The Sequence Dependence of Photoinduced Single Strand Break in 5‑Bromo-2′-deoxyuridine Labeled DNA Supports That Electron Transfer Is Responsible for the Damage

The UVB irradiation of DNA labeled with 5-bromo-2′-deoxyuridine (BrdU) leads to single-strand breaks (SSBs) as a major photochemical damage. Some time ago, we demonstrated that SSB is a secondary damage forming due to thermal dissociation of 2′-deoxyribonolactone generated photochemically in DNA labeled with BrdU. For the first time, we study here the variation of the yield of UVB generated SSBs with the alteration of 3′-neighbor nucleobase of electron donor (2′-deoxyguanine (dG)) and acceptor (excited BrdU) in double-stranded DNA. We showed that the experimental damage yields can be explained by the calculated ionization potentials of dG and electron affinities of excited BrdU via a kinetic scheme based on the Marcus model of electron transfer (ET). Hence, our studies on the sequence dependence of photochemical damage in DNA labeled with BrdU constitute a further argument that photochemically generated SSBs occur as a result of long-range ET

UV-Induced Strand Breaks in Double-Stranded DNA Labeled with 5‑Bromouracil: Frank or Secondary?

Some literature reports suggest that in DNA labeled with 5-bromouracil (5BrU), near-UV photons lead to strand breaks that are formed due to the formation of a reactive uracil-5-yl radical capable of abstracting a hydrogen atom from its own or adjacent sugar moiety, which results in a direct strand break. However, other reports propose the formation of 2′-deoxyribonolactone rather than a strand break during the photodamage of 5BrU-substituted DNA. In order to resolve these contradictions, we carried out a series of experiments where 25 nucleotides-long DNA duplexes labeled with 5BrU were irradiated with 300 nm light. Two experimental methods were used to detect and separate the degradation products generated under experimental conditions, DHPLC (completely denaturing high-performance liquid chromatography) and denaturing PAGE electrophoresis. In addition, the identity of the particular products was confirmed with negative ion mass spectrometry. Our studies demonstrate that direct strand breaks reported in the past for 5BrU-labeled oligonucleotides are rather secondary breaks

Photoinduced Single Strand Breaks and Intrastrand Cross-Links in an Oligonucleotide Labeled with 5‑Bromouracil

5-Bromouracil (BrU) is photoreactive toward near UVB photons and can be introduced into genomic DNA during its biosynthesis in cells. However, PCR seems to be a simpler approach, which can be used to obtain labeled DNA similar to that synthesized within the cell. In the current work, PCR has been employed and optimized in order to substitute all thymines (besides those present in starters) with BrU in the dsDNA fragment of 80 base pairs (bp) in length. The modified oligonucleotide was irradiated with 300 nm photons in a buffered aqueous solution (pH = 7) and digested with a cocktail of enzymes specific to the phosphodiester bond cleavage. Initially, the extent of damage in the intact photolyte was measured with DHPLC. Then, the digested reaction mixture was subjected to HPLC and MS analyses and, in addition to the formation of 5-bromo-2′-deoxuyridine, which proves the occurrence of single strand breaks (SSBs) due to irradiation, U∧U and U∧C dimers were found, whose molecular structure was confirmed by MS/MS analysis. Although the abundance of such tandem lesions is lower than that of the SSB type, they pose a potent threat to genome integrity. Thus, our findings shed new light on the photosensitizing properties of BrU toward DNA

5‑Selenocyanatouracil: A Potential Hypoxic Radiosensitizer. Electron Attachment Induced Formation of Selenium Centered Radical

The propensity of 5-selenocyanatouracil (SeCNU) to decomposition induced by attachment of electron was scrutinized with the G3B3 composite quantum-chemical method and radiolytic studies. Favorable thermodynamic (Gibbs free reaction energy of −13.65 kcal/mol) and kinetic (Gibbs free activation energy of 1.22 kcal/mol) characteristics revealed by the G3B3 free energy profile suggest SeCNU to be sensitive to electron attachment. The title compound was synthesized in the reaction between uracil and selenocyanogen chloride in acetic acid. Then, an aqueous and deoxygenated solution of the HPLC purified compound containing <i>tert</i>-butanol as a hydroxyl radical scavenger was irradiated with X-rays. SeCNU radio-degradation results in two major products: the U–Se–Se–U dimer and the adduct of the ^●OtBu radical to the U–Se^● radical, U–Se–OtBu. The effects of radiolysis as well as the results of G3B3 calculations point to U–Se^● as the primary product of dissociative electron attachment to SeCNU. The MTT test shows that SeCNU is nontoxic <i>in vitro</i> in concentrations equal to or lower than 10^–6 M. Ionizing radiation will probably induce cytotoxic intra- and interstrand DNA cross-links as well as protein–DNA cross-links in the genomic DNA labeled with SeCNU

Photoelectron Spectroscopy and Computational Modeling of Thymidine Homodimer Anions

The intact thymidine homodimer anion (dT₂^–) was generated in the gas phase using an infrared desorption/photoemission source and recorded by a pulsed photoelectron spectrometer. The photoelectron spectrum (PES) revealed a broad signal with the maximum at electron binding energy ∼2.0 eV and the threshold value at 1.1 eV. The relative energies and vertical detachment energies of the possible anion structures were calculated at the B3LYP/6-31++G­(d,p) level. Here we report that the most stable anion radical homodimer geometries observed in the PES are the anionic nucleoside coordinated by the O8 atom of thymine to the deoxyribose of the second neutral nucleoside. Unlike previous experimental–computational studies on anionic complexes involving nucleobases with proton donors, the electron-induced proton-transferred structures are not responsible for the shape of the PES of dT₂^–

Excess Electron Attachment to the Nucleoside Pair 2′-Deoxyadenosine (dA)–2′-Deoxythymidine (dT)

The 2′-deoxyadenosine···2′-deoxythymidine (dAdT^•–) radical anion nucleoside pair has been investigated both experimentally and theoretically in the gas phase. The vertical detachment energy (VDE) and adiabatic electron affinity (AEA) were determined by anion photoelectron spectroscopy (PES). The measured photoelectron spectrum features a broad band having an onset at ∼1.1 eV and a maximum at the electron binding energy (EBE) ranging from 1.7 to 1.9 eV. Calculations performed at the M06-2X/6-31++G** level reveal that the observed PES signal is probably due to a dAdT^•– complex in which the thymine of the dT nucleoside forms hydrogen bonds that engage its O7 and O8 atoms as well as the 3′- and 5′-hydroxyl groups of 2′-deoxyadenosine (dA), while dT’s 3′-hydroxyl group interacts with the N1 of dA. In this heterodimer, the excess electron is entirely located on thymine. The biologically relevant Watson–Crick arrangement of the dAdT^•– dimer was found to be substantially less stable (by ∼19 kcal mol^–1 in Gibbs free energy scale) than the above-mentioned configuration; hence, it is not populated in the gas phase

Fundamental Mechanisms of DNA Radiosensitization: Damage Induced by Low-Energy Electrons in Brominated Oligonucleotide Trimers

The replacement of nucleobases with brominated analogs enhances DNA radiosensitivity. We examine the chemistry of low-energy electrons (LEEs) in this sensitization process by experiments with thin films of the oligonucleotide trimers TBrXT, where BrX = 5-BrU (5-bromouracil), 5-BrC (5-bromocytosine), 8-BrA (8-bromoadenine), or 8-BrG (8-bromoguanine). The products induced from irradiation of thin (∼ 2.5 nm) oligonucleotide films, with 10 eV electrons, under ultrahigh vacuum (UHV) are analyzed by HPLC-UV. The number of damaged brominated trimers ranges from about 12 to 15 × 10^–3 molecules per incident electron, whereas under the identical conditions, these numbers drop to 4–7 × 10^–3 for the same, but nonbrominated oligonucleotides. The results of HPLC analysis show that the main degradation pathway of trinucleotides containing brominated bases involve debromination (i.e., loss of the bromine atom and its replacement with a hydrogen atom). The electron-induced sum of products upon bromination increases by factors of 2.1 for the pyrimidines and 3.2 for the purines. Thus, substitution of any native nucleobase with a brominated one in simple models of DNA increases LEE-induced damage to DNA and hence its radiosensitivity. Furthermore, besides the brominated pyrimidines that have already been tested in clinical trials, brominated purines not only appear to be promising sensitizers for radiotherapy, but could provide a higher degree of radiosensitization

Dominant Pathways of Adenosyl Radical-Induced DNA Damage Revealed by QM/MM Metadynamics

Brominated nucleobases sensitize double stranded DNA to hydrated electrons, one of the dominant genotoxic species produced in hypoxic cancer cells during radiotherapy. Such radiosensitizers can therefore be administered locally to enhance treatment efficiency within the solid tumor while protecting the neighboring tissue. When a solvated electron attaches to 8-bromoadenosine, a potential sensitizer, the dissociation of bromide leads to a reactive C8 adenosyl radical known to generate a range of DNA lesions. In the current work, we propose a multiscale computational approach to elucidate the mechanism by which this unstable radical causes further damage in genomic DNA. We employed a combination of classical molecular dynamics conformational sampling and QM/MM metadynamics to study the thermodynamics and kinetics of plausible reaction pathways in a realistic model, bridging between different time scales of the key processes and accounting for the spatial constraints in DNA. The obtained data allowed us to build a kinetic model that correctly predicts the products predominantly observed in experimental settingscyclopurine and β-elimination (single strand break) lesionswith their ratio and yield dependent on the effective lifetime of the radical species. To date, our study provides the most complete description of purine radical reactivity in double stranded DNA, explaining the radiosensitizing action of electrophilic purines in molecular detail as well as providing a conceptual framework for the computational modeling of competing reaction pathways in biomolecules

How to Find Out Whether a 5‑Substituted Uracil Could Be a Potential DNA Radiosensitizer

Incorporated into genomic DNA, 5-substituted uracils could be employed in human cancer radiotherapy if they could be sensitized to dissociate upon reaction with hydrated electrons. Using the B3LYP/6-31++G­(d,p) method, we calculate electron affinities and energy profiles related to the dissociation of the respective anions for a series of uracil derivatives. We demonstrate that for a uracil analogue to be an efficient electron acceptor the uracil substituent has to possess significant electron-withdrawing power. On the other hand, in order to ensure effective dissociation of the anion, the chemical bond holding together the substituent and uracil residue should be relatively weak. Our theoretical predictions are in excellent agreement with the results of our negative ion photoelectron spectroscopy experiments. We propose two new potential sensitizers that seem to possess the required properties, although they have never been tested in radiobiological experiments

Dissociative Electron Attachment to 5‑Iodo-4-thio-2′-deoxyuridine: A Potential Radiosensitizer of Hypoxic Cells

In the search for effective radiosensitizers for tumor cells, halogenated uracils have attracted more attention due to their large cross section for dissociation upon the attachment of low-energy electrons. In this study, we investigated dissociative electron attachment (DEA) to 5-iodo-4-thio-2′-deoxyuridine, a potential radiosensitizer using a crossed electron-molecule beam experiment coupled with quadrupole mass spectrometry. The experimental results were supported by calculations on the threshold energies of formed anions and transition state calculations. We show that low-energy electrons with kinetic energies near 0 eV may effectively decompose the molecule upon DEA. The by far most abundant anion observed corresponds to the iodine anion (I–). Due to the associated bond cleavage, a radical site is formed at the C5 position, which may initiate strand break formation if the molecule is incorporated into a DNA strand. Our results reflect the conclusion from previous radiolysis studies with the title compound, suggesting its potential as a radiosensitizer