Dye Displacement Research Articles

Interactions of New Synthesized Fluorescent Cationic Amphiphiles Bearing Pyrene Hydrophobe with Plasmid DNA: Binding Affinities, Aggregation and Intracellular Uptake

In this work, we prepared a novel series of cationic amphiphiles denoted as the Py-cations (Py-Gly, Py-Ala, Py-Cap, Py-G(1)-Lys and Py-G(2)-Lys) bearing fluorescent pyrene and various hydrocarbon linkers between the pyrene hydrophobe and cationic block. Employing these new cationic amphiphiles with pyrene as the fluorescent probe, the interactions between these Py-cations and plasmid DNA (pDNA) in distilled water and 0.1 M PBS buffer solution have been explored by means of UV-vis and fluorescent spectrometers, and ethidium bromide dye displacement and agarose-gel retardant assays were also implemented to evaluate their pDNA binding affinities in aqueous solution. Furthermore, the average sizes and morphologies of self-assembled Py-cation/pDNA lipoplex aggregates were examined by dynamic laser light scattering (DLS) and atomic force microscopy (AFM). It was found that these fluorescent cationic amphiphiles showed blue fluorescence emission of pyrene probe at λ = 340 nm in distilled water while their interactions with pDNA led to new strong green emission at λ = 490 nm, and this may be due to the stacking of pyrene and new formation of excimers via the rigid pDNA templated self-assembly. It was also revealed that the binding between new Py-cations and pDNA in aqueous solution was strongly influenced by the Py-cation hydrophobicity, charges of the cation and the presence of electrolytes. With respect to the Py-cation/pDNA aggregate morphologies, very interesting 1-D hybrid nanofibers were predominantly observed by AFM for the Py-Cap/pDNA aggregates. In addition, utilizing a COS-7 cell-line, in-vitro cellular uptakes of new cationic amphiphiles with pyrene probe were studied and visualized by fluorescent microscopy. As a result, this may provide a new approach to investigate the interactions between synthetic cationic lipids and nucleic acids, and pave an alternative clue to design new organic gene delivery carriers.

Binding of Functionalized Paramagnetic Nanoparticles to Bacterial Lipopolysaccharides And DNA

Magnetite and metallic cobalt-based nanoparticles with sizes ranging from 10 to 300 nm and surface-functionalized with poly(hexamethylene biguanide) (PHMBG) are introduced as capable lipopolysaccharide (LPS)-sequestering agents. The nanoparticles efficiently bind to whole E. coli cells and can be used to separate the cells effectively from suspension using a magnet. A fluorescence dye displacement assay shows strong affinities of the nanoparticles for lipid A, the glycolipid component of LPS responsible for septic shock. The particle-lipid A affinity is of the same order of magnitude or higher than that of polymyxin B. The affinity of smaller (< 50 nm) magnetite particles modified with PHMBG to lipid A is several-fold higher than that of their larger counterparts (> 100 nm) due to their higher surface area to volume ratio. The nanoparticles possess high saturation capacity for double-stranded lambdaDNA from E. coli, with which particle-polyelectrolyte complexes are formed. The PHMBG-modified nanoparticles are potent bactericides, inhibiting E. coli viability and growth at concentrations at < or = 10 microg/mL.

Substrate-Selective Supramolecular Tandem Assays: Monitoring Enzyme Inhibition of Arginase and Diamine Oxidase by Fluorescent Dye Displacement from Calixarene and Cucurbituril Macrocycles

A combination of moderately selective host-guest binding with the impressive specificity of enzymatic transformations allows the real-time monitoring of enzymatic reactions in a homogeneous solution. The resulting enzyme assays ("supramolecular tandem assays") exploit the dynamic binding of a fluorescent dye with a macrocyclic host in competition with the binding of the substrate and product. Two examples of enzymatic reactions were investigated: the hydrolysis of arginine to ornithine catalyzed by arginase and the oxidation of cadaverine to 5-aminopentanal by diamine oxidase, in which the substrates have a higher affinity to the macrocycle than the products ("substrate-selective assays"). The depletion of the substrate allows the fluorescent dye to enter the macrocycle in the course of the enzymatic reaction, which leads to the desired fluorescence response. For arginase, p-sulfonatocalix[4]arene was used as the macrocycle, which displayed binding constants of 6400 M(-1) with arginine, 550 M(-1) with ornithine, and 60,000 M(-1) with the selected fluorescent dye (1-aminomethyl-2,3-diazabicyclo[2.2.2]oct-2-ene); the dye shows a weaker fluorescence in its complexed state, which leads to a switch-off fluorescence response in the course of the enzymatic reaction. For diamine oxidase, cucurbit[7]uril (CB7) was used as the macrocycle, which showed binding constants of 4.5 x 10(6) M(-1) with cadaverine, 1.1 x 10(5) M(-1) with 1-aminopentane (as a model for the thermally unstable 1-aminopentanal), and 2.9 x 10(5) M(-1) with the selected fluorescent dye (acridine orange, AO); AO shows a stronger fluorescence in its complexed state, which leads to a switch-on fluorescence response upon enzymatic oxidation. It is demonstrated that tandem assays can be successfully used to probe the inhibition of enzymes. Inhibition constants were estimated for the addition of known inhibitors, i.e., S-(2-boronoethyl)-L-cysteine and 2(S)-amino-6-boronohexanoic acid for arginase and potassium cyanide for diamine oxidase. Through the sequential coupling of a "product-selective" with a "substrate-selective" assay it was furthermore possible to monitor a multistep biochemical pathway, namely the decarboxylation of lysine to cadaverine by lysine decarboxylase followed by the oxidation of cadaverine by diamine oxidase. This "domino tandem assay" was performed in the same solution with a single reporter pair (CB7/AO).

Dye displacement assay for saccharide detection with boronate hydrogels

Hydrogel spheres, fashioned from an operationally simple mould, that incorporate boronate units were shown to function as saccharide sensors.

Supramolecular Tandem Enzyme Assays for Multiparameter Sensor Arrays and Enantiomeric Excess Determination of Amino Acids

The coupling of an enzymatic transformation with dynamic host-guest exchange allows the unselective binding of macrocycles to be used for highly selective analyte sensing. The resulting supramolecular tandem enzyme assays require the enzymatic substrate and its corresponding product to differ significantly in their affinity for macrocycles, for example, cation receptors, and to show a differential propensity to displace a fluorescent dye from its host-guest complex. The enzymatic transformation results in a concomitant dye displacement that can be accurately followed by optical spectroscopy, specifically fluorescence. By exploiting this label-free continuous enzyme assay principle with the fluorescent dye Dapoxyl and the macrocyclic host cucurbit[7]uril, a multiparameter sensor array has been designed, which is capable of detecting the presence of amino acids (e.g. histidine, arginine, lysine, and tyrosine) and their decarboxylases. Only in the presence of both, the particular amino acid and the corresponding decarboxylase, is the amine or diamine product formed. These products are more highly positively charged than the substrate, have a higher affinity for the macrocycle and, therefore, displace the dye from the complex. The extension of the high selectivity and muM sensitivity of the tandem assay principle has also allowed for the accurate measurement of D-lysine enantiomeric excesses of up to 99.98 %, as only the L-enantiomer is accepted by the enzyme as a substrate and is converted to the product that is responsible for the observed fluorescence signal.

Synthesis and Photophysical Investigation of Squaraine Rotaxanes by “Clicked Capping”

Pseudorotaxane complexes of squaraine dyes and tetralactam macrocycles are converted into permanently interlocked rotaxane structures using copper-catalyzed and copper-free cycloaddition reactions with bulky stopper groups. The photophysical properties of the encapsulated squaraine depend on the structure of the macrocycle. In one case, squaraine rotaxanes are produced in near-quantitative yields and with intense near-IR fluorescence. In another case, squaraine fluorescence is greatly diminished upon macrocyclic encapsulation but the signal can be restored by dye displacement with anions.

Glutathione-mediated release of functional plasmid DNA from positively charged quantum dots

DNA was efficiently bound to water-soluble positively charged CdTe quantum dots (QDs) through complementary electrostatic interaction. These QDs–DNA complexes were disrupted and DNA was released by glutathione (GSH) at intracellular concentrations. Interestingly, there was almost no detectable DNA released by extracellular concentration of GSH. The formation of QDs–DNA complexes and GSH-mediated DNA release from the complexes were confirmed by dye displacement assay, electrophoretic mobility shift assay (EMSA), transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS) experiments. The released DNA retained transcriptional activity and expressed enhanced green fluorescent protein (EGFP) after being transfected into HEK 293 cells. The transfection efficiency measured by flow cytometry (FCM) was comparable with the positive control. The obvious difference of GSH concentration in nature between the intra- and extracellular environments as well as the GSH concentration-dependent triggered release suggests potential applications of these positively QDs in selective unpacking of payload in living cells in a visible manner.

Study of thiazole orange in aptamer-based dye-displacement assays

We screened a series of RNA and DNA aptamers for their ability to serve in the dye displacement assays in which analytes compete with TO dye. We conclude that, while the performance of the TO dye displacement approach is not always predictable, it is still a simple and sensitive assay to detect binding between RNA aptamers and small molecules. In particular, we describe efficient assays for tobramycin and theophylline, with up to 90% displacement of TO observed, and we describe the first aptameric assay for cAMP.

DNA affinity binding studies using a fluorescent dye displacement technique: the dichotomy of the binding site

We have observed a number of discrepancies and contradictions in the use of a fluorescent intercalator displacement assay in surveying the binding affinities of dinuclear polypyridyl ruthenium(II) complexes with DNA. By a modification of the assay using the fluorescent minor-groove binder 4',6-diamidino-2-phenylindole, rather than intercalating dyes (ethidium bromide or thiazole orange), results were obtained for all complexes studied which were consistent with relative affinities and stereoselectivities observed with other techniques, including NMR, affinity chromatography and equilibrium dialysis. It is believed that the difference in binding mode between the minor groove-binding Ru(II) complexes and the intercalating fluorescent dyes they are displacing may contribute to these discrepancies.

Mechanisms of Helicases

Mechanisms of Helicases

Lipoplexes with biotinylated transferrin accessories: Novel, targeted, serum-tolerant gene carriers

Novel transfecting assemblies comprising biotinylated cationic liposomes, DNA and tribiotinylated transferrin-streptavidin (streptavidin(bio 3-transferrin)) accessories have been prepared, characterized and evaluated for toxicity and DNA delivery capability in human cervical carcinoma cells (HeLa). Two new lipophilic cholesteryl-based biotin derivatives, biotinylcholesterylformylhydrazide (MSB1) and aminohexanoylbiotinylcholesterylformylhydrazide (MSB2) provided docking points for streptavidin(bio 3-transferrin) on cationic liposomes which were formulated with N, N-dimethylaminopropylaminylsuccinylcholesterylformylhydrazide (MS09) and dioleoylphosphatidylethanolamine (DOPE) in a 2:48:50 molar ratio. Ethidium dye displacement assays and gel retardation studies suggest that in ternary complexes, the DNA is electrostatically bound to the cationic liposomes while transferrins remain liposome-bound through streptavidin–biotin interactions. Assemblies fully protected plasmid DNA from serum nuclease digestion over a range of liposome:pGL3 DNA ratios (3–8:1, w/w) and exhibited low growth inhibition of HeLa cells (circa 5%) at the optimal transfection composition for streptavidin(bio 3-transferrin):liposome:pGL3 DNA of 10:6:1 (w/w/w). Transfection levels, which were twice those of untargeted lipoplexes containing MSB1 or MSB2, were not significantly diminished in the presence of 10% foetal bovine serum. Excess transferrin (200 μg per well) reduced transfection levels to those of untargeted complexes, supporting the notion that at least 50% of ternary complexes gained entry into the cervical carcinoma cells by receptor mediation. Conversely, transfection levels with untargeted lipoplexes were only slightly reduced in the presence of transferrin at the same concentration.

Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme

We recently demonstrated that the RecBCD enzyme is a bipolar DNA helicase that employs two single-stranded DNA motors of opposite polarity to drive translocation and unwinding of duplex DNA. We hypothesized that this organization may explain the exceptionally high rate and processivity of DNA unwinding catalyzed by the RecBCD enzyme. Using a stopped-flow dye displacement assay for unwinding activity, we test this idea by analyzing mutant RecBCD enzymes in which either of the two helicase motors is inactivated by mutagenesis. Like the wild-type RecBCD enzyme, the two mutant proteins maintain the ability to bind tightly to blunt duplex DNA ends in the absence of ATP. However, the rate of forward translocation for the RecB motor-defective enzyme is only approximately 30% of the wild-type rate, whereas for the RecD motor-defective enzyme, it is approximately 50%. More significantly, the processivity of translocation is substantially reduced by approximately 25- and 6-fold for each mutant enzyme, respectively. Despite retaining the capacity to bind blunt dsDNA, the RecB-mutant enzyme has lost the ability to unwind DNA unless the substrate contains a short 5'-terminated single-stranded DNA overhang. The consequences of this observation for the architecture of the single-stranded DNA motors in the initiation complex are discussed.

Effect of exopolysaccharides on the adsorption of metal ions by Pseudomonas sp. CU-1

Pseudomonas sp. CU-1, which was isolated from an interfacial biofilm of a sludge sample collected from an electroplating company, had a relatively high ability to adsorb Cu2 + in solution. The bacterium grown in broth culture produced a large amount of capsular exopolymers mainly consisting of polysaccharides. The exopolysaccharides (EPS) were partially purified. The adsorption isotherm experiments showed that cells and EPS of Pseudomonas sp. CU-1 had similar Q0 and b for the dye, Janus Green, and Cu2+. The adsorption of Cu2 + by cells could be monitored by the amount of dye displaced, due to the binding of metal ions onto the cell surface. The order of adsorption ability of metal ions and dye displacement by metal ion of the bacterium was: Cu2 + &amp;gt; Cd2 + &amp;gt; Zn2 + &amp;gt; Ni2 + . The results of the dye displacement by metal ions binding onto the surfaces of cells, EPS-removed cells, and EPS suggest that EPS produced by Pseudomonas sp. CU-1 plays an important role in preventing metal ions in the surrounding environment from contact with the bacterial cells. The possible role of the metal ion adsorption by the EPS of this biofilm bacterium was discussed.

Colorimetric Molecularly Imprinted Polymer Sensor Array using Dye Displacement

A colorimetric sensor array composed of seven molecularly imprinted polymers was shown to accurately identify seven different aromatic amines. The response patterns were systematically classified using linear discriminant analysis with 94% classification accuracy. Analyses of the response patterns of the analytes to the imprinted polymer array suggest that the different selectivity patterns, although subtle, appear to arise from the imprinting process. The molecular imprinting process enabled the rapid preparation of the polymers in the array from ethylene glycol dimethacrylate and methacrylic acid (80:20) in the presence of six different template molecules plus a blank nonimprinted polymer. The response of the imprinted polymer array was coupled to a colorimetric response, using a dye displacement strategy. A benzofurazan dye was selected and shown to give an accurate measure of the binding properties of the imprinted polymer array to all seven analytes. The colorimetric response also enabled the inclusion of analytes that are not spectroscopically active and were not among the original analytes that were used as template molecules. This broadens the potential utility of the imprinted polymer sensor array strategy to a wider range of analytes and applications.

Adsorption of dyes to cotton and inhibition by surfactants, polymers and surfactant–polymer mixtures

A wide‐ranging investigation has been made of the adsorption of direct dyes to cotton and of inhibition by surfactants, polymers and polymer–surfactant mixtures. Generally, the selected polymers are extremely effective at inhibiting adsorption of most of the direct dyes to cotton but are less effective at inhibiting adsorption of small, model azo dyes. Micellar solutions of zwitterionic and cationic surfactants can inhibit adsorption of both small dyes and commercial dyes. It is shown that anionic surfactants at sub‐micellar concentrations can inhibit polymer‐dye interactions due to displacement of dye and/or relocation into micelle‐like polymer–surfactant complexes. New insights have been obtained into the interactions of dyes with cotton and with polymers, surfactants or their mixtures, particularly into observed dye selectivities.

Solubilisation of dyes by surfactant micelles. Part 3; A spectroscopic study of azo dyes in surfactant solutions†

A spectroscopic study (UV–vis and adsorption) has been made of the interactions of select model azo dyes with a range of surfactant types or their mixtures both above and below their respective critical micelle concentrations. All surfactants inhibit adsorption of the dyes to cotton above their critical micelle concentrations due to incorporation in micelles. However, formation of 1;1 complexes between dyes and cationic or zwitterionic surfactants in sub‐micellar regions results in enhanced deposition on cotton. It is shown that attractive or repulsive electrostatic interactions play a key role in dye binding to micelles. Unusually, spectra of complexes formed between the dye and cationic surfactant are typical of those of the azo tautomeric form as opposed to the hydrazone form that is prevalent in aqueous media. Addition of anionic surfactant to micellar solutions of nonionic or zwitterionic surfactants results in successive displacement of dye from the respective micelles, i.e. binding is competitive.

A Fluorescence Study of the Structure and Accessibility of Plasmid DNA Condensed with Cationic Gene Delivery Vehicles

The cationic lipids 1,2‐dioleoyl‐3‐trimethylammonium‐propane and dimethyldioctadecylammonium bromide, with or without the helper lipids 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine or cholesterol, and the cationic polymer polyethyleneimine, were compared for their ability to displace fluorescent dyes from DNA. Differences in displacement of the intercalating dyes ethidium bromide and ethidium homodimer correlate with their relative affinities with DNA, with the extent of ethidium homodimer displacement significantly less. Differences in ethidium homodimer and ethidium bromide displacement as a function of the ratio of polycation to DNA and the charge density of the polycation suggest a greater sensitivity of the former to topological changes in condensed DNA. Marked differences in the ability of these cationic delivery systems to displace the minor groove binding dyes 4′,6‐diamidino‐2‐phenylindole and Hoechst 33258 upon interaction with DNA are also apparent, with the majority of Hoechst 33258 remaining bound to DNA. Changes in the spectral properties of Hoechst 33258 were further used to characterize polycation‐induced changes in solvent accessibility of the DNA minor groove. Taken together, these studies demonstrate differences in the interaction of various cationic lipids and polyethyleneimine in terms of regional displacement of dyes, polycation‐induced structural changes in DNA, as well as polycation‐mediated changes in solvent accessibility of the minor groove. The relevance of these studies to current models of the structure and assembly of polycation/DNA complexes are discussed. © 2003 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:1272–1285, 2003

Influence of polymer architecture on the structure of complexes formed by PEG–tertiary amine methacrylate copolymers and phosphorothioate oligonucleotide

The influence of polymer structure on the characteristics of complexes of a phosphorothioate antisense oligonucleotide (ISIS 5132) was studied, using well-defined cationic copolymers based on 2-(dimethylamino) ethyl methacrylate (DMAEMA) and poly(ethylene glycol) (PEG). The three related copolymer structures were: DMAEMA–PEG (a diblock copolymer) DMAEMA–OEGMA 7 (a brush-type copolymer), DMAEMA–stat-PEGMA (a comb-type copolymer); each of these were examined together with DMAEMA homopolymer, which served as a control. The results revealed that all the polymers exhibited good binding ability with the oligonucleotide (ON). Interestingly, the comb-type polymer DMAEMA–stat-PEGMA demonstrated the highest binding ability and DMAEMA homopolymer the lowest, as judged by a dye displacement assay. DMAEMA homopolymer produced large agglomerates of smaller individual complexes as observed by optical density, photon correlation spectroscopy and transmission electron microscopy studies. In contrast, two PEG-block copolymers, DMAEMA–PEG and DMAEMA–OEGMA 7, formed compact complexes of 80–150 nm which had good long-term colloidal stability. This is attributed to the steric stabilisation effect of the PEG chains on the ON–copolymer complexes. These two copolymers are believed to form complexes with ON that have a micellar structure. Comb-type DMAEMA–stat-PEGMA copolymer formed highly soluble complexes with the ON that did not phase separate from the buffer solution. This study clearly demonstrates that varying the copolymer architecture allows access to a range of ON complexes. In vitro cytotoxicity experiments on HepG2 cells showed that all of the tertiary amine methacrylate copolymers displayed lower cytotoxicity than the control poly( l-lysine).

Processive translocation and DNA unwinding by individual RecBCD enzyme molecules.

RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 +/- 172 base pairs per second (0.30 microm s(-1)), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.

Characterization of a targeted gene carrier, lactose-polyethylene glycol-grafted poly-L-lysine and its complex with plasmid DNA.

The physicochemical properties and gene transfer ability of lactose-polyethylene glycol-grafted poly-L-lysine (Lac-PEG-PLL) were investigated. A dye displacement assay showed that plasmid DNA self-assembled with Lac-PEG-PLL, and condensation began at a <1:1 charge ratio of plasmid DNA to polymer. In atomic force microscopy, spontaneously assembled Lac-PEG-PLL/DNA complexes revealed a compact structure, with a size of about 100-200 nm. Circular dichroism spectra of Lac-PEG-PLL/DNA complexes revealed that the secondary structure of DNA was altered by complex formation and was similar to that of the poly-L-lysine/DNA complex. Lac-PEG-PLL was shown to protect DNA against nuclease action in a DNase I protection assay. The cytotoxicity test demonstrated that the complex composed of plasmid DNA and Lac-PEG-PLL had little influence on the viability of HepG2 cells, especially in comparison with that of poly-L-lysine/DNA complexes. In conclusion, our copolymer, Lac-PEG-PLI, formed complexes with plasmid DNA (on average, 150 nm), gave little cytotoxicity, and showed increased efficiency of gene transfer into hepatoma cells in vitro. Lactose-polyethylene glycol was grafted to poly-L-lysine to be used as a gene carrier for hepatoma cell targeting and to improve the solubility of the polyplexes. The average size of the carrier/DNA complexes was about 150 nm. The complexes also proved to have high resistance against nuclease attack and little cytotoxicity. The polymer also delivered plasmid DNA efficiently into a HepG2 cell line. Lac-PEG-PLL was more efficient than Lipofectin or galactose-PEG-PLL in transfection efficiency.