63 research outputs found
Unraveling the Complexity of DNA Radiation Damage Using DNA Nanotechnology
Funding Information: I.B. acknowledges financial support from the German Research Foundation (project no. 450169704). Publisher Copyright: © 2024 The Authors. Published by American Chemical Society.Radiation cancer therapies use different ionizing radiation qualities that damage DNA molecules in tumor cells by a yet not completely understood plethora of mechanisms and processes. While the direct action of the radiation is significant, the byproducts of the water radiolysis, mainly secondary low-energy electrons (LEEs, <20 eV) and reactive oxygen species (ROS), can also efficiently cause DNA damage, in terms of DNA strand breakage or DNA interstrand cross-linking. As a result, these types of DNA damage evolve into mutations hindering DNA replication, leading to cancer cell death. Concomitant chemo-radiotherapy explores the addition of radiosensitizing therapeutics commonly targeting DNA, such as platinum derivatives and halogenated nucleosides, to enhance the harmful effects of ionizing radiation on the DNA molecule. Further complicating the landscape of DNA damage are secondary structures such as G-quadruplexes occurring in telomeric DNA. These structures protect DNA from radiation damage, rendering them as promising targets for new and more selective cancer radiation treatments, rather than targeting linear DNA. However, despite extensive research, there is no single paradigm approach to understanding the mysterious way in which ionizing radiation causes DNA damage. This is due to the multidisciplinary nature of the field of research, which deals with multiple levels of biological organization, from the molecular building blocks of life toward cells and organisms, as well as with complex multiscale radiation-induced effects. Also, intrinsic DNA features, such as DNA topology and specific oligonucleotide sequences, strongly influence its response to damage from ionizing radiation. In this Account, we present our studies focused on the absolute quantification of photon- and low-energy electron-induced DNA damage in strategically selected target DNA sequences. Our methodology involves using DNA origami nanostructures, specifically the Rothemund triangle, as a platform to expose DNA sequences to either low-energy electrons or vacuum-ultraviolet (VUV, <15 eV) photons and subsequent atomic force microscopy (AFM) analysis. Through this approach, the effects of the DNA sequence, incorporation of halogenated radiosensitizers, DNA topology, and the radiation quality on radiation-induced DNA strand breakage have been systematically assessed and correlated with fundamental photon- and electron-driven mechanisms underlying DNA radiation damage. At lower energies, these mechanisms include dissociative electron attachment (DEA), where electrons attach to DNA molecules causing strand breaks, and dissociative photoexcitation of DNA. Additionally, further dissociative processes such as photoionization and electron impact contribute to the complex cascade of DNA damage events induced by ionizing radiation. We expect that emerging DNA origami-based approaches will lead to a paradigm shift in research fields associated with DNA damage and suggest future directions, which can foster the development of technological applications in nanomedicine, e.g., optimized cancer treatments or the molecular design of optimized radiosensitizing therapeutics.publishersversionpublishe
Special issue: Dynamics of systems on the nanoscale (2018). Editorial
The structure, formation and dynamics of both animate and inanimate matter on the nanoscale are a highly interdisciplinary ïŹeld of rapidly emerging research engaging a broad community encompassing experimentalists, theorists, and technologists. It is relevant for a large variety of molecular and nanosystems of diïŹerent origin and composition and concerns numerous phenomena originating from physics, chemistry, biology, or materials science. This Topical Issue presents a collection of original research papers devoted to diïŹerent aspects of structure and dynamics on the nanoscale. Some of the contributions discuss speciïŹc applications of the research results in several modern technologies and in next generation medicine. Most of the works of this topical issue were reported at the Fifth International Conference on Dynamics of Systems on the Nanoscale (DySoN) â the premier forum for the presentation of cutting-edge research in this ïŹeld that was held in Potsdam, Germany in October of 2018
Molecular states and spin crossover of hemin studied by DNA origami enabled single-molecule surface-enhanced Raman scattering
The study of biologically relevant molecules and their interaction with external stimuli on a single molecular scale is of high importance due to the availability of distributed rather than averaged information. Surface enhanced Raman scattering (SERS) provides direct chemical information, but is rather challenging on the single molecule (SM) level, where it is often assumed to require a direct contact of analyte molecules with the metal surface. Here, we detect and investigate the molecular states of single hemin by SM-SERS. A DNA aptamer based G-quadruplex mediated recognition of hemin directs its placement in the SERS hot-spot of a DNA Origami Nanofork Antenna (DONA). The configuration of the DONA structure allows the molecule to be trapped at the plasmonic hot-spot preferentially in no-contact configuration with the metal surface. Owing to high field enhancement at the plasmonic hot spot, the detection of a single folded G-quadruplex becomes possible. For the first time, we present a systematic study by SM-SERS where most hemin molecule adopt a high spin and oxidation state (III) that showed state crossover to low spin upon strong-field-ligand binding. The present study therefore, provides a platform for studying biologically relevant molecules and their properties at SM sensitivity along with demonstrating a conceptual advancement towards successful monitoring of single molecular chemical interaction using DNA aptamers
Decomposition of halogenated nucleobases by surface plasmon resonance excitation of gold nanoparticles
Halogenated uracil derivatives are of great interest in modern cancer therapy, either as chemotherapeutics or radiosensitisers depending on their halogen atom. This work applies UV-Vis spectroscopy to study the radiation damage of uracil, 5-bromouracil and 5-fluorouracil dissolved in water in the presence of gold nanoparticles upon irradiation with an Nd:YAG ns-pulsed laser operating at 532 nm at different fluences. Gold nanoparticles absorb light efficiently by their surface plasmon resonance and can significantly damage DNA in their vicinity by an increase of temperature and the generation of reactive secondary species, notably radical fragments and low energy electrons. A recent study using the same experimental approach characterized the efficient laser-induced decomposition of the pyrimidine ring structure of 5-bromouracil mediated by the surface plasmon resonance of gold nanoparticles. The present results show that the presence of irradiated gold nanoparticles decomposes the ring structure of uracil and its halogenated derivatives with similar efficiency. In addition to the fragmentation of the pyrimidine ring, for 5-bromouracil the cleavage of the carbon-halogen bond could be observed, whereas for 5-fluorouracil this reaction channel was inhibited. Locally-released halogen atoms can react with molecular groups within DNA, hence this result indicates a specific mechanism by which doping with 5-bromouracil can enhance DNA damage in the proximity of laser irradiated gold nanoparticles. Graphical abstract
Kinetics of molecular decomposition under irradiation of gold nanoparticles with nanosecond laser pulsesâA 5-Bromouracil case study
Laser illuminated gold nanoparticles (AuNPs) efficiently absorb light and heat up the surrounding medium, leading to versatile applications ranging from plasmonic catalysis to cancer photothermal therapy. Therefore, an in-depth understanding of the thermal, optical, and electron induced reaction pathways is required. Here, the electrophilic DNA nucleobase analog 5-Bromouracil (BrU) has been used as a model compound to study its decomposition in the vicinity of AuNPs illuminated with intense ns laser pulses under various conditions. The plasmonic response of the AuNPs and the concentration of BrU and resulting photoproducts have been tracked by ultraviolet and visible (UVâVis) spectroscopy as a function of the irradiation time. A kinetic model has been developed to determine the reaction rates of two parallel fragmentation pathways of BrU, and their dependency on laser fluence and adsorption on the AuNP have been evaluated. In addition, the size and the electric field enhancement of the decomposed AuNPs have been determined by atomic force microscopy and finite domain time difference calculations, respectively. A minor influence of the direct photoreaction and a strong effect of the heating of the AuNPs have been revealed. However, due to the size reduction of the irradiated AuNPs, a trade-off between laser fluence and plasmonic response of the AuNPs has been observed. Hence, the decomposition of the AuNPs might be limiting the achievable temperatures under irradiation with several laser pulses. These findings need to be considered for an efficient design of catalytic plasmonic systems
Controlling Plasmonic Chemistry Pathways through Specific Ion Effects
Plasmon-driven dehalogenation of brominated purines has been recently explored as a model system to understand fundamental aspects of plasmon-assisted chemical reactions. Here, it is shown that divalent Ca2+ ions strongly bridge the adsorption of bromoadenine (Br-Ade) to Ag surfaces. Such ion-mediated binding increases the molecule's adsorption energy leading to an overlap of the metal energy states and the molecular states, enabling the chemical interface damping (CID) of the plasmon modes of the Ag nanostructures (i.e., direct electron transfer from the metal to Br-Ade). Consequently, the conversion of Br-Ade to adenine almost doubles following the addition of Ca2+. These experimental results, supported by theoretical calculations of the local density of states of the Ag/Br-Ade complex, indicate a change of the charge transfer pathway driving the dehalogenation reaction, from Landau damping (in the lack of Ca2+ ions) to CID (after the addition of Ca2+). The results show that the surface dynamics of chemical species (including water molecules) play an essential role in charge transfer at plasmonic interfaces and cannot be ignored. It is envisioned that these results will help in designing more efficient nanoreactors, harnessing the full potential of plasmon-assisted chemistry
Die Rolle der Zucker- und Phosphateinheit bei DNA-StrangbrĂŒchen
Title
1 Introduction 1
2 Theoretical considerations 4
2.1 Electron-molecule interactions 4
2.2 Fundamental processes in radiation damage 17
2.3 Laser desorption techniques 24
3 Experiments 29
3.1 Dissociative Electron Attachment Spectroscopy in the Gas Phase 29
3.2 DEA Spectroscopy using Laser Induced Acoustic Desorption (LIAD) 34
3.3 Matrix-Assisted Laser Desorption/Ionisation (MALDI) 39
3.4 Calculations 41
4 Results and discussion 43
4.1 Sites selective fragmentation of D-ribose anions 43
4.2 Improving the model of sugar in DNA: DEA to peracetylated D-ribose 72
4.3 The phosphate moiety: DEA to phosphate esters 81
4.4 DEA to labile biomolecules using LIAD 88
4.5 DEA to hexafluoroacetone azine: Selective CN - formation 99
5 Summary 111
6 Zusammenfassung 113
Bibliography 117
Appendix 127
A Heats of formation 127
B Structures of 72 and 101 amu anions 129
C Electron energy loss spectroscopy (EELS) of furan 131
D Publications 139
E Conference contributions 143To elucidate the molecular mechanisms of low energy electron induced DNA
strand breaks, dissociative electron attachment (DEA) to different model
compounds of the DNA (and RNA) backbone was investigated in the gas phase
including the sugar and the phosphate moiety.
It was found that the free sugar D-ribose efficiently captures electrons close
to zero eV and dissociates subsequently into various fragment anions by loss
of neutral water and formaldehyde molecules. The use of the isotope labelled
analogues 5-13C-D-ribose, 1-13C-D-ribose and C -1-D-D-ribose enabled an
unambiguous assignment of the generated fragment ions. Furthermore it was
demonstrated that C5 is selectively excised from the sugar ring as a
formaldehyde molecule close to zero eV electron energy. In DNA C5 of the sugar
unit is directly connected to a phosphate group. The threshold signals are
explained by the initial formation of a dipole bound state that serves as a
doorway to dissociation.
An essential question is to which degree the results that are obtained on a
single building block of DNA can be transferred to the situation when the
corresponding unit is coupled in the DNA/RNA molecular network. In that
respect tetraacetyl-D-ribose was investigated, which can be regarded as
substantially improved model for the sugar bound within DNA. It was found that
the threshold signals of the isolated sugar are preserved leading to an
abstraction of all acetate groups following electron attachment. In addition
to the signals that are attributed to the sugar ring, a Ï* shape resonance was
identified at 1 3 eV located on the acetate groups, which results in further
fragmentation products.
The response of the phosphate group to low energy electrons was studied by
means of phosphate esters, viz., dibutylphosphate and triethylphosphate. It
was observed that electron attachment into the Ï* orbitals of the phosphate
group occurs at energies below 3 eV accompanied with P-O and C-O bond
breaking, and a core excited resonance is located at 7 10 eV. The
abstraction of a whole butyl group from dibutylphosphate at 2 4 eV and 7
10 eV is due to a C-O bond cleavage, which would correspond to a strand break
in DNA.
To judge which resonances and reactions of the individual parts of DNA are
preserved in larger systems, it is vitally important to study DEA to molecules
such as sugar-phosphates, nucleosides and nucleotides. Due to the thermal
fragility of these compounds a new experimental setup was constructed that
allows the investigation of DEA to thermal labile biomolecules employing laser
induced acoustic desorption (LIAD). To evaluate the performance of the new
experiment, DEA to bromouracil and thymidine was studied. Finally, electron
attachment to D-ribose-5-phosphate was investigated and an attachment of near
0 eV electrons to both the phosphate group and the sugar unit was found
followed by a cleavage of the phosphate-sugar linkage leading to the formation
of H2PO4- and [Ribosephosphate-H2PO3]- , respectively.
In the work presented here it was demonstrated that all building blocks of the
DNA backbone capture electrons at 0 12 eV with subsequent dissociation. The
highest ion yields were found at subexcitation energies (< 4 eV). Different
reaction mechanisms were suggested that could explain the previously
determined ( Phys. Rev. Lett. , 2004, 93 , 068101), well-structured yield
functions of low energy electron induced strand breaks in plasmid DNA.Um die molekularen Mechanismen von DNA-StrangbrĂŒchen aufzuklĂ€ren, die durch
niederenergetische Elektronen induziert werden, wurde die dissoziative
Elektronenanlagerung (DEA) an Modellsubstanzen fĂŒr das DNA- (und RNA-)
RĂŒckgrat untersucht. Das beinhaltet sowohl die Zucker- als auch die
Phosphateinheit.
Es wurde herausgefunden, dass der isolierte Zucker D-Ribose effektiv
Elektronen nahe 0 eV einfÀngt und danach durch Verlust von neutralen Wasser-
und FormaldehydmolekĂŒlen in verschiedene Fragment-Anionen zerfĂ€llt. Die
Verwendung der isotopenmarkierten Analoga 5-13C-D-ribose, 1-13C-D-ribose und C
-1-D-D-ribose ermöglichte eine eindeutige Zuordnung der beobachteten
Fragmentionen. AuĂerdem konnte gezeigt werden, dass das Kohlenstoffatom C5 des
Zuckers selektiv in Form eines FormaldehydmolekĂŒls bei Elektronenenergien nahe
0 eV abgespalten wird. In der DNA ist das Kohlenstoffatom C5 direkt an die
benachbarte Phosphatgruppe gebunden. Die beobachteten 0 eV - Signale können
durch die anfÀngliche Bildung eines dipolgebundenen Zustands erklÀrt werden,
der dann zur Dissoziation fĂŒhrt.
Eine zentrale Fragestellung betrifft die Ăbertragbarkeit von Ergebnissen, die
von einer einzelnen Untereinheit der DNA erhalten wurden, auf die Situation,
wenn die entsprechende Einheit im molekularen Netzwerk der DNA/RNA eingebunden
ist. Um diesem Problem nachzugehen, wurde Tetraacetyl-D-Ribose untersucht, die
als wesentlich besseres Modell fĂŒr den Zuckerbaustein in DNA dient als der
isolierte Zucker. Es konnte gezeigt werden, dass die Signale bei 0 eV erhalten
bleiben und in diesem MolekĂŒl nach Elektroneneinfang sogar zur Abspaltung
aller Acetylgruppen fĂŒhren. ZusĂ€tzlich zu den Signalen, die auf den Zuckerring
zurĂŒckzufĂŒhren sind, wurden Ï* shape \- Resonanzen bei 1 3 eV nachgewiesen,
die auf den Acetylgruppen lokalisiert sind und weitere Fragmentierungen zur
Folge haben.
Die Wechselwirkung von niederenergetischen Elektronen mit der Phosphatgruppe
wurde mit Hilfe der organischen Phosphatester Dibutylphosphat und
Triethylphosphat untersucht. Es wurde ein Elektroneneinfang vom Ï * - Orbital
der Phosphatgruppe unterhalb von 3 eV beobachtet, der zu verschiedenen C-O und
P-O-BindungsbrĂŒchen fĂŒhrt. ZusĂ€tzlich wurde eine core excited \- Resonanz bei
Energien von 7 10 eV nachgewiesen. Die Abspaltung einer gesamten Butylgruppe
aus Dibutylphosphat bei 2 4 eV und 7 10 eV kommt durch einen
C-O-Bindungsbruch zustande, der im Falle einer in der DNA gebundenen
Phosphatgruppe einem Strangbruch entspricht.
Um zu beurteilen, welche Resonanzen und Reaktionsmechanismen der einzelnen
DNA-Bausteine auch in gröĂeren Systemen erhalten bleiben, ist die Untersuchung
von MolekĂŒlen wie Zucker-Phosphaten, Nukleosiden und Nukleotiden dringend
erforderlich. Aufgrund der thermischen Empfindlickeit dieser Substanzen wurde
ein neues Experiment konstruiert, das die Untersuchung von DEA an thermisch
labile BiomolekĂŒle durch Anwendung von Laser-induzierter akustischer
Desorption (LIAD) erlaubt. Um die LeistungsfÀhigkeit des neuen Experiments zu
testen, wurde DEA an 5-Bromuracil und Thymidin gemessen. SchlieĂlich wurde die
Elektronenanlagerung an D-Ribose-5-Phosphat studiert, bei der ein Einfang von
Elektronen nahe 0 eV sowohl von der Phosphatgruppe als auch von der
Zuckereinheit festgestellt wurde, der zur Bildung der Fragmentionen H2PO4-
beziehungsweise [Ribosephosphat-H2PO3]- fĂŒhrt.
Es konnte gezeigt werden, dass alle Untereinheiten des DNA-GerĂŒstes Elektronen
im Energiebereich von 0 bis 12 eV einfangen und dann vielfÀltige
Dissoziationsreaktionen eingehen. Die gröĂten Ionenausbeuten wurden bei
Energien unterhalb von elektronischer Anregung, also unter 4 eV, erhalten. Es
wurden verschiedene Reaktionsmechanismen fĂŒr die Entstehung von StrangbrĂŒchen
in Plasmid-DNA durch niederenergetische Elektronen vorgeschlagen, die die von
Sanche et al. ( Phys. Rev. Lett. , 2004, 93 , 068101) gemessenen
Ausbeutekurven erklÀren können
Length and Energy Dependence of Low-Energy Electron-Induced Strand Breaks in Poly(A) DNA
The DNA in living cells can be effectively damaged by high-energy radiation, which can lead to cell death. Through the ionization of water molecules, highly reactive secondary species such as low-energy electrons (LEEs) with the most probable energy around 10 eV are generated, which are able to induce DNA strand breaks via dissociative electron attachment. Absolute DNA strand break cross sections of specific DNA sequences can be efficiently determined using DNA origami nanostructures as platforms exposing the target sequences towards LEEs. In this paper, we systematically study the effect of the oligonucleotide length on the strand break cross section at various irradiation energies. The present work focuses on poly-adenine sequences (d(A4), d(A8), d(A12), d(A16), and 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. When going from d(A4) to d(A16), the strand break cross section increases with oligonucleotide length, but only at 7.0 and 8.4 eV, i.e., close to the maximum of the negative ion resonance, the increase in the strand break cross section with the length is similar to the increase of an estimated geometrical cross section. For d(A20), a markedly lower DNA strand break cross section is observed for all electron energies, which is tentatively ascribed to a conformational change of the dA20 sequence. The results indicate that, although there is a general length dependence of strand break cross sections, individual nucleotides do not contribute independently of the absolute strand break cross section of the whole DNA strand. The absolute quantification of sequence specific strand breaks will help develop a more accurate molecular level understanding of radiation induced DNA damage, which can then be used for optimized risk estimates in cancer radiation therapy
Molecular Processes Studied at a Single-Molecule Level Using DNA Origami Nanostructures and Atomic Force Microscopy
DNA origami nanostructures allow for the arrangement of different functionalities such as proteins, specific DNA structures, nanoparticles, and various chemical modifications with unprecedented precision. The arranged functional entities can be visualized by atomic force microscopy (AFM) which enables the study of molecular processes at a single-molecular level. Examples comprise the investigation of chemical reactions, electron-induced bond breaking, enzymatic binding and cleavage events, and conformational transitions in DNA. In this paper, we provide an overview of the advances achieved in the field of single-molecule investigations by applying atomic force microscopy to functionalized DNA origami substrates
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