Studies on amikhellin: I. Intercalative binding to double-stranded DNA

The present paper reports that amikhellin, a drug so far used as a coronary vasodilator, binds to double-stranded DNA by an intercalation process which does not depend upon DNA base composition. The binding to DNA was established by spectrophotometry, ultracentrifugation and competition with ethidium bromide. The parameters of the binding equilibrium were calculated by these two latter methods. Evidence for intercalation was obtained from the observation by viscosimetric experiments of the length increase of sonicated calf thymus DNA and of the untwisting of circular PM2 DNA. The unwinding angle was measured to be 6° per bound drug molecule.     

1H NMR study of an ethidium dimer poly(dA-dT) complex: evidence of a transition between bis and monointercalation

Comparative 1H NMR and optical studies of the interaction between poly(dA-dT), ethidium bromide (Et) and ethidium dimer (Et2) in 0.7 M NaCl are reported as a function of the temperature. Denaturation of the complexes followed at both polynucleotide and drug levels leads to a biphasic melting process for poly(dA-dT) complexed with ethidium dimer (t1/2 = 75 degrees C; 93 degrees C) but a monophasic one in poly(dA-dT): ethidium bromide complex (t1/2 = 74 degrees C). In both cases drug signals exhibit monophasic thermal dependence (Et = 81 degrees C; Et2 = 95 degrees C). Evidence is presented showing that the ethidium dimer bisintercalates into poly(dA-dT) in high salt, based on the observation that i) dimer and monomer ring protons exhibit similar upfield shifts upon DNA binding, ii) upfield shifts of DNA sugar protons are twice as large with the dimer than with ethidium bromide. Comparison between native DNA fraction and bound drug fraction indicates that ethidium covers, n = 2.5-3 base pairs. The dimer bisintercalates and covers, n = 5.7 base pairs when the helix fraction is high but as the number of available sites decreases the binding mode changes and the drug monointercalates (n = 2.9).     

Figure-8 configuration of dimers of S13 and φX174 replicative form DNA

Evidence from electron microscopy of the replicative form of S13 and φX174 DNA shows the presence of a “figure-8” configuration. This species consists of two monomer length and one dimer length circular strands in covalently closed circular form and containing a fused junction that divides the molecule into two equal circular segments. Its existence is supported by the demonstration that it is converted by digestion with the restriction endonuclease of Hemophilus influenzae strain Rd to α- and X-shaped forms that retain the fused junction, and by examination by electron microscopy in the presence of ethidium bromide, which eliminates tangling and accidental overlays of parts of the DNA molecules. Kgure-8s are present to the extent of about 5% of the dimers present in replicative form DNA. They are proposed to be intermediates in genetic recombination in S13 and φX174.     

Comparison of the effect of ultraviolet radiation and ethidium bromide intercalation on the conformation of superhelical phiX174 replicative form DNA

Irradiation of Superhelical bacteriophage φX174 replicative form DNA with ultraviolet light increases the sedimentation coefficient of the DNA in 1 m-NaCl. Evidence that this is caused by the formation of pyrimidine dimers is provided by the observation that treatment of the DNA with photoreactivating enzyme restores the sedimentation coefficient of the Superhelical DNA to its original value. Also, there is an approximate correlation between the fraction of thymines dimerized and the increase in s value.Titration of the Superhelical twists in the irradiated and unirradiated DNA with ethidium bromide indicates that the ultraviolet light-induced increase in s value is accompanied by a decrease in the number of Superhelical turns. The appearance of the Superhelical DNA in electron micrographs is consistent with this conclusion.These experiments suggest that dimer formation and intercalation have the same effect on the structure of the DNA double helix, most probably that of partially unwinding the duplex. Each dimer formed appears to untwist the helix by approximately 5 to 6 °.     

Comparative microcalorimetric and dilatometric analysis of the interactions of quinacrine,chloroquine, and ethidium bromide with DNA

The enthalpies of binding of chloroquine and quinacrine to DNA at different molar ratios of drug to DNA and at different ionic strengths have been measured. The limiting values obtained with quinacrine fall in the range found for typical intercalating agents (e.g., ethidium, proflavin, adriamycin), whereas the value obtained with chloroquine is always zero, independent of the ratio of drug to DNA and ionic strength. The dilatometric measurements performed on the same systems and on the ethidium–DNA system show that when ethidium and quinacrine bind to DNA at low drug/DNA ratios, a volume decrease of about 16 mL/mol of bound drug occurs. No change in volume is observed when the two drugs bind to DNA through external, electrostatic forces. The volume change can be attributed to the loss of structured water around hydrophobic moieties of the drug molecules, following intercalation. In contrast, chloroquine binding to DNA at low drug/DNA ratios is characterized by a volume change distinctly smaller than that shown by quinacrine. The low ΔV_B and ΔH_B values shown by chloroquine are discussed in terms of the mechanism of interaction with DNA.     

Structure of the complex between lac repressor headpiece and operator DNA from measurements of the orientation relaxation and the electric dichroism.

The complex between lac repressor headpiece and short rodlike DNA fragments containing the lac operator sequence is characterised by measurements of the rotation diffusion. Using the method of electric dichroism we measure the rotation relaxation and determine changes in the length of the DNA upon ligand binding with high accuracy. According to these measurements any change in the length of the operator DNA upon binding of the first two headpiece molecules remains below 1A; the electric dichroism also remains virtually unchanged. At high degrees of (unspecific) binding we observe an increase in the rotation relaxation time, which is attributed to an increase of the apparent mean radius of the complex. As a control of our procedure for the determination of length changes we use the intercalation of ethidium bromide and arrive at an increase of the DNA length per bound ethidium of 3.2A (at 3.4A rise per base pair). The results obtained for the headpiece operator complex are not consistent with models assuming large changes of the DNA structure or intercalation of tyrosine residues.     

Effects of DNA charge and length on the electrophoretic mobility of intercalated DNA

DNA molecules ranging in size from 1 to 630 kilobase pair and intercalated with either ethidium bromide (EtBr) or propidium iodide (PI) were electrophoresed in 1% agarose at four different electric field strengths. The extent of intercalation of EtBr under the conditions of our electrophoresis experiments was determined by a spectroscopic technique, whereas the extent of intercalation of PI was inferred from previous studies. The effects of the increase in DNA contour length and the concomitant decrease of linear charge density were separated based on our analysis of the mobility data. We conclude that the main factor responsible for the reduced electrophoretic mobility of intercalated DNA is the diminished linear charge density and not the increased contour length. © 1994 John Wiley & Sons, Inc.     

Torsional sensing of small-molecule binding using magnetic tweezers

DNA-binding small molecules are widespread in the cell and heavily used in biological applications. Here, we use magnetic tweezers, which control the force and torque applied to single DNAs, to study three small molecules: ethidium bromide (EtBr), a well-known intercalator; netropsin, a minor-groove binding anti-microbial drug; and topotecan, a clinically used anti-tumor drug. In the low-force limit in which biologically relevant torques can be accessed (<10 pN), we show that ethidium intercalation lengthens DNA ∼1.5-fold and decreases the persistence length, from which we extract binding constants. Using our control of supercoiling, we measure the decrease in DNA twist per intercalation to be 27.3 ± 1° and demonstrate that ethidium binding delays the accumulation of torsional stress in DNA, likely via direct reduction of the torsional modulus and torque-dependent binding. Furthermore, we observe that EtBr stabilizes the DNA duplex in regimes where bare DNA undergoes structural transitions. In contrast, minor groove binding by netropsin affects neither the contour nor persistence length significantly, yet increases the twist per base of DNA. Finally, we show that topotecan binding has consequences similar to those of EtBr, providing evidence for an intercalative binding mode. These insights into the torsional consequences of ligand binding can help elucidate the effects of small-molecule drugs in the cellular environment.     

Modulation of DNA intercalation by resveratrol and genistein

Time correlated Single Photon Counting study (TCSPC) was performed for the first time to evaluate the effect of resveratrol (RES) and genistein (GEN) at 10–100 μM and 10–150 μM respectively, in modulating the DNA conformation and the variation induced due to intercalation by the dyes, ethidium bromide (EtBr) and acridine orange (AO). It is demonstrated using UV-absorption and fluorescence spectroscopy that RES and GEN, at 50 μM and 100 μM respectively can bind to DNA resulting in significant de-intercalation of the dyes, preventing their further intercalation within DNA. Hyperchromicity with red/blue shifts in DNA when bound to dyes was reduced upon addition of RES and GEN. DNA-dependent fluorescence of EtBr and AO was quenched in the presence of RES by 87.97% and 79.13% respectively, while similar quenching effect was observed for these when interacted with GEN (85.52% and 83.85%). It is found from TCSPC analysis that the higher lifetime component or constituent of intercalated dyes (τ₂, A ₂) decreased with the subsequent increase in smaller component or constituent of free dye (τ₁, A ₁) after the interaction of drugs with the intercalated DNA. Thus these findings signify that RES and GEN can play an important role in modulating DNA intercalation, leading to the reduction in DNA-directed toxicity.     

Conformational dynamics of DNA affected by intercalation and minor groove binding: direct observation of large DNA.

Using fluorescence microscopy, we have observed moving DNA molecules in solution and analyzed the "higher-order" structure in a quantitative manner. It was found that EB (ethidium bromide), an intercalator, has the effect to increase the persistent length. In other words, EB expands DNA. Whereas, DAPI (4',6-diamidino-2-phenylindole), a minor groove binding drug, decreases the persistent length. It is demonstrated that the direct observation of DNA molecules with fluorescence microscopy is quite useful to study the interaction of various chemical compounds with DNA molecules.     

Ethidium bromide interactions with DNA: an exploration of a classic DNA–ligand complex with unbiased molecular dynamics simulations

Visualization of double stranded DNA in gels with the binding of the fluorescent dye ethidium bromide has been a basic experimental technique in any molecular biology laboratory for >40 years. The interaction between ethidium and double stranded DNA has been observed to be an intercalation between base pairs with strong experimental evidence. This presents a unique opportunity for computational chemistry and biomolecular simulation techniques to benchmark and assess their models in order to see if the theory can reproduce experiments and ultimately provide new insights. We present molecular dynamics simulations of the interaction of ethidium with two different double stranded DNA models. The first model system is the classic sequence d(CGCGAATTCGCG)₂ also known as the Drew–Dickerson dodecamer. We found that the ethidium ligand binds mainly stacked on, or intercalated between, the terminal base pairs of the DNA with little to no interaction with the inner base pairs. As the intercalation at the terminal CpG steps is relatively rapid, the resultant DNA unwinding, rigidification, and increased stability of the internal base pair steps inhibits further intercalation. In order to reduce these interactions and to provide a larger groove space, a second 18-mer DNA duplex system with the sequence d(GCATGAACGAACGAACGC) was tested. We computed molecular dynamics simulations for 20 independent replicas with this sequence, each with ∼27 μs of sampling time. Results show several spontaneous intercalation and base-pair eversion events that are consistent with experimental observations. The present work suggests that extended MD simulations with modern DNA force fields and optimized simulation codes are allowing the ability to reproduce unbiased intercalation events that we were not able to previously reach due to limits in computing power and the lack of extensively tested force fields and analysis tools.     

Interactions of molecules with nucleic acids. IV. Binding energies and conformations of acridine and phenanthridine compounds in the two principal and in several unconstrained dimer-duplex intercalation sites

The binding positions and relative minimum binding energies are calculated for complexes of 9-aminoacridine, proflavine, N-methylphenanthridinium, and ethidium in theoretically determined intercalation sites in B-DNA (sites I and II) and in unconstrained dimer-duplex sites. The selection of site I in B-DNA by these compounds agrees with the theoretical interpretation of studies of unwinding angles in closed circular DNA in all cases but ethidium, which is predicted to select site II. The most stable binding positions of the acridines and ethidium in unconstrained dimer-duplex units agree with experimental results of intercalation complexes of dinucleoside monophosphate units. Base-pair specificity for Watson-Crick pairing is examined. The energy of an intercalation complex is partitioned into ΔE₂₃, the energy required to open base pairs BP₂ and BP₃ in B-DNA to a site, and ΔE_In, the energy change when a free molecular intercalates. ΔE₂₃ depends strongly on the base-pair sequence, whereas ΔE_In for the four molecules studied does not. The three most stable sequences contain (pyrimidine)p(purine) units, and this provides a rationale for the exclusive formation of crystals of intercalation complexes with these units. In spite of this selectivity, the distribution of G?C and A?T base pairs is equal for these three units and persists as the more unstable sequences are included. Therefore, specificity arises from the interaction between the base pairs and the 2′-deoxyribose 5′-monophosphate backbone for the opening of B-DNA to an intercalation site and not from the interaction between the chromophore and the DNA.     

Effect of B-Z transition and nucleic acid structure on the conformational dynamics of bound ethidium dimer measured by hydrogen deuterium exchange kinetics.

Ethidium dimer is shown to bind by intercalation, almost equally well, to the B and Z form of poly[(dG-m5dC)].poly[(dG-m5dC)], whereas the ethidium monomer shows a strong preference for the B form. The hydrogen-deuterium (H-D) exchange kinetics of the ethidium dimer bound to the B and Z form of poly [(dG-m5dC)].poly[(dG-m5dC)] could then be compared. The kinetics of the H-D exchange were strikingly slower when the dye was bound to Z DNA as compared to B DNA. The exchange kinetics were also modified when ethidium dimer was bound to tRNA and to a triple stranded structure. It is proposed that a dynamic fluctuation at the level of the nucleic acid could modulate the dynamic fluctuation at the level of the bound ligand.     

Interaction of intercalating and non-intercalating agents with DNA: use of hydroxyapatite chromatography and S1 nuclease

We have used hydroxyapatite (HA) chromatography and S1 nuclease hydrolysis to study the modification in the secondary structure of DNA caused by certain intercalating and non-intercalating ligands. The principal conclusions of HA experiments were as follows: (1) when native DNA, complexed with drugs believed to bind to DNA by intercalation (ethidium bromide, acridine orange, actinomycin D and acriflavin), is chromatographed on HA a lower affinity of DNA for HA is observed; also, the DNA elutes from HA columns as a drug-DNA complex; (ii) ligands that are known to interact with DNA by surface interactions do not show these effects; (iii) it may be possible to quantitate the binding of the intercalating drug to DNA and to determine its degree of binding by HA chromatography. Possibly, intercalation causes a change in the configuration of the sugarphosphate backbone of DNA, resulting in an altered steric orientation or 'burial' of phosphate groups with reduced availability for surface interactions with HA. S1 nuclease was used to determine the thermal melting profiles of DNA complexed with ethidium bromide and acridine orange. The melting profile in both cases was found to be biphasic with considerably reduced denaturation even at 95 degrees C. This is accounted for by the property of intercalating agents of stabilizing the secondary structure of DNA and the reported preference in binding to G-C base pairs.     

Atomic force microscopy study of DNA conformation in the presence of drugs

Binding of ligands to DNA gives rise to several relevant biological and biomedical effects. Here, through the use of atomic force microscopy (AFM), we studied the consequences of drug binding on the morphology of single DNA molecules. In particular, we quantitatively analyzed the effects of three different DNA-binding molecules (doxorubicin, ethidium bromide, and netropsin) that exert various pharmacologic and therapeutic effects. The results of this study show the consequences of intercalation and groove molecular binding on DNA conformation. These single-molecule measurements demonstrate morphological features that reflect the specific modes of drug–DNA interaction. This experimental approach may have implications in the design of therapeutically effective agents.     

Restrained torsional dynamics of nuclear DNA in living proliferative mammalian cells

Physical parameters, describing the state of chromatinized DNA in living mammalian cells, were revealed by in situ fluorescence dynamic properties of ethidium in its free and intercalated states. The lifetimes and anisotropy decays of this cationic chromophore were measured within the nuclear domain, by using the ultra-sensitive time-correlated single-photon counting technique, confocal microscopy, and ultra-low probe concentrations. We found that, in living cells: 1) free ethidium molecules equilibrate between extracellular milieu and nucleus, demonstrating that the cation is naturally transported into the nucleus; 2) the intercalation of ethidium into chromatinized DNA is strongly inhibited, with relaxation of the inhibition after mild (digitonin) cell treatment; 3) intercalation sites are likely to be located in chromatin DNA; and 4) the fluorescence anisotropy relaxation of intercalated molecules is very slow. The combination of fluorescence kinetic and fluorescence anisotropy dynamics indicates that the torsional dynamics of nuclear DNA is highly restrained in living cells.     

An assay for proteinases and their inhibitors based on DNA/ethidium bromide fluorescence

Proteinases and their inhibitors have become the subject of intense research interest recently, since they control a multitude of very important biological processes, from the development of lambda phage to hypertension in humans. We have developed a simple and sensitive assay for detecting the activity of proteinases and of their proteinase inhibitors. The assay is based on ethidium bromide fluorescence, according to the following principles: (i) Ethidium bromide increases its fluorescence by 25-fold when it intercalates between base pairs of double-stranded DNA. (ii) Histones prevent this large increase in fluorescence by binding with high affinity to DNA thus blocking ethidium bromide intercalation. (iii) A proteinase that digests histones will make more DNA available for ethidium bromide intercalation, thereby producing an increase of fluorescence. Proteinase activity can easily be determined, in the presence of a DNA/histone complex, from the rate of ethidium fluorescence increase. In contrast, activity of a proteinase inhibitor is quantitated by the inhibition of fluorescence gain in the presence of a known amount of proteinase. This assay is rapid, simple, inexpensive, and, at the same time, accurate and sensitive enough to allow quantitation of nanogram amounts of various broad-specificity proteinases and their inhibitors. We show some possible applications of the assay (i) in testing column fractions during protein purifications, (ii) quantitation of alpha 1-antitrypsin in human serum, and (iii) detection of proteinase activity in cell extracts.     

Structure and Stability of Z* DNA

Abstract

The structure and stability of the left handed Z* DNA aggregate was examined by spectroscopic methods and by electron microscopy. Poly(dGdC), upon heating in the presence of Mn⁺⁺, forms a large aggregate which may be sedimented at 12,000 X g, with a circular dichroism spectrum characteristic of left handed DNA Aggregation gives rise to turbidity changes at visible wavelengths, providing a convenient means of monitoring the transition in solution. The wavelength dependence of turbidity is consistent with the scattering behavior of a long thin rod. Electron microscopy shows that Z* DNA is a large fibrous structure of indeterminant length, with a uniform diameter of approximately 20 nm. The results obtained in solution and under the requisite conditions for electron microscopy are mutually consistent Poly(dGdC) preparations with average lengths of 60,240,500, and 2000 base pairs all form Z* DNA Poly(dGm⁵dC) forms Z* DNA in the presence of Mn⁺⁺ without heating, but poly(dAdC)-poly(dGdT) and calf thymus DNA cannot be induced to the Z* form under any conditions tried. Kinetic studies, monitored by turbidity changes, provide evidence that the formation of Z* DNA proceeds by a nucleated condensation mechanism. Dissolution of the Z* aggregate results from the chelation of Mn⁺⁺ or by the addition of the intercalator ethidium bromide. The allosteric conversion of Z* DNA to an intercalated, right handed form by ethidium is demonstrated by kinetic studies, equilibrium binding studies and circular dichroism spectroscopy. Electron microscopy provides a striking visualization of the dissolution of the Z* aggregate by ethidium.     

Intercalative binding to DNA of antitumour drugs derived from 3-nitro-1,8-naphthalic acid.

Two new antitumour drugs, imide derivatives of 3-nitro-1,8-naphthalic acid having different basic side chains linked to the imide nitrogen, have been shown to bind to double-helical DNA by intercalation. At ionic strength 0.01 mol/litre, pH 7, their intrinsic association constants are about 1.45 x 10(5) M-1 and each bound ligand molecule occludes about 3.4 nucleotides of the DNA lattice. They remove and reverse the supercoiling of closed circular duplex PM2 DNA with apparent unwinding angles of 11-12 degrees per bound drug molecule, referred to an assumed unwinding angle of 26 degrees for ethidium. They increase the viscosity of sonicated rod-like DNA fragments, each bound drug molecule producing a calculated increment in length of 2.2 - 2.5 A. No important differences between the DNA-binding characteristics of the two drugs were detected, though one appears marginally more active than the other in certain biological tests.     

Anticooperative Binding Governs the Mechanics of Ethidium-Complexed DNA

DNA intercalators bind nucleic acids by stacking between adjacent basepairs. This causes a considerable elongation of the DNA backbone as well as untwisting of the double helix. In the past few years, single-molecule mechanical experiments have become a common tool to characterize these deformations and to quantify important parameters of the intercalation process. Parameter extraction typically relies on the neighbor-exclusion model, in which a bound intercalator prevents intercalation into adjacent sites. Here, we challenge the neighbor-exclusion model by carefully quantifying and modeling the force-extension and twisting behavior of single ethidium-complexed DNA molecules. We show that only an anticooperative ethidium binding that allows for a disfavored but nonetheless possible intercalation into nearest-neighbor sites can consistently describe the mechanical behavior of intercalator-bound DNA. At high ethidium concentrations and elevated mechanical stress, this causes an almost complete occupation of nearest-neighbor sites and almost a doubling of the DNA contour length. We furthermore show that intercalation into nearest-neighbor sites needs to be considered when estimating intercalator parameters from zero-stress elongation and twisting data. We think that the proposed anticooperative binding mechanism may also be applicable to other intercalating molecules.