DNA interactions and photocatalytic strand cleavage by artificial nucleases based on water-soluble gold(III) porphyrins.

The novel gold porphyrin complex (5,10,15-tris(N-methylpyridinium-4-yl)-20-(1-pyrenyl)-porphyrinato)gold(III) chloride, [Au(III)(TMPy3Pyr1P)]Cl4, was prepared and characterized by optical spectroscopy, high-resolution nuclear magnetic resonance (NMR), and electrospray mass spectrometry. This cationic multichromophore compound exhibits excellent water solubility and does not form aggregates under physiological conditions. Binding interactions of this complex and related model compounds with nucleic acid substrates have been studied and characterized by NMR and circular dichroism spectroscopy. The photoreactivity of [Au(III)(TMPy3Pyr1P)]Cl4 was investigated under anaerobic and aerobic conditions in the presence of an excess of purine nucleoside, guanosine, and plasmid DNA. Photocatalytic oxidative degradation of guanosine and the change from supercoiled to circular plasmid DNA upon monochromatic irradiation and polychromatic blue-light exposure with a maximum at 420 nm was explored. The potential of the novel water-soluble cationic metallointercalator complex [Au(III)(TMPy3Pyr1P)]Cl4 to serve as a catalytic photonuclease for the cleavage of DNA has been demonstrated.

Fast repair activities towards dGMP hydroxyl radical adducts by silybin and its analogues.

The repair activities of silybin (SLB) and its analogues towards the oxidizing deoxyguanosine monophosphate (dGMP) hydroxyl radical adducts are investigated by pulse radiolytic techniques. On pulse irradiation of nitrous oxide saturated 2.0 mM dGMP aqueous solution containing 0.1 mM silybin at neutral pH, the transient absorption spectrum of the dGMP hydroxyl radical adducts decreases with the formation of the phenoxyl radical of silybin within tens of microseconds, indicating that there is a repair reaction between the dGMP hydroxyl radical adduct and silybin. The rate constant of the repair reaction is calculated to be 1.0 x 10(9) M(-1)s(-1) for silybin. The repair activity of hesperetin (HESP), naringin (NAN) and naringenin (NAR) towards hydroxyl radical adducts of dGMP are also studied.

Radicals produced by gamma-irradiation of hyperquenched glassy water containing 2'-deoxyguanosine-5'-monophosphate.

Hyperquenched glassy water (HGW) has been suggested as the best model for liquid water, to be used in low-temperature studies of indirect radiation effects on dissolved biomolecules (Bednarek et al. J. Am. Chem. Soc. 1996, 118, 9387). In the present work, these effects are examined by X-band electron spin resonance spectroscopy (ESR) in gamma-irradiated HGW matrix containing 2'-deoxyguanosine-5'-monophosphate. Analysis of the complex ESR spectra indicates that, in addition to OH(*) and HO2(*) radicals generated by water radiolysis, three species are trapped at 77 K:(i) G(C8)H(*) radical, the H-adduct to the double bond at C8; (ii) G(- *) radical anion, the product of electron scavenging by the aromatic ring of the base; and (iii) dR(-H)(*) radicals formed by H abstraction from the sugar moiety, predominantly at the C'5 position. We discuss the yields of the radicals, their thermal stability and transformations, as well as the effect of photobleaching. This study confirms our earlier suggestion that in HGW the H atom addition/abstraction products are created at 77 K in competition with HO2(*) radicals, in a concerted process following ionization of water molecule at L-type defect sites of the H-bonded matrix. The lack of OH(*) reactivity toward the solute suggests that the H-bonded structure in HGW is much more effective in recombining OH(*) radicals than that of aqueous glasses obtained from highly concentrated electrolyte solutions. Furthermore, complementary experiments for the neat matrix have provided evidence that HO2(*) radicals are not the product of H atom reaction with molecular oxygen, possibly generated by ultrasounds used in the process of sample preparation.

Oxidation of 2'-deoxyguanosine 5'-monophosphate photoinduced by pterin: type I versus type II mechanism.

UV-A radiation (320-400 nm) induces damage to the DNA molecule and its components through different photosensitized reactions. Among these processes, photosensitized oxidations may occur through electron transfer or hydrogen abstraction (type I) and/or the production of singlet molecular oxygen ((1)O2) (type II). Pterins, heterocyclic compounds widespread in biological systems, participate in relevant biological processes and are able to act as photosensitizers. We have investigated the photosensitized oxidation of 2'-deoxyguanosine 5'-monophosphate (dGMP) by pterin (PT) in aqueous solution under UV-A irrradiation. Kinetic analysis was employed to evaluate the participation of both types of mechanism under different pH conditions. The rate constant of (1)O2 total quenching (k(t)) by dGMP was determined by steady-state analysis of the (1)O2 NIR luminescence, whereas the rate constant of the chemical reaction between (1)O2 and dGMP (k(r)) was evaluated from kinetic analysis of concentration profiles obtained by HPLC. The results show that the oxidation of dGMP photosensitized by PT occurs through two competing mechanisms that contribute in different proportions depending on the pH. The dominant mechanism in alkaline media involves the reaction of dGMP with (1)O2 produced by energy transfer from the PT triplet state to molecular oxygen (type II). In contrast, under acidic pH conditions, where PT and the guanine moiety of dGMP are not ionized, the main pathway for dGMP oxidation involves an initial electron transfer between dGMP and the PT triplet state (type I mechanism). The biological implications of the results obtained are also discussed.

Cooperative effects in the photophysical properties of self-associated triguanosine diphosphates.

The present study deals with the photophysical properties of triguanosine diphosphate in aqueous solutions, which are compared with those of the 2'-deoxyguanosine monophosphate. They are studied by steady-state absorption and fluorescence spectroscopy as well as by time-resolved fluorescence spectroscopy with femtosecond resolution. The temperature, salt and concentration dependence of the absorption and fluorescence spectra reveal that association of the trimers takes place. The resulting aggregates could correspond to a tetraplex structure. The aggregate fluorescence quantum yield is higher and the fluorescence lifetime much longer than those of the monomer. These results show the interaction between guanosine residues that may manifest itself via self-solvation, hydrogen bonding and/or delocalization of the excitation.

Oxidative damage to nucleic acids photosensitized by titanium dioxide.

The semiconductor TiO2 is known to have photobiological activity in prokaryotic and eukaryotic cells. Applications of this photobiological activity have been suggested including sterilization of waste water and phototherapy of malignant cells. Here, several model and cellular systems were used to study the mechanism of photocatalysis by TiO2. Treatment of TiO2 (anatase, 0.45 microns), suspended in water containing a spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), with UV radiation (320 nm) resulted in an electron spin resonance (ESR) signal characteristic of the hydroxyl radical. Irradiation of solutions containing calf thymus DNA and TiO2 with UVA (320-400 nm) radiation resulted in hydroxylation of guanine bases. The degree of hydroxylation was dependent on both UVA fluence and amount of TiO2 in suspension. Human skin fibroblasts, preincubated 18 h with 10 micrograms/cm2 TiO2 and then UVA-irradiated (0-58 KJ/m2), showed dose dependent photocytoxicity. RNA, isolated from similarly treated fibroblasts, contained significant levels of photooxidation, measured as hydroxylation of guanine bases. However, no oxidative damage was detectable in cellular DNA. These results suggest that nucleic acids are a potential target for photooxidative damage sensitized by TiO2, and support the view that TiO2 photocatalyzes free radical formation.

Reaction of cysteamine with individual DNA base radicals in gamma-irradiated nucleotides at low temperature.

An ESR investigation of the individual DNA base radicals produced by gamma-irradiation of frozen solutions of the nucleotides TMP, dCMP, dGMP and dAMP and their reactions with cysteamine upon annealing is reported. The results show that water radicals in bulk ice do not lead to the formation of DNA or cysteamine radicals. Radicals from the oxidation pathway which include the DNA base one electron oxidized radicals and their successors, G(C8)OH., A(C8)OH. and thymine dimers (.Tdi) and/or T(C6)OH; readily react with cysteamine to form RS. and ultimately RSSR-. Reactions of dGMP and dCMP radicals from the oxidation pathway with cysteamine occur at lower temperatures than those of dAMP and TMP, suggesting hole migration. Both T(C6)H. and C(N3)H. react with cysteamine to form RS. and diamagnetic products, but G(C8)H. and A(C8)H. do not. Subtraction of the anion radical T-. and its proton adduct T(C6)H. from the total radical yield of TMP (with or without cysteamine) suggests that somewhat less than half of the total TMP radicals found are a result of the oxidative pathway. Similar results are found in the other nucleotides. The total spectral intensity derived from the radicals from the oxidative pathway such as G(C8)OH., A(C8)OH. and .Tdi/T(C6)OH. are somewhat less than that for the protonated anion radicals. Only one non-base radical is identified, a sugar radical at the C(1)' site on the deoxyribose portion of dAMP. This species, S(A)., is also found to react with cysteamine or its disulfide radical anion. Analyses performed in the presence and absence of a thiol are found to allow for a clear separation of oxidative and reductive pathways.

Photoinduced interaction of Ru(bpy)3 2+ with nucleotides and nucleic acids in the presence of S2O8 2-; a transient conductivity study.

The photochemical reactions of Ru(bpy)3(2+) with single- and double-stranded DNA, polynucleotides and purine-containing nucleotides in argon-saturated aqueous solution in the presence of S2O8(2-) were studied using time-resolved absorption and conductivity methods. The conversion of Ru(bpy(3(3+) to Ru(bpy)3(2+), monitored spectroscopically either after rapid mixing with substrate or after laser flash excitation (lambda exc = 353 nm) is quantitative at nucleotide-to-sensitizer ratios [N]/[S] of 1-2 for DNA and other guanine-containing compounds. Conductivity measurements following the laser pulse revealed a fast conductivity increase (rise time, less than 0.1 ms) due to the formation of protons and, to a lesser degree, to charged species of much lower ion mobility. A slower component in the 0.01-1 s range was observed for nucleic acids; its amplitude is markedly reduced at pH 6-9. In buffered neutral solution the signal is replaced by a slight decrease in conductivity. Electronically excited Ru(bpy)3(2+) bound to DNA reacts with S2O8(2-) to form Ru(bpy)3(3+) and SO4(.-) as primary oxidizing species both of which react with bases. The resulting base radicals react subsequently with Ru(bpy)3(3+) and Ru(bpy)3(2+) or the ligands in the ruthenium complex, producing protons which give rise to the slower conductivity increase. The formation of single-strand breaks and the ensuing release of condensed counterions does not appear to contribute significantly to the slow component. The transient conductivity behaviour is sensitive to the single- or double-stranded nature of DNA.

Free radical formation in single crystals of 2'-deoxyguanosine 5'-monophosphate tetrahydrate disodium salt: an EPR/ENDOR study.

Single crystals of 2'-deoxyguanosine 5'-monophosphate were X-irradiated at 10 K and at 65 K, receiving doses between 4.5 and 200 kGy, and studied using K-band EPR, ENDOR, and field-swept ENDOR (FSE) spectroscopy. Evidence for five base-centered and more than nine sugar-centered radicals was found at 10 K following high radiation doses. The base-centered radicals were the charged anion, the N10-deprotonated cation, the C8 H-addition radical, a C5 H-addition radical, and finally a stable radical so far unidentified but with parameters similar to those expected for the charged cation. The sugar-centered radicals were the H-abstraction radicals centered at C1', C2', C3', and C5', an alkoxy radical centered at O3', a C5'-centered radical in which the C5'-O5' phosphoester bond appears to be ruptured, a radical tentatively assigned to a C4'-centered radical involving a sugar-ring opening, as well as several additional unidentified sugar radicals. Most radicals were formed regardless of radiation doses. All radicals formed following low doses (4.5-9 kGy) were also observed subsequent to high doses (100-200 kGy). The relative amount of some of the radicals was dose dependent, with base radicals dominating at low doses, and a larger relative yield of sugar radicals at high doses. Above 200 K a transformation from a sugar radical into a base radical occurred. Few other radical transformations were observed. In the discussion of primary radicals fromed in DNA, the presence of sugar-centered radicals has been dismissed since they are not apparent in the EPR spectra. The present data illustrate how radicals barely traceable in the EPR spectra may be identified due to strong ENDOR resonances. Also, the observation of a stable radical with parameters similar to those expected for the charge guanine cation is interesting with regard to the nature of the primary radicals stabilized in X-irradiated DNA.

Radiation chemistry of 2'-deoxycytidylyl-(3'-5')-2'-deoxyguanosine and its sequence isomer in N2O- and O2-saturated solutions.

The radiation chemistry of the dinucleoside monophosphate d(CpG) and its sequence isomer, d(GpC), has been examined in aqueous solutions saturated with either N2O or O2. The products were isolated using HPLC, and the major products were identified using proton NMR spectroscopy and mass spectrometry. The major products include 5,6-dihydroxy-5,6-dihydrouracil (glycol) derivatives, 5- and 6-hydroxycytosine substitution products, 1-carbamoyl-2-oxo-4,5-dihydroxyimidazolidine products, and the 8-hydroxyguanine substitution product. Both trans stereoisomers of the imidazolidine derivatives are obtained from d(CpG) as well as from its sequence isomer. These are prominent products when the irradiation is carried out in the presence of oxygen, but they are not observed in the absence of oxygen.

Free radical formation in crystals of 2'-deoxyguanosine 5'-monophosphate irradiated at 15 K: an ESR study.

Radiation-induced radicals in single crystals of 2'-deoxyguanosine 5'-monophosphate (5'-dGMP) at 15 K have been studied by electron spin resonance (ESR) spectroscopy. At low temperatures three radicals were analyzed in detail. The negatively charged pi anion of the guanine base completely dominated the spectra. Weaker resonances were due to an alkoxy radical with the spin density in the C3'-O3' region of the sugar moiety as well as another sugar-centered radical. The anion rapidly decayed upon exposure to uv light at 15 K or by annealing above 25 K. In both cases no successor radical was observed. The second sugar-centered radical decays at 200 K with a concomitant appearance of the resonance from the C8 H-addition radical. By annealing at 295 K the latter resonance was the only one observed. After irradiation at 295 K, however, an additional resonance from a sugar-centered radical, which has been analyzed previously by B. Rakvin and J. N. Herak (Radiat. Res. 88, 240-250 (1981)) was observed. A reinvestigation of this resonance was performed.

Free radicals from single crystals of deoxyguanosine 5'-monophosphate (Na salt) irradiated at low temperatures.

In single crystals of the DNA nucleotide 2'deoxyguanosine-5'phosphate (5'dGMP) X- or gamma-irradiated at 4.2 K or 15 K, two primary radical species can be discriminated and assigned to the cation and anion of the guanine base, G(+) and G(-). Both species are unstable. G(-) partially transforms into a secondary radical at 4.2 K, the latter being the precursor to the dominant 300 K species formed by net H-addition to carbon C8. The secondary radical, together with another intermediate appearing at 77 K and perhaps connected with the anion decay could not be structurally identified. The guanine cation G(+) transforms upon annealing to temperatures above 77 K into a more stable species by deprotonation at position N1.

Pulse radiolytic study of the interaction of thiols and ascorbate with OH adducts of dGMP and dG: implications for DNA repair processes.

Using the technique of pulse radiolysis, the interaction of .OH radicals with 2'-deoxyguanosine (dG) and dG-5'-monophosphate (dGMP) has been shown to result in the production of intermediates with different redox properties as demonstrated by their reactions with tetranitromethane (TNM) and N,N,N',N'-tetramethyl-p-phenylenediamine. The ratio of the yields of oxidizing to reducing-type .OH radical adducts of dGMP was determined to be about 1:1 and independent of pH (6-11). The nature of the intermediates produced on reaction of .OH with dGMP are discussed. The thiols, cysteine, glutathione, mercaptoacetic acid, and ascorbate have been shown to interact with those .OH adducts of dGMP and dG with oxidizing properties preferentially via an electron transfer process (k approximately 3 X 10(7)-1.4 X 10(9) dm3 mole-1 sec-1) as implied from the pH dependence of the rate constants. It is further demonstrated that oxygen and TNM do not interact with those .OH adducts of the purines with oxidizing properties. The implications of these findings are discussed with reference to the mechanistic aspects of radioprotection and especially of radiosensitization.