@phdthesis{Ebel2021, author = {Ebel, Kenny}, title = {Quantification of low-energy electron induced single and double strand breaks in well-defined DNA sequences using DNA origami nanostructures}, doi = {10.25932/publishup-50449}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-504499}, school = {Universit{\"a}t Potsdam}, pages = {111}, year = {2021}, abstract = {Ionizing radiation is used in cancer radiation therapy to effectively damage the DNA of tumors leading to cell death and reduction of the tumor tissue. The main damage is due to generation of highly reactive secondary species such as low-energy electrons (LEE) with the most probable energy around 10 eV through ionization of water molecules in the cells. A simulation of the dose distribution in the patient is required to optimize the irradiation modality in cancer radiation therapy, which must be based on the fundamental physical processes of high-energy radiation with the tissue. In the present work the accurate quantification of DNA radiation damage in the form of absolute cross sections for LEE-induced DNA strand breaks (SBs) between 5 and 20 eV is done by using the DNA origami technique. This method is based on the analysis of well-defined DNA target sequences attached to DNA origami triangles with atomic force microscopy (AFM) on the single molecule level. The present work focuses on poly-adenine sequences (5'-d(A4), 5'-d(A8), 5'-d(A12), 5'-d(A16), and 5'- d(A20)) irradiated with 5.0, 7.0, 8.4, and 10 eV electrons. Independent of the DNA length, the strand break cross section shows a maximum around 7.0 eV electron energy for all investigated oligonucleotides confirming that strand breakage occurs through the initial formation of negative ion resonances. Additionally, DNA double strand breaks from a DNA hairpin 5'-d(CAC)4T(Bt-dT)T2(GTG)4 are examined for the first time and are compared with those of DNA single strands 5'-d(CAC)4 and 5'- d(GTG)4. The irradiation is made in the most likely energy range of 5 to 20 eV with an anionic resonance maximum around 10 eV independently of the DNA sequence. There is a clear difference between σSSB and σDSB of DNA single and double strands, where the strand break for ssDNA are always higher in all electron energies compared to dsDNA by the factor 3. A further part of this work deals with the characterization and analysis of new types of radiosensitizers used in chemoradiotherapy, which selectively increases the DNA damage upon radiation. Fluorinated DNA sequences with 2'-fluoro-2'-deoxycytidine (dFC) show an increased sensitivity at 7 and 10 eV compared to the unmodified DNA sequences by an enhancement factor between 2.1 and 2.5. In addition, light-induced oxidative damage of 5'-d(GTG)4 and 5'-d((CAC)4T(Bt-dT)T2(GTG)4) modified DNA origami triangles by singlet oxygen 1O2 generated from three photoexcited DNA groove binders [ANT994], [ANT1083] and [Cr(ddpd)2][BF4]3 illuminated in different experiments with UV-Vis light at 430, 435 and 530 nm wavelength is demonstrated. The singlet oxygen induced generation of DNA damage could be detected in both aqueous and dry environments for [ANT1083] and [Cr(ddpd)2][BF4]3.}, language = {en} } @phdthesis{Schuermann2017, author = {Sch{\"u}rmann, Robin Mathis}, title = {Interaction of the potential DNA-radiosensitizer 8-bromoadenine with free and plasmonically generated electrons}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-407017}, school = {Universit{\"a}t Potsdam}, pages = {xi, 120}, year = {2017}, abstract = {In Germany more than 200.000 persons die of cancer every year, which makes it the second most common cause of death. Chemotherapy and radiation therapy are often combined to exploit a supra-additive effect, as some chemotherapeutic agents like halogenated nucleobases sensitize the cancerous tissue to radiation. The radiosensitizing action of certain therapeutic agents can be at least partly assigned to their interaction with secondary low energy electrons (LEEs) that are generated along the track of the ionizing radiation. In the therapy of cancer DNA is an important target, as severe DNA damage like double strand breaks induce the cell death. As there is only a limited number of radiosensitizing agents in clinical practice, which are often strongly cytotoxic, it would be beneficial to get a deeper understanding of the interaction of less toxic potential radiosensitizers with secondary reactive species like LEEs. Beyond that LEEs can be generated by laser illuminated nanoparticles that are applied in photothermal therapy (PTT) of cancer, which is an attempt to treat cancer by an increase of temperature in the cells. However, the application of halogenated nucleobases in PTT has not been taken into account so far. In this thesis the interaction of the potential radiosensitizer 8-bromoadenine (8BrA) with LEEs was studied. In a first step the dissociative electron attachment (DEA) in the gas phase was studied in a crossed electron-molecular beam setup. The main fragmentation pathway was revealed as the cleavage of the C-Br bond. The formation of a stable parent anion was observed for electron energies around 0 eV. Furthermore, DNA origami nanostructures were used as platformed to determine electron induced strand break cross sections of 8BrA sensitized oligonucleotides and the corresponding nonsensitized sequence as a function of the electron energy. In this way the influence of the DEA resonances observed for the free molecules on the DNA strand breaks was examined. As the surrounding medium influences the DEA, pulsed laser illuminated gold nanoparticles (AuNPs) were used as a nanoscale electron source in an aqueous environment. The dissociation of brominated and native nucleobases was tracked with UV-Vis absorption spectroscopy and the generated fragments were identified with surface enhanced Raman scattering (SERS). Beside the electron induced damage, nucleobase analogues are decomposed in the vicinity of the laser illuminatednanoparticles due to the high temperatures. In order to get a deeper understanding of the different dissociation mechanisms, the thermal decomposition of the nucleobases in these systems was studied and the influence of the adsorption kinetics of the molecules was elucidated. In addition to the pulsed laser experiments, a dissociative electron transfer from plasmonically generated "hot electrons" to 8BrA was observed under low energy continuous wave laser illumination and tracked with SERS. The reaction was studied on AgNPs and AuNPs as a function of the laser intensity and wavelength. On dried samples the dissociation of the molecule was described by fractal like kinetics. In solution, the dissociative electron transfer was observed as well. It turned out that the timescale of the reaction rates were slightly below typical integration times of Raman spectra. In consequence such reactions need to be taken into account in the interpretation of SERS spectra of electrophilic molecules. The findings in this thesis help to understand the interaction of brominated nucleobases with plasmonically generated electrons and free electrons. This might help to evaluate the potential radiosensitizing action of such molecules in cancer radiation therapy and PTT.}, language = {en} } @misc{WinkelbeinerWandtEbertetal.2020, author = {Winkelbeiner, Nicola Lisa and Wandt, Viktoria Klara Veronika and Ebert, Franziska and Lossow, Kristina and Bankoglu, Ezgi E. and Martin, Maximilian and Mangerich, Aswin and Stopper, Helga and Bornhorst, Julia and Kipp, Anna Patricia and Schwerdtle, Tanja}, title = {A Multi-Endpoint Approach to Base Excision Repair Incision Activity Augmented by PARylation and DNA Damage Levels in Mice}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1021}, issn = {1866-8372}, doi = {10.25932/publishup-48483}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-484831}, pages = {21}, year = {2020}, abstract = {Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2'-deoxyguanosine (8-oxodG), 5-hydroxy-2'-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery.}, language = {en} }