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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 Si3N4 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 (SSB) and double-strand breaks (DSB), was determined by electrophoresis. The median lethal dose of D1/2 = 1.7 ± 0.3 Gy was found to be much smaller compared to partially or fully hydrated DNA irradiated under vacuum conditions. The ratio of DSB to SSB 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, cosolutes) for an electron energy range which is difficult to probe by standard methods.
The determination of the microscopic dose-damage relationship for DNA in an aqueous environment is of a fundamental interest for dosimetry and applications in radiation therapy and protection. We combine geant4 particle-scattering simulations in water with calculations concerning the movement of biomolecules to obtain the energy deposit in the biologically relevant nanoscopic volume. We juxtaposition these results to the experimentally determined damage to obtain the dose-damage relationship at a molecular level. This approach is tested for an experimentally challenging system concerning the direct irradiation of plasmid DNA (pUC19) in water with electrons as primary particles. Here a microscopic target model for the plasmid DNA based on the relation of lineal energy and radiation quality is used to calculate the effective target volume. It was found that on average fewer than two ionizations within a 7.5-nm radius around the sugar-phosphate backbone are sufficient to cause a single strand break, with a corresponding median lethal energy deposit being E1/2=6±4 eV. The presented method is applicable for ionizing radiation (e.g., γ rays, x rays, and electrons) and a variety of targets, such as DNA, proteins, or cells.
Electron beam therpy is used to treat tumor cells by irradiation with high energy electrons (HEE). The irradiation is performed by medical linear accelerators.
By interaction of the HEE with the irradiated tissue a broad spectra of secondary electrons and water dissoziation products is generated.
These products interact with biomolecules, especially DNA, by various processes. The disentanglement of the resulting direct- and indirect damage to DNA and other cellular components is still under debate.
To increase the efficiency of future therapies a better understanding of the microscopic damaging processes is highly important.
Especially the various contributions of the secondary species produced, such as low energy electrons (LEE) and radicals is far from understood and quantified.
To increase the understanding we present a combination of a microdosimetric simulations and experiments to quantify the damage by means of electron scattering and diffusion simulations within the Geant4-DNA framework.
In combination with in-liquid irradiation of plasmid DNA it possible to determine microdosimetric quantities for biomolecules in liquid environment.
The presented method was applied to the irradiation of plasmid DNA (pUC19) in water.
This opens up new possibilities in radiation research to quantify the dosage-damage relationship for microscopic plasmid volumes under well defined physiological condition.
The damage caused by ionizing radiation to biomolecules, especially DNA, is the reason to treat cancer via radiation therapy. A better understanding of the molecular processes and the quantification of the various damaging mechanisms is the prerequisit to develope efficient therapies. Hereby the understanding of the processes involved in the damage to DNA are of key interest due to its central role in reproduction and mutation. Due to the high amount of water in biological tissue, most of the damage is caused by the secondary particles produced by the interaction of the IR with water. Thereby a multitude of species are produced, e.g. kinetic low energy electrons, prehydrated electrons, OH-radicals and ions. The quantification of the contribution to DNA damage of the various species is of interest. Here we present an experimental approach to disentangle their relative DNA strand break yield. Plasmid DNA is irradiated in water with electrons under the presence of different scavengers. With the presented method it will be possible to reveal the relative contributions of OH-radicals, low energy electrons and prehydrated electrons to the DNA single and double strand break yield.
We report on the design, preparation and application of immobilized Oligonucleotides as biological sensors for dosimetry and protein activity. Oligonucleotides can be custom tailored in terms of sequence and length to the needs of the specific application. The preparation can be scaled from small lab-scales to higher output via spotting technology on various surfaces such as glass, silicon-dioxide or gold. They can be applied for the sensing of DNA-protein interaction and surface-plasmon resonance to detect the influence of cosolutes on the protein activity. This can be applied in the testing of pharmaceutical or cosmsetic applications and incorporated in microfluidic devices. Other important use cases are the usage in dosimetric application where the oligonucleotides are subjected to ionizing radiation. They can directly act as a sensor for biological damage which is detected and quantified by the subsequent hybridization of complementary strands who carry a fluorescence dye. The quantification can be performed by an array scanner or fluorescence microscope. Investigation on a molecular level can be performed with the AFM-IR technique. Applications are calibration of radiation sources and the investigation of interaction of ionizing radiation with biomolecules.
Strand breaks and conformational changes of DNA have consequences for the physiological role of DNA. The natural protecting molecule ectoine is beneficial to entire bacterial cells and biomolecules such as proteins by mitigating detrimental effects of environmental stresses. It was postulated that ectoine-like molecules bind to negatively charged spheres that mimic DNA surfaces. We investigated the effect of ectoine on DNA and whether ectoine is able to protect DNA from damages caused by ultraviolet radiation (UV-A). In order to determine different isoforms of DNA, agarose gel electrophoresis and atomic force microscopy experiments were carried out with plasmid pUC19 DNA. Our quantitative results revealed that a prolonged incubation of DNA with ectoine leads to an increase in transitions from supercoiled (undamaged) to open circular (single-strand break) conformation at pH 6.6. The effect is pH dependent and no significant changes were observed at physiological pH of 7.5. After UV-A irradiation in ectoine solution, changes in DNA conformation were even more pronounced and this effect was pH dependent. We hypothesize that ectoine is attracted to the negatively charge surface of DNA at lower pH and therefore fails to act as a stabilizing agent for DNA in our in vitro experiments.
Ectoine, a compatible solute and osmolyte, is known to be an effective protectant of biomolecules and whole cells against heating, freezing and extreme salinity. Protection of cells (human keratinocytes) by ectoine against ultraviolet radiation has also been reported by various authors, although the underlying mechanism is not yet understood. We present the first electron irradiation of DNA in a fully aqueous environment in the presence of ectoine and at high salt concentrations. The results demonstrate effective protection of DNA by ectoine against the induction of single-strand breaks by ionizing radiation. The effect is explained by an increase in low-energy electron scattering at the enhanced free-vibrational density of states of water due to ectoine, as well as the use of ectoine as an ˙OH-radical scavenger. This was demonstrated by Raman spectroscopy and electron paramagnetic resonance (EPR).
Ectoine plays an important role in protecting biomolecules and entire cells against environmental stressors such as salinity, freezing, drying and high temperatures. Recent studies revealed that ectoine also provides effective protection for human skin cells from damage caused by UV-A radiation. These protective properties make ectoine a valuable compound and it is applied as an active ingredient in numerous pharmaceutical devices and cosmetics. Interestingly, the underlying mechanism resulting in protecting cells from radiation is not yet fully understood. Here we present a study on ectoine and its protective influence on DNA during electron irradiation. Applying gel electrophoresis and atomic force microscopy, we demonstrate for the first time that ectoine prevents DNA strand breaks caused by ionizing electron radiation. The results presented here point to future applications of ectoine for instance in cancer radiation therapy.
In dosimetry the determination of the effectiveness of the damaging processes is standardized and accounted for by the radiation and tissue weighting factor. For the underlying constituents of the tissue, that is the various biomolecules, such a systematic approach doesn't exist. This makes it difficult to compare results obtained under different experimental conditions. In the following work, we will describe a method to obtain comparable values for the radiation-biomolecule interaction, measured under different conditions. This approach can lead to standardization of dosedamage relationship at the molecular level. Such approach is necessary for a better understanding of the relations between the damage of the single constituents of biological tissue and the whole – finally gaining a more complete picture of irradiation damage.