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.     

Environment-induced changes in DNA conformation as probed by ethidium bromide fluorescence

The interaction of the ethidium cation with calf thymus DNA is investigated in solutions of different ionic strength and temperature by observation of the enhancement of fluorescence of ethidium upon intercalation in the duplex structure. The quantum yield of the fluorescence of the intercalated dye is found to increase either upon lowering the Na+ concentration or upon increasing the temperature. The existence of a correlation between the geometry of the intercalation complex and the features of the secondary structure of DNA is suggested. Binding isotherms under corresponding environmental conditions are also quantitated by fluorescence enhancement and interpreted in terms of the neighbor exclusion model. Large contributions from change in hydration to the thermodynamics of binding are demonstrated by the temperature dependences of the equilibrium constants. The neighbor exclusion range is found to be practically independent of the salt concentration but its value increases from an average of 2.4 around room temperature to 4-5 at 80 degrees C, as inferred from the binding curves in 0.15 and 0.5 M [Na+] or from the DNA hypochromism vs temperature profiles of complexes at 10(-3) M [Na+]. All the data point to a possible sequence-conformation specificity in the intercalation of ethidium which in heterogeneous DNA is mediated by environmental changes.     

Differential binding of the enantiomers of chloroquine and quinacrine to polynucleotides: Implications for stereoselective metabolism

Interaction of the antimalarial drugs quinacrine and chloroquine with DNA has been studied extensively in order to understand the origin of their biological activity. These studies have shown that they bind to DNA through an intercalative mode and show little sequence specificity. All previous experiments were carried out using the racemic form of these drugs. We have investigated the binding of the enantiomeric forms of quinacrine and chloroquine to synthetic polynucleotides poly (dA-dT) · poly(dA-dT) and poly (dG-dC) · poly(dG-dC), and found interesting differences in their binding parameters. Quinacrine enantiomers have a much higher binding affinity for the two polynucleotides compared to those of chloroquine. The negative enantiomers were found to have higher binding affinity than the positive ones. The binding constant for the binding of quinacrine (?) to poly(dG-dC) · poly(dG-dC) was found to be about 3 times that of quinacrine (+). The differences in these binding affinities were further confirmed by equilibrium dialysis of the complexes of the polynucleotides with the racemic form of the drugs, which resulted in the enrichment of the dialysate with the positive enantiomer. CD spectra of the enantiomers and their polynucleotide complexes are reported. Changes in the fluorescence properties of quinacrine in the presence of the two polynucleotides are also described. Biological implications of these findings are discussed. © 1993 John Wiley & Sons, Inc.     

31P NMR spectra of ethidium, quinacrine, and daunomycin complexes with poly(adenylic acid).poly(uridylic acid) RNA duplex and calf thymus DNA

31P NMR provides a convenient monitor of the phosphate ester backbone conformational changes upon binding of the intercalating drugs ethidium, quinacrine, and daunomycin to sonicated poly(A).poly(U) and calf thymus DNA. 31P chemical shifts can also be used to assess differences in the duplex unwinding angles in the presence of the drug. Thus a new 31P signal, 1.8-2.2 ppm downfield from the double-stranded helix signals, is observed in the ethidium ion-poly(A).poly(U) complex. This signal arises from phosphates which are in perturbed environments due to intercalation of the drug. This is in keeping with the hypothesis that the P-O ester torsional angle in phosphates linking the intercalated base pairs is more trans-like. Similar though smaller deshielding of the 31P signals is observed in sonicated poly(A).poly(U)-quinacrine complexes as well as in the daunomycin complexes. The effect of added ethidium ion, quinacrine, and daunomycin on the 31P spectra of sonicated calf thymus DNA is consistent with Wilson and Jones' (1982) earlier study. In these drug-DNA complexes the drug produces a gradual downfield shift in the DNA 31P signal without the appearance of a separate downfield peak. These differences are attributed to differences in the rate of chemical exchange of the drug between free and bound duplex states. The previous correlation of 31P chemical shift with drug duplex unwinding angle (Wilson & Jones, 1982) is confirmed for both the RNA and DNA duplexes.     

The effect of ionic strength on DNA-ligand unwinding angles for acridine and quinoline derivatives.

We have quantitatively examined the unwinding angles for the complexes of a related series of acridine and quinoline derivatives with DNA. Ethidium bromide was used as a control for determining superhelix densities at different ionic strengths. Relative to ethidium, 9-aminoacridine and quinacrine had an essentially constant unwinding angle of approximately 17 degrees at all ionic strengths tested. The apparent unwinding angle for chloroquine and 9-amino-1,2,3,4-tetrahydroacridine was found to be ionic strength dependent, increasing with increasing ionic strength. This suggests that competitive nonintercalative binding at low ionic strengths causes an apparent lowering of the quinoline unwinding angle. This can also explain why 4-aminoquinaldine, examined at low ionic strength, gives a quite low apparent unwinding angle. Quinacrine along with chloroquinine and 9-aminoacridine approaches a limiting value for their unwinding angle of approximately 17 degrees. 4-aminoquinaldine and 9-amino-1,2,3,4-tetrahydroacridine could not be examined at an ionic strength above 0.03 because of their very low equilibrium binding constants.     

Interaction of chloroquine with linear and supercoiled DNAs. Effect on the torsional dynamics, rigidity, and twist energy parameter

The magnitude and uniformity of the torsion elastic constant (alpha) of linear pBR322 DNA and supercoiled pBR322 DNAs with high-twist (sigma = -0.083) and normal-twist (sigma = -0.48) are measured in 0.1 M NaCl as a function of added chloroquine/base-pair ratio (chl/bp) by studying the fluorescence polarization anisotrophy (FPA) of intercalated ethidium dye. The time-resolved FPA is measured by using a picosecond dye laser for excitation and time-correlated single-photon counting detection. A general theory is developed for the binding of ligands that unwind superhelical DNAs, and the simultaneous binding of two different intercalators is treated in detail. The equilibrium constant (K) for binding chloroquine to linear pBR322 DNA and the number (r) of bound chloroquines per base pair are determined from the relative amplitude ratio of the slow (normally intercalated) and fast (free) components in the decay of the (probe) ethidium fluorescence intensity as a function of chl/bp. For chloroquine binding to supercoiled pBR322 DNAs, the intrinsic binding constant is assumed to be the same as for the linear DNA, but the twist energy parameter ET (N times the free energy to change the linking number from 0 to 1 in units of kBT) is regarded as adjustable. Using the best-fit ET, the binding ratios r are calculated for each chl/bp ratio. Twist energy parameters are also determined for ethidium binding to these supercoiled DNAs by competitive dialysis. For chloroquine binding, we obtain ET = 360 and 460 respectively for the normal-twist and high-twist supercoiled DNAs. For ethidium binding the corresponding values are ET = 280 +/- 70 and 347 +/- 50. Like other dye-binding values, these are substantially lower than those obtained by ligation methods. In the absence of chloroquine, the torsion constants of all three DNAs are virtually identical, alpha = (5.0 +/- 0.4) x 10(-12) dyn.cm. For linear pBR322 DNA, the magnitude and uniformity of alpha remain unaltered by intercalated chloroquine up to r = 0.19. This finding argues that the FPA is not significantly relaxed by diffusion of any kinks or solitons. If alpha d denotes the torsion constant between a dye and a base pair and alpha 0 that between two base pairs, then our data imply that alpha d/alpha 0 lies in the range 0.65-1.64, with a most probable value of 1.0.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biophysical and transfection studies of an amine-modified poly(vinyl alcohol) for gene delivery

Novel, multifunctional polymers remain an attractive objective for drug delivery, especially for hydrophilic macromolecular drugs candidates such as peptides, proteins, RNA, and DNA. To facilitate intracellular delivery of DNA, new amine-modified poly(vinyl alcohol)s (PVAs) were synthesized by a two-step process using carbonyl diimidazole activated diamines to produce PVAs with different degrees of amine substitution. The resulting polymers were characterized using NMR, thermogravimetric analysis (TGA), and gelpermation chromatography (GPC). Atomic force microscopy (AFM), dynamic light scattering photon correlation spectroscopy (PCS), and zeta-potential were used to investigate polyplexes of DNA with PVA copolymers. These studies suggest an influence of the polycation structure on the morphology of condensed DNA in polyplexes. Significant differences were observed by changing both the degrees of amine substitution and the structure of the PVA backbone, demonstrating that both electrostatic and hydrophobic interactions affect DNA condensation. DNA condensation measured by an ethidium bromide intercalation assay showed a higher degree of condensation with pDNA with increasing degrees of amine substitution and more hydrophobic functional groups. These findings are in line with transfection experiments, in which a good uptake of these polymer DNA complexes was noted, unfortunately, with little endosomal escape. Co-administration of chloroquine resulted in increased endosomal escape and higher transfection efficiencies, due to disruption of the endosomal membrane. In this study, the structural requirements for DNA complexation and condensation were characterized to provide a basis for rational design of nonviral gene delivery systems.     

Mechanisms of quinacrine binding and fluorescence in nuclei and chromosomes

The mechanisms has been investigated whereby quinacrine binds to the DNA of nuclei and chromosomes in cytological preparations fixed in methanol-acetic acid. A variety of evidence is consistent with the idea that the quinacrine binds by intercalation. This is supported by a high value for the affinity of quinacrine for DNA, together with a saturation value of 0.2 quinacrine molecules/nucleotide; binding in the presence of strong salt solutions; and inhibition of fluorescence and banding by denaturation or depurination of DNA. At high quinacrine concentrations, weak binding of quinacrine to nuclei and chromosomes also occurs, but this is not relevant to the production of strong fluorescence or Q-banding patterns. A number of factors were tested which might have affected quinacrine fluorescence and banding. These included: pH; blocking protein amino groups by acetylation or benzoylation; introduction of hydrophobic groups by benzoylation; and dephosphorylation. All these treatments were without effect. However, comparison of the quinacrine fluorescence of human and onion nuclei, which differ substantially in the base composition of their DNA, shows that quinacrine fluorescence can be enhanced in cytological preparations by AT-rich DNA.     

Mechanisms of quinacrine binding and fluorescence in nuclei and chromosomes

Summary The mechanism has been investigated whereby quinacrine binds to the DNA of nuclei and chromosomes in cytological preparations fixed in methanol-acetic acid. A variety of evidence is consistent with the idea that the quinacrine binds by intercalation. This is supported by a high value for the affinity of quinacrine for DNA, together with a saturation value of 0.2 quinacrine molecules/nucleotide; binding in the presence of strong salt solutions; and inhibition of fluorescence and banding by denaturation or depurination of DNA. At high quinacrine concentrations, weak binding of quinacrine to nuclei and chromosomes also occurs, but this is not relevant to the production of strong fluorescence or Q-banding patterns.A number of factors were tested which might have affected quinacrine fluorescence and banding. These included: pH; blocking protein amino groups by acetylation or benzoylation; introduction of hydrophobic groups by benzoylation; and dephosphorylation. All these treatments were without effect. However, comparison of the quinacrine fluorescence of human and onion nuclei, which differ substantially in the base composition of their DNa, shows that quinacrine fluorescence can be enhanced in cytological preparations by AT-rich DNA.In honour of Prof. P. van Duijn     

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.     

The number of superhelical turns in native virion SV40 DNA and minicol DNA determined by the band counting method.

By a method of overlapping the results obtained after agarose gel electrophoresis under two different sets of conditions, it has become possible to determine the number of superhelical turns in a given DNA by counting the bands present after partially relaxing the DNA (Keller and Wendel, 1974) with highly purified nicking-closing (N-C) enzyme from LA9 mouse cell nuclei. Because native supercoiled DNA is heterogeneous with respect to superhelix density, an average number of superhelical turns was determined. Virion SV40 DNA contains 26 +/- 0.5 superhelical turns, and native Minicol DNA contains 19 +/- 0.5 superhelical turns. The above are values at 0.2 M NaCl and at 37 degrees C, the condition under which the enzymatic relaxations were performed. The superhelix densities determined by the band counting method have been compared with superhelix densities determined by buoyant equilibrium in PDl-CsCl gradients. The Gray, Upholt, and Vinograd (1971) calculation procedure has been used for evaluating the superhelix densities by the latter method with the new statement, however, that relaxed DNA has zero superhelical turns. Comparison of the superhelix densities obtained by both methods permits a calculation of an unwinding angle for ethidium. The mean value from experiments with SV40 DNA is 23 +/- 3 degree. The average number of superhelical turns in SV40, 26, combined with the value, 21, obtained by both Griffith (1975) and Germond et al. (1975) for the average number of nucleosomes per SV40 genome, yields an average of 1.25 superhelical turns per 1/21 of the SV40 genome. If the regions of internucleosomal DNA are fully relaxed, 1.25 correesponds to the average number of superhelical turns with a nucleosome. When analyzed under identical conditions, the limit product generated by ligating a nicked circular substrate in the presence of 0.001 M Mg2+ at 37 degrees C (ligation conditions) is slightly more positively supercoiled than the limit product obtained when the N-C reaction is performed in 0.2 M NaCl at 37 degrees C. The difference in superhelix density as measured in gels between the two sets of limit products for both Minicol and SV40 DNAs is 0.0059 +/- 0.0005. This result indicates that the DNA duplex is overwound in the ligation solvent relative to its state in 0.2 M NaCl.     

Viscosity dependence of ethidium-DNA intercalation kinetics

The kinetics of ethidium intercalation into double-stranded poly[d(G-C)] were investigated by use of repetitive pressure-jump chemical relaxation at 20 degrees C in low ionic strength (0.1 M NaCl) aqueous buffers containing either glycerol or methanol. The viscosity of the various solvents differed by more than an order of magnitude while other physical properties (e.g., dielectric constant) remained approximately constant. The single-reciprocal kinetic relaxation time (tau -1) increases linearly with DNA concentration. The observed association rate constant is lower in all organic-aqueous mixtures than in water and is inversely proportional to the viscosity. These results provide evidence for an additional step in the intercalation mechanism which is identified as an obligatory DNA conformational change preceding ethidium intercalation. From the data presented, the equilibrium constant of this local conformational change is approximately 10(-3), i.e., greatly favoring the structure incapable of intercalation. The corresponding kinetics were not directly determined; however, in order to be consistent with all of the data the forward and/or reverse rate constants of the conformational change must be larger than the rate of the intercalation reaction. Thus, it is proposed that the rate of the conformational change back to the nonintercalating B-DNA structure is greater than approximately 500 s-1, implying a rate of opening greater than approximately 0.5 s-1, in agreement with other hydrogen exchange and NMR data. The observed overall rate constant for the dissociation of ethidium is inversely proportional to the solvent density, possibly reflecting a dependence on the solvent free volume. The overall volume change of intercalation is less negative in the organic-aqueous solvent mixtures than in water.     

Mechanisms of chromosome banding. V. Quinacrine banding.

A series of biochemical investigations were undertaken to determine the mechanism of Q-banding. The results were as follows: 1. In agreement with previous studies, highly AT-rich DNA, such as poly(dA)-poly(dT), markedly enhanced quinacrine fluorescence while GC containing DNA quenched fluorescence. These effects persisted at DNA concentrations comparable to those in the metaphase chromosome. 2. Studies of quinacrine-DNA complexes in regard to the hypochromism of quanacrine, DNA Tm, DNA viscosity, and equilibrium dialysis, indicated the quinacrine was bound be intercalation with relatively little sid binding. 3. Single or double stranded nucleotide polymers, in the form of complete or partial helices, were 1000-fold more effective in quenching than solutions of single nucleotides, suggesting that base stacking is required for quenching. 4. Studies of polymers in the A conformation, such as transfer RNA and DNA-RNA hybrids, indicated that marked base tilting does not affect the ability of nuclei acids to cause quenching or enhancement of quinacrine fluorescence. 5. Salts inhibit the binding of quinacrine to DNA. 6. Spermine, polylysine and polyarginine, which bind in the small groove of DNA, inhibited quinacrine binding and quenching, while histones, which probably bind in the large groove, had little effect. This correlated with the observation that removal of histones with acid has no effect on Q-banding. 7. Mouse liver chromatin was separated into five fractions. At concentrations of quinacrine from 2 times 10-6 to 2 times 10-5 M all fractions inhibited to varying degrees the ability of the chromatin DNA to bind quinacrine and quench quinacrine fluorescence. At saturating levels of quinacrine two fractions, the 400 g pellet (rich in heterochromatin) and a dispersed euchromatin supernatant fraction, showed a decreased number of binding sites for quinacrine. These two fractions were also the richest in non-histone proteins. 8. DNA isolated from the different fractions all showed identical quenching of quinacrine fluorescenc. 9. Mouse GC-rich, mid-band, AT-rich, and satellite DNA, isolated by CsCL AND Cs-2SO-4-Ag+ centrifugation all showed identical quenching of quinacrine fluorescence, indicating that within a given organism, except for very AT or GC-rich satellites, the variation in base composition is not adequate to explain Q-banding.We interpret these results to indicate that: (a) quinacrine binds to chromatin by intercalation of the three planar rings with the large group at position 9 lying in the small groove of DNA, (b) most pale staining regions are due to a decrease binding of quinacrine, and (c) this inhibition of binding is predominately due to non-histone proteins.     

Pressure-jump study of the kinetics of ethidium bromide binding to DNA

Pressure-jump chemical relaxation has been used to investigate the kinetics of ethidium bromide binding to the synthetic double-stranded polymers poly[d(G-C)] and poly[d(A-T)] in 0.1 M NaCl, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, and 1 mM ethylenediaminetetraacetic acid, pH 7.2, at 24 degrees C. The progress of the reaction was followed by monitoring the fluorescence of the intercalated ethidium at wavelengths greater than 610 nm upon excitation at 545 nm. The concentration of DNA was varied from 1 to 45 microM and the ethidium bromide concentration from 0.5 to 25 microM. The data for both polymers were consistent with a single-step bimolecular association of ethidium bromide with a DNA binding site. The necessity of a proper definition of the ethidium bromide binding site is discussed: it is shown that an account of the statistically excluded binding phenomenon must be included in any adequate representation of the kinetic data. For poly[d(A-T)], the bimolecular association rate constant is k1 = 17 X 10(6) M-1 s-1, and the dissociation rate constant is k-1 = 10 s-1; in the case of poly[d(G-C)], k1 = 13 X 10(6) M-1 s-1, and k-1 = 30 s-1. From the analysis of the kinetic amplitudes, the molar volume change, delta V0, of the intercalation was calculated. In the case of poly[d(A-T)], delta V0 = -15 mL/mol, and for poly[d(G-C)], delta V0 = -9 mL/mol; that is, for both polymers, intercalation is favored as the pressure is increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of the expected DNA lengthening caused by mono-and bisintercalating drugs using electron microscopy

PM₂ DNA molecules were treated with intercalating reagents (ethidium bromide, ethidium dimer, acridine dimer) and observed by electron microscopy. The adaptation of different electron microscopy techniques has enabled the determination of DNA lengthening upon drug intercalation. A 50% length increase was generally obtained for DNA saturated with the drugs. This result is in agreement with the intercalation model proposed by Lerman. In some cases (ethidium dimer), an increase of length larger than 50% can be obtained. Experimental conditions of DNA spreading strongly interfere with the DNA–drug interaction. In some cases it was possible to estimate the apparent binding constants and also to distinguish the mono- from the bisintercalating derivatives in their reaction with DNA.     

Mechanisms of chromosome banding

A series of biochemical investigations were undertaken to determine the mechanism of Q-banding. The results were as follows: 1. In agreement with previous studies, highly AT-rich DNA, such as poly(dA)-poly(dT), markedly enhanced quinacrine fluorescence while GC containing DNA quenched fluorescence. These effects persisted at DNA concentrations comparable to those in the metaphase chromosome. 2. Studies of quinacrine-DNA complexes in regard to the hypochromism of quinacrine, DNA Tm, DNA viscosity, and equilibrium dialysis, indicated the quinacrine was bound by intercalation with relatively little side binding. 3. Single or double stranded nucleotide polymers, in the form of complete or partial helices, were 1000-fold more effective in quenching than solutions of single nucleotides, suggesting that base stacking is required for quenching. 4. Studies of polymers in the A conformation, such as transfer RNA and DNA-RNA hybrids, indicated that marked base tilting does not affect the ability of nucleic acids to cause quenching or enhancement of quinacrine fluorescence. 5. Salts inhibit the binding of quinacrine to DNA. 6. Spermine, polylysine and polyarginine, which bind in the small groove of DNA, inhibited quinacrine binding and quenching, while histones, which probably bind in the large groove, had little effect. This correlated with the observation that removal of histones with acid has no effect on Q-banding. 7. Mouse liver chromatin was separated into five fractions. At concentrations of quinacrine from 2×10^?6 to 2×10^?5 M all fractions inhibited to varying degrees the ability of the chromatin DNA to bind quinacrine and quench quinacrine fluorescence. At saturating levels of quinacrine two fractions, the 400 g pellet (rich in heterochromatin) and a dispersed euchromatin supernatant fraction, showed a decreased number of binding sites for quinacrine. These two fractions were also the richest in non-histone proteins. 8. DNA isolated from the different fractions all showed identical quenching of quinacrine fluorescence. 9. Mouse GC-rich, mid-band, AT-rich, and satellite DNA, isolated by CsCl and Cs₂SO₄-Ag⁺ centrifugation all showed identical quenching of quinacrine fluorescence, indicating that within a given organism, except for very AT or GC-rich satellites, the variation in base composition is not adequate to explain Q-banding. — We interpret these results to indicate that: (a) quinacrine binds to chromatin by intercalation of the three planar rings with the large group at position 9 lying in the small groove of DNA, (b) most pale staining regions are due to a decrease binding of quinacrine, and (c) this inhibition of binding is predominately due to non-histone proteins.     

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.     

Binding of ethidium derivatives to natural DNA: a 300 MHz 1H NMR study.

The binding of three ethidium derivatives, ethidium (1), des-3-amino ethidium (2) and des-8-amino ethidium (3), to short (approximately 35 base pairs), random sequence DNA has been investigated using 300 MHz proton NMR. At 35 degrees C all three drugs cause upfield shifts of the resonances from the exchangeable imino protons, as expected for intercalative binding to DNA. However, the lineshapes vary significantly with the nature of the drug. The temperature dependence of the spectra of the DNA shows that differences between spectra observed at 35 degrees C with ethidium and with des-3-amino ethidium are primarily due to differences in the drug binding kinetics rather than to differences in mode of binding. Removal of the amino group at position 3, but not at position 8, on the parent ethidium shortens the lifetime of the intercalative state; this implies that the 3-NH2 group is involved in stabilization of the drug-DNA complex. Analysis of the drug-DNA spectra indicates that there is a preference for binding of the drugs adjacent to G.C base pairs.     

Fluorescence-determined preferential binding of quinacrine to DNA.

Quinacrine complexes with native DNA (Calf thymus, Micrococcus lysodeikticus, Escherichia coli, Bacillus subtilis, and Colstridium perfringens) and synthetic polynucleotides (poly(dA) . poly(dT), poly[d(A-T)] . poly[d(A-T)], poly(dG) . poly(dC) and poly[d(G-C)] . poly[d(G-C)]) has been investigated in solution at 0.1 M NaCl, 0.05 M Tris HCl, 0.001 M EDTA, pH 7.5, at 20 degrees C. Fluorescence excitation spectra of complexes with dye concentration D = 5-30 microM and DNA phosphate concentration P = 400 microM have been examined from 300 to 500 nm, while collecting the emission above 520 nm. The amounts of free and bound quinacrine in the dye-DNA complexes have been determined by means of equilibrium dialysis experiments. Different affinities have been found for the various DNAs and their values have been examined with a model that assumes that the binding constants associated with alternating purine and pyrimidine sequences are larger than those relative to nonalternating ones. Among the alternating nearest neighbor base sequences, the Pyr(3'-5')Pur sequences, i.e., C-G, T-G, C-A and T-A seem to bind quinacrine stronger than the remaining sequences. In particular the three sites, where a G . C base pair is involved, are found to display higher affinities. Good agreement is found with recent calculations on the energetics of intercalation sites in DNA. The analysis of the equilibrium shows also that the strength of the excitation spectrum of bound dye depends strongly upon the ratio of bound quinacrine to DNA. This effect can be attributed to dye-dye energy transfer along DNA.