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 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.
Single-stranded DNA oligonucleotides (33-mers) containing different numbers of guanines (n = 1−4) were tethered to a gold surface and exposed to 1 eV electrons. The electrons induced DNA damage, ...which was analyzed with fluorescence and infrared spectroscopy methods. The damage was identified as strand breaks and found to correlate linearly with the number of guanines in the sequence. This sequence dependence indicates that the electron capture by the DNA bases plays an important role in the damage reaction mechanism.
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.
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.
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.
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► Irradiation of immobilized DNA in solution by direct injection of electrons through a membrane. ► DNA damage detected in in-situ by means of fluorescence. ► Tuning into the low-energy electron DNA damage mode by changing the electron impact energy. ► Potential for development of new dosage concepts and detectors.
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 ν(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.
Nanometer-sized zero-dimensional (0D) cavities, 5–10 nm in diameter and 3–4 atomic layers deep, were produced locally on a Au(111) surface in a nitrate electrolyte by applying short voltage pulses ...(80 ns, −2.8 V) to a scanning tunnelling microscope (STM) tip. In the system Au(111)/BiO
+,NO
3
−, localised electrochemical underpotential deposition (upd) of 0D bismuth dots was observed in these nano-cavities by in-situ STM. This contrasted to the behaviour of the Au(111)/Ag
+,NO
3
− system in which the 0D cavities remained unfilled in the entire upd range, and only were filled during layer-by-layer growth of Ag on the (111) terraces at overpotentials. The difference between the two systems are discussed in respect to the different and nearly equal atomic radii of the Bi/Au(111) and Ag/Au(111) adsorbate–substrate systems, respectively, and the influence of the anion adsorption on the stability of close-packed 2D ad-layers relative to the formation of 3D phases. 0D deposits can be generated when a sufficiently wide stability region for a dense monolayer existed so that concurrent growth of 2D (terraces) and 3D (bulk) phases were avoided.
We have examined the adsorption of thymine on (111), (100), and (210) gold single-crystal surfaces. The adsorption behavior on these three surfaces has been investigated by classical electrochemical ...methods like cyclic voltammetry and capacitance−potential measurements. Additionally in situ scanning tunneling microscopy (STM) and ex situ photoelectron spectroscopy (XPS) measurements have been performed for the adsorption of thymine on the (111) surface. The capacitance measurements as well as cyclic voltammetry investigations show the three adsorption states of thymine on all Au electrodes. The first adsorption state refers to a random adsorption of thymine molecules at negative surface charges. The second state can be characterized as a condensed but weakly adsorbed adlayer on the (100) and (111) crystals, whereas a noncondensed state has been found on the (210) surface. The condensed thymine film is stabilized mainly by hydrogen bonding. High-resolution STM images for this film on the (111) electrode point to an ordered adlayer with a unit cell which is incommensurate with the underlying Au surface. The images indicate flat adsorbing thymine molecules in this state. The third adsorption state is characterized by charge transfer from deprotonated thymine molecules to the gold surface. XPS data show one chemically modifed nitrogen atom for the chemisorbed thymine film. This adsorption state shows a commensurate 2√3 × 2√3 overstructure in the STM image. The STM images are interpreted by stacks of adsorbed thymine molecules with the molecular plane perpendicular to the surface. The stacks are connected by coadsorbed water molecules. The molecules are bound by a deprotonated nitrogen to the surface.
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