel electrophoresis. With increasing irradiation time, the fraction of undamaged DNA decreased approximately exponentially, suggesting that DNA damage followed a pattern consistent with random radical attacks by plasma-generated species such as hydroxyl radicals ( • OH). DNA damage was dominated by strand break formation, while oxidative base lesions were present at relatively lower levels. DSB signals were not detected on the gel, which may be caused by DNA fragmentation following strand break formation, resulting in the loss of detectable bands. Increasing the applied frequency from 1 to 4 kHz increased the SSB yield approximately 2-fold, while increasing the applied voltage from 6 to 12 kV increased the SSB yield approximately 4-fold. Changes in these parameters did not alter the ratio of enzyme-sensitive sites (ESSs) to SSBs, which remained greater than 0 but below unity across all tested conditions, suggesting that the relative distribution of plasma-induced oxidative base damage is independent of the discharge parameters tested.