Direct electron irradiation of DNA in a fully aqueous environment. Damage determination in combination with Monte Carlo simulations

Abstract

We report on a study in which plasmid DNA in water was irradiated with 30 keV electrons generated by a scanning electron microscope and passed through a 100 nm thick SiN membrane. The corresponding Monte Carlo simulations suggest that the kinetic energy spectrum of the electrons throughout the water is dominated by low energy electrons (<100 eV). The DNA radiation damage, single-strand breaks (SSBs) and double-strand breaks (DSBs), was determined by gel electrophoresis. The median lethal dose of D = 1.7 ± 0.3 Gy was found to be much smaller as compared to partially or fully hydrated DNA irradiated under vacuum conditions. The ratio of the DSBs to SSBs was found to be 1 : 12 as compared to 1 : 88 found for hydrated DNA. Our method enables quantitative measurements of radiation damage to biomolecules (DNA, proteins) in solutions under varying conditions (pH, salinity, co-solutes) for an electron energy range which is difficult to probe by standard methods.

DNA protection by ectoine from ionizing radiation: molecular mechanisms

“…Compared to our previous studies, 19,20 where DNA was irradiated in pure water, we find a decrease of the DNA damage in PBS. Such behavior is expected, as the positive ions of the buffer are well known to stabilize the DNA backbone.…”

“…4). 19,20 They are deexcited by multiple inelastic scattering processes before they get fully hydrated. 33 Here, the most common process is the ionization of water, whereby further damaging species such as OH-radicals and secondary electrons are produced.…”

“…19,20 Briefly, DNA damage is predominantly by the actions of the secondary particles produced by the interaction of ionizing radiation with water. 33 Thereby, OH-radicals, secondary (kinetic) low energy electrons (LEE) and prehydrated electrons can be assumed to be the most lethal agents.…”

“…The relative distribution of the kinetic energies doesn't change significantly between 20 and 1000 mm depth. 19 Details on the simulation can be found in our previous studies.…”

“…The solutions were irradiated under a scanning electron microscope (FEI XL30) by (4.7 AE 0.2) Â 10 13 primary electrons (30 keV) within (100 AE 4) s. A custom setup and a sample holder have been extensively described in our previous work. 19,20 Before and after irradiation, the electron current was measured using a Faraday cup and a picoammeter (Keithley 6485). After irradiation the samples were analyzed by gel electrophoresis with a 0.8% agarose gel and ethidium bromide (0.5 mg L…”