DNA analysis by microfabricated capillary electrophoresis device. (25/618)

The LIGA (Lithographie Galvanoformung Abformung) process using synchrotron radiation lithography is applied to the microfabrication of capillary array electrophoresis (CAE) device. Laser-induced fluorescence detection system for the CAE device has been constructed by the modification of laser confocal fluorescence microscopy. DNA molecules were detected during migrating in the microchannels filled with polymer separation matrices under electric field to optimize the separation conditions for DNA analysis. Based on this observation, we demonstrated that microfabricated CAE device is realized the fast separation of DNA.  (+info)

Immobilization of DNA by UV irradiation and its utilization as functional materials. (26/618)

The water-insoluble DNA film was successfully prepared by UV irradiation. The DNA film was stable in water. It could effectively accumulated the DNA-binding intercalating materials, such as ethidium bromide, dibenzo-p-dioxin and benzo[a]pyrene, in their aqueous solutions. On the other hand, DNA was immobilized onto nonwoven cellulose fabrics, also by the UV irradiation. The DNA immobilized cloth was found to bind silver ions. The DNA-cloth containing silver ion showed antibacterial activity. The water-insoluble DNA prepared by UV irradiation has a potential ability to serve as biomaterials for medical, engineering and environmental objects.  (+info)

Interaction of a novel antitumor agent TAS-103 with DNA. (27/618)

Interaction of a novel antitumor agent TAS-103 with DNA has been studied by a variety of methods including thermal melting study, UV-Visible spectroscopy, 1H- and 31P-NMR spectroscopy. Thermal melting study indicated that TAS-103 stabilizes the double stranded form of DNA and the relative binding strength of TAS-103 is equal to that of ethidium bromide (EtBr). UV-Visible spectroscopy demonstrated that titration curves are nearly identical with all DNA oligomers producing a hypochromic and hypsochromic effect. A hypsochromic effect of TAS-103 is differ from typical intercalators such as EtBr and Actinomycin D that exhibit a bathochromic effect. 1H- and 31P-NMR spectroscopy revealed that TAS-103 has mainly two binding modes. Major binding mode is outside binding and minor binding mode is intercalation.  (+info)

The bleomycin amplification assay in V79 cells predicts frameshift mutagenicity of intercalative agents. (28/618)

We have recently reported on the use of a cell-based bleomycin amplification assay for the detection of DNA intercalating agents. In order to further validate this assay, two series of proprietary compounds were evaluated for frameshift mutagenesis in the Ames bacterial reversion system and for bleomycin amplification in the Chinese hamster V79 micronucleus system. It is shown that 10 of 11 frameshift-positive compounds were bleomycin amplifiers. These studies indicate that positive frameshift mutagenicity findings are consistent with expectations from the results of the bleomycin amplification assay, providing additional validation of the amplification assay for the detection of DNA intercalating agents. The studies also demonstrate that intercalation is necessary but not sufficient for frameshift mutagenesis since bleomycin amplifiers lacking frameshift mutagenic activity were also identified.  (+info)

Flavopiridol binds to duplex DNA. (29/618)

Flavopiridol, the first potent cyclin-dependent kinase inhibitor to enter clinical trials, was recently found to be cytotoxic to noncycling cells. The present studies were performed to examine the hypothesis that flavopiridol, like several other antineoplastic agents that kill noncycling cells, might also interact with DNA. Consistent with this possibility, treatment of A549 human lung cancer cells with clinically achievable concentrations of flavopiridol resulted in rapid elevations of the DNA damage-responsive protein p53. In further studies, the binding of flavopiridol to DNA was examined in vitro by four independent techniques. Absorption spectroscopy revealed that addition of DNA to aqueous flavopiridol solutions resulted in a red shift of the flavopiridol lambda(max) from 311 to 344 nm, demonstrating an isosbestic point typical of changes seen with DNA-binding compounds. Reverse-phase high-performance liquid chromatography demonstrated that flavopiridol binds to genomic DNA to a similar extent as ethidium bromide and Hoechst 33258. Nuclear magnetic resonance spectroscopy revealed that DNA caused extreme broadening of flavopiridol 1H nuclear magnetic resonance signals that could be reversed by addition of ethidium bromide or by DNA melting, suggesting that flavopiridol binds to (and likely intercalates into) duplex DNA. Equilibrium dialysis demonstrated that the equilibrium dissociation constant of the flavopiridol-DNA complex (5.4+/-3.4 x 10(-4) M) was in the same range observed for binding of the intercalators doxorubicin and pyrazoloacridine to DNA. Molecular modeling confirmed the feasibility of flavopiridol intercalation into DNA and analysis of the effects of flavopiridol in the National Cancer Institute tumor cell line panel using the COMPARE algorithm demonstrated that flavopiridol most closely resembles cytotoxic antineoplastic intercalators. Collectively, these data suggest that DNA might be a second target of flavopiridol, providing a potential explanation for the ability of this agent to kill noncycling cancer cells.  (+info)

Femtosecond linear dichroism of DNA-intercalating chromophores: solvation and charge separation dynamics of [Ru(phen)2dppz]2+ systems. (30/618)

The DNA-intercalating chromophore [Ru(phen)(2)dppz](2+) has unique photophysical properties, the most striking of which is the "light-switch" characteristic when binding to DNA. As a dimer, it acts as a molecular staple for DNA, exhibiting a remarkable double-intercalating topology. Herein, we report femtosecond dynamics of the monomeric and the covalently linked dimeric chromophores, both free in aqueous solution and complexed with DNA. Transient absorption and linear dichroism show the electronic relaxation to the lowest metal-to-ligand charge-transfer (CT) state, and subpicosecond kinetics have been observed for this chromophore for what is, to our knowledge, the first time. We observe two distinct relaxation processes in aqueous solution with time constants of 700 fs and 4 ps. Interestingly, these two time constants are very similar to those observed for the reorientational modes of bulk water. The 700-fs process involves a major dichroism change. We relate these observations to the change in charge distribution and to the time scales involved in solvation of the CT state. Slower processes, with lifetimes of approximately 7 and 37 ps, were observed for both monomer and dimer when bound to DNA. Such a difference can be ascribed to the change of the structural and electronic relaxation experienced in the DNA intercalation pocket. Finally, the recombination lifetime of the final metal-to-ligand CT state to the ground state, which is a key in the light-switch process, is found in aqueous solution to be sensitive to structural modification, ranging from 260 ps for [Ru(phen)(2)dppz](2+) and 360 ps for the monomer chromophore derivative to 2.0 ns for the dimer. This large change reflects the direct role of solvation in the light-switch process.  (+info)

Potent inhibition of werner and bloom helicases by DNA minor groove binding drugs. (31/618)

Maintenance of genomic integrity is vital to all organisms. A number of human genetic disorders, including Werner Syndrome, Bloom Syndrome and Rothmund-Thomson Syndrome, exhibit genomic instability with some phenotypic characteristics of premature aging and cancer predisposition. Presumably the aberrant cellular and clinical phenotypes in these disorders arise from defects in important DNA metabolic pathways such as replication, recombination or repair. These syndromes are all characterized by defects in a member of the RecQ family of DNA helicases. To obtain a better understanding of how these enzymes function in DNA metabolic pathways that directly influence chromosomal integrity, we have examined the effects of non-covalent DNA modifications on the catalytic activities of purified Werner (WRN) and Bloom (BLM) DNA helicases. A panel of DNA-binding ligands displaying unique properties for interacting with double helical DNA was tested for their effects on the unwinding activity of WRN and BLM helicases on a partial duplex DNA substrate. The levels of inhibition by a number of these compounds were distinct from previously reported values for viral, prokaryotic and eukaryotic helicases. The results demonstrate that BLM and WRN proteins exhibit similar sensitivity profiles to these DNA-binding ligands and are most potently inhibited by the structurally related minor groove binders distamycin A and netropsin (K(i) +info)

Robust charge transport in DNA double crossover assemblies. (32/618)

BACKGROUND: Multiple-stranded DNA assemblies, encoded by sequence, have been constructed in an effort to self-assemble nanodevices of defined molecular architecture. Double-helical DNA has been probed also as a molecular medium for charge transport. Conductivity studies suggest that DNA displays semiconductor properties, whereas biochemical studies have shown that oxidative damage to B-DNA at the 5'-G of a 5'-GG-3' doublet can occur by charge transport through DNA up to 20 nm from a photo-excited metallointercalator. The possible application of DNA assemblies, in particular double crossover (DX) molecules, in electrical nanodevices prompted the design of a DNA DX assembly with oxidatively sensitive guanine moieties and a tethered rhodium photo-oxidant strategically placed to probe charge transport. RESULTS: DX assemblies support long-range charge transport selectively down the base stack bearing the intercalated photo-oxidant. Despite tight packing, no electron transfer (ET) crossover to the adjacent base stack is observed. Moreover, the base stack of a DX assembly is well-coupled and less susceptible than duplex DNA to stacking perturbations. Introducing a double mismatch along the path for charge transport entirely disrupts long-range ET in duplex DNA, but only marginally decreases it in the analogous stack within DX molecules. CONCLUSIONS: The path for charge transport in a DX DNA assembly is determined directly by base stacking. As a result, the two closely packed stacks within this assembly are electronically insulated from one another. Therefore, DX DNA assemblies may serve as robust, insulated conduits for charge transport in nanoscale devices.  (+info)