Natural DNA assisted white light generation and stimuli responsive colour tuning.

The unique structure of a natural nucleic acid, calf thymus DNA, which can provide an appropriate scaffold for an efficient cascaded energy transfer among organic chromophores, has been used for the generation of bright and pure white light on UV light excitation. Two most commonly used DNA stains, 4',6-diamidino-2-phenylindole (DAPI) and ethidium bromide (EB) have been used as a part of the donor-acceptor pairs. We have judiciously selected 10-anthracene-10-yl-3-methylbenzothiazol-3-ium chloride (AnMBTZ), an ultrafast molecular rotor, to act as a bridge between DNA bound DAPI and EB for the cascaded flow of energy. The unique molecular rotor properties of AnMBTZ and its exceptional binding ability with natural DNA help to form a distinct tri-chromophoric system in DNA template which can produce bright and pure white light on UV excitation. Detailed flow of energy from photoexcited DAPI to EB via AnMBTZ has been explored using steady state and time-resolved emission spectroscopy. Further, unique binding nature of AnMBTZ with DNA molecules has been used to modulate the colour of the emission from the present tri-chromophoric system by external stimuli, like salt and temperature. Such unique stimuli responsive multi-chromophoric system in a bio-template has great potential for different lightening applications.

Decomposition of DNA staining agent ethidium bromide by gamma irradiation: Conditions, kinetics, by-products, biological activity, and removal from wastewater.

Ethidium Bromide (Eth-Br) is an intercalating agent commonly used in medical and biological laboratories as a DNA staining dye. Despite its popular use, aqueous solutions containing Eth-Br showed high toxicity, mutagenic capacity, and deactivate DNA transcription. In this study, the removal of Eth-Br from aqueous solutions by gamma irradiation has been fully investigated. Gamma irradiation was capable of achieving a near complete removal of Eth-Br in neutral and non-buffered aqueous solutions at an absorbed dose of 15 kGy. Various experimental conditions were studied and showed that the removal efficiency is not diminished. The addition of hydrogen peroxide (2 %) to the irradiated solutions reduced the D50 and D90 by 50 %. Modeling Eth-Br decomposition showed that the reaction followed pseudo first-order kinetics and reaches at least 90 % removal under all experimental conditions. TOC and HPLC measurements confirmed that Eth-Br is fully mineralized when the absorbed dose reaches 15 kGy. The biological activity of Eth-Br after irradiation treatment was investigated with synthetic DNA and natural DNA. The biological activity of Eth-Br was deactivated at an absorbed dose as low as 5 kGy. Toxicity measurement with E-coli bacteria also confirmed that the absorbed dose of 5 kGy was sufficient to remove Eth-Br toxicity.

Visible light irradiation of ethidium bromide-stained interphase nuclei causes DNA-protein linking and structural stabilization of nucleoprotein complexes.

Fixation of DNA and proteins in the isolated rat hepatocyte nuclei stained with ethidium bromide and irradiated with visible light was analyzed in this study. It was shown that irradiation results in the following modifications of higher-level nucleoprotein complexes of interphase chromatin: (1) the complexes acquire resistance to decondensing treatments, which may be indicative of the formation of links between proteins or proteins and DNA in the chromatin; (2) the linking rate for both DNA and proteins is dose dependent; (3) the irradiation induces intermolecular link formation between DNA molecules, which brings about an increase in the average molecular weight of DNA fragments; (4) some modifications (dimerization, etc.) of histones and nonhistone proteins occur; and (5) histone proteins are not effectively cross-linked to DNA. The structural stabilization of interphase chromatin is possibly mediated by free radical-based mechanisms, whereas disulfide bonds seem to play no significant role in the cross-linking.

Luminescence quenching by DNA-bound viologens: effect of reactant identity on efficiency and dynamics of electron transfer in DNA.

Photoinduced electron transfer from two intercalating photoactive donors, Ru(phen)2dppz2+ and ethidium, to intercalating viologen acceptors of the N,N'-dialkyl-6-(2'-pyridiniumyl)phenanthridinium family has been investigated through steady-state and time-resolved luminescence quenching measurements. Efficient quenching of the emission from these donors bound to DNA is observed at low concentrations of acceptor (1-10 eq.), and in time-resolved emission experiments it is determined that electron transfer occurs on the nanosecond time scale. Furthermore, transient absorption measurements confirm that the quenching is the result of a charge-transfer process; upon photoreaction of intercalated Ru(phen)2dppz2+ with a viologen acceptor, an intermediate with spectral properties resembling the expected charge-separated pair is observed. The quenching yields and kinetics obtained with this quencher are in marked contrast to those observed with these same donors paired with Rh(phi)2bpy3+ as an acceptor. The differing efficiencies of electron transfer for these donor/acceptor pairs bound to DNA as compared to others previously described are discussed qualitatively in terms of the structural and electronic properties of the different reactants.

Long-range and short-range oxidative damage to DNA: photoinduced damage to guanines in ethidium-DNA assemblies.

Short-range and long-range photoreactions between ethidium and DNA have been characterized. While no DNA reaction is observed upon excitation into the visible absorption band of ethidium, higher-energy irradiation (313-340 nm) leads both to direct strand cleavage at the 5'-G of 5'-GG-3' doublets and to piperidine-sensitive lesions at guanine. This reactivity is not consistent with oxidation of guanine by either electron transfer or singlet oxygen as shown by comparison with reactions of a rhodium intercalator and methylene blue, respectively. By covalently tethering ethidium to one end of a DNA duplex, we demonstrate the presence of two distinct reactions, one short-range and the other long-range. The short-range reaction involves a covalent modification of guanine by ethidium, based upon HPLC analysis of the nucleoside products and studies with ethidium derivatives. The long-range reaction is entirely consistent with oxidation of guanine by DNA-mediated electron transfer. The yield of this electron-transfer reaction is not attenuated with distance; equal yields of guanine damage are observed at a proximal (17 A Et-GG separation) and distal (44 A Et-GG separation) site. These results are quite similar to those previously observed with a covalently tethered rhodium photooxidant and underscore the unique ability of the DNA base stack to facilitate long-range electron transfer so as to effect oxidative damage from a distance.

Single UV excitation of Hoechst 33342 and ethidium bromide for simultaneous cell cycle analysis and viability determinations on in vitro cultures of murine B lymphocytes.

Assessment of DNA content by flow cytometry has largely depended on staining techniques which do not permit exclusion of dead cells from the data set. During studies of B cell activation in vitro, the large number of nonviable cells greatly affects the cell cycle distribution and thus the accurate evaluation of proliferation flow cytometry. This report describes the development of two dual staining techniques which use Hoechst 33342 and ethidium bromide excited by a single UV source to eliminate dead cells from the DNA histogram of the viable cells in murine B cell cultures. Hoechst 33342 and 0.62 micrograms/ml of ethidium bromide permit the evaluation of cell cycle distributions on the viable cells with a ratio gate. The combination of Hoechst 33342 and 6.2 micrograms/ml ethidium bromide results in the resolution of the two populations due to fluorescence energy transfer with a single PMT. Using this technique we demonstrated the simultaneous determination of DNA and RNA content on viable cells using only two PMTs. Both these techniques can be performed on either a laser or an arc lamp flow cytometer where CVs of less than 7% and as low as 3.2% are normally achieved. Determination of the S phase using these techniques produces a high correlation with DNA synthesis determined by radiolabeled precursor determination. These techniques permit the use of flow cytometry to determine proliferation during B cell activation.

Mutation analysis of genetic diseases by asymmetric-PCR SSCP and ethidium bromide staining: application to neurofibromatosis and cystic fibrosis.

The Single Strand Conformation Polymorphism (SSCP) technique is widely used in mutation analysis. We have introduced several modifications to the SSCP method, which overcome the problem of incomplete denaturation or reannealing of DNA during electrophoresis. The modifications consist of asymmetrical PCR amplification of the sequence of interest, electrophoresis with a higher concentration of acrylamide, and the analysis of the DNA fragments under u.v. light. We have applied this method to the analysis of two specific diseases: neurofibromatosis type 1 (NF1) and cystic fibrosis (CF) from PCR amplified exons. Two single nucleotide changes were observed with this method.

Cytotoxicity and SOS-inducing ability of ethidium and photoactivable analogs on E. coli ethidium-bromide-sensitive (Ebs) strains.

In a recently-characterized ethidium-bromide-sensitive E. coli strain, DNA appears to be much more accessible to DNA-binding agents. This strain therefore appears to be of interest for studying the mutagenic properties of chemicals. For this purpose, a series of ethidium-sensitive E. coli strains (Ebs) with normal and defective DNA-repair capacity was constructed and made lysogenic for lambda (sfiA::lacZ). These strains were used to study the cytotoxicity and SOS-inducing ability of ethidium and its two photoactivable analogs 8-azido- and 3,8-diazido-ethidium. When non-covalent DNA complexes are formed, these dyes elicit only a bacteriostatic effect in the Ebs strains, which is almost independent of the strain's DNA-repair capacity. The SOS system is not induced. When covalent DNA adducts are formed after photoactivation of ethidium azido analogs, the effects are quite different. The formation of about 5 DNA monoadducts per cell induces a lethal hit in the Ebs uvrB recA strain and measurable SOS induction in the Ebs uvrB (lambda (sfiA::lacZ) strain. The formation of more than 1000 DNA adducts in the Ebs strain with normal DNA-repair capacity does not induce any measurable cytotoxic effect.

[Inductive-resonance energy transfer between chromophores localized in different parts of irradiated and nonirradiated erythrocyte ghosts]. / Induktivno-rezonansnyi perenos énergii mezhdu khromoforami, lokalizovannymi v raznykh uchastkakh obluchennykh i neobluchennykh tenei éritrotsitov.

A study was made of the inductive-resonance energy transfer between chromophore pairs, tryptophan--pyren, tryptophan--1.8-anilinonaphthalene sulfonate (ANS), puren--1.8-ANS, diphenylhexatrien--ethidium, and 1,8-ANS--ethidium, in irradiated (250 Gy) and nonirradiated preparations of erythrocytic membranes. The radiation--induced decrease in the energy transfer was noted in the pairs in which one of the chromophores was localized in a lipid phase. On the basis of the data obtained it is suggested that irradiation of membranes causes the lesions in them which lead to a reduction of the effective thickness of a hydrophobic part of the lipid bilayer.

Interaction of ethidium bromide with heparin.

The reactions of hydrated electrons produced during pulse radiolysis have been utilized to investigate the binding of ethidium bromide to heparin. Complexes of ethidium bromide and heparin can be dissociated with salt. Divalent cations are more effective than monovalent cations in this respect. Pulse-radiolysis investigations at different temperatures indicate that the thermodynamic parameters governing the interaction of ethidium bromide with heparin are deltaH' = 11-6 kcal mole-1 and deltaS' = 42-6 cal deg-1 mole-1.

Application of pulse radiolysis to the study of drug interactions with biological macromolecules.

Pulse radiolysis has been used to study the interactions of a variety of small molecules with DNA. The diffusion-controlled rate constants for the reactions of e(aq) with dyes, cations and other small molecules including radioprotectors and radiosensitizers, were found to decrease remarkably upon association with DNA. These kinetic data can be used to quantitate the degree and extent of such interactions. This technique may have wide practical application for the in situ study of many small molecule-macromolecule associations, under physiological conditions.