Time-resolved electrophoretic analysis of mobility shifts for dissociating DNA ligands

Intercalative binding of ligands to DNA can be demonstrated by helix unwinding, monitored by gel electrophoresis of supercoiled DNA, as electrophoretic mobility is sensitive to the topological DNA state. However, we show that an apparent lack of unwinding in an electrophoretic assay could be due to dissociation of the (intercalated) ligand during the analysis, rather than evidence for a nonintercalative mode of binding to DNA. Repetitive scanning during the electrophoresis ensures that release of the ligand during electrophoresis does not affect the measured degree of unwinding, based on the electrophoretic velocity being determined as a function of time. We use this assay to establish intercalation as a mode of binding to DNA for the cyanine dyes YO, YO-PRO as well as two enantiomeric forms of the ruthenium complexes [(phen)2 Ru(tatpp)Ru(phen)2]4+, and to support groove-binding for the new unsymmetrical cyanine dyes BOXTO and BOXTO-PRO. Groove-binding could be concluded from a lack of unwinding, because we could rule out that it is caused by release of the dye during the electrophoresis. The gel electrophoresis has the advantage over hydrodynamic techniques that much smaller sample amounts are required, and our time-resolved approach can be employed in all mobility-shift assays when applied to dissociating complexes.     

Effects of condensing agent and nuclease on the extent of ejection from phage lambda

We have recently demonstrated, that DNA ejection from bacteriophage lambda can be partially or completely suppressed in vitro by external osmotic pressure. This suggests that DNA ejection from phage is driven by an internal mechanical force consisting of DNA bending and DNA-DNA electrostatic repulsion energies. In the present work we investigate the extent to which DNA ejection is incomplete at zero osmotic external pressure when phage is opened with its receptor in vitro. The DNA fragment remaining in the capsid and the tail that is no longer bent or compressed -and hence for which there is no internal driving force for ejection- is shown not to be ejected. We also demonstrate that DNA can be "pulled" out from the capsid by DNase I acting as a DNA binding protein or spermine acting as a DNA condensing agent. In particular, cryo electron microscopy and gel electrophoresis experiments show the following: (i) DNA ejection from bacteriophage lambda incubated in vitro with its receptor is incomplete at zero external osmotic force, with several persistence lengths of DNA remaining inside the phage capsid, if no nuclease (DNase I) or DNA condensing agent (spermine) is present in the host solution; (ii) in the presence of both DNase I and spermine in the host solution, 60% (approximately 29 kbp) of wild-type lambda DNA (48.5 kbp) remains unejected inside the phage capsid, in the form of an unconstrained toroidal condensate; (iii) with DNase I added, but no spermine, the ejection is complete; (iv) with spermine, but without DNase I added, all the DNA is again ejected, and organized as a toroidal condensate outside.     

Photophysical and photochemical effects of dye binding

Abstract— A discussion is given of the photophysical and photochemical consequences of the binding of dyes and of pigments of biological importance to polymeric substrates. The modification of the photochemical properties induced by dye binding can in large part be ascribed to the known changes in photophysical properties of dyes engendered by such interactions. Principally, these involve enhanced formation of metastable species of dye molecules and decreased opportunity for self-quenching. In photochemical terms, dye binding thus enhances susceptibility to photoreduction, causes an increase in the quantum yield of photoreduction with increasing concentration of bound dye, and induces enhanced ability to act as a sensi-tizer in photoreduction. Paradoxically, dye binding decreases the ability of the bound dye to act as a sensitizer in photoxidation.     

Adsorption behavior of methylene blue and its congeners on a stainless steel surface

Methylene blue and its congeners as model dyes were adsorbed onto stainless steel particles at different ionic strengths, pH values, and ethanol contents, and the adsorption mechanism was investigated. A Fourier transform infrared spectroscopy (FTIR) analysis of the dyes adsorbed on the stainless steel plate was carried out to determine the orientations of the adsorbed dyes on stainless steel surface. The adsorption isotherms for all the dyes tested were approximated by a Langmuir equation (Q=Kq(m)C/(1+KC)) in most cases except under strongly basic conditions. From the ionic strength and ethanol content dependencies of the K value in the Langmuir equations, both the electrostatic and hydrophobic interactions were indicated to contribute to the adsorption of the dyes at neutral pH. By comparing the K and q(m) values for the methylene blue congeners and with the aid of the FTIR analyses, it was found that the kind of substituent groups at most positions of the polyheterocycles of methylene blue strongly affects the adsorption behavior, particularly the area occupied by an adsorbed dye molecule, the affinity for the stainless steel surface, and the orientation of the adsorbed dye molecule on the stainless steel surface.     

Evolution of peptides that modulate the spectral qualities of bound, small-molecule fluorophores

Background: Fluorophore dyes are used extensively in biomedical research to sensitively assay cellular constituents and physiology. We have created, as proof of principle, fluorophore dye binding peptides that could have applications in fluorescent dye-based approaches in vitro and in vivo.Results: A panel of Texas red, Rhodamine red, Oregon green 514 and fluorescein binding peptides, termed here ‘fluorettes’, was selected via biopanning of a combinatorial library of 12-mer peptides fused to a minor coat plll protein of the filamentous bacteriophage M 13, The ‘best’ fluorette sequences from each of the groups were subjected to further mutagenesis, followed by a second biopanning to select a new generation of improved fluorettes. Phage were selected that had higher avidity for each fluorophore except Rhodamine red, Of these, peptides were characterized that could specifically and with high affinity bind at least one dye, Texas red, in solution, In addition, the binding of certain peptides to Texas red shifted the peak excitation and/or the emission spectra of the bound dye.Conclusions: Peptides in the context of phage display could readily be selected that could bind to small-molecule fluorophores. The affinities of selected mutant fluorettes could be increased by mutation and further selection, Only a subset of the free peptides could bind free dyes in solution, suggesting that phage context contributed to the selection and ability of certain peptidic regions to independently bind the dyes, Future screens might lead to the creation of other dye-binding peptides with novel characteristics or Texas red derivatives with cross-linking substituents might be designed to increase the utility of the system.     

Interaction of Polymethine Dyes with Detonation Nanodiamonds

Among cationic, anionic, and merocyanine polymethine dyes, the binding to detonation nanodiamond (DND) colloid particles in hydrosol occurs only for negatively charged dye species. This, in view of the positive ζ-potential of the DND used in this study, suggests the predominance of electrostatic interactions over other intermolecular forces in such systems. Indeed, after decorating the merocyanine and the cationic dye by one and two negatively charged sulfopropyl groups, respectively, so that the net charge of their colored species becomes negative, the compounds also demonstrate affinity to the DND particles. In all cases, the binding of dyes to DND is accompanied by a decrease in fluorescence intensity and a bathochromic shift of their absorption and fluorescence bands. A quantitative study of the dyes adsorption on the DND nanoparticles as performed using the Küster-Freundlich and Langmuir equations reveals some peculiarities of their attaching to the DND aggregates and allows estimating the specific area of the DND particles at a concentration of 0.0212 wt/vol %.     

Coherent interactions of dyes assembled on DNA

The optical behavior of an organized dye assembly is different from that of the isolated dye; this difference is explained using molecular exciton theory. The theory predicts that mutual orientation, the number of dyes in the cluster, and combinations of different dyes should display given characteristic spectroscopic behaviors due to coherent interactions. Comparison of theoretical predictions with experimental results has been limited so far. One of the reasons is the absence of a rigid and well-organized system that can control the orientation and size of the dye assembly. Recently, the DNA duplex has been used to assemble chromophores in a programmed manner. Use of DNA allows organized dye assembly with a given size and particular orientation. In this review, we evaluate the spectroscopic behavior of the H-type aggregate based on molecular exciton theory and compare it with actual dye assembly with DNA duplex. Furthermore, we demonstrate the importance of coherent interactions on the observed optical properties of dyes assembled in a DNA duplex.     

A study of interaction of cationic dyes with anionic polyelectrolytes

The interactions of Acridine Orange with Sodium Alginate and Pinacyanol Chloride with Heparin have been investigated by spectrophotometric method. The polymers induce metachromasy in the dye as evidenced from the considerable blue shift in the absorption maxima of the corresponding dyes. The interaction constant and thermodynamic parameters of polymer–dye interactions have been determined. The effect of additives such as alcohols, and urea on the reversal of metachromasy has been studied. The data has been used to determine the stability of the metachromatic complex and the nature of binding. The thermodynamic parameters of interaction revealed that binding between Acridine Orange and Sodium Alginate involved only electrostatic forces while that between Pinacyanol Chloride involved both electrostatic and hydrophobic forces. The reversal studies using surfactants indicated the involvement of both electrostatic and hydrophobic forces in binding. Based on the results it can be concluded that Pinacyanol Chloride is more effective inducing metachromasy than Acridine Orange.     

Molecular Docking approach on the effect of Site- Selective and Site-Specific Drugs on the Molecular Interactions of Human Serum Albumin (HSA) -Acridinedione dye complex

Molecular Docking (Mol.dock) of resorcinol based acridinedione dyes (ADR1 and ADR2) with a globular protein, Human Serum Albumin (HSA) were carried out. Docking studies reveal that ADR2 dye binding with HSA is energetically more stable and feasible than ADR1 dye. ADR1 dye predominantly resides in site I and III of HSA rather than binding site II wherein, ADR1 dye acts as hydrogen bonding (HB) acceptor through its carbonyl oxygen. On the contrary, ADR2 dye resides in all the binding sites of HSA such that the dye acts as the HB donor through the NH hydrogen atom and the carbonyl oxygen of the amino acid acts as the HB acceptor. The stability of dye-protein complex in the presence of several non-steroidal anti-inflammatory drugs (NSAIDs) was carried out by employing specific site selective drugs (Sudlow binding site drugs). The energetics and the bimolecular interactions of various drugs with ADR1-HSA and ADR2-HSA were generated to ascertain the influence of drug and its governance on the binding affinity of dye-protein complex. Sudlow site I binding drugs were effective in decreasing the energetics of ADR1 dye-HSA complex whereas site II binding drugs predominantly decreases the affinity of ADR2 dye with HSA. However, the dyes efficiently displaces the site specific drugs from their specific binding sites of HSA which was not observed in the case of drugs on the displacing ability over dyes situated in different domains of protein. Mol.dock studies are employed as an authentic, reliable and most effective tool to ascertain the binding stability of host–guest complex as well as to ascertain the most probable location of several competing ligands in various domains of HSA.     

Binding interaction of cationic phenazinium dyes with calf thymus DNA: a comparative study

Absorption, steady-state fluorescence, steady-state fluorescence anisotropy, and intrinsic and induced circular dichroism (CD) have been exploited to explore the binding of calf thymus DNA (ctDNA) with three cationic phenazinium dyes, viz., phenosafranin (PSF), safranin-T (ST), and safranin-O (SO). The absorption and fluorescence spectra of all the three dyes reflect significant modifications upon interaction with the DNA. A comparative study of the dyes with respect to modification of fluorescence and fluorescence anisotropy upon binding, effect of urea, iodide-induced fluorescence quenching, and CD measurements reveal that the dyes bind to the ctDNA principally in an intercalative fashion. The effect of ionic strength indicates that electrostatic attraction between the cationic dyes and ctDNA is also an important component of the dye-DNA interaction. Intrinsic and induced CD studies help to assess the structural effects of dyes binding to DNA and confirm the intercalative mode of binding as suggested by fluorescence and other studies. Finally it is proposed that dyes with bulkier substitutions are intercalated into the DNA to a lesser extent.     

Energies and pressures in viruses: contribution of nonspecific electrostatic interactions

We summarize some aspects of electrostatic interactions in the context of viruses. A simplified but, within well defined limitations, reliable approach is used to derive expressions for electrostatic energies and the corresponding osmotic pressures in single-stranded RNA viruses and double-stranded DNA bacteriophages. The two types of viruses differ crucially in the spatial distribution of their genome charge which leads to essential differences in their free energies, depending on the capsid size and total charge in a quite different fashion. Differences in the free energies are trailed by the corresponding characteristics and variations in the osmotic pressure between the inside of the virus and the external bathing solution.     

ON THE COMPLEX FORMATION OF ACRIDINE DYES WITH DNA-IV. THE EQUILIBRIUM CONSTANTS OF SUBSTITUTED PROFLAVINE AND ACRIDINE ORANGE DERIVATIVES*

Abstract— The binding constants of the complex formation of several acridine dyes with calf thymus DNA are reported. The results indicate, that the binding tendency increases with the basicity of the dyes in the order acridine < 3–amino-acridine < 3.6–bis-monocetamino-acridine < 3.6–diamino-acridine ? 3.6–bis-dimethylamino-acndine < 3.6–diamino-10–methyl-acridine. The ring-N alkylated amino-acridines give in general the highest brinding constants, but there is no significant difference in the extrapolated values, K₀, at diminishing dye concentration if the chain length of the alkyl groups is increased from methyl to amyl. Only substitution with phenyl seems to inhibit the binding. On the other hand, the weaker binding of the acndine itself is sensitive to the ring-N alkylation and the binding tendency decreases in the order acridine > 10–methyl-acridine > 10–propyl-acridine. These findings could be explained, if the electrostatic binding involving the positively charged ring nitrogen, which seems to be more important for the more weakly interacting acridines, is complemented by other binding mechanisms, including possibly non-electrostatic interactions in the case of those amino-acridines having the strong binding effect.     

Multilinear regression and comparative molecular field analysis (CoMFA) of azo dye-fiber affinities. 2. Inclusion of solution-phase molecular orbital descriptors

For a data set with 30 direct azo dyes taken from literature, quantitative structure-activity relationship (QSAR) analyses have been performed to model the affinity of the dye molecules for the cellulose fiber. The electronic structure of the compounds was characterized using quantum chemical gas-phase (AM1) and continuum-solvation molecular orbital parameters. As regards the solution phase, COSMO appears to be better suited than SM2 in quantifying relative trends of the aqueous solvation energy. For the dye-fiber affinity, the leave-one-out prediction capability of multilinear regression equations is superior to CoMFA, with predictive squared correlation coefficients ranging from 0.63 (pure CoMFA) to 0.89. At the same time, solution-phase CoMFA is superior to previously derived AM1-based CoMFA models. As a general trend, the dye-fiber affinity increases with increasing electron donor capacity that corresponds to an increasing hydrogen bond acceptor strength of the azo dyes. The discussion includes the consideration of structural features that are likely to be involved in dye-fiber and dye-dye hydrogen bonding interactions, and possible links between CoMFA electrostatic results and the atomic charge distribution of the compounds.     

Influence de la conformation des polyions sur la fixation de colorants—III. Etude de la fixation de l'auramine o sur des polyacides synthetiques par equilibres de dialyse

The binding of auramine O (AuO), a cationic dye which does not dimerize, to poly(methacrylic acid) (PMA) and poly(acrylic acid) (PAA) has been measured by dialysis experiments. The effects of ionization, ionic strength and polymer to dye ratio (P/D) have been systematically investigated. At low ionization and P/D = 100, the bound fraction (q) of AuO is distinctly higher with PMA than with PAA at all ionic strength; this difference can be attributed to non-electrostatic interactions. Increase of ionic strength leads to displacement of the bound dye; on the other hand, high P/D ratio favours binding. We were able to describe the binding isotherms by an ion-exchange process at constant ionization and ionic strength. Water-methanol mixtures, known to destroy the coiled conformations of PMA, lead to a decrease in q; the same is observed in unionized acidic PMA solutions where ionic interactions are suppressed. These results indicate a strong binding due to the insertion of the dye into the polyion compact core and also a weak electrostatic one; they agree with the observations of spectrofluorescence; it must be emphasized that the bound fraction of dye is not, in itself, a significant parameter of conformational states.     

Thermodynamic studies on the interaction of folic acid with bovine serum albumin

Binding of the vitamin folic acid with bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC) in combination with fluorescence and circular dichroism spectroscopies. The thermodynamic parameters of binding have been evaluated as a function of temperature, ionic strength, in the presence of nonionic surfactants triton X-100, tetrabutylammonium bromide, and sucrose. The values of the van’t Hoff enthalpy calculated from the temperature dependence of the binding constant agree with the calorimetric enthalpies indicating that the binding of folic acid to the BSA is a two state process without involving intermediates. These observations are supported by the intrinsic fluorescence and circular dichroism spectroscopic measurements. With increase in the ionic strength, reduction in the binding affinity of folic acid to BSA is observed suggesting predominance of electrostatic interactions in the binding. The contribution of hydrophobic interactions in the binding is also demonstrated by decrease in the binding affinity in the presence of tetrabutylammonium bromide (TBAB). The value of binding affinity in the presence of sucrose indicates that hydrogen bonding also plays a significant contribution in the complexation process. The calorimetric and spectroscopic results provide quantitative information on the binding of folic acid to BSA and suggest that the binding is dominated by electrostatic interactions with contribution from hydrogen bonding.     

Monitoring viral DNA release with capillary electrophoresis

Viral DNA injection into host cells is one of the primary mechanisms of viral propagation. Drug development that targets viral propagation requires fast and sensitive methods for monitoring the release of viral DNA in vitro. Here we demonstrate the use of capillary electrophoresis (CE) for monitoring DNA release from virus particles. As a model for this study, we used T5 bacteriophages that infect the bacterium Escherichia coli K-12 by binding to the outer membrane FhuA receptor and then injecting DNA. DNA release from the T5 phages in vitro was induced by either elevated temperature or by interaction with the purified FhuA receptor. After DNA release, the viral samples were stained with the high affinity fluorescent dye YOYO-1, injected into the capillary and subjected to electrophoresis. YOYO-1-stained DNA generated a well-defined peak, allowing reliable detection of viral DNA from as few as 10(5) viral particles. The staining to track T5 phage DNA release exemplifies the great versatility that CE offers in studying viral systems. This CE-based method can be used to study molecular mechanisms of viral infections and to evaluate anti-viral drug candidates.     

Spectral and thermodynamic studies of the interaction of binding site probes with poly(vinylpyrrolidone)

The binding of two ionic azo dyes (4-phenylazo-1-naphthol mono-and disulfonate) and a fluorescent probe (2-p-toluidinonaphthalene-6-sulfonic acid, TNS) to poly(vinylpyrrolidone) (PVP) was studied to obtain information on the nature of the interaction, binding isotherm, and binding site. Sorption of the dyes followed a Langmuir isotherm only at low polymer saturation. Apparent cooperativity in binding was seen at higher saturation. The polymer had a higher intrinsic binding constant but lower binding capacity for the doubly charged dye than for the structurally similar singly charged dye. Both dyes consisted of tautomeric mixtures of hydrazone and azonaphthol forms in equilibrium in the bound and unbound state. The preferential binding of the azonaphthol tautomer of the disulfonate was highly exothermic and accompanied by an entropy decrease. The binding of the hydrazone form was less favored by 1.8 kcal/mol, was weakly exothermic, and accompained by an entropy increase. Increased preference for the azonaphthol tautomer accompanied chain extension from charging the polymer. Chain extension had no effect on the emission frequency of bound TNS. Large differences in binding capacities for similarly charged dyes indicated the existence of specific dye-site interactions. Arguments are presented against nonspecific hydrophobic interactions as predominant forces responsible for binding.     

Structure–affinity relationships for the binding of actinomycin D to DNA

Molecular models of the complexes between actinomycin D and 14 different DNA hexamers were built based on the X-ray crystal structure of the actinomycin–d(GAAGCTTC)2 complex. The DNA sequences included the canonical GpC binding step flanked by different base pairs, nonclassical binding sites such as GpG and GpT, and sites containing 2,6-diamino- purine. A good correlation was found between the intermolecular interaction energies calculated for the refined complexes and the relative preferences of actinomycin binding to standard and modified DNA. A detailed energy decomposition into van der Waals and electrostatic components for the interactions between the DNA base pairs and either the chromophore or the peptidic part of the antibiotic was performed for each complex. The resulting energy matrix was then subjected to principal component analysis, which showed that actinomycin D discriminates among different DNA sequences by an interplay of hydrogen bonding and stacking interactions. The structure–affinity relationships for this important antitumor drug are thus rationalized and may be used to advantage in the design of novel sequence-specific DNA-binding agents.     

Phagemid encoded small molecules for high throughput screening of chemical libraries

A new strategy for monovalently displaying small molecules on phage surfaces was developed and applied to high throughput screening for molecules with high binding affinity to the target protein. Peptidyl carrier protein (PCP) excised from nonribosomal peptide synthetase was monovalently displayed on the surface of M13 phage as pIII fusion proteins. Small molecules of diverse structures were conjugated to coenzyme A (CoA) and then covalently attached to the phage displayed PCP by Sfp phosphopantetheinyl transferase. Because Sfp is broadly promiscuous for the transfer of small molecule linked phosphopantetheinyl moieties to apo PCP domains, this approach will enable displaying libraries of small molecules on phage surfaces. Unique 20-base-pair (bp) DNA sequences were also incorporated into the phagemid DNA so that each compound displayed on the phage surface was encoded by a DNA bar code encapsulated inside the phage coat protein. Single round selection of phage displayed small molecules achieved more than 2000-fold enrichment of small molecules with nM binding affinity to the target protein. The selection process is further accelerated by the use of DNA decoding arrays for identifying the selected small molecules.