Filtern
Erscheinungsjahr
Dokumenttyp
Schlagworte
- Low energy electrons (7)
- DNA (5)
- Dosimetry (4)
- Ectoine (4)
- Raman spectroscopy (4)
- Aqueous solution (3)
- Compatible solute (3)
- DNA damage (3)
- DNA radiation damage (3)
- Electron irradiation (3)
- Geant4 (3)
- Hydroxyl radicals (3)
- Microdosimetry (3)
- Radiation damage (3)
- XPS (3)
- Biological structure (2)
- Cancer (2)
- DNA hybridization (2)
- Double-strand break (DSB) (2)
- Ectoin (2)
- Gasfluorierung (2)
- Hydroxyectoine (2)
- IR (2)
- LET (2)
- Lethal dose (2)
- Monte Carlo simulation (2)
- Organic osmolytes (2)
- Plasmid DNA in water (2)
- Radiation damage to biomolecules (2)
- Radiation protection (2)
- Radiation therapy (2)
- Radical scavenger (2)
- Salt (2)
- Single-strand break (SSB) (2)
- Solutions (pH, salinity, cosolutes) (2)
- Water structure (2)
- -COOH und NF2-Gruppen (1)
- 266nm (1)
- AFM (1)
- AFM intermittent contact (1)
- Abasic site (1)
- Agarose gel electrophorese (1)
- Amino acid (1)
- Biomolecules (1)
- CPD (1)
- Cancer therapy (1)
- Counterions (1)
- DEA (1)
- DET (1)
- DFG (1)
- DNA base damage (1)
- DNA immobilization (1)
- DNA melting temperature (1)
- DNA oligonucleotide (1)
- DNA protection (1)
- DNA strand-break (1)
- DNS (1)
- Damage (1)
- Ectoine DNA interaction (1)
- Ectoine DNA protection (1)
- Ectoine UV absorption (1)
- Ectoine radiation protection (1)
- Ectoine-DNA binding (1)
- Effective irradiation dose 0.2-16 [Gy] (1)
- Electron irradiation 30 [kV] (1)
- Electron irradiation of DNA (1)
- Excited states (1)
- FTIRS (1)
- Fluorescence (1)
- Fluorescence imaging (1)
- Geant4-DNA (1)
- Gel electrophoresis (1)
- Gene-5 protein (1)
- Guanine content (1)
- High throughput (1)
- Hydroxyl radical (1)
- Ionizing radiation (1)
- LEE (1)
- Linear energy transfer (1)
- Low voltage SEM (1)
- Low-energy electrons (1)
- Monte Carlo (1)
- NF2-Gruppe (1)
- OH radicals (1)
- OH scavenger (1)
- OH-radical (1)
- PBS (1)
- Plasmid DNA pUC19 (1)
- Polyamid-6 (1)
- Preferential exclusion (1)
- Radiation (1)
- Radiation chemistry (1)
- Radioprotector ectoine (1)
- SSB (1)
- SYBR gold (1)
- Secondary electrons (1)
- Silanized DNA (1)
- Single stranded DNA (1)
- Sodium chloride (1)
- Sun (1)
- Sunscreen (1)
- Therapy (1)
- UV absorption (1)
- UV irradiation (1)
- UV photons (1)
- UV protection (1)
- UV radiation (1)
- UV-A (1)
- UV-B (1)
- UV-C (1)
- UV-Vis (1)
- Zwitterion (1)
- g5p protein (1)
Organisationseinheit der BAM
Bulk samples and thin films of polyamides (PA6 and PA12) were exposed to fluorine (1 - 10 vol.-% F2 in N2) and analysed with photoelectron (XPS) and infrared spectroscopy. Fluorination affects both, the amide and the hydrocarbon parts of the polymers. However, only the carbon atom next to the carbonyl is readily fluorinated. Chemical modification of the amide group is apparent in a large binding energy shift (+5 eV) of the N1s level and the appearance of a CO band at 1734 cm-1. It is concluded that the amide C-N bond is cleaved in the fluorination process and that COOH and NF2 end groups are formed. This conclusion is corroborated by the appearance of ester oxygen in the XPS and by the 19F NMR spectra of the volatile products that show fluorine signals chemically shifted about 200 ppm towards lower field as compared with the CHF environment.
Thin polyamide-6 films were exposed to fluorine gas and analysed with photoelectron and infrared spectroscopies. Fluorine cleaves the amide CN bond resulting in the formation of the COOH and NF2 terminal groups. This is evident from large shifts in the N1s binding energy (+5 eV) and C=O stretching frequency (~80 cm-1), appearance of ester oxygen in the XPS spectra, as well as by the 19F nmr spectra of volatile products consistent with a terminal NF2 group.
Efficient formation of difluoramino (NF2) groups within the polymer matrix upon exposure of polyamides (PA6 and PA12) to elemental fluorine is reported. The reaction was assessed on bulk and thin-film samples by means of RA-FTIR (reflection–absorption FTIR), XPS (X-ray photoelectron spectroscopy), and NMR (nuclear magnetic resonance) techniques. Direct fluorination causes cleavage of the amide C–N bond and concomitant formation of the NF2 chain-end functionalities as evident from an exceptionally large shift (+5 eV) of the N 1s binding energy and an increase of the v(CO) frequency by about 80 cm-1. The structural model is supported by the 19F NMR spectra of volatile reaction products that clearly reveal the presence of the NF2 group.
Low-energy secondary electrons are the most abundant radiolysis species which are thought to be able to attach to and damage DNA via formation and decay of localized molecular resonances involving DNA components. In this study, we analyze the consequences of low-energy electron impact on the ability of DNA to hybridize (i.e., to form the duplex). Specifically, single-stranded thymine DNA oligomers tethered to a gold surface are irradiated with very low-energy electrons (E = 3 eV, which is below the 7.5 eV ionization threshold of DNA) and subsequently exposed to a dye-marked complementary strand to quantify by a fluorescence method the electron induced damage. The damage to (dT)25 oligomers is detected at quite low electron doses with only about 300 electrons per oligomer being sufficient to completely preclude its hybridization. In the microarray format, the method can be used for a rapid screening of the sequence dependence of the DNA-electron interaction. We also show for the first time that the DNA reactions at surfaces can be imaged by secondary electron (SE) emission with both high analytical and spatial sensitivity. The SE micrographs indicate that strand breaks induced by the electrons play a significant role in the reaction mechanism.
Immobilization of silanized DNA on glass: influence of the silane tether on the DNA hybridization
(2010)
Two trifunctional (trimethoxy and triethoxy) and one difunctional (methyldimethoxy) 3-mercaptopropyl-alkoxysilanes were covalently tethered to thiolated DNA oligonucleotides in solution. After deposition as microarrays onto glass, the immobilized DNA probes were tested for hybridization ability by a florescence-based method. The results demonstrate a large enhancement in the fluorescence signal when the functionality of the silane tether is reduced from three to two. An XPS analyses revealed that this is not due to a higher DNA surface density. FTIR spectra of the spin-coated silanes showed that the trifunctional silanes form branched and cyclic siloxane moieties, whereas the difunctional silane generates predominantly short straight siloxane chains. Therefore, the propensity of trifunctional silanes to form more complex networks leads to conformations of the bound DNA which are less favorable for the specific interaction with the complementary strand. The data implicate that further significant improvements in the DNA hybridization ability are possible by adroit choice of the silane system.
We report surface plasmon resonance (spr) and confocal fluorescence results concerning the interaction of a gene-5-protein (g5p) with single-stranded DNA oligonucleotides (dT25) tethered to a gold surface. The spr data show that a highly stable g5pssDNA complex is readily formed on a gold surface with koff = 1.7 × 10-3 s-1. The extent of the complexion indicated involvement of the cooperative proteinprotein interactions within the binding to DNA. In the experiments where dT25 coexist with g5p in the solution, the fluorescence data show that g5p also mediates the binding between the non-complementary oligonucleotides in the solution and those immobilised on the surface.
In fields involving irradiated aqueous solutions, such as radiotherapy and nuclear waste remediation, it is often unclear whether the principal reactive species are OH° radicals or secondary (low-energy) electrons. This is mostly because both are rapidly attenuated in water. Presently a large part of the evidence for the involvement of low-energy electrons in biological radiation damage is based on 'dry' DNA samples. We demonstrate irradiation of DNA in solution by direct injection of electrons through a 40-nm thin SiO2 membrane, followed by in-situ detection of the DNA damage by a fluorescence-based method. Corresponding Monte Carlo simulations show that the spatial distribution of ionizing events in water with respect to the membrane is controlled by the electron impact energy. By immobilizing DNA to the solution side of the membrane, and because dynamics and reaction ranges of OH° radicals and low-energy electrons are dramatically different, it is possible to tune into the OH° radical or into the electron 'reaction modes' by simply changing the electron impact energy. Such experiments have the potential to provide important information on the radio-sensitivity at a level of a single biomolecule and to contribute to the development of new dosage concepts.
Microorganisms accumulate molar concentrations of compatible solutes like ectoine to prevent proteins from denaturation. Direct structural or spectroscopic information on the mechanism and about the hydration shell around ectoine are scarce. We combined surface plasmon resonance (SPR), confocal Raman spectroscopy, molecular dynamics simulations, and density functional theory (DFT) calculations to study the local hydration shell around ectoine and its influence on the binding of a gene-S-protein (G5P) to a single-stranded DNA (dT(25)). Due to the very high hygroscopicity of ectoine, it was possible to analyze the highly stable hydration shell by confocal Raman spectroscopy. Corresponding molecular dynamics simulation results revealed a significant change of the water dielectric constant in the presence of a high molar ectoine concentration as compared to pure water. The SPR data showed that the amount of protein bound to DNA decreases in the presence of ectoine, and hence, the protein-DNA dissociation constant increases in a concentration-dependent manner. Concomitantly, the Raman spectra in terms of the amide I region revealed large changes in the protein secondary structure. Our results indicate that ectoine strongly affects the molecular recognition between the protein and the oligonudeotide, which has important consequences for osmotic regulation mechanisms.
Ectoine is an important osmolyte, which allows microorganisms to survive in extreme environmental salinity. The hygroscopic effects of ectoine in pure water can be explained by a strong water binding behavior whereas a study on the effects of ectoine in salty solution is yet missing. We provide Raman spectroscopic evidence that the influence of ectoine and NaCl are opposing and completely independent of each other. The effect can be explained by the formation of strongly hydrogen-bonded water molecules around ectoine which compensate the influence of the salt on the water dynamics. The mechanism is corroborated by first principles calculations and broadens our understanding of zwitterionic osmolytes in aqueous solution. Our findings allow us to provide a possible explanation for the relatively high osmolyte concentrations in halotolerant bacteria.
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.
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.
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.
Ectoine is a small zwitterionic osmolyte and compatible solute, which does not interfere with cell metabolism even at molar concentrations. Plasmid DNA (pUC19) was irradiated with ultraviolet radiation (UV-C at 266 nm) under quasi physiological conditions (PBS) and in pure water in the presence and absence of ectoine (THP(B)) and hydroxyectoine (THP(A)). Different types of UV induced DNA damage were analysed: DNA single-strand breaks (SSBs), abasic sites and cyclobutane pyrimidine dimers (CPDs). A complex interplay between these factors was observed with respect to the nature and occurrence of DNA damage with 266 nm photons. In PBS, the cosolutes showed efficient protection against base damage, whilst in pure water, a dramatic shift from SSB damage to base damage was observed when cosolutes were added. To test whether these effects are caused by ectoine binding to DNA, further experiments were conducted: small-angle X-ray scattering (SAXS), surface-plasmon resonance (SPR) measurements and Raman spectroscopy. The results show, for the first time, a close interaction between ectoine and DNA. This is in stark contrast to the assumption made by preferential exclusion models, which are often used to interpret the behaviour of compatible solutes within cells and with biomolecules. It is tentatively proposed that the alterations of UV damage to DNA are attributed to ectoine influence on nucleobases through the direct interaction between ectoine and DNA.
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