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The change of DNA radiation damage upon hydration: In-situ observations by near-ambient-pressure XPS
(2023)
Ionizing radiation damage to DNA plays a fundamental role in cancer therapy. X-ray photoelectron-spectroscopy (XPS) allows simultaneous irradiation and damage monitoring. Although water radiolysis is essential for radiation damage, all previous XPS studies were performed in vacuum. Here we present near-ambient-pressure XPS experiments to directly measure DNA damage under water atmosphere. They permit in-situ monitoring of the effects of radicals on fully hydrated double-stranded DNA. The results allow us to distinguish direct damage, by photons and secondary low-energy electrons (LEE), from damage by hydroxyl radicals or hydration induced modifications of damage pathways. The exposure of dry DNA to x-rays leads to strand-breaks at the sugar-phosphate backbone, while deoxyribose and nucleobases are less affected. In contrast, a strong increase of DNA damage is observed in water, where OH-radicals are produced. In consequence, base damage and base release become predominant, even though the number of strand-breaks increases further.
X-ray photoelectron-spectroscopy (XPS) allows simultaneous irradiation and damage monitoring. Although water radiolysis is essential for radiation damage, all previous XPS studies were performed in vacuum. Here we present near-ambient-pressure XPS experiments to directly measure DNA damage under water atmosphere. They permit in-situ monitoring of the effects of radicals on fully hydrated double-stranded DNA. Our results allow us to distinguish direct damage, by photons and secondary low-energy electrons (LEE), from damage by hydroxyl radicals or hydration induced modifications of damage pathways. The exposure of dry DNA to x-rays leads to strand-breaks at the sugar-phosphate backbone, while deoxyribose and nucleobases are less affected. In contrast, a strong increase of DNA damage is observed in water, where OH-radicals are produced. In consequence, base damage and base release become predominant, even though the number of strand-breaks increases further. Furthermore, first data about the degradation of single-stranded DNA binding-proteins (G5P / GV5 and hmtSSB) under vacuum and NAP-XPS conditions are presented.
X-ray photoelectron-spectroscopy (XPS) allows simultaneous irradiation and damage monitoring. Although water radiolysis is essential for radiation damage, all previous XPS studies were performed in vacuum. Here we present near-ambient-pressure XPS experiments to directly measure DNA damage under water atmosphere. They permit in-situ monitoring of the effects of radicals on fully hydrated double-stranded DNA. Our results allow us to distinguish direct damage, by photons and secondary low-energy electrons (LEE), from damage by hydroxyl radicals or hydration induced modifications of damage pathways. The exposure of dry DNA to x-rays leads to strand-breaks at the sugar-phosphate backbone, while deoxyribose and nucleobases are less affected. In contrast, a strong increase of DNA damage is observed in water, where OH-radicals are produced. In consequence, base damage and base release become predominant, even though the number of strand-breaks increases further.
To enhance the biological effects of radiation damage in cancerous cells, we present an alternative approach to the use of gold nanoparticles (AuNP), focusing on the synthesis and characterization of highly monodisperse, spherical radioactive gold nanoparticles 198AuNP. The size of the AuNP size was optimized with the help of Geant4/TOPAS particle scattering simulations, and energy deposition per nm3 per decay for varying radii (2–10 nm) was evaluated. This work is the foundation for ongoing experimental work to evaluate cell death induced by 198AuNP which aims for the use of radioactive gold nanoparticles in cancer treatment.
Dose enhancement by gold nanoparticles (AuNP) was shown to increase the biological effectiveness of radiation damage in biomolecules and tissue. Most of the current studies focus on external beam therapy on combination with AuNP. Here we present a Monte-Carlo study (Geant4) to characterise radioactive AuNP. Radioactive ¹⁹⁸Au emits beta and gamma rays and is considered for applications with solid tumours. To effectively apply ¹⁹⁸AuNP their energy deposit characteristics have to be determined in terms of intrinsic and extrinsic properties e.g. AuNP diameter, AuNP density, and their clustering behaviour. After each decay process, the energy deposit, inelastic scattering events, kinetic energy spectrum of secondary particles within the AuNP themselves and in a spherical target volume of water up to 1 μm radius were determined. Simulations were performed for AuNP radii ranging from 2.5 nm to 20 nm radius, different cluster sizes and densities. The results show an increase of the energy deposit in the vicinity of the AuNP up to 150 nm. This effect nearly vanishes for distances up to one micron. For the case of AuNP clusters and the same activity, the enhancement of the energy deposit increases with the relative gold mass percentage and therefore can be adjusted by changing AuNP radius or clustering behaviour.
Dose enhancement by gold nanoparticles (AuNP) was shown to increase the biological effectiveness of radiation damage in biomolecules and tissue. Most of the current studies focus on external beam therapy on combination with AuNP. Here we present a Monte-Carlo study (Geant4) to characterise radioactive AuNP. Radioactive ¹⁹⁸Au emits beta and gamma rays and is considered for applications with solid tumours. To effectively apply ¹⁹⁸AuNP their energy deposit characteristics have to be determined in terms of intrinsic and extrinsic properties e.g. AuNP diameter, AuNP density, and their clustering behaviour. After each decay process, the energy deposit, inelastic scattering events, kinetic energy spectrum of secondary particles within the AuNP themselves and in a spherical target volume of water up to 1 μm radius were determined. Simulations were performed for AuNP radii ranging from 2.5 nm to 20 nm radius, different cluster sizes and densities. The results show an increase of the energy deposit in the vicinity of the AuNP up to 150 nm. This effect nearly vanishes for distances up to one micron. For the case of AuNP clusters and the same activity, the enhancement of the energy deposit increases with the relative gold mass percentage and therefore can be adjusted by changing AuNP radius or clustering behaviour.
We give an overview about recent work concerning ionizing radiation damage to Oligonucleotides, plasmid DNA, DNA binding proteins (G5P), and DNA-protein complexes.
We focus on combining new experimental setups with Geant4/TOPAS particle scattering simulations to understand the effets of ionizing radiation.
Radiation biophysics
(2023)
Ionizing radiation damage to DNA plays a fundamental role in cancer therapy. X-ray photoelectron-spectroscopy (XPS) allows simultaneous irradiation and damage monitoring. Although water radiolysis is essential for radiation damage, all previous XPS studies were performed in vacuum. Here we present near-ambient-pressure XPS experiments to directly measure DNA damage under water atmosphere. They permit in-situ monitoring of the effects of radicals on fully hydrated double-stranded DNA. The results allow us to distinguish direct damage, by photons and secondary low-energy electrons (LEE), from damage by hydroxyl radicals or hydration induced modifications of damage pathways. The exposure of dry DNA to x-rays leads to strand-breaks at the sugar-phosphate backbone, while deoxyribose and nucleobases are less affected. In contrast, a strong increase of DNA damage is observed in water, where OH-radicals are produced. In consequence, base damage and base release become predominant, even though the number of strand-breaks increases further.
X-ray photoelectron-spectroscopy (XPS) allows simultaneous irradiation and damage monitoring. Although water radiolysis is essential for radiation damage, all previous XPS studies were performed in vacuum. Here we present near-ambient-pressure XPS experiments to directly measure DNA damage under water atmosphere. They permit in-situ monitoring of the effects of radicals on fully hydrated double-stranded DNA. Our results allow us to distinguish direct damage, by photons and secondary low-energy electrons (LEE), from damage by hydroxyl radicals or hydration induced modifications of damage pathways. The exposure of dry DNA to x-rays leads to strand-breaks at the sugar-phosphate backbone, while deoxyribose and nucleobases are less affected. In contrast, a strong increase of DNA damage is observed in water, where OH-radicals are produced. In consequence, base damage and base release become predominant, even though the number of strand-breaks increases further.
Ionizing radiation damage to DNA plays a fundamental role in cancer therapy. X-ray photoelectron-spectroscopy (XPS) allows simultaneous irradiation and damage monitoring. Although water radiolysis is essential for radiation damage, all previous XPS studies were performed in vacuum. Here we present near-ambient-pressure XPS experiments to directly measure DNA damage under water atmosphere. They permit in-situ monitoring of the effects of radicals on fully hydrated double-stranded DNA. The results allow us to distinguish direct damage, by photons and secondary low-energy electrons (LEE), from damage by hydroxyl radicals or hydration induced modifications of damage pathways. The exposure of dry DNA to x-rays leads to strand-breaks at the sugar-phosphate backbone, while deoxyribose and nucleobases are less affected. In contrast, a strong increase of DNA damage is observed in water, where OH-radicals are produced. In consequence, base damage and base release become predominant, even though the number of strand-breaks increases further.
Showcasing research from the Federal Institute for Material Research and Testing Berlin and Fraunhofer Institute for Celltherapy and Immunology Branch Bioanalytics and Bioprocesses Potsdam.
Bio-SAXS of single-stranded DNA-binding proteins: Radiation protection by the compatible solute ectoine.
We aimed to increase the possible undisturbed exposure time during bio-SAXS measurements of single-stranded DNA-binding proteins. Therefore small angle X-ray scattering was performed on Gene-V Protein (G5P/GVP), which is involved in DNA repair processes. To achieve this, irradiations were performed in presence and absence of the hydroxyl-radical scavenger and osmolyte Ectoine, which showed efficient radiation protection and prevented protein aggregation, thus allows for a non-disturbing way to improve structure-determination of biomolecules.
In-situ monitoring of water dependent DNA and protein radiation damage by near-ambient-pressure XPS
(2023)
X-ray photoelectron-spectroscopy (XPS) allows simultaneous irradiation and damage monitoring. Although water radiolysis is essential for radiation damage, all previous XPS studies were performed in vacuum. Here we present near-ambient-pressure XPS experiments to directly measure DNA damage under water atmosphere. They permit in-situ monitoring of the effects of radicals on fully hydrated double-stranded DNA. Our results allow us to distinguish direct damage, by photons and secondary low-energy electrons (LEE), from damage by hydroxyl radicals or hydration induced modifications of damage pathways. The exposure of dry DNA to x-rays leads to strand-breaks at the sugar-phosphate backbone, while deoxyribose and nucleobases are less affected. In contrast, a strong increase of DNA damage is observed in water, where OH-radicals are produced. In consequence, base damage and base release become predominant, even though the number of strand-breaks increases further.
Ionizing radiation damage to DNA plays a fundamental role in cancer therapy. X-ray photoelectron-spectroscopy (XPS) allows simultaneous irradiation and damage monitoring. Although water radiolysis is essential for radiation damage, all previous XPS studies were performed in vacuum. Here we present near-ambient-pressure XPS xperiments to directly measure DNA damage under water atmosphere. They permit in-situ monitoring of the effects of radicals on fully hydrated double-stranded DNA. The results allow us to distinguish direct damage, by photons and secondary low-energy electrons (LEE), from damage by hydroxyl radicals or hydration induced modifications of damage pathways. The exposure of dry DNA to x-rays leads to strand-breaks at the sugar-phosphate backbone, while deoxyribose and nucleobases are less affected. In contrast, a strong increase of DNA damage is observed in water, where OH-radicals are produced. In consequence, base damage and base release become predominant, even though the number of strand-breaks increases further.
Small-angle X-ray scattering (SAXS) can be used for structural de- termination of biological macromolecules and polymers in their na- tive states. To improve the reliability of such experiments, the re- duction of radiation damage occurring from exposure to X-rays is needed.One method, is the use of scavenger molecules that protect macromolecules against radicals produced by radiation exposure.In this study we investigate the feasibility to apply the compatible solute, osmolyte and radiation protector Ectoine (THP(B)) as a scavenger throughout SAXS measurements of single-stranded DNA-binding protein Gene-V Protein (G5P/GVP). Therefore we monitor the radiation induced changes of G5P during bio-SAXS. The resulting microscopic energy-damage relation was determined by particle scattering simu- lations with TOPAS/Geant4. The results are interpreted in terms of radical scavenging as well as post-irradiation effects, related to preferential-exclusion from the protein surface. Thus, Ectoine provides an non-disturbing way to improve structure-determination of proteins via bio-SAXS in future studies.
The compatible solute and osmolyte ectoine is an effective protectant of biomolecules and whole cells against heating, freezing and high salinity. The protection of cells (human Keratinocytes) by ectoine against ultraviolet radiation was also reported by various authors, although the underlying mechanism is not yet understood. We present results on the irradiation of biomolecules (DNA) with ionizing radiation (high energy electrons) in fully aqueous environment in the presence of ectoine and high salt concentrations. The results demonstrate an effective radiation protection of DNA by ectoine against the induction of single strand breaks by ionizing radiation. The effect is explained by an increased in low-energy electron scattering at the enhanced free-vibrational density of states of water due to ectoine, as well as the action of ectoine as an OH-radical scavenger. This was demonstrated by Raman spectroscopy, electron paramagnetic resonance (EPR) and Monte-Carlo simulations (Geant4).
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.
The damage caused by ionizing radiation to DNA and proteins is the reason to treat cancer by radiation therapy. A better understanding of the molecular processes and quantification of the different damaging mechanisms is the prerequisite to develop more 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.
For radiation with low linear energy transfer (LET), most of the damage is caused by the secondary particles produced by scattering of the ionizing radiation with water. Thereby a multitude of species are produced, whereby especially kinetic low energy electrons, prehydrated electrons, OH-radicals and ions are of importance. With higher LET the relative amount of the direct damaging effects increases. This is especially important considering the increased usage of high LET nucleons in radiation therapy. Therefore, the quantification of the contribution to DNA damage of direct and indirect effects and the different secondary species is of high interest due to the increase of radio biological efficiency when applying high LET radiation.
Here we present an approach to investigate the relative contributions to DNA strand break yield for radiation of different LET within a single electron microscope in combination with electron scattering simulations.
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.[1] We juxtaposition these results to the experimentally determined damage to obtain the microscopic 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.[2] 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.[1] 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.
[1] Hahn, M. B., Meyer, S., Kunte, H.-J., Solomun, T. & Sturm, H. Measurements and simulations of microscopic damage to DNA in water by 30 keV electrons: A general approach applicable to other radiation sources and biological targets. Phys. Rev. E 95, 052419 (2017).
[2] Hahn, M. B. et al. Direct electron irradiation of DNA in a fully aqueous environment. Damage determination in combination with Monte Carlo simulations. Phys. Chem. Chem. Phys. 19, 1798–1805 (2017).
Dose enhancement by gold nanoparticles (AuNP) increases the biological effectiveness of Radiation damage in biomolecules and tissue. To apply them effectively during cancer therapy their influence on the locally delivered dose has to be determined. Hereby, the AuNP locations strongly influence the energy deposit in the nucleus, mitochondria, membrane and the cytosol of the targeted cells. To estimate these effects, particle scattering simulations are applied. In general, different approaches for modeling the AuNP and their distribution within the cell are possible. In this work, two newly developed continuous and discrete-geometric models for simulations of AuNP in cells are presented.
These models are applicable to simulations of internal emitters and external radiation sources. Most of the current studies on AuNP focus on external beam therapy. In contrast, we apply the presented models in Monte-Carlo particle scattering simulations to characterize the energy deposit in cell organelles by radioactive 198AuNP. They emit beta and gamma rays and are therefore considered for applications with solid tumors. Differences in local dose enhancement between randomly distributed and nucleus targeted nanoparticles are compared. Hereby nucleus targeted nanoparticels showed a strong local dose enhancement in the radio sensitive nucleus. These results are the foundation for future experimental work which aims to obtain a mechanistic understanding of cell death induced by radioactive 198Au.
Dose enhancement by gold nanoparticles (AuNP) increases the biological effectiveness of radiation damage in biomolecules and tissue. To apply them effectively during cancer therapy their influence on the locally delivered dose has to be determined.[1] Hereby, the AuNP locations strongly influence the energy deposit in the nucleus, mitochondria, membrane and the cytosol of the targeted cells. To estimate these effects, particle scattering simulations are applied. In general, different approaches for modeling the AuNP and their distribution within the cell are possible. In this work, two newly developed continuous and discrete-geometric models for simulations of AuNP in cells are presented. [2] These models are applicable to simulations of internal emitters and external radiation sources. Most of the current studies on AuNP focus on external beam therapy. In contrast, we apply the presented models in Monte-Carlo particle scattering simulations to characterize the energy deposit in cell organelles by radioactive 198AuNP. They emit beta and gamma rays and are therefore considered for applications with solid tumors. Differences in local dose enhancement between randomly distributed and nucleus targeted nanoparticles are compared. Hereby nucleus targeted nanoparticels showed a strong local dose enhancement in the radio sensitive nucleus. These results are the foundation for ongoing experimental work which aims to obtain a mechanistic understanding of cell death induced by radioactive 198Au.
Dose enhancement by gold nanoparticles (AuNP) increases the biological effectiveness of radiation damage in biomolecules and tissue. To apply them effectively during cancer therapy their influence on the locally delivered dose has to be determined.[1] Hereby, the AuNP locations strongly influence the energy deposit in the nucleus, mitochondria, membrane and the cytosol of the targeted cells. To estimate these effects, particle scattering simulations are applied. In general, different approaches for modeling the AuNP and their distribution within the cell are possible. In this work, two newly developed continuous and discrete-geometric models for simulations of AuNP in cells are presented. [2] These models are applicable to simulations of internal emitters and external radiation sources. Most of the current studies on AuNP focus on external beam therapy. In contrast, we apply the presented models in Monte-Carlo particle scattering simulations to characterize the energy deposit in cell organelles by radioactive 198AuNP. They emit beta and gamma rays and are therefore considered for applications with solid tumors. Differences in local dose enhancement between randomly distributed and nucleus targeted nanoparticles are compared. Hereby nucleus targeted nanoparticels showed a strong local dose enhancement in the radio sensitive nucleus. These results are the foundation for ongoing experimental work which aims to obtain a mechanistic understanding of cell death induced by radioactive 198Au.
Ionizing radiation damage to DNA plays a fundamental role in cancer therapy. X-ray photoelectron-spectroscopy (XPS) allows simultaneous irradiation and damage monitoring. Although water radiolysis is essential for radiation damage, all previous XPS studies were performed in vacuum. Here we present near-ambient-pressure XPS experiments to directly measure DNA damage under water atmosphere. They permit in-situ monitoring of the effects of radicals on fully hydrated double-stranded DNA. The results allow us to distinguish direct damage, by photons and secondary low-energy electrons (LEE), from damage by hydroxyl radicals or hydration induced modifications of damage pathways. The exposure of dry DNA to x-rays leads to strand-breaks at the sugar-phosphate backbone, while deoxyribose and nucleobases are less affected. In contrast, a strong increase of DNA damage is observed in water, where OH-radicals are produced. In consequence, base damage and base release become predominant, even though the number of strand-breaks increases further.
Dose enhancement by gold nanoparticles (AuNP) increases the biological effectiveness of radiation damage in biomolecules and tissue.
To apply them effectively during cancer therapy their influence on the locally delivered dose has to be determined. Hereby, the AuNP locations strongly influence the energy deposit in the nucleus, mitochondria, membrane and the cytosol of the targeted cells. In this work, two newly developed continuous and discrete-geometric models for simulations of AuNP in cells are presented. We apply the presented models in Monte-Carlo particle scattering simulations to characterize the energy deposit in cell organelles by radioactive 198AuNP. They emit beta and gamma rays and are therefore considered for applications
with solid tumors. Differences in local dose enhancement between randomly distributed and nucleus targeted nanoparticles are compared.
Hereby nucleus targeted nanoparticels showed a strong local dose enhancement in the radio sensitive nucleus.
Small-angle X-ray scattering (SAXS) can be used for structural determination of biological macromolecules and polymers in their native states (e.g. liquid phase). This means that the structural changes of (bio-)polymers, such as proteins and DNA, can be monitored in situ to understand their sensitivity to changes in chemical environments. In an attempt to improve the reliability of such experiments, the reduction of radiation damage occurring from exposure to X-rays is required. One such method, is to use scavenger molecules to protect macromolecules against radicals produced during radiation exposure, such as reactive oxygen species (ROS). In this study we investigate the feasibility of applying the compatible solute, osmolyte and radiation protector Ectoine (THP(B)), as a scavenger molecule during SAXS measurements of the single-stranded DNA-binding protein Gene-V Protein (G5P/GVP). In this case, we monitor the radiation induced changes of G5P during bio-SAXS measurments and the resulting microscopic energy-damage relation was determined from microdosimetric calculations by Monte-Carlo based particle scattering simulations with TOPAS/Geant4 and a custom target-model. This resulted in a median-lethal energy deposit of pure G5P at 4 mg mL−1 of E1/2 = 7 ± 5 eV, whereas a threefold increase of energy-deposit was needed under the presence of Ectoine to reach the same level of damage. This indicates that Ectoine increases the possible exposure time before radiation-damage to G5P is observed. Furthermore, the dominant type of damage shifted from aggregation in pure solutions towards a fragmentation for solutions containing Ectoine as a cosolute. These results are interpreted in terms of indirect radiation damage by reactive secondary species, as well as post-irradiation effects, related to preferential-exclusion of the cosolute from the protein surface. Hence, Ectoine is shown to provide a non-disturbing way to improve structure-determination of proteins via bio-SAXS in future studies.
Bei der Behandlung von Krebs mittels Strahlentherapie sollen Tumorzellen abgetötet werden ohne das umliegende gesunde Gewebe zu zerstören. Um Strahlentherapien für Patienten verträglicher zu machen, ist ein besseres Verständnis der zugrundeliegenden Prozesse auf der molekularen Ebene nötig. Dabei sind der Energieeintrag und die Streuprozesse der Strahlung in der Umgebung der DNA von besonderem Interesse. Durch Streuung von hochenergetischer Strahlung in Wasser werden besonders viele Sekundärelektronen mit niedriger Energie erzeugt. Zur Untersuchung der Schädigungseffizienz dieser Elektronen wurde ein Verfahren zur direkten Bestrahlung von Lösungen mittels Elektronen variabler Energien enwtickelt. Dies wurde durch einen neu entwickelten Probenhalter mit einer für Elektronen durchlässigen Nanomembran ermöglicht. Mit diesem können Bestrahlungen an DNA, Proteinen, und Zellen bei verschiedenen pH-Werten oder Salzkonzentrationen durchgeführt werden. Parallel dazu wurde der ortsabhängige Energieeintrag innerhalb des Wassers durch Elektronenstreusimulationen bestimmt. Diese neuartige Kombination von Experiment und Simulation ermöglicht die Bestimmung der Schaden-Dosis-Relation für Elektronenbestrahlung von biologischen Systemen unter realistischen physiologischen Bedingungen. So konnten für die genutzten Primärelektronen wie die mittlere letale Dosis, bei der 50 Prozent der DNA geschädigt sind, mit 1,7 Gy bestimmt. Ebenfalls wurde das für mikrodosimetrische Modellierungen und Betrachtungen der sogenannten Linear energy transfer (LET) Effekte, wichtige Verhältnis von DNA Einzelstrangbrüchen (SSB) zu Doppelstrangbrüchen (DSB) als SSB/DSB = 12/1 bestimmt. Mit Hilfe eines Modells für das Targetvolumen der DNA wurde der mittlere mikroskopische letale Energieeintrag berechnet als E1/2 = 6 ± 4 eV . Es wurde gefolgert, dass weniger als zwei Ionisationsprozesse im sensitiven Targetvolumen der DNA im Mittel zu einem Einzelstrangbruch führen. Diese Methode ist unabhängig von den Primärpartikel und geometrischen Bedingungen. Deshalb ermöglicht sie die Vergleichbarkeit experimenteller Systeme mit inhomogenen Energieverteilungen, welches sonst nicht gegeben ist. Des weiteren wurden die Strahlenschutzfunktionen des Zellschutzmoleküls Ectoines und sein Einfluss auf Wasser und Biomoleküle untersucht. Seine Schutzfunktion gegen ionisierende Strahlung wurde auf die Erhöhung des Streuquerschnitts niederenergetischer Elektronen und seine Eigenschaft als OH-Radikalfänger zurückgeführt. Aufbauend auf unseren Erkenntnissen finden in klinischen Arbeitsgruppen Untersuchungen zu Einsatzmöglichkeiten im Umfeld der Strahlentherapie statt.
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 245767821
Small-angle X-ray scattering (SAXS) can be used for structural determination of biological macromolecules and polymers in their native states (e.g. liquid phase). This means that the structural changes of (bio-)polymers, such as proteins and DNA, can be monitored in situ to understand their sensitivity to changes in chemical environments. In an attempt to improve the reliability of such experiments, the reduction of radiation damage occurring from exposure to X-rays is required. One such method, is to use scavenger molecules to protect macromolecules against radicals produced during radiation exposure, such as reactive oxygen species (ROS). In this study we investigate the feasibility of applying the compatible solute, osmolyte and radiation protector Ectoine (THP(B)), as a scavenger molecule during SAXS measurements of the single-stranded DNA-binding protein Gene-V Protein (G5P/GVP). In this case, we monitor the radiation induced changes of G5P during bio-SAXS measurments and the resulting microscopic energy-damage relation was determined from microdosimetric calculations by Monte-Carlo based particle scattering simulations with TOPAS/Geant4 and a custom target-model. This resulted in a median-lethal energy deposit of pure G5P at 4 mg mL−1 of E1/2 = 7 ± 5 eV, whereas a threefold increase of energy-deposit was needed under the presence of Ectoine to reach the same level of damage. This indicates that Ectoine increases the possible exposure time before radiation-damage to G5P is observed. Furthermore, the dominant type of damage shifted from aggregation in pure solutions towards a fragmentation for solutions containing Ectoine as a cosolute. These results are interpreted in terms of indirect radiation damage by reactive secondary species, as well as post-irradiation effects, related to preferential-exclusion of the cosolute from the protein surface. Hence, Ectoine is shown to provide a non-disturbing way to improve structure-determination of proteins via bio-SAXS in future studies.
Small-angle X-ray scattering (SAXS) can be used for structural determination of biological macromolecules and polymers in their native states (e.g. liquid phase). This means that the structural changes of (bio-)polymers, such as proteins and DNA, can be monitored in situ to understand their sensitivity to changes in chemical environments. In an attempt to improve the reliability of such experiments, the reduction of radiation damage occurring from exposure to X-rays is required. One such method, is to use scavenger molecules to protect macromolecules against radicals produced during radiation exposure, such as reactive oxygen species (ROS). In this study we investigate the feasibility of applying the compatible solute, osmolyte and radiation protector Ectoine (THP(B)), as a scavenger molecule during SAXS measurements of the single-stranded DNA-binding protein Gene-V Protein (G5P/GVP). In this case, we monitor the radiation induced changes of G5P during bio-SAXS measurments and the resulting microscopic energy-damage relation was determined from microdosimetric calculations by Monte-Carlo based particle scattering simulations with TOPAS/Geant4 and a custom target-model. This resulted in a median-lethal energy deposit of pure G5P at 4 mg mL−1 of E1/2 = 7 ± 5 eV, whereas a threefold increase of energy-deposit was needed under the presence of Ectoine to reach the same level of damage. This indicates that Ectoine increases the possible exposure time before radiation-damage to G5P is observed. Furthermore, the dominant type of damage shifted from aggregation in pure solutions towards a fragmentation for solutions containing Ectoine as a cosolute. These results are interpreted in terms of indirect radiation damage by reactive secondary species, as well as post-irradiation effects, related to preferential-exclusion of the cosolute from the protein surface. Hence, Ectoine is shown to provide a non-disturbing way to improve structure-determination of proteins via bio-SAXS in future studies.
The damage caused by ionizing radiation to DNA and proteins is the reason to treat cancer by radiation therapy. A better understanding of the molecular processes and quantification of the different damaging mechanisms is the prerequisite to develop more 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.
For radiation with low linear energy transfer (LET), most of the damage is caused by the secondary particles produced by scattering of the ionizing radiation with water. Thereby a multitude of species are produced, whereby especially kinetic low energy electrons, prehydrated electrons, OH-radicals and ions are of importance. With higher LET the relative amount of the direct damaging effects increases. This is especially important considering the increased usage of high LET nucleons in radiation therapy. Therefore, the quantification of the contribution to DNA damage of direct and indirect effects and the different secondary species is of high interest due to the increase of radio biological efficiency when applying high LET radiation.
Here we present an approach to investigate the relative contributions to DNA strand break yield for radiation of different LET within a single electron microscope in combination with electron scattering simulations.