Clustered DNA Damages induced by 0.5 to 30 eV Electrons.

Low-energy electrons (LEEs) of energies ≤30 eV are generated in large quantities by ionizing radiation. These electrons can damage DNA; particularly, they can induce the more detrimental clustered lesions in cells. This type of lesions, which are responsible for a large portion of the genotoxic stress generated by ionizing radiation, is described in the Introduction. The reactions initiated by the collisions of 0.5-30 eV electrons with oligonucleotides, duplex DNA, and DNA bound to chemotherapeutic platinum drugs are explained and reviewed in the subsequent sections. The experimental methods of LEE irradiation and DNA damage analysis are described with an emphasis on the detection of cluster lesions, which are considerably enhanced in DNA-Pt-drug complexes. Based on the energy dependence of damage yields and cross-sections, a mechanism responsible for the clustered lesions can be attributed to the capture of a single electron by the electron affinity of an excited state of a base, leading to the formation of transient anions at 6 and 10 eV. The initial capture is followed by electronic excitation of the base and dissociative attachment-at other DNA sites-of the electron reemitted from the temporary base anion. The mechanism is expected to be universal in the cellular environment and plays an important role in the formation of clustered lesions.

Recent developments in reversible photoregulation of oligonucleotide structure and function.

There is a growing interest in the photoregulation of biological functions, due to the high level of spatiotemporal precision achievable with light. Additionally, light is non-invasive and waste-free. In particular, the photoregulation of oligonucleotide structure and function is a rapidly developing study field with relevance to biological, physical and material sciences. Molecular photoswitches have been incorporated in oligonucleotides for 20 years, and the field has currently grown beyond fundamental studies on photochemistry of the switches and DNA duplex stability, and is moving towards applications in chemical biology, nanotechnology and material science. Moreover, the currently emerging field of photopharmacology indicates the relevance of photocontrol in future medicine. In recent years, a large number of publications has appeared on photoregulation of DNA and RNA structure and function. New strategies are evaluated and novel, exciting applications are shown. In this comprehensive review, the key strategies for photoswitch inclusion in oligonucleotides are presented and illustrated with recent examples. Additionally the applications that have emerged in recent years are discussed, including gene regulation, drug delivery and materials design. Finally, we identify the challenges that the field currently faces and look forward to future applications.

Efficient UV-induced charge separation and recombination in an 8-oxoguanine-containing dinucleotide.

During the early evolution of life, 8-oxo-7,8-dihydro-2'-deoxyguanosine (O) may have functioned as a proto-flavin capable of repairing cyclobutane pyrimidine dimers in DNA or RNA by photoinduced electron transfer using longer wavelength UVB radiation. To investigate the ability of O to act as an excited-state electron donor, a dinucleotide mimic of the FADH2 cofactor containing O at the 5'-end and 2'-deoxyadenosine at the 3'-end was studied by femtosecond transient absorption spectroscopy in aqueous solution. Following excitation with a UV pulse, a broadband mid-IR pulse probed vibrational modes of ground-state and electronically excited molecules in the double-bond stretching region. Global analysis of time- and frequency-resolved transient absorption data coupled with ab initio quantum mechanical calculations reveal vibrational marker bands of nucleobase radical ions formed by electron transfer from O to 2'-deoxyadenosine. The quantum yield of charge separation is 0.4 at 265 nm, but decreases to 0.1 at 295 nm. Charge recombination occurs in 60 ps before the O radical cation can lose a deuteron to water. Kinetic and thermodynamic considerations strongly suggest that all nucleobases can undergo ultrafast charge separation when π-stacked in DNA or RNA. Interbase charge transfer is proposed to be a major decay pathway for UV excited states of nucleic acids of great importance for photostability as well as photoredox activity.

Radiation damage to single stranded oligonucleotide trimers labelled with 5-iodopyrimidines.

The radiolysis of deoxygenated aqueous solution containing trimeric oligonucleotides labelled with iodinated pyrimidines and Tris-HCl as the hydroxyl radical scavenger leads to electron attachment to the halogenated bases that mainly results in single strand breaks. The iodinated trimers are 2-fold more sensitive to solvated electrons than the brominated oligonucleotides, which is explained by the barrier-free dissociation of the iodinated base anions. The present study fills the literature gap concerning the chemistry triggered by ionizing radiation in the iodinated pyrimidines incorporated into DNA.

5-Bromo-2'-deoxycytidine-a potential DNA photosensitizer.

A double-stranded oligonucleotide, 80 base pairs in length, was multiply labeled with 5-bromo-2'-deoxycytidine (BrdC) using polymerase chain reaction (PCR). The modified oligonucleotide was irradiated with 300 nm photons and its damage was assayed by employing DHPLC, LC-MS and denaturing polyacrylamide gel electrophoresis (PAGE). Two types of damage were demonstrated, namely, single strand breaks (SSBs) and intrastrand cross-links (ICLs); the ICLs were in the form of d(G^C) and d(C^C) dimers. The former species are probably formed due to photoinduced electron transfer between the photoexcited BrdC and the ground state 2'-deoxyguanosine (dG), whereas the latter is a result of a cycloaddition reaction. Since SSBs and ICLs are potentially lethal to the cell, BrdC could be considered as a nucleoside with possible clinical applications.

Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA.

Replication Protein A is a single-stranded (ss) DNA-binding protein that is highly conserved in eukaryotes and plays essential roles in many aspects of nucleic acid metabolism, including replication, recombination, DNA repair and telomere maintenance. It is a heterotrimeric complex consisting of three subunits: RPA1, RPA2 and RPA3. It possesses four DNA-binding domains (DBD), DBD-A, DBD-B and DBD-C in RPA1 and DBD-D in RPA2, and it binds ssDNA via a multistep pathway. Unlike the RPA1 and RPA2 subunits, no ssDNA-RPA3 interaction has as yet been observed although RPA3 contains a structural motif found in the other DBDs. We show here using 4-thiothymine residues as photoaffinity probe that RPA3 interacts directly with ssDNA on the 3'-side on a 31 nt ssDNA.

Low-energy electron-induced DNA damage: effect of base sequence in oligonucleotide trimers.

DNA damage induced by low-energy electrons (LEEs) has attracted considerable attention in recent years because LEEs represent a large percentage of the total energy deposited by ionizing radiation and because LEEs have been shown to damage DNA components. In this article, we have studied the effect of base sequences in a series of oligonucleotide trimers by the analysis of damage remaining within the nonvolatile condensed phase after LEE irradiation. The model compounds include TXT, where X represents one of the four normal bases of DNA (thymine (T), cytosine (C), adenine (A), and guanine (G)). Using HPLC-UV analysis, several known fragments were quantified from the release of nonmodified nucleobases (T and X) as well as from phosphodiester C-O bond cleavage (pT, pXT, Tp, and TXp). The total damage was estimated by the disappearance of the parent peaks in the chromatogram of nonirradiated and irradiated samples. When trimers were irradiated with LEE (10 eV), the total damage decreased 2-fold in the following order: TTT > TCT > TAT > TGT. The release of nonmodified nuclobases (giving from 17 to 24% of the total products) mainly occurred from the terminal sites of trimers (i.e., T) whereas the release of central nucleobases was minor (C) or not at all detected (A and G). In comparison, the formation of products arising from phosphodiester bond cleavage accounted for 9 to 20% of the total damage and it partitioned to the four possible sites of cleavage present in trimers. This study indicates that the initial LEE capture and subsequent bond breaking within the intermediate anion depend on the sequence and electron affinity of the bases, with the most damage attributed to the most electronegative base, T.

The formation of DNA photodamage: the role of exciton localization.

The electronic structure during the formation of a cyclobutane pyrimidine dimer (CPD) between two thymine bases is investigated using semi-empirical and first-principles approaches. The dimerization of two isolated thymine bases is found to have no barrier or a very small barrier in agreement with previous studies suggesting low photostability of DNA. The well-known high photostability of DNA can only be explained taking other factors into account. We investigate the role of the exciton location in the particular environment. Different model systems, from isolated thymine bases to an oligonucleotide in aqueous solution, are discussed. Analysis of the frontier orbitals allows one to understand the connection between the location of the exciton, the relative orientation of the thymine bases, and the observed reactivity.

DNA damage and interstrand cross-link formation upon irradiation of aryl iodide C-nucleotide analogues.

The 5-halopyrimidine nucleotides damage DNA upon UV-irradiation or exposure to gamma-radiolysis via the formation of the 2'-deoxyuridin-5-yl sigma-radical. The bromo and iodo derivatives of these molecules are useful tools for probing DNA structure and as therapeutically useful radiosensitizing agents. A series of aryl iodide C-nucleotides were incorporated into synthetic oligonucleotides and exposed to UV-irradiation and gamma-radiolysis. The strand damage produced upon irradiation of DNA containing these molecules is consistent with the generation of highly reactive sigma-radicals. Direct stand breaks and alkali-labile lesions are formed at the nucleotide analogue and flanking nucleotides. The distribution of lesion type and location varies depending upon the position of the aryl ring that is iodinated. Unlike 5-halopyrimidine nucleotides, the aryl iodides produce interstrand cross-links in duplex regions of DNA when exposed to gamma-radiolysis or UV-irradiation. Quenching studies suggest that cross-links are produced by gamma-radiolysis via capture of a solvated electron, and subsequent fragmentation to the sigma-radical. These observations suggest that aryl iodide C-nucleotide analogues may be useful as probes for excess electron transfer and radiosensitizing agents.

Synthesis and hybridization properties of iridium(III) polypyridyl complex-conjugated oligonucleotide.

An Ir(III) polypyridyl complex-conjugated 14-mer oligonucleotide (IrIII-DNA) was synthesized and its hybridization properties with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) were evaluated by UV-melting experiments. The stabilities of the duplexes of IrIII-DNA with 14-, 20-, and 26-mer ssDNAs were higher than those of the unconjugated oligonucleotides. The triplex of IrIII-DNA with 14-mer dsDNA was also stabilized. However, the triplexes of IrIII-DNA with 20- and 26-mer dsDNAs, flanked by 3 and 6 base pairs at the both ends of 14-mer dsDNA target, were destabilized. This is presumably because of steric repulsion between the Ir(III) complex and the protruding 3- and 6-mer dsDNA moieties which are inflexible compared to ssDNA.

Hydrated electrons induce the formation of interstrand cross-links in DNA modified by cisplatin adducts.

Double-stranded oligonucleotides containing cisplatin adducts, with and without a mismatched region, were exposed to hydrated electrons generated by gamma-rays. Gel electrophoresis analysis demonstrates the formation of cisplatin-interstrand crosslinks from the cisplatin-intrastrand species. The rate constant per base for the reaction between hydrated electrons and the double-stranded oligonucleotides with and without cisplatin containing a mismatched region was determined by pulse radiolysis to be 7 × 109 and 2 × 109 M-1 s-1, respectively. These results provide a better understanding of the radiosensitizing effect of cisplatin adducts in hypoxic tumors and of the formation of interstrand crosslinks, which are difficult for cells to repair.

DNA damage by low-energy electron impact: dependence on guanine content.

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.

Multi-level patterning nucleic acid photolithography.

The versatile and tunable self-assembly properties of nucleic acids and engineered nucleic acid constructs make them invaluable in constructing microscale and nanoscale devices, structures and circuits. Increasing the complexity, functionality and ease of assembly of such constructs, as well as interfacing them to the macroscopic world requires a multifaceted and programmable fabrication approach that combines efficient and spatially resolved nucleic acid synthesis with multiple post-synthetic chemical and enzymatic modifications. Here we demonstrate a multi-level photolithographic patterning approach that starts with large-scale in situ surface synthesis of natural, modified or chimeric nucleic acid molecular structures and is followed by chemical and enzymatic nucleic acid modifications and processing. The resulting high-complexity, micrometer-resolution nucleic acid surface patterns include linear and branched structures, multi-color fluorophore labeling and programmable targeted oligonucleotide immobilization and cleavage.

Light-Induced Reversible DNA Ligation of Gold Nanoparticle Superlattices.

DNA-mediated self-assembly of nanoparticles has been of great interest because it enables access to nanoparticle superstructures that cannot be synthesized otherwise. However, the programmability of higher order nanoparticle structures can be easily lost under DNA denaturing conditions. Here, we demonstrate that light can be employed as an external stimulus to master the stability of nanoparticle superlattices (SLs) via the promotion of a reversible photoligation of DNA in SLs. The oligonucleotides attached to the nanoparticles are encoded to ligate using 365 nm light, effectively locking the SLs and rendering them stable under DNA denaturing conditions. The reversible process of unlocking these structures is possible by irradiation with light at 315 nm, recovering the structures to their natural state. Our work inspires an alternative research direction toward postassembly manipulation of nanoparticle superstructures using external stimuli as a tool to enrich the library of additional material forms and their application in different media and environments.

Coupling across a DNA helical turn yields a hybrid DNA/organic catenane doubly tailed with functional termini.

We describe the synthesis of a hybrid DNA/organic macrocycle that is prepared by formation of an amide linkage across one full turn of DNA. Formation of a catenane proved that the linkage crossed a turn rather than running along the phosphodiester backbone contour. The product, a doubly tailed catenane, contains 5'- and 3'-termini that can be functionalized further or used to incorporate the catenane structure into other DNA assemblies.

Ultrafast reversible photo-cross-linking reaction: toward in situ DNA manipulation.

We describe a novel ultrafast reversible DNA interstrand photo-cross-linking reaction via 3-cyanovinylcarbazole nucleoside ( (CNV)K). Oligodeoxynucleotide (ODN) containing (CNV)K can be photo-cross-linked by irradiation at 366 nm for 1 s, and the photo-cross-linked ODN can be split by irradiation at 312 nm for 60 s.

Unfolding thermodynamics of DNA pyrimidine triplexes with different molecularities.

Nucleic acid oligonucleotides (ODNs), as drugs, present an exquisite selectivity and affinity that can be used in antigene and antisense strategies for the control of gene expression. In this work we try to answer the following question: How does the molecularity of a DNA triplex affect its overall stability and melting behavior? To this end, we used a combination of temperature-dependent UV spectroscopy and calorimetric (differential scanning calorimetry) techniques to investigate the melting behavior of DNA triplexes with a similar helical stem, TC+TC+TC+T/AGAGAGA/TCTCTCT, but formed with different strand molecularity. We determined standard thermodynamic profiles and the differential binding of protons and counterions accompanying their unfolding. The formation of a triplex is accompanied by a favorable free energy term, resulting from the typical compensation of favorable enthalpy-unfavorable entropy contributions, i.e., the folding of a particular triplex is enthalpy driven. The magnitude of the favorable enthalpy contributions corresponds to the number and strength of the base-triplet stacks formed, which are helped by stacking contributions due to the incorporation of dangling ends or loops. Triplex stability is in the following order: monomolecular > bimolecular > trimolecular; this is explained in terms of additional stacking contributions due to the inclusion of loops. As expected, acidic pH stabilized all triplexes by allowing protonation of the cytosines in the third strand; however, the percentage of protonation increases as the molecularity decreases. The results help to choose adequate solution conditions for the study of triplexes containing different ratios of CGC+ and TAT base triplets and to aid in the design of oligonucleotide sequences as targeting reagents that could effectively react with mRNA sequences involved in human diseases, thereby increasing the feasibility of using the antisense strategy for therapeutic purposes.

Synthesis and application of molecular probe for detection of hydroxyl radicals produced by Na(125)I and gamma-rays in aqueous solution.

PURPOSE: To synthesize N-(3-(3-aminopropylamino)propyl)-2-oxo-2H-chromene-3-carboxamide (7), a novel DNA-binding, coumarin-based, fluorescent hydroxylradical ((*)OH) indicator and to assess its quantum efficiency compared with that of coumarin-3-carboxylic acid (1) and N1,N12-bis[2-oxo-2H-chromene-3-carbonyl]- 1,12-diamine-4,9-diazadodecane (9). MATERIALS AND METHODS: Using computer-generated molecular modeling, 7 and 9 and their respective 7-hydroxylated derivatives 8 and 10 were docked onto DNA dodecamer d(CGCGAATTCGCG)2, the ligand-DNA complexes were energy minimized, and binding free energies and inhibition constants were calculated. Compound 7 was judged an appropriate target molecule and was synthesized. Compounds 1, 7, and 9 were incubated with Na(125)I or irradiated with (137)Cs gamma-rays, and the influence of pH, dose, type of radiation, and the concentration of indicator on fluorescence yield were determined. RESULTS: Non-fluorescent 7 and 9 are converted to fluorescent, 7-hydroxylated derivatives 8 and 10 after interaction with (*)OH in aqueous solution. For 1, 7, and 9, hydroxylation yield increases linearly with both Na(125)I dose (0-700 x 10(6) decays) and (137)Cs dose (0-11.0 Gy). Fluorescence induction is significantly reduced at acidic pH and the fluorescent quantum yield of 8 is approximately 3 times that of 2 or 10 at pH 7.0. With Na(125)I incubation and gamma-ray irradiation, the fluorescence signal of 7 increases linearly with concentration and saturates at approximately 50 microM. CONCLUSION: Compound 7 quantifies lower concentrations of (*)OH than do 1 and 9. This detector is therefore likely to be a good reporter of (*)OH produced within a few nanometers of DNA.

Radiation damage during in situ electron microscopy of DNA-mediated nanoparticle assemblies in solution.

Oligonucleotide-nanoparticle conjugates, also called programmable atom equivalents, carry promise as building blocks for self-assembled colloidal crystals, reconfigurable or stimuli responsive functional materials, as well as bio-inspired hierarchical architectures in wet environments. In situ studies of the DNA-mediated self-assembly of nanoparticles have so far been limited to reciprocal space techniques. Liquid-cell electron microscopy could enable imaging such systems with high resolution in their native environment but to realize this potential, radiation damage to the oligonucleotide linkages needs to be understood and conditions for damage-free electron microscopy identified. Here, we analyze in situ observations of DNA-linked two-dimensional nanoparticle arrays, along with control experiments for different oligonucleotide configurations, to identify the mechanisms of radiation damage for ordered superlattices of DNA-nanoparticle conjugates. In a biological context, the results point to new avenues for studying direct and indirect radiation effects for small ensembles of DNA in solution by tracking conjugated nanoparticles. By establishing low-dose conditions suitable for extended in situ imaging of programmable atom equivalents, our work paves the way for real-space observations of DNA-mediated self-assembly processes.

Mechanisms of direct radiation damage in DNA, based on a study of the yields of base damage, deoxyribose damage, and trapped radicals in d(GCACGCGTGC)(2).

Dose-response curves were measured for the formation of direct-type DNA products in X-irradiated d(GCACGCGTGC)(2)prepared as dry films and as crystalline powders. Damage to deoxyribose (dRib) was assessed by HPLC measurements of strand break products containing 3' or 5' terminal phosphate and free base release. Base damage was measured using GC/ MS after acid hydrolysis and trimethylsilylation. The yield of trappable radicals was measured at 4 K by EPR of films X-irradiated at 4 K. With exception of those used for EPR, all samples were X-irradiated at room temperature. There was no measurable difference between working under oxygen or under nitrogen. The chemical yields (in units of nmol/J) for trapped radicals, free base release, 8-oxoGua, 8-oxoAde, diHUra and diHThy were G(total)(fr) = 618 +/- 60, G(fbr) = 93 +/- 8, G(8-oxoGua) = 111 +/- 62, G(8-oxoAde) = 4 +/- 3, G(diHUra) = 127 +/- 160, and G(diHThy) = 39 +/- 60, respectively. The yields were determined and the dose-response curves explained by a mechanistic model consisting of three reaction pathways: (1) trappable-radical single-track, (2) trappable-radical multiple-track, and (3) molecular. If the base content is projected from the decamer's GC:AT ratio of 4:1 to a ratio of 1:1, the percentage of the total measured damage (349 nmol/J) would partition as follows: 20 +/- 16% 8-oxoGua, 3 +/- 3% 8-oxoAde, 28 +/- 46% diHThy, 23 +/- 32% diHUra, and 27 +/- 17% dRib damage. With a cautionary note regarding large standard deviations, the projected yield of total damage is higher in CG-rich DNA because C combined with G is more prone to damage than A combined with T, the ratio of base damage to deoxyribose damage is approximately 3:1, the yield of diHUra is comparable to the yield of diHThy, and the yield of 8-oxoAde is not negligible. While the quantity and quality of the data fall short of proving the hypothesized model, the model provides an explanation for the dose-response curves of the more prevalent end products and provides a means of measuring their chemical yields, i.e., their rate of formation at zero dose. Therefore, we believe that this comprehensive analytical approach, combined with the mechanistic model, will prove important in predicting risk due to exposure to low doses and low dose rates of ionizing radiation.