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We demonstrate a rapid and sensitive method for DNA detection without the need for fluorescence. This is based on carbon-coated magnetic iron (Fe) microparticles with a covalent surface attachment of DNA. We show that these magnetic microparticles can capture complementary DNA. Significantly, the DNA covalent surface bonds are robust to high temperatures and can be included in a sample during polymerase chain reaction (PCR). This method is employed for the detection of targeted DNA sequences (40-50 bp). Hybridization probes on the surface of the magnetically susceptible Fe microparticle recognize the target DNA sequence-specifically. The double-stranded DNA (dsDNA) microparticles are then quickly captured with a magnet from the sample matrix. This foregoes postpurification processes, such as electrophoresis, which make our technique time- and cost-effective. Captured dsDNA can be detected with intercalating dyes such as ethidium bromide through a loss in the UV absorption signal with a limit of detection (LOD) of 24 nM within 15 min. Likewise, surface-bound DNA can act as a primer in PCR to decrease the LOD to 5 pM within 2 h. This is the first instance of a nucleotide-modified magnetically susceptible carbon substrate that is PCR-compatible. Besides DNA capture, this strategy can eventually be applied to sequence-specific nucleic acid purification and enrichment, PCR cleanup, and single-strand generation. The DNA-coated particles are stable under PCR conditions (unlike commonly used polystyrene or gold particles).

The graphene-based materials with unique, versatile, and tunable properties have brought new opportunities for the leading edge of advanced nanobiotechnology. In this regard, the use of graphene in gene delivery applications is still at early stages. In this study, we successfully designed a new complex of carboxylated-graphene (G-COOH) with ethidium bromide (EtBr) and used it as a nanovector for efficient gene delivery into the AGS cells. G-COOH, with carboxyl functions on its surface, in the presence of EtBr, formaldehyde, and cyclohexylisocyanide were participated in Ugi four component reaction to fabricate a stable amphiphilic graphene-EtBr (AG-EtBr) composite. The coupling reaction was confirmed by further analyses with FT-IR, AFM, UV-vis, Raman, photoluminescence, EDS, and XPS. The AG-EtBr nanocomposite was able to interact with a plasmid DNA (pDNA). This nanocomposite has been applied for transfection of cultured mammalian cells successfully. Moreover, the AG-EtBr composites showed a remarkable decreased cytotoxicity in compared to EtBr. Interestingly, the advantages of AG-EtBr in cell transfection are more dramatic (3-fold higher) than Lipofectamine2000 as a commercial nonviral vector. To the best of our knowledge, this is the first report in which EtBr is used as an intercalating agent along with graphene to serve as a new vehicle for gene delivery application. [ABSTRACT FROM AUTHOR]

Mimicking proton conduction mechanism of Nafion to construct novel proton-conducting materials with low cost and high proton conductivity is of wide interest. Herein, we have designed and synthesized a cationic covalent organic framework with high thermal and chemical stability by combining a cationic monomer, ethidium bromide (EB) (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide), with 1,3,5-triformylphloroglucinol (TFP) in Schiff base reactions. This is the first time that the stable cationic crystalline frameworks allowed for the fabrication of a series of charged COFs (EB-COF:X, X = F, Cl, Br, I) through ion exchange processes. Exchange of the extra framework ions can finely modulate the COFs' porosity and pore sizes at nanoscale. More importantly, by introducing PW12O403- into this porous cationic framework, we can greatly enhance the proton conductivity of ionic COF-based material. To the best of our knowledge, EB-COF:PW12 shows the best proton conductivity at room temperature among ever reported porous organic materials. [ABSTRACT FROM AUTHOR]

Celedon et al. reported an unexpectedly low slope of applied torque vs turns (or apparent torsional rigidity) for a long DNA subject to 0.8 pN tension and modest negative torques (up to approximately -5 pN nm) in 3.4 × 10 -9 M ethidium ( J. Phys. Chem. B 2010 , 16929-16935). Extrusion of inverted repeat sequences to create cruciforms with anomalously large association constants for binding 4 ethidiums to the cruciform arms is investigated as a possible explanation for this observation and also for its compatibility with other observations of Celedon et al. The equilibrium between the linear main chain and cruciform states of an inverted repeat sequence under the prevailing tension, torque, and ethidium concentration is treated by first computing the free energy per bp of the linear main chain. This is done for a complex model, wherein every bp in the linear main chain participates in both the recently reviewed cooperative two-state 114 , 16929-16935). Extrusion of inverted repeat sequences to create cruciforms with anomalously large association constants for binding 4 ethidiums to the cruciform arms is investigated as a possible explanation for this observation and also for its compatibility with other observations of Celedon et al. The equilibrium between the linear main chain and cruciform states of an inverted repeat sequence under the prevailing tension, torque, and ethidium concentration is treated by first computing the free energy per bp of the linear main chain. This is done for a complex model, wherein every bp in the linear main chain participates in both the recently reviewed cooperative two-state a ⇔ b equilibrium ( Quarterly Reviews of Biophysics , e5, 1-25) and in ethidium binding with a modest relative preference for either the 2021 , 54 , e5, 1-25) and in ethidium binding with a modest relative preference for either the a M ethidium. Besides a large drop in b -state. Plausible assumptions are made concerning the relative populations of the cruciform and linear main chain states of an inverted repeat, and also the relative populations of cruciform states with and without 4 bound ethidiums in the presence of tension, torque, and 3.4 × 10 -9 M ethidium. Besides a large drop in slope M ethidium, this theory also predicts maxima between 6.4 × 10 -9 to 10 -8 M ethidium, a region where no measurements were made. Overall agreement between theoretical and experimental values of the -8 (or apparent torsional rigidity), and also the number of negative turns due to bound ethidium at zero torque, is fairly good for all of the ethidium concentrations studied by Celedon et al., provided that there is a modest preference for binding to the -7 M ethidium, a region where no measurements were made. Overall agreement between theoretical and experimental values of the slope -state, the theory significantly underestimates experimental values at the higher ethidium concentrations, likely ruling out that possibility.b -state. When there is a modest preference for binding to the a -state, the theory significantly underestimates experimental values at the higher ethidium concentrations, likely ruling out that possibility.

Ionic liquids (ILs) are salts with poor ionic coordination, resultantly remaining in liquid state below 100 °C and some may retain liquid state even at room temperature. ILs are known to provide a conducive environment for many biological enzymatic reactions, but their interaction with biomacromolecules are poorly understood. In the present study, we investigate the effect of various ionic liquids on DNA-small molecule interaction using calf thymus DNA (ctDNA)-ethidium bromide (EB) as a model system. The effect of various ionic liquids on these interactions is studied by an array of techniques such as circular dichroism (CD), UV melting, fluorescence exclusion and isothermal titration calorimetry. Interestingly, we observed that presence of IL increased the stability of ctDNA without altering its structure. The binding affinities Kbs for EB binding to ctDNA in the presence of 300 mM ILs are about half order of magnitude smaller than the Kbs in absence of ILs and correspond to a less favorable free energy. We noted that, when adjusted to corresponding buffer condition, the unfavorable shift in ΔG of ctDNA-EB interaction is attributed to decreased entropy in the case of ILs, whereas the same effect by NaCl was due to increased enthalpy. [ABSTRACT FROM AUTHOR]

In this work we focus on the use of novel homo and block copolymers based on poly(vinyl benzyl trimethylammonium chloride) as gene delivery vectors. The homopolymers and block copolymers were synthesized by RAFT polymerization schemes and molecularly characterized. DNA/polymer complexes (polyplexes) in a wide range of N/P (amino-to-phosphate groups) ratios were prepared. The ability of the novel polymers to form complexes with linear DNA was investigated by light scattering, zeta potential, and ethidium bromide fluorescence quenching measurements. The resulting polyplexes were in the size range of 80-300 nm and their surface potential changed from negative to positive depending on the N/P ratio. The stability of polyplexes was monitored by changes in their hydrodynamic parameters in the presence of salt. The novel vector systems were visualized by transmission electron microscopy. The influence of factors such as molar mass, content, and chemical structure of the polycationic moieties as well as presence of a hydrophilic poly[oligo(ethylene glycol) methacrylate] block on the structure and stability of the polyplexes, kinetics of their formation, and effectiveness of the (co)polymers to shrink and pack DNA was discussed. [ABSTRACT FROM AUTHOR]

We propose a simple and fast method for detecting arbitrary DNA mutations. Single-stranded DNA probes labeled with fluorescein amidite (FAM-ssDNA), ethidium bromide (EB), and graphene oxide (GO) are employed in the sensing system. The detection is achieved in two steps. In the first step, the sensing system contains FAM-ssDNA probes and EB molecules. It exhibits different fluorescence emissions in the presence of perfectly matched, mismatched, and random DNA sequences. With the addition of GO in the second step, the fluorescence signal for perfectly matched and random DNA does not vary greatly, which, however, experiences a significant change for mismatched DNA targets. The signal ratio before and after the addition of GO can clearly distinguish mutations from normal and random DNA sequences. The detection method works well regardless of the mutation positions and only requires "mix-and-detect" steps, which are completed within 15 min. [ABSTRACT FROM AUTHOR]

Activation of apoptosis, the cell death machinery, in tumor cells by organelle-specific delivery of antitumor theranostic agent is the utmost challenge in cancer therapy. Herein, we developed a highly efficient mitochondria-targeting antitumor theranostic prodrug 7 that contained two molecules of drug 5'-deoxy-5-fluorouridine and an apoptotic marker ethidium for self-monitoring intrinsic (mitochondrial) apoptosis after its activation in tumor cells. Theranostic 7 was activated by endogenously produced mitochondrial-overexpressed H2O2 and released drug 5'-deoxy-5-fluorouridine and apoptotic marker ethidium to the tumor cells. The in vitro and in vivo drug release was monitored by observing the fluorescence changes of ethidium. Theranostic 7 exhibited an enhanced cytotoxicity over commercial 5-fluorouracil (an active drug of 5'-deoxy-5-fluorouridine) leading to intrinsic apoptosis monitored by in situ generated ethidium. Enhanced expression of mitochondria-mediated apoptotic genes (NOXA, PUMA, BID, BAX, and BAK), Cyt C, Caspase-3 and -9, and cell surface death receptors was observed after theranostic 7 activation in tumor cells. In vivo and ex vivo xenografts revealed that theranostic 7 significantly inhibited tumor progression and cured the tumor-bearing mice. Such organelle-specific theranostic strategies have great potential for the early diagnosis and precise treatment of cancer. [ABSTRACT FROM AUTHOR]

Inthis work we have characterized the effects of the intercalatorethidium bromide (EtBr) on the DNA condensation process by using forcespectroscopy and gel electrophoresis. We have tested two condensingagents: spermine (spm4+), a tetravalent cationic aminewhich promotes cation-induced DNA condensation, and poly(ethyleneglycol) (PEG), a neutral polymer which promotes DNA ψ-condensation.Two different types of experiments were performed. In the first type,bare DNA molecules disperse in solution are first treated with EtBrfor intercalation, and then the condensing agent is added to the samplewith the purpose of verifying the effects of the intercalator in hinderingDNA condensation. In the second experiment type, the bare DNA moleculesare first condensed, and then the intercalator is added to the samplein order to verify its influence on the previously condensed DNA.The results obtained with the two different experimental techniquesused agree very well, indicating that previously intercalated EtBrcan hinder both cation-induced and ψ-condensation, being moreefficient in the first case. On the other hand, EtBr has little effecton the previously formed cation-induced condensates, but is efficientin unfolding the ψ-condensates. [ABSTRACT FROM AUTHOR]

DNA intercalation has found many diagnostic and therapeutic applications. Here, we propose the use of simple DNA intercalators, such as ethidium bromide, as tools to facilitate the error-free self-assembly of DNA nanostructures. We show that ethidium bromide can influence DNA self-assembly, decrease the formation of oligomeric side products, and cause libraries of multiple equilibrating structures to converge into a single product. Using a variety of 2D- and 3D-DNA systems, we demonstrate that intercalators present a powerful alternative for the adjustment of strand-end alignment, favor the formation of fully duplexed "closed" structures, and create an environment where the smallest, most stable structure is formed. A new 3D-DNA motif, the ninja star, was self-assembled in quantitative yield with this method. Moreover, ethidium bromide can be readily removed using isoamyl alcohol extractions combined with intercalator-specific spin columns, thereby yielding the desired ready-to-use DNA structure. [ABSTRACT FROM AUTHOR]

Sulfonated graphene (SG) and graphene oxide (GO) areused as anethidium bromide (EtBr, E) binding platform, to preferentially senseDNA (D) among the other biomolecules such as RNA (R), bovine serumalbumin (BSA, P) and glucose (G) using spectroscopic techniques. EtBrloses its intrinsic fluorescence property after binding with SG. DNAcan “turn on” the quenched fluorescence of an SG–EtBrhybrid to a greater extent compared to the RNA, BSA, and glucose.UV–vis absorption spectra and circular dichroism (CD) spectraalso support the higher ability of DNA to release adsorbed EtBr fromthe SG surface in comparison to the above-mentioned biomolecules.Compared to GO–EtBr, the SG–EtBr hybrid is superiorto preferentially sense DNA, as the enhancement of fluorescence intensityis 16 times in the later but it is 4.5 times in the former from theirrespective complexes. An analysis of Raman spectral data indicatesthat the interaction of EtBr in its adsorbed state on an SG templateis greater with DNA than with RNA. [ABSTRACT FROM AUTHOR]

Traditional in vitro nanotoxicity researches are conducted on cultured two-dimensional (2D) monolayer cells and thereby cannot reflect organism response to nanoparticle toxicities at tissue levels. This paper describes a new, high-throughput approach to test in vitro nanotoxicity in three-dimensional (3D) microtissue array, where microtissues are formed by seeding cells in nonsticky microwells, and cells are allowed to aggregate and grow into microtissues with defined size and shape. Nanoparticles attach and diffuse into microtissues gradually, causing radial cytotoxicity among cells, with more cells being killed on the outer layers of the microtissue than inside. Three classical toxicity assays [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT), glucose-6-phosphate dehydrogenase (G6DP), and calcein AM and ethidium homodimer (calcein AM/EthD-1)] have been adopted to verify the feasibility of the proposed approach. Results show that the nanotoxicities derived from this method are significantly lower than that from traditional 2D cultured monolayer cells (p < 0.05). Equipped with a microplate reader or a microscope, the nanotoxicity assay could be completed automatically without transferring the microtissue, ensuring the reliability of toxicity assay. The proposed approach provides a new strategy for high-throughput, simple, and accurate evaluation of nanoparticle toxicities by combining 3D microtissue array with a panel of classical toxicity assays. [ABSTRACT FROM AUTHOR]

In the present study we explore the efficacy of caffeinein dissociatingthe ethidium (Et) molecule, a model DNA-intercalator as well as apotential mutagen, from nanometer sized micelles of various charges.Steady-state and picosecond-resolved spectroscopic studies on thedetachment of Et from various biomimicking micelles of different charges(cationic hexadecyltrimethylammonium bromide (CTAB), neutral (polar)Triton X-100 (TX-100), and anionic sodium dodecyl sulfate (SDS)) revealthe specificity of the caffeine molecule for carrying out such dissociation.The picosecond-resolved Förster resonance energy transfer (FRET)studies between a DNA minor groove binder dye Hoeschst 33258 (H258,donor) and Et (acceptor) have been employed to investigate the alterationin their association in the presence of caffeine at the molecularlevel. Analysis of our experimental results employing both the generalizedand the extended version of the well-known “Infelta–Tachiyamodel” vividly illustrates how the distribution of Et alongwith the equilibrium constant of its solubilization in the micellechanges in the presence of caffeine in aqueous solution. Finally,our fluorescence micrographs of squamous epithelial cells validatethe alteration of FRET efficiency between the donor and the acceptordue to the release of the latter in the presence of caffeine. [ABSTRACT FROM AUTHOR]

Binding geometry to DNA and the efficiency as a donorfor energytransfer of various metallo- and nonmetallo-porphyrins were investigatedmainly by polarized light spectrscopy and fluorescence measurements.Planar porphyrins including nonmetallo meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP), CuTMPyP, andNiTMPyP produced large red-shift and hypochromism in absorption spectrumand a negative circular dichroism (CD) in the Soret band suggestingthat these porphyrins intercalate between DNA base-pairs as expected.In the intercalation pocket, the molecular plane of these porphyrinstilts to a large extent. From a linear dichroism (LD) study, the anglebetween the two electric transition moments in the Soret band were16°, 12°, and 11° for TMPyP, NiTMPyP, and CuTMPyP,respectively. On the other hand, porphyrins with axial ligands namely,VOTMPyP, TiOTMPyP, and CoTMPyP, produced a positive CD signal in theSoret band. Hyperchromism and less red-shift were apparent in theabsorption spectrum. These observations indicated that the porphyrinswith an axial ligand bind outside of the DNA. The angles of both the Bxand Bytransition with respect to the local DNAhelix were 39°∼46° for all porphyrins. From theseresults, the conceivable binding site of porphyrins with axial ligandsis suggested to be the minor groove. All porphyrins were able to quenchthe fluorescence of intercalated ethidium. Strong overlap betweenemission spectrum of ethidium and the absorption spectrum of porphyrinswhen they simultaneously bound to DNA was found suggesting the mechanismbehind energy transfer is, at least in part, the Förster typeresonance energy transfer (FRET). The minimum distance in base pairsbetween ethidium and porphyrin required to permit the excited ethidiumto emit a photon was the longest for CoTMPyP being 17.6 base pairsand was the shortest for CuTMPyP and NiTMPyP at 8.0 base pairs. Thevariation in the distance was almost proportional to the extent ofthe spectral overlap, the common area under emission spectrum of ethidiumand absorption spectrum of porphyrin, supporting the FRET mechanism,whereas the effect of the orientation factor which was consideredby relative binding geometry was limited. [ABSTRACT FROM AUTHOR]

Chemical pollution is known to affect microbial community composition but it is poorly understood how toxic compounds influence physiology of single cells that may lay at the basis of loss of reproductive fitness. Here we analyze physiological disturbances of a variety of chemical pollutants at single cell level using the bacterium Pseudomonas fluorescens in an oligotrophic growth assay. As a proxy for physiological disturbance we measured changes in geometric mean ethidium bromide (EB) fluorescence intensities in subpopulations of live and dividing cells exposed or not exposed to different dosages of tetradecane, 4-chlorophenol, 2-chlorobiphenyl, naphthalene, benzene, mercury chloride, or water-dissolved oil fractions. Because ethidium bromide efflux is an energy-dependent process any disturbance in cellular energy generation is visible as an increased cytoplasmic fluorescence. Interestingly, all pollutants even at the lowest dosage of 1 nmol/mL culture produced significantly increased ethidium bromide fluorescence compared to nonexposed controls. Ethidium bromide fluorescence intensities increased upon pollutant exposure dosage up to a saturation level, and were weakly (r² = 0.3905) inversely correlated to the proportion of live cells at that time point in culture. Temporal increase in EB fluorescence of growing cells is indicative for toxic but reversible effects. Cells displaying high continued EB fluorescence levels experience constant and permanent damage, and no longer contribute to population growth. The procedure developed here using bacterial ethidium bromide efflux pump activity may be a useful complement to screen sublethal toxicity effects of chemicals. [ABSTRACT FROM AUTHOR]

This article presents a novel approach to increase the detection sensitivity of trace amounts of DNA in a sample by employing Förster resonance energy transfer (FRET) between intercalating dyes. Two intercalators that present efficient FRET were used to enhance sensitivity and improve specificity in detecting minute amounts of DNA. Comparison of steady-state acceptor emission spectra with and without the donor allows for simple and specific detection of DNA (acceptor bound to DNA) down to 100 pg/μL. When utilizing as an acceptor a dye with a significantly longer lifetime (e.g., ethidium bromide bound to DNA), multipulse pumping and time-gated detection enable imaging/visualization of picograms of DNA present in a microliter of an unprocessed sample or DNA collected on a swab or other substrate materials.

Atomic force microscopy (AFM) enables determination of physical properties from single DNA molecules. Insertion of aromatic molecules into the structure of DNA results in morphological changes. However, the accompanying changes to elastic properties due to this insertion are not fully understood. AFM was used to examine the morphological effects of intercalator binding and report changes in the elastic properties of intrinsically straight DNA molecules. The persistence length and polymer extension were characterized in the presence of three intercalating molecules: ethidium bromide and the less well studied chloroquine and acridine. It was found that all three intercalators significantly increased the bending persistence length. In addition, an analysis of the normal bending modes of the static molecules corroborated these results. This approach of measuring binding effects of intercalators on DNA physical properties using a model system of intrinsically straight DNA is applicable to other DNA binding ligands and other modes of DNA interaction.

The reaction of [Au(dien)(N-heterocycle)](3+) (AuN4) coordination compounds with simple amino acids and zinc finger proteins is reported. Compared to [AuCl(dien)](2+), NMR studies show that the presence of a more substitution-inert N-donor as the putative leaving group slows the reaction with the sulfur-containing amino acids N-acetylmethionine (NAcMet) and N-acetylcysteine (NAcCys). Lack of ligand dissociation upon reaction with NAcCys indicates, to our knowledge, the first long-lived N-heterocycle-Au-S species in solution. Reactions with zinc finger proteins show a higher reactivity with the Cys3His zinc finger than with Cys2His2, likely due to the presence of fewer aurophilic cysteines in the latter. Of the Au(III) compounds studied, [Au(dien)(DMAP)](3+) (DMAP = 4-dimethylaminopyridine) appears to be the least reactive, with ESI-MS studies showing the presence of intact zinc fingers at initial reaction times. These results, in combination with previously reported characterization and pH dependency studies, will further aid in optimizing the structure of these AuN4 species to obtain a substitution-reactive yet selective compound for targeting zinc finger proteins.

The compaction of DNA by cationic liposomes constituted by a mixture of a cationic lipid, dioctadecyldimethylammonium bromide (DODAB), and a zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), has been evaluated by means of experimental studies (electrophoretic mobility, conductometry, cryogenic electron transmission microscopy or cryo-TEM, and fluorescence spectroscopy) as well as theoretical calculations. This information reveals that DODAB/DOPE and DODAB/DLPC liposomes are mostly spherical and unilamellar, with a mean diameter of around 70 and 61 nm, respectively, a bilayer thickness of 4.5 nm, and gel-to-fluid transition temperatures, T(m), of around 19 and 28 degrees C, respectively. Their positively charged surfaces efficiently compact the negatively charged DNA by means of a strong entropically driven surface interaction that yields DODAB/DOPE-DNA and DODAB/DLPC-DNA lipoplexes as confirmed by zeta potential and ethidium bromide fluorescence intercalation assays. These experiments have permitted as well the evaluation of the different microenvironments of varying polarity of the DNA helix, liposomes, and/or lipoplexes. DODAB/DOPE-DNA and DODAB/DLPC-DNA lipoplexes have been characterized by isoneutrality ratios (L/D)(phi) of around 4.7 and 4.8, respectively, a more fluid membrane than that of the parent liposomes, and T(m) around 24 and 28 degrees C, respectively, as revealed by fluorescence anisotropy. Cryo-TEM micrographs reveal a rich scenario of nanostructures and morphologies, from unilamellar DNA-coated liposomes to multilamellar lipoplexes passing through cluster-like structures. Phase diagrams (aggregation and re-entrant condensation phenomena), calculated by means of a phenomenological theory, have confirmed the experimental concentration domains and the isoneutrality conditions. The influence of helper lipid in the compaction process, as well as the optimum choice among those herein chosen, has been analyzed.

This work is the first in-depth study of osmium binding to DNA and confirms the pharmacological activity of a new class of anticancer metallodrugs. We investigated the interactions between the potential biological target DNA and four osmium(II) arene complexes, of the type [(eta 6-arene)Os(LL)Cl]n+, where arene = biphenyl or p-cymene and LL = ethylenediamine, picolinate, or oxinate in an effort to understand their mechanism of action. Most notably we show that these complexes bind to DNA. DNA adducts of the OsII complexes that exhibit promising cytotoxic effects in ovarian tumor cell lines largely distort its conformation. The data are consistent with DNA binding of the complexes containing biphenyl as the arene ligand that involves combined coordination to guanine residues and noncovalent interactions between the arene ligand and DNA. The results also indicate both a mechanism of action and a detoxification mechanism for OsII arene compounds different from those of cisplatin.

Here, we present a direct method for determining mitochondrial DNA (mtDNA) copy numbers in individual mitochondrial particles, isolated from cultured cells, by means of capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. We demonstrate that this method can detect a single molecule of PicoGreen-stained mtDNA in intact DsRed2-labeled mitochondrial particles isolated from human osteosarcoma 143B cells. This ultimate limit of mtDNA detection made it possible to confirm that an individual mitochondrial nucleoid, the genetic unit of mitochondrial inheritance, can contain a single copy of mtDNA. The validation of this approach was achieved via monitoring chemically induced mtDNA depletion and comparing the CE-LIF results to those obtained by quantitative microscopy imaging and multiplex real-time PCR analysis. Owing to its sensitivity, the CE-LIF method may become a powerful tool for investigating the copy number and organization of mtDNA in mitochondrial disease and aging, and in molecular biology techniques requiring manipulation and quantitation of DNA molecules such as plasmids.

Glycated DNA is considered to be a pathogenic factor for diabetes mellitus. Here we present a novel and preliminary study on normal and glycated (with fructose and glucose-6-phosphate as reducing sugars) human placenta DNA using agarose gel electrophoresis and photon correlation spectroscopy. The former is used to find structural alterations, while the latter is exploited to observe differences in the dynamics between normal (i.e., pure) and glycated DNA molecules. For scattering angles up to 90 degrees , we obtained a quasi-single-exponential relaxation process for the pure DNA, whereas at higher scattering angles the relaxation of pure DNA becomes broader with a stretching parameter beta approximately 0.6 at 130 degrees. Interestingly, for both the glycated DNAs stretched relaxation profiles and higher relaxation rates (Omega) are observed for all scattering angles. Moreover, a separate and very fast relaxation (e.g., relaxation time tau approximately 2 micros at 90 degrees ) can be noticed for both the glycated DNAs at all the studied scattering angles. Thus, the dramatic changes in the relaxation parameters (Omega, tau, and beta) of the glycated DNA show at the molecular level, for the first time, that the structure and dynamics of DNA are strongly affected by glycation. Implications of the results are discussed.

Structural effects of binding the intercalating drug ethidium bromide (EtBr) to 160 base pair (bp) fragments of nucleosomal calf thymus DNA have been probed by the method of Raman difference spectroscopy. With the use of a near-infrared (NIR) laser source to excite the Raman spectrum at 752 nm, vibrational signatures of both the EtBr intercalant and DNA target have been identified in spectra of the drug-DNA complexes. Analysis of the results obtained on complexes consisting of 1 EtBr bound/10 bp leads to the following conclusions: (i) Raman markers diagnostic of DNA phosphodiester conformation are converted from the B type to the A type with EtBr binding, commensurate with the proportion of ethidium-bound nucleotides in the complex. (ii) Ethidium binding converts deoxynucleoside sugar puckers from the C2'-endo to the C3'-endo conformation, also consistent with binding stoichiometry. Both pyrimidine and purine deoxynucleoside sugar puckers are perturbed by the phenanthridinium ring intercalation. (iii) Phenanthridinium insertion between bases is accomplished with no apparent change in hypochromicities of purine or pyrimidine Raman markers, indicating that base-phenanthridinium interactions provide compensatory hypochromic effects. (iv) Novel Raman markers of helix unwinding have been identified and assigned primarily to methylene deformation modes of the deoxyribosyl C2'H(2) and C5'H(2) groups. The present study provides new insights into drug-DNA recognition in solution and demonstrates the feasibility of NIR-Raman spectroscopy for structural studies of highly chromophoric DNA complexes.

Complete biophysical characterization of complexes (polyplexes) of cationic polymers and DNA is needed to understand the mechanism underlying nonviral therapeutic gene transfer. In this article, we propose a new series of synthesized random cationic polymers (RCPs) from methoxy poly(ethylene glycol) monomethacrylate (MePEGMA) and (3-(methacryloylamino)propyl)trimethylammonium chloride with different mole ratios (32:68, 11:89, and 6:94) which could be used as a model system to address and answer the basic questions relating to the mechanism of the interaction of calf thymus DNA (CT-DNA) and cationic polymers. The solubility of the complexes of CT-DNA and RCP was followed by turbidity measurements. It has been observed that complexes of RCP with 68 mol % MePEGMA precipitate near the charge neutralization point, whereas complexes of the other two polymers are water-soluble and stable at all compositions. Dnase 1 digestion experiments show that DNA is inaccessible when it forms complexes with RCP. Ethidium bromide exclusion and gel electrophoretic mobility show that both polymers are capable of binding with CT-DNA. Atomic force microscopy images in conjunction with light scattering experiments showed that the complexes are spherical in nature and 75-100 nm in diameter. Circular dichroism spectroscopy studies indicated that the secondary structure of DNA in the complexes is not perturbed due to the presence of poly(ethylene glycol) segments in the polymer. Furthermore, we used a combination of spectroscopic and calorimetric techniques to determine complete thermodynamic profiles accompanying the helix-coil transition of CT-DNA in the complexes. UV and differential scanning calorimetry melting experiments revealed that DNA in the complexes is more stable than in the free state and the extent of stability depends on the polymer composition. Isothermal titration calorimetry experiments showed that the binding of these RCPs to CT-DNA is associated with small exothermic enthalpy changes. A complete thermodynamic profile showed that the RCP/DNA complex formation is entropically favorable. Much broader opportunities to vary the architecture of the polymers studied here make these systems promising in addressing various basic and practical problems in gene delivery systems.

The optimization of DNA-cationic polymer complexation is crucial for nonviral gene delivery. Although physicochemical characterization of the interaction between DNA and cationic polymers has recently attracted more attention in the nonviral DNA delivery field, the literature on the effect of varying polycation charge density on DNA-cationic polymer complexation is still scarce. Thus, the aim of this study was to systematically assess the influence of the degree of ionization of a weak cationic polyelectrolyte (poly[2-(dimethylamino)ethyl methacrylate] or DMAEMA homopolymer) on its ability to form complexes with DNA. This was achieved by varying the solution pH from 4.0 to 8.0 and analyzing the resulting effects on the binding affinity, thermodynamic properties, complex size, and morphology. Lowering the solution pH led to higher degrees of ionization for the cationic polymer and hence greater binding affinities with DNA, as judged by the increased propensity of the former to displace ethidium bromide from DNA and also by relatively low monomer:nucleotide molar ratio (0.8:1) required to retard the migration of free DNA. Isothermal titration microcalorimetry studies further confirmed that a stronger interaction occurred at low pH than at high pH. By decreasing the pH from 8.0 to 6.6, K(obs) increased from 7.8 x 10(5) to 20.4 x 10(5) M(-1). More efficient condensation at low pH was demonstrated by the reduction of ethidium bromide fluorescence in the loading wells from gel electrophoresis, decreased complex sizes without agglomeration occurring at high polymer/DNA ratios, together with discrete and dense spherical complexes observed in TEM studies. This may be attributed to the presence of electrostatic stabilization from excess cationic polymer chains, which provide a repulsive shell around the polymer/DNA complex. The physicochemical data indicate that the increased degree of ionization for the DMAEMA homopolymer at lower pH results in higher binding affinity, smaller and more compact complexes, and more efficient condensation. These findings therefore highlight the importance of the degree of ionization on DNA complex formation for weak cationic polyelectrolytes.

The synthesis, chemical characterization, and interaction with cells of new sterically hindered trans- and cis-diaminedichloroplatinum(II) complexes are described. The amine ligands include monofunctional piperidine (pip) and piperazine (pz). The poor solubility of trans-diaminedichloroplatinum complexes was overcome by introducing the positively charged pz ligand, which allows retaining of the classic platinum coordination sphere. In vitro evaluation in OV-1063 and C-26 tumor cells revealed that replacing one NH3 of the inactive transplatin by an aromatic planar ligand (4-picoline, 4-pic) or by an aliphatic nonplanar heterocyclic ligand (pip) or replacing both NH3 groups with a 4-pic ligand and a pip or pz ligand significantly increases the cytotoxic activity of these complexes. The unsymmetric complexes trans-[PtCl2(4-pic)(pip)] and trans-[PtCl2(4-pic)(pz)]HCl were the most cytotoxic compounds against the cisplatin-sensitive tumor cell line C-26 (IC50 = 4.5 and 5.5 microM, respectively) and the cisplatin-sensitive tumor cell line OV-1063 (IC50 = 6.5 and 7.4 microM, respectively). In contrast, replacing one NH3 of the cis isomer by an aromatic planar ligand (4-pic) or by an aliphatic amine lowered their cytotoxiciy in comparison to cisplatin. Cell penetration and Pt-DNA adduct formation were also evaluated, and it was clearly shown that both trans-[PtCl2(4-pic)(pip)] and trans-[PtCl2(4-pic)(pz)]HCl penetrate efficiently the cellular membrane of the tumor cells and platinate the cellular DNA. When comparing cellular DNA platination, positively charged trans-[PtCl2(4-pic)(pz)]HCl was 7-fold higher than both cisplatin and its neutral analogue trans-[PtCl2(4-pic)(pip)]. Moreover, in contrast to cisplatin, in the cell lines used, cell death caused by both complexes appeared to be apoptotic according to several criteria including early phosphatidylserine exposure, activation of caspases, and characteristic morphological changes. Our results suggest that these novel mixed nonclassical trans-Pt(II) complexes are biologically and mechanistically distinct from known Pt complexes and deserve evaluation of their efficacy in tumor-bearing animals.

In this work, four different fluorescent intercalating dyes are compared for the ultrasensitive CGE-LIF detection of DNA from transgenic maize in flours. The fluorescent intercalating dyes compared are YOPRO-1, SYBR-Green-I, Ethidium bromide (EthBr), and EnhanCE. For all the four dyes optimum concentrations are established, and efficient separations of DNA fragments ranging in size from 80 to 1000 bp are obtained. The comparative study demonstrates that SYBR-Green-I and YOPRO-1 provide better limits of detection (LODs) than EnhanCE or EthBr (i.e., LODs are, respectively, 700, 1000, 11300, and 97400 zmol, calculated for a 200-bp DNA fragment). Separations using YOPRO-1 are faster than those using SYBR-Green-I (30 min vs 47 min for the analysis of the 80-1000 bp DNA fragments). Also, separations using YOPRO-1 are more efficient than those using SYBR-Green-I (e.g., 2.4 x 10(6) plates/m vs 1.6 x 10(6) plates/m, respectively, calculated for the 200-bp fragment). Also, buffer depletion and cost per analysis are worse with SYBR-Green-I than with YOPRO-1. Therefore, YOPRO-1 was selected as the preferred intercalating dye. Using this fluorescent compound, analysis time reproducibility for the CGE-LIF separation of the DNA fragments is determined to be better than 1.7% (% RSD, n = 10) within the same day, and better than 1.9% (% RSD, n = 30) for three different days. Moreover, the fluorescence signal obtained using this dye is shown to vary linearly with the DNA concentration in the range studied, i.e., 1-500 ng/microL. It is demonstrated that by using this method 0.01% of transgenic maize can be detected in flour by direct injection of the PCR-amplified sample.

The molecular structure of the cationic lipids used in gene transfection strongly influences their transfection efficiency. High transfection efficiencies of non-glycerol-based simple monocationic transfection lipids with hydroxyethyl headgroups recently reported by us (Banerjee et al. J. Med. Chem. 1999, 42, 4292-4299) are consistent with the earlier observations that the presence of hydroxyl functionalities in the headgroup region of a cationic lipid contributes favorably in liposomal gene delivery. Using simple sugar molecules as the source of multiple hydroxyl functionalities in the headgroup region of the transfection lipids, we have synthesized four novel simple monocationic transfection lipids, namely, 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-xylitol (1), 1-deoxy-1-[methyl(ditetradecyl)ammonio]-D-arabinitol (2), 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-arabinitol (3) and 1-deoxy-1-[methyl(dioctadecyl)ammonio]-D-arabinitol (4), containing hydrophobic aliphatic tails and the hydrophilic arabinosyl or xylose sugar groups linked directly to the positively charged nitrogen atom. Syntheses, chemical characterizations, and the transfection biology of these novel transfection lipids 1-4 are described in this paper. Lipid 1, the xylosyl derivative, showed maximum transfection on COS-1 cells. All the lipids showed transfection with cholesterol as colipid and not with dioleoylphosphatidylethanolamine (DOPE). Radioactive quantitation of free and complexed DNA combined with ethidium bromide exclusion measurements suggest that though nearly 70% of the DNA exists as complexed DNA, the DNA may not have condensed as was observed with other cationic lipids. Presence of additional (more than two) hydroxyl functionalities in the headgroup of the cationic lipids appears to have improved the transfection efficiency and made these lipids less cytotoxic compared to two-hydroxyl derivatives.

Novel polyamine carbamates have been designed and prepared from cholesterol. Our synthesis uses an orthogonal protection strategy based upon trifluoroacetyl and Boc-protecting groups. These unsymmetrical polyamine carbamates have been prepared from symmetrical (e.g., spermine and thermine) polyamines. Detailed interpretations of (1)H and (13)C NMR spectroscopic data led to the unambiguous assignment of these polyamine carbamates. These target conjugates contain a variety of positive charges distributed along methylene chains. Their pK(a)s have been determined potentiometrically for conjugates substituted with up to five amino functional groups. Condensation of calf thymus DNA into particles was monitored using light scattering at 320 nm. Salt-dependent binding affinity for calf thymus DNA was determined using an ethidium bromide fluorescence quenching assay. These cholesteryl polyamine carbamates are models for lipoplex formation with respect to gene delivery (lipofection), a key first step in gene therapy.

A tetracationic anthraquinone derivative (27AQS2) binds to hairpin DNA and irradiation of the bound quinone leads to selective strand cleavage. NMR spectroscopy reveals that 27AQS2 binds at the loop and to the stem-loop junction of hairpin DNA. UV irradiation of the bound quinone causes cleavage of the DNA in the loop region and at guanines in the stem region. Inclusion of ethidium bromide in the reaction mixture leads to a greatly increased selectivity for loop cleavage. Spectroscopic and chemical evidence suggests a three component mechanism for reaction. The ability to target single-stranded regions of DNA structures is an important property of this photonuclease.

Because actin can form a complex in vitro containing both gelsolin and DNase I, gelsolin and DNase I have been assumed to bind independently to actin. Although this assumption is consistent with the known crystalline structures of gelsolin with one actin and of actin with DNase I, which suggest that the binding sites on actin for both gelsolin and DNase I are distinct and separate, we propose that a second actin binding site on gelsolin competes with DNase I for actin. Since actin is an inhibitor of DNase I, competition at the second binding site results in activation of DNase I by gelsolin. Covalent cross-linking experiments confirm that DNase I prevents dimerization of actin by gelsolin, consistent with displacement of one actin from gelsolin by DNase I. Activation of DNase I by gelsolin is a novel function for a cytoskeletal protein and could have broad implications for biology, such as a role in initiating apoptosis. These results also may explain why both gelsolin and DNase I decrease sputum viscosity in cystic fibrosis (CF). While the activity of DNase I had originally been attributed to fragmentation of DNA, subsequent data suggested that both gelsolin and DNase I may affect viscosity by depolymerizing filamentous actin. The current results alternatively suggest aht dissociation of the actin-DNase I complex by gelsolin in CF sputum results in activation of the nuclease activity of constitutive DNase I. The nuclease activity of DNase I alone is therefore sufficient to explain the effects of both gelsolin and DNase I on CF sputum.

The arrangement and stacking of noncovalently contiguous double-helical sections are increasingly invoked in single-stranded DNA and RNA tertiary structure. These tertiary structures of nucleic acids are defined by their double stranded regions, and their orientation in the molecular frame constitutes an important component of the nucleic acid structure. A direct view of these tertiary structures can be obtained by fluorescence polarization anisotropy of bound ethidium bromide (EB). The orientation of the dye in the molecular frame of the nucleic acid yields the orientation of the helix. The complete anisotropy function for EB intercalated in genome-derived DNA duplexes was derived by Allison and Schurr (1979) and accounts for base-pair twisting and DNA bending. Single-stranded ribozymes, ribosomal and transfer RNAs, and model DNA junctions contain double-stranded regions shorter than 35 bp in length, for which bending is not significant. We developed and experimentally verified an expression of the anisotropy function for short DNA duplexes which is theoretically compatible with the existing theory, originally developed for long nucleic acids (Schurr et al., 1992). Simulations showed that for DNA duplexes shorter than 35 bp, our expression of the anisotropy function is equivalent to Schurr's and is consistent with experiments carried out on eight DNA duplexes. Modeling the eight duplexes as cylinders, we calculate a duplex diameter of 1.91 +/- 0.15 nm when EB makes a 90 degrees angle with the DNA helix axis and undergoes anisotropic wobbling and 1.97 +/- 0.15 nm when EB makes a 70.5 degrees angle and undergoes isotropic wobbling, respectively. We used this treatment to establish the conformation of five DNA oligonucleotides made of single and tethered hairpins, some designed to exhibit coaxial stacking. Analysis of the fluorescence anisotropy decays shows that the tethered hairpins take an extended rather than parallel conformation. It also shows that the DNA oligonucleotides made of two tethered hairpins exhibit freedom compatible with two independent hairpins. When the linker between hairpins is shortened, the two hairpins are not independent anymore as probed by fluorescence anisotropy, suggesting coaxial stacking of the two helices.

An N-methyl-N-nitrosourea (MNU) moiety [CH3N(N=O)C(=O)NH-] linked to the C4'-position of the 5-substituted phenyl ring of phenyl neutral red (PNR), 2-methyl-3-amino-5-[p-[[2-[(N-nitroso-N-methylcarbamoyl)amino]ethy l] carbamoyl]phenyl]-7-(dimethylamino)phenazenium chloride (MNU-PNR), has been synthesized as an approach to design a molecule that will deliver alkylating agents with some preference to guanine (Gua) in the major groove of DNA. The PNR nucleus was chosen because previous studies suggested the following: (1) PNR binds with a slight preference for G/C rich sequences; and (2) PNR intercalates into DNA from the major groove with the 5-phenyl ring pointing out into the major groove (Müller, W., Bünemann, H., and Dattagupta, N. (1975) Eur. J. Biochem. 54, 279-291). It is demonstrated that MNU-PNR yields 2.6 and 6.0 times more N7-methylguanine (7-MeGua) than MNU at low salt (10 mM Tris buffer) and high salt (10 mM Tris buffer + 200 mM NaCl), respectively. It is also shown that the ratio of 7-MeGua (a major groove adduct) to N3-methyladenine (a minor groove adduct) is approximately 5 times higher for MNU-PNR than for MNU. The yield of the 7-MeGua adduct is decreased by the coaddition of a nonmethylating analogue of MNU-PNR or NaCl, but increased in the presence of the minor groove intercalator, ethidium bromide. Using a 32P-end-labeled restriction fragment, the enhanced methylation by MNU-PNR at 7-Gua is confirmed, and it is demonstrated that the sequence-dependent formation of 7-MeGua from MNU-PNR is the same as that seen with MNU. UV, circular dichrosism, and viscosity studies are consistent with MNU-PNR binding to DNA via an intercalation-based process.

To understand how DNA is released from cationic liposome/DNA complexes in cells, we investigated which biomolecules mediate release of DNA from a complex with cationic liposomes. Release from monovalent[1,2-dioleoyl-3(1)-1(trimethylammonio)propane] or multivalent (dioctadecylamidoglycylspermine) lipids was quantified by an increase of ethidium bromide (EtBr) fluorescence. Plasmid sensitivity to DNAse I degradation was examined using changes in plasmid migration on agarose gel electrophoresis. Physical separation of the DNA from the cationic lipid was confirmed and quantified on sucrose density gradients. Anionic liposomes containing compositions that mimic the cytoplasmic-facing monolayer of the plasma membrane (e.g. phosphatidylserine) rapidly released DNA from the complex. Release occurred near a 1/1 charge ratio (-/+) and was unaffected by ionic strength or ion type. Water soluble molecules with a high negative linear charge density such as dextran sulfate or heparin also released DNA. However, ionic water soluble molecules such as ATP, tRNA, DNA, poly(glutamic acid), spermidine, spermine, or histone did not, even at 100-fold charge excess (-/+). On the basis of these results, we propose that after the cationic lipid/DNA complex is internalized into cells by endocytosis it destabilizes the endosomal membrane. Destabilization induces flip-flop of anionic lipids from the cytoplasmic-facing monolayer, which laterally diffuse into the complex and form a charge neutral ion pair with the cationic lipids. This results in displacement of the DNA from the cationic lipid and release of the DNA into cytoplasm. This mechanism accounts for a variety of observations on cationic lipid/DNA complex-cell interactions.

The synthesis, characterization, inhibitory activity against topoisomerase I, and biological evaluation of a series of oligopeptide-substituted bisindolylmaleimides 7-12 are described. Compounds 7-9, which contain a basic C-terminus function such as (dimethylamino)propyl and bind to DNA with C(50) values of 200, 160, and 135 microM, respectively, exhibited inhibition of topoisomerase I in a concentration dependent manner. Also, the relative order of observed topoisomerase I inhibition is 9 > 8 > 7 at < or = 100 microM concentration, corresponding to the increase of the number of pyrrole units in the oligopeptide moiety. Compounds 10-12, which contain an electrostatically neutral moiety, such as methyl ester, did not bind to DNA templates nor inhibit topoisomerase I. However, the cytotoxicity activities of these compounds were 1.5 times greater than those of compounds 7-9.

The interaction of histones H3 and H4 with highly positively coiled DNA has been studied. To carry out this study, it was necessary to develop a protocol for the preparation of large quantities of highly positively coiled DNA. Such preparations were obtained by maximizing transcription-induced stress in cells containing PBR322 and then applying a short-term treatment with novobiocin, which inhibits gyrase. Fractionation on CsCl-EtBr gradients gave PBR322 plasmids with topological states ranging from +0.15 to -0.043 superhelical density. In competition experiments between negatively and positively coiled DNA, histones H3,H4 preferentially bound the positively coiled DNA when the superhelical density was greater than +0.10. This preference was shown on the basis of sedimentation rate on sucrose gradients, selective aggregation by H3,H4 binding, and cross-linking experiments in which the histone-DNA content was characterized on CsCl density gradients. An analysis of the DNA helical pitch in 1.1 M NaCl, a condition in which moderately positively coiled DNA preferentially binds H3,H4 indicated that the preferential binding may be due to a decrease in helical pitch that approximates 0.06 bp/turn. Consistent with this observation is the ability of histones H3,H4 to transiently hold this altered pitch in the presence of topoisomerase I. A possible explanation for this preference is based on the known observation that histones overwind the DNA helix when in a nucleosome. These data provide an explanation for the in vivo observation that histones H3,H4 are rarely displaced during the transcription process. These observations are discussed with regard to mechanisms for transcription through nucleosomes.

Using antibodies that specifically recognize the acetylated forms of histone H4, we show that it is possible to immunoprecipitate newly assembled (acetylated) nucleosomes. Newly replicated HeLa cell chromatin was labeled for 5-30 min with [3H]thymidine in the presence of sodium butyrate (thus inhibiting the deacetylation of newly deposited H4); bulk chromatin DNA was labeled for 24 h with [14C]thymidine. When soluble nucleosomes were incubated with immobilized antibodies, a comparison of the bound and unbound fractions showed up to a 65-fold enrichment for new chromatin DNA in the immunoprecipitate (bound), relative to the supernatant (unbound). No enrichment for new DNA was observed when preimmune control serum was used in a similar fashion. The enrichment for new DNA in the immunopellet was paralleled by a similar enrichment for all four newly synthesized histones. Acetylation was required for antibody recognition: When chromatin was replicated in the absence of butyrate (permitting histone deacetylation and chromatin maturation), equally low levels of new and old chromatin were immunoprecipitated, and no enrichment for new DNA was observed. Competition experiments confirmed these results. Analyses of histone deposition during the inhibition of DNA replication established that acetylated chromatin is the preferential target for H2A/H2B exchange. These experiments provide evidence for the highly selective assembly of newly synthesized H3, H2A, and H2B with acetylated H4, and for the involvement of histone acetylation in dynamic chromatin remodeling. In addition, immunoprecipitations of radiolabeled cytosolic extracts identified a possible somatic chromatin preassembly complex, containing newly synthesized H3 and new (acetylated) H4.

The DNA binding properties of a series of imidazole-containing and C-terminus-modified analogues 4-7 of distamycin are described. These analogues contain one to four imidazole units, respectively. Data from the ethidium displacement assay showed that these compounds bind in the minor groove of DNA, with the relative order of binding constants of 6 (Im3) > 7 (Im4) > 5 (Im2) > 4 (Im1). The reduced binding constants of these compounds for poly(dA-dT) relative to distamycin, while they still interact strongly with poly(dG-dC), provided evidence of GC sequence acceptance. The preferences for GC-rich sequences by these compounds were established from a combination of circular dichroism (CD) titration, proton nuclear magnetic resonance (1H-NMR), and methidiumpropylethylenediaminetetraacetate-iron(II) [MPE.Fe-(II)] footprinting studies. In the CD studies, these compounds produced significantly larger DNA-induced ligand bands with poly(dG-dC) than poly(dA-dT) at comparable ligand concentrations. 1H-NMR studies of the binding of 5 to d-[CATGGCCATG]2 provided further evidence of the recognition of GC sequences by these compounds, and suggested that the ligand was located on the underlined sequence in the minor groove with the C-terminus oriented over the T residue. MPE footprinting studies on a GC-rich BamHI/SalI fragment of pBR322 provided unambiguous evidence for the GC sequence selectivity for some of these compounds. Compounds 4 and 7 produced poor footprints on the gels; however, analogues 5 and 6 gave strong footprints.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of intercalation into DNA double helices by ethidium has been analyzed by temperature-jump relaxation and stopped-flow measurements using fluorescence detection. Artifacts due to field- or flow-induced alignment have been eliminated by measurements under magic angle conditions; the theoretical basis for suppression of orientation effects resulting from external forces is given for the case of fluorescence measurements. Excluded site effects have been avoided by restriction to low degrees of binding. The temperature-jump relaxation observed for ethidium binding to DNA could be described by single exponentials under most conditions. The reciprocal time constants increased linearly with the DNA concentration, leading to association rate constants of 2.7 x 10(6) M-1 s-1 at 12 degrees C. These rate constants are virtually independent of the DNA chain length for samples with 200, 500, 4228, and 30,000 base pairs, showing that the rate is controlled by reaction and not by a diffusive process. At high DNA concentrations around 200 microM, an additional relaxation effect with an amplitude opposite to the main one is observed which is probably due to some conformational change of the DNA-ethidium complex. The results obtained by stopped-flow measurements are consistent with those from T-jump measurements, but owing to higher amplitudes and better signal to noise ratios, the stopped-flow data clearly require two exponentials for satisfactory representation. The reciprocal time constants for both processes increase linearly with the DNA concentration. The simplest mechanism consistent with this result involves parallel formation of two different complexes with a direct transfer of ethidium between the binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

We have 5'-end-labeled one polymerase chain reaction (PCR) primer with the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). After performing PCR in the presence of another unlabeled primer, we separated the 362 bp PCR product with 2% low melting point agarose gel electrophoresis. The gel was then immersed into a Eu3+ solution. During soaking, Eu3+ diffuses into the gel and associates with BCPDA to form a fluorescent complex of long fluorescence lifetime. This complex can be quantified by scanning the gel with a time-resolved fluorometric reader. Because BCPDA and Eu3+ are not fluorescent by themselves, background signals are very low. The detection limit was about 5 ng of DNA. We have also shown that the BCPDA-labeled product could be blotted and detected on the membrane by using an anti-BCPDA antibody. These two technologies may find applications other than in PCR, e.g. in fluorescence-based DNA sequencing and in solution hybridization.

We report differences in the interaction of two structurally similar phenanthroline intercalators, ethidium and propidium, with poly(dA).poly(dT) and poly[d(A-T)] as a function of ionic strength based on titration microcalorimetry, fluorescence titration, and hydrostatic pressure measurements. Both ethidium and propidium bind more strongly to poly[d(A-T)].poly[d(A-T)] than to poly(dA).poly(dT). Ethidium intercalation into the latter polymer displays titrations with positive cooperativity; this is not found with propidium. The enthalpy of intercalation (delta H degrees) is exothermic for both dyes with poly[d(A-T)].poly[d(A-T)]; however, the value of this parameter is nearly zero in the case of poly(dA).poly(dT). The molar volume change (delta V degrees) accompanying dye intercalation is negative under all conditions for poly[d(A-T)].poly[d(A-T)] whereas it is positive for poly(dA).poly(dT). The changes observed in delta V degrees correlate well with the entropy changes derived from the titration and calorimetric data for this reaction. The results, interpreted in terms of the relative hydration of these two polymers, are consistent with a higher extent of hydration of poly(dA).poly(dT) relative to poly[d(A-T)].poly[d(A-T)].

We have used 31P and 1H NMR to show that DNA internal motions are stopped or greatly hindered when ethidium bromide intercalates into DNA. We show that the effect is localized to a 2 base pair long DNA region at the binding site and that internal motions outside the binding site are nearly unaffected. Based upon the nearly independent behavior of bound and free DNA regions, we conclude that in B-DNA individual base pairs experience internal motion which is independent of the motion of their neighbors.

The nonprotein chromophore of neocarzinostatin was found to share many of the characteristics of classical intercalators in its interaction with DNA. Viscosity studies with PM2 DNA indicated that the DNA helix unwinding induced by the chromophore was 0.82 times that of ethidium or 21 degrees. Electric dichroism of the chromophore--DNA complex showed that each bound chromophore molecule lengthened DNA by 3.3 A and that absorbance transitions of the chromophore at 315--385 nm were oriented approximately parallel to DNA bases, as expected for an intercalated aromatic ring. Binding to DNA induced strong hypochromicity and a pronounced red shift in the absorbance spectrum of the chromophore. Spectrophotometric titrations suggested at least two types of chromophore binding sites on DNA; one type of site was saturated at rb = 0.125 chromophore molecule/nucleotide, but binding to additional sites continued to at least rb = 0.3. These physical--chemical studies were performed at pH 4--5 in order to keep the chromophore stable, but chromophore bound to an excess of DNA at pH 7 showed a stable absorbance spectrum identical with that seen at pH 4--5, suggesting that a similar type of binding occurs at neutral pH. Chromophore which had spontaneously degraded in pH 8 buffer did not bind to DNA at all, as judged by absorbance spectroscopy. The degree of protection afforded by DNA against spontaneous chromophore degradation implied a dissociation constant of approximately 5 microM for the DNA--chromophore complex at neutral pH and physiological ionic strength. Supercoiled DNA was nearly twice as effective as relaxed DNA in protecting chromophore from degradation, providing additional evidence for intercalation at neutral pH. Comparison of absorbance, fluorescence, and dichroism spectra suggests that the naphthalene ring system is the intercalating moiety.

Clone H6-15/163 is a clone of cells, originally derived from SV-40 transformed 3T3 cells, which express the transformed phenotype at low (32 degrees C) but not at high (39 degrees C) temperature. Chromatin was isolated from these cells grown at either temperature and studied by circular dichroism and for its ability to bind the intercalating dye, ethidium bromide. During the exponential phase of growth the chromatins of cells grown at either 32 or 39 degrees C are undistinguishable. Cessation of growth in confluent cultures results in marked changes in circular dichroism spectra and in ethidium bromide binding capacity of chromatin. The changes are much are much more pronounced at 39 degrees C (where the cells truly become quiescent) than at 32 degrees C (where cell proliferation continues although the number of cells per culture remains stationary). Temperature shifts and medium replacement also cause changes in chromatin structure, but the changes are again related to the extent of cell proliferation. It is concluded that the chromatin changes occurring in H6-15/163 cells and detectable by circular dichroism and ethidium bromide binding can be related to the proliferating activity of the cultured cells rather than to the expression of the transformed or untransformed phenotype.

The interactions between the fluorescent probe ethidium and acetylcholine receptor enriched membranes from Torpedo californica are described. One class of saturable ethidium sites was blocked by alpha-bungarotoxin and therefore reflects direct binding to the receptor (Kd approximately 3 micrometers; stoichiometry--one ethidium site per two alpha-bungarotoxin sites). The second class of sites was nonsaturable and unaffected by alpha-toxin and was therefore considered nonspecific in nature. The increase in fluorescence intensity observed upon addition of cholinergic agonists and antagonists accurately reflects the dissociation constant and stoichiometry of the high-affinity receptor sites for these ligands. The effects of local anaesthetics are complex in nature and depend on the structure of the ligand. For carbamylcholine, the increase in flourescence intensity was due to an increase in the quantum yield of the dye bound to the membrane rather than a dye uptake. In general, ethidium appears not to strongly alter the properties of the membrane-bound acetylcholine receptor and can therefore be profitably used as a spectroscopic probe.

Internal motion of the DNA in chicken erythrocyte chromatin fibers was studied by measurement of the fluorescence anisotropy decay of ethidium intercalated in the linker region. A comparison of the decay curves of the dye in chicken erythrocyte chromatin with those of calf thymus chromatin [Ashikawa, I., Kinosita, K., Jr., Ikegami, A., Nishimura, Y., Tsuboi, M., Watanabe, K., Iso, K., & Nakano, T. (1983) Biochemistry 22, 6018-6026] revealed greater suppression of nucleosome movement in chicken erythrocyte chromatin. Furthermore, the transition of this chromatin to the compact (solenoidal) structure occurred at lower solvent concentrations of Na+ or Mg2+ than those for calf thymus chromatin. These results demonstrated increased stability of the higher order structure (the solenoid) of chicken erythrocyte chromatin, which may be related to the reduction of nuclear activity in the chicken erythrocyte cell. In addition to intact chicken erythrocyte chromatin, we studied the structural transitions of H1-depleted and H1,H5-depleted chromatins. The result indicated that histone H5 of this chromatin stabilizes the higher order structure in the presence of magnesium (or divalent) cation and did not induce the transition in the solution containing only sodium cation.

The interaction of platinum and palladium complexes with closed and nicked circular and linear DNAs was investigated by a variety of methods. Cationic metal complexes containing flat, aromatic ligands, such as 2,2',2''-terpyridine, o-phenanthroline, and 2,2'-bipyridine, interfere with the usual fluorescence enhancement of ethidium bromide by competing for intercalation sites on calf-thymus DNA. Metal complexes having kinetically exchangable ligands, including the antitumor drugs cis-[(NH3)2PtCl2] and [(en)PtC12], inhibit noncompetitively the DNA-associated ethidium fluorescence enhancement by binding covalently to the bases and blocking potential intercalation sites. Only the metallointercalators were capable of altering the DNA duplex winding, as judged by the effects of these reagents upon the electrophoretic mobility and sedimentation behavior of PM-2 DNAs. Long-term (t greater than 120 h) interactions of metal complexes with PM-2 DNAs I, I0, and II, corresponding to superhelical, closed relaxed, and nicked circles, respectively, showed that covalent binding occurs the most readily to DNA I, possibly because of the presence of underwound duplex regions in this tightly wound superhelical DNA. The active antitumor drugs cis-[(NH3)2PtC12] and [(en)PtC12] bind covalently to DNA I under conditions where the inactive trans- [(NH3)2PtC12] does not. Most of the complexes studied were capable of producing chain scissions in PM-2DNA I. Exceptions are the kinetically inert complexes [(bipy)Pt(en)]2+ and (terpy)Pt(SCH2CH2OH)]+, suggesting that covalent binding might be a prerequisite for nicking.

The mechanism of the enhancement of the fluorescence of ethidium bromide on binding to double helical RNA and DNA has been investigated. From an examination of the effect of different solvents on the fluorescence lifetime, quenching of fluorescence by proton acceptors, and the substantial lengthening of lifetime observed upon deuteration of the amino protons, regardless of the medium, we conclude that proton transfer from the excited singlet state is the process primarily responsible for the approximately equal to 3.5-fold increase in the lifetime of free ethidium bromide in going from H2O to D2O; the fact that addition of small amounts of water to nonaqueous solvents decreases the fluorescence whereas addition of small amounts of D2O enhances the fluorescence; and the enhancement of the ethidium bromide triplet state yield on binding to DNA. Other proposed mechanisms are shown to be inconsistent with our findings.