Interaction of molecules with nucleic acids. II. Two pairs of families of intercalation sites, unwinding angles, and the neighbor-exclusion principle

Intercalation-site geometries are generated for a tetramer duplex extracted from B-DNA. Glycosidic angles and puckers of the deoxyribose sugar groups bonded to base pairs BP₁ and BP₄, namely, those at either end of the tetramer duplex, are assumed to be those of B-DNA to insure continuity. All possible geometrical conformations for combinations of C(2′)-endo, C(3′)-endo, C(2′)-exo, and C(3′)-exo sugar puckers are determined for the tetranucleotide backbone. Those with minimum energy are selected as candidates for intercalation sites. Calculations reveal two pairs of physically meaningful families of intercalation sites which occur in two distinct regions, I and II, of helical angles which orient BP₂ relative to BP₃ and with the helical axis disjointed between these base pairs. For each site I and II within BP₂ and BP₃, there are two distinct backbone conformations, A and B, connecting BP₃ to BP₄ or BP₁ to BP₂ which do not disrupt backbone conformations connecting BP₂ to BP₃. Hence two pairs, IA and IB, and IIA and IIB, of intercalation sites exist in which the sugar puckers along the backbone of the tetramer alternate from C(2′)-endo to C(3′)-endo on the backbone (5′p3′) connecting BP₂ to BP₃. The glycosidic angles of the C(3′)-endo sugar χ₃^γ are, coincidentally, 80° ± 2° for both conformations γ = A and B connecting BP₃ to BP₄ along the phosphate backbone (5′p3′). Consistent with the theoretical results, the experimental unwinding angles can be grouped into two categories with absolute values of 18° and 26°. The theoretical unwinding angles for sites IA and IB of 16° and for sites IIA and IIB of 20° occur for a displacement of -0.8 Å in the helical axes of BP₂ and BP₃ and for a 100% G·C composition, with a decrease depending on the amount of A·T base pairs present. Ratios of theoretical unwinding angles of sites I and II, which range from 0.75 to 0.84 for the two principal sites, compare well with the experimental value of 0.71. The theoretical results, in agreement with experimental observation, provide a new interpretation of the nature and conformation of the possible binding sites. Conformations obtained from these studies of intercalation sites in a tetramer duplex are used to rationalize the well-known neighbor-exclusion principle. The possibility of violation of this principle is demonstrated by the existence of two families of physically meaningful conformations. Conformations of unconstrained dimer duplexes are also obtained, one of which corresponds to the experimental crystal structure of ethidium–dinucleoside complexes, but these cannot be joined to the B-DNA structure. Backbone conformations of the tetramer duplex can be constructed until the base-pair separation reaches 8.25 Å, which may limit the molecules that can intercalate.     

Interactions of molecules with nucleic acids. III. Steric and electrostatic energy contours for the principal intercalation sites,prerequisites for binding,and the exclusion of essential metabolites from intercalation

Based on steric and electrostatic considerations, the prerequisites for binding to DNA via the intercalation mechanism are proposed. Steric contour energy curves are presented to demonstrate the region inaccessible to an intercalant. They are calculated with a 6-n (n = 14) potential. This method is a soft potential analog of an excluded-volume approach. Electrostatic contours on the steric surface illustrate the relatively positive and negative regions of the binding site. The principal intercalation sites, predicted to fit into B-DNA via a tetramer-duplex unit, and the unconstrained dimer-duplex units, obtained in crystal structures, are examined. These contours illustrate the requirements of size, conformation, and net atomic charges necessary for intercalation and optimum binding. Based on the limited space available for intercalation by the presence of the backbone and the maximum base-pair separation of 8.25 Å, an Essential Metabolite Exclusion Hypothesis is presented.     

Interactions of molecules with nucleic acids. VII. Intercalation and T.A specificity of daunomycin in DNA

Intercalation complexes of daunomicin(+1) with tetramer duplexes in DNA are studied with the theoretically determined intercalation sites (I, ?0.4), (II, ?0.4), and (III, ?1.4). These sites occur with base pairs separated by 6.76 Å for helical angles of 26°, 22°, and 8° about the intercalation site. Site I is preferred, and this is in agreement with experimental unwinding angles. Optimum binding positions and conformations are established, and these are in agreement with experimental results from crystal structures. A systematic procedure is devised to study base-pair and base-sequence specificity, which results in the demonstration that the most stable sequences are mainly ↑BP₁, T·A, DAUN, A·T, BP₄↓ and ↑BP₁, T·A, DAUN, G·C, BP₄↓, i.e., with the TpA and CpG (pyrimidine)p(purine) sequences about the intercalation site. These 32 possible sequences are found among the 40 most stable complexes. These theoretical calculations of intercalation complexes with daunomicin(+1) provide the first example in which a drug specifically selects the base pair T·A and prefers it in a particular sequence about the intercalation site. This specificity is in agreement with some experimental results. Problems associated with the interpretation of specificity are discussed in terms of the base, base-pair, and base-sequence resulting from the DNA site and the DNA–drug interactions. T·A specificity is rationalized by noting that the 2′deoxyribo-5′-monophosphate backbone attached to A is slightly more negative than that on the other nucleotides. Hence, a preference exists for binding to the protonated daunosamine (+1) groups. Stereographic projections of daunomycinone and daunomycin(+1) in a bond model and in a space-filling model with steric contours illustrate the results.     

A molecular mechanics investigation of RNA complexes. I. Ethidium intercalation in an HIV-1 TAR RNA sequence with an unpaired adenosine.

Nucleic acid complexes with ethidium intercalated into different sites in a segment of HIV-1 TAR RNA with an unpaired A base, along with corresponding complexes with a normal RNA sequence without an unpaired base were studied by molecular mechanics energy minimization methods. Different intercalation geometries as well as different orientations of the ethidium molecule in the intercalation sites were tested. A general binding affinity enhancement for the ethidium binding to the bulge sequence compared with the normal RNA segment was obtained. With the unpaired adenosine base stacked in the duplex, the binding site adjacent to the 3' side of the bulge was found to be the most energetically favorable binding site, and the intercalation site 5' to the bulge in the same sequence is much less favorable. Unique correlated backbone conformational changes on binding of ethidium to the intercalation site 3' to the bulge were found to relieve backbone strains caused by the stacking of the unpaired base into the helix. These backbone conformational changes present a plausible molecular basis for the experimentally observed ethidium binding preference in this bulge RNA segment (L.S. Ratmeyer, R. Vinayak, G. Zon and W.D. Wilson, J. Med. Chem. 35, 966, 1992).     

Thermodynamic characterization of ethidium bromide binding to a unique site on yeast tRNAphe.

A self-consistent thermodynamic characterization of the binding of ethidium to yeast phenylalanine-specific tRNA at 25°C, pH 7.0, in 11 nM MgCl₂, 375 nM NaCl, and 25 mM sodium phosphate has been obtained. Two ethidium molecules bind per tRNA under these conditions. The stronger site has a dissociation constant equal to 1.9 ± 0.5 μM and ΔH_dis°′ = 12 ± 1 Kcal/mol, and the weaker sites has a dissociation constant equal to 24 ± 9 μM and ΔH_dis°′ = 8.9 ± 1.5 Kcal/mol. The average calorimetric ΔH_dis°′ for the to sites 10.6 ± 0.4 kcal/mol. The thermodynamics of binding to the stranger sites are most probably the thermodynamics of interaction between A·U (6) and A·U (7), the unique site identified by Jones and Kearns. The binding is enthalpically driven and classical hydrophobic interactions do not appear to be important in the binding reaction.     

Enthalpy and entropy changes for the intercalation of small molecules to DNA. II. Ethidium and propidium fluoride

Enthalpy changes (ΔH_B) for the binding of ethidium (a monocation) and propidium (a dication) to calf thymus DNA have been determined calorimetrically in piperazine-N, N′-bis(2-ethanesulfonic acid) buffer with the fluoride ion as the counterion. Heats of dilution for the fluoride salts of ethidium and propidium were substantially less than the corresponding values found for other halide salts of these cations. At a Na⁺ ion concentrations of 0.019, ΔH_B = ?8.3 and ?7.9 ± 0.3 kcal mol^?1 for ethidium and propidium, respectively. For these two cations, just as was observed for the naphthalene monoimide (monocation) and diimide (dication) [H. P. Hopkins, K. A. Stevenson, and W. D. Wilson, (1986) J. Sol. Chem. 15 , 563–579], ΔH_B is within the same experimental error for both cations. Apparently, charge–charge interactions in DNA–cation complexes produce only small changes in the enthalpy for the system. In the concentration range 0.019–0.207, the ΔH_B values for propidium did not depend appreciably on the Na⁺ ion concentration, and a similar pattern was shown to exist for ethidium. When these results were combined with ΔG_B values for the binding of these cations to DNA, we found the variation of ΔS_B with Na⁺ ion concentration to be remarkably close to the predictions of modern polyelectrolyte theory, i.e., propidium binding to DNA causes approximately twice as many Na⁺ ions to be released into the bulk solution as does the binding of ethidium. The much stronger binding of propidium, relative to ethidium, at low ionic strengths is thus seen to be primarily due to entropic effects.     

Interacalation of ethidium ion into DNA and RNA oligonucleotides

The thermodynamics of ethidium ion binding to the double strands formed by the ribooligonucleotides rCA₅G + rCU₅G and the analogous deoxyribo-oligonucleotides dCA₅G + dCT₅G were determined by monitoring the absorbance versus temperature at 260 and 283 nm at several concentrations of oligonucleotides and ethidium bromide. A maximum of three ethidium ions bind to the oligonucleotides, which is consistent with intercalation and nearest-neighbor exclusion. For the ribo-oligonucleotide the binding mechanism is complex. Either two sites (assumed to be the intercalation sites at the two ends of the oligonucleotide) bind more strongly by a factor of 140 than the third site, or all sites are identical, but there is strong anticooperativity on binding (cooperativity parameter, 0.1). In sharp contrast, the binding to the same sequence (with thymine substituted for uracil) in the deoxyribo-oligonucleotide showed all sites equivalent and no cooperativity. For the ribo-oligonucleotides the enthalpy for ethidium binding is ?14 kcal/mol. The equilibrium constants at 25°C depend on the model; either K = 6 × 10⁵M^?1 for the two strong sites (4 × 10³M^?1 for the weak site) or K = 2.5 × 10⁵M^?1 for the intrinsic constant of the anticooperative model. For the equivalent deoxyribo-oligonucleotide the enthalpy of binding is -9 kcal/mol and the equilibrium constant at 25°C is a factor of 10 smaller (K = 2.5 × 10⁴M^?1).     

On the interaction of daunomycin with synthetic alternating DNAs: sequence specificity and polyelectrolyte effects on the intercalation equilibrium

The interaction of daunomycin with ctDNA and six purine–pyrimidine alternating poly-deoxynucleotides has been studied using fluorometric and uv-visible absorption methods. In the explored binding range of r > 0.05, the intercalation of the drug into the DNAs proved to be anticooperative, as indicated by the pronounced upward curvature of all the Scatchard plots obtained. The experimental data have been analyzed according to the recent theory of Friedman and Manning, which describes the polyelectrolyte effects on the site binding equilibria, drug intercalation included. We found that, accounting for the polyelectrolyte effects in the neighbor site exclusion model, the experimental data were nearly equally well described, in a wide range of binding ratios, by assuming the presence of sequence specificity effects (site size = 2 base pairs, exclusion parameter n = 1) or its absence (site size = 1 base pair, n = 1.7). The relevant results are as follows: (a) Daunomycin binds to all the DNAs considered with a stoichiometry of approximately 1 drug for every two base pairs. (b) The anticooperative nature of the interaction is essentially polyelectrolytic in origin. (c) The binding affinity shown by the drug for the different sites considered decreases in the order of Gm⁵C > AT > AC-GT > IC > GC > AU, indicating a stabilizing effect of the —CH₃ group in position 5 of the pyrimidines. (d) The extent of quenching of the intrinsic fluorescence of daunomycin in the presence of DNA is bound to the presence, at the intercalation site, of a guanine residue, since GC, Gm⁵C, and AC-GT sites induce a nearly total quenching, whereas AT, AU, and IC sites act only partially in this respect. The structural results obtained from the daunomycin-d[(CGTACG)]₂ crystal suggest that the 2-NH₂ group of guanine might be responsible for such a phenomenon. The influence of both the temperature and the ionic strength on the free energy of drug intercalation into ctDNA, poly[d(G-C)] : poly[d(G-C)], and poly[d(A-C)] : poly[d(G-T)] is examined and discussed.     

Interactions of molecules with nucleic acids. XI. Generalization of techniques to generate nucleic acid structures with applications to intercalation sites and kinked structures

A generalized procedure to generate nucleic acid structures is presented. In this procedure, the bases of a base pair are oriented first for characterization of particular DNA receptor sites. The resultant sites are then used in the study of specific molecule–DNA interactions. For example, intercalation sites, kinked DNA, and twisted and tilted bases are envisioned. Alterations of structures via anti → syn orientations of bases, as well as crankshaft motion about collinear bonds, provide additional conformations without disrupting the overall backbone structure. These approaches to the generation of nucleic acid structures are envisioned as required in studies of the intercalation phenomenon, minor adjustments of DNA to accommodate denaturation, binding of carcinogens to DNA, complex formation of transition metals with DNA, and antitumor agents as ligands. For these base-pair and base orientations, backbone orientations are calculated by the AGNAS technique to yield physically meaningful conformations, namely, those conformations for which nonbonded contacts are favourable. A procedure is presented to generate dimer duplex units that are physically meaningful and to assemble these units into a polynucleotide duplex. Double helices that begin with B-DNA, undergo a transition to one of the above-mentioned receptor sites, and return to B-DNA can be assembled from a catalog of dimer duplexes. Stereographic projections of the various receptor sites already being used to model binding to DNA are presented.     

Electrostatic factors in DNA intercalation

The factors that determine the binding of a chromophore between the base pairs in DNA intercalation complexes are dissected. The electrostatic potential in the intercalation plane is calculated using an accurate ab initio based distributed multipole electrostatic model for a range of intercalation sites, involving different sequences of base pairs and relative twist angles. There will be a significant electrostatic contribution to the binding energy for chromophores with a predominantly positive electrostatic potential, but this varies significantly with sequence, and somewhat with twist angle. The usefulness of these potential maps for understanding the binding of intercalators is explored by calculating the electrostatic binding energy for 9-aminoacridine, ethidium, and daunomycin in a variety of model binding sites. The electrostatic forces play a major role in the positioning of an intercalating 9-aminoacridine and a significant stabilizing role in the binding of ethidium in its sterically constrained position, but the intercalation of daunomycin is determined by the side-chain binding. Sequence preferences are likely to be determined by a complex and subtle mixture of effects, with electrostatics being just one component. The electrostatic binding energy is also unlikely to be a major determinant of the twist angle, as its variation with angle is modest for most intercalation sites. Overall, the electrostatic potential maps give guidance on how positively charged chromophores can be chemically adapted by heteroatomic substitution to optimise their binding.     

Imino 1H- and 31P-NMR analysis of the interaction of propidium and ethidium with DNA

At low temperature and low salt concentration, both imino proton and ³¹p-nmr spectra of DNA complexes with the intercalators ethidium and propidium are in the slow-exchange region. Increasing temperature and/or increasing salt concentration results in an increase in the site exchange rate. Ring-current effects from the intercalated phenanthridinium ring of ethidium and propidium cause upfield shifts of the imino protons of A · T and G · C base pairs, which are quite similar for the two intercalators. The limiting induced chemical shifts for propidium and ethidium at saturation of DNA binding sites are approximately 0.9 ppm for A · T and 1.1 ppm for G · C base pairs. The similarity of the shifts for ethidium and propidium, in both the slow- and fast-exchange regions over the entire titration of DNA, shows that a binding model for propidium with neighbor-exclusion binding and negative ligand cooperativity is correct. The fact that a unique chemical shift is obtained for imino protons at intercalated sites over the entire titration and that no unshifted imino proton peaks remain at saturation binding of ethidium and propidium supports a neighbor-exclusion binding model with intercalators bound at alternating sites rather than in clusters on the double helix. Addition of ethidium and propidium to DNA results in downfield shifts in ³¹P-nmr spectra. At saturation ratios of intercalator to DNA base pairs in the titration, a downfield shoulder (approximately ?2.7 ppm) is apparent, which accounts for approximately 15% of the spectral area. The main peak is at ?3.9 to ?4.0 ppm relative to ?4.35 in uncomplexed DNA. The simplest neighbor-binding model predicts a downfield peak with approximately 50% of the spectral area and an upfield peak, near the chemical shift for uncomplexed DNA, with 50% of the area. This is definitely not the case with these intercalators. The observed chemical shifts and areas for the DNA complexes can be explained by models, for example, that involve spreading the intercalation-induced unwinding of the double helix over several base pairs and/or a DNA sequence- and conformation-dependent heterogeneity in intercalation-induced chemical shifts and resulting exchange rates.     

Proton and phosphorus NMR studies of d-CpG(pCpG)n duplexes in solution. Helix-coil transition and complex formation with actinomycin-D.

The Watson–Crick imino and amino exchangeable protons, the nonexchangeable base and sugar protons, and the backbone phosphates for d-CpG(pCpG)_n, n = 1 and 2, have been monitored by high-resolution nmr spectroscopy in aqueous solution over the temperature range 0°–90°C. The temperature dependence of the chemical shifts of the tetramer and hexamer resonances is consistent with the formation of stable duplexes at low temperature in solution. Comparison of the spectral characteristics of the tetranucleotide with those of the hexanucleotide with temperature permits the differentiation and assignment of the cytosine proton resonances on base pairs located at the end of the helix from those in an interior position. There is fraying at the terminal base pairs in the tetranucleotide and hexanucleotide duplexes. The Watson–Crick ring imino protons exchange at a faster rate than the Watson–Crick side-chain amino protons, with exchange occurring by transient opening of the double helix. The structure of the d-CpG(pCpG)_n double helices has been probed by proton relaxation time measurements, sugar proton coupling constants, and the proton chemical shift changes associated with the helix–coil transition. The experimental data support a structural model in solution, which incorporates an anti conformation about the glycosyl bonds, C(3) exo sugar ring pucker, and base overlap geometries similar to the B-DNA helix. Rotational correlation times of 1.7 and 0.9 × 10^?9 sec have been computed for the hexanucleotide and tetranucleotide duplexes in 0.1 M salt, D₂O, pH 6.25 at 27°C. The well-resolved ³¹P resonances for the internucleotide phosphates of the tetramer and hexamer sequences at superconducting fields shift upfield by 0.2–0.5 ppm on helix formation. These shifts reflect a conformational change about the ω,ω′ phosphodiester bonds from gauche-gauche in the duplex structure to a distribution of gauche-trans states in the coil structure. Significant differences are observed in the transition width and midpoint of the chemical shift versus temperature profiles plotted in differentiated form for the various base and sugar proton and internucleotide phosphorous resonances monitoring the d-CpG(pCpG)_n helix–coil transition. The twofold symmetry of the d-CpGpCpG duplex is removed on complex formation with the antibiotic actinomycin-D. Two phosphorous resonances are shifted downfield by ～2.6 ppm and ～1.6 ppm on formation of the 1:2 Act-D:d-CpGpCpG complex in solution. Model studies on binding of the antibiotic to dinucleotides of varying sequence indicate that intercalation of the actinomycin-D occurs at the GpC site in the d-CpGpCpG duplex and that the magnitude of the downfield shifts reflects strain at the O-P-O backbone angles and hydrogen bonding between the phenoxazone and the phosphate oxygens. Actinomycin-D is known to bind to nucleic acids that exhibit a B-DNA conformation; this suggests that the d-CpG(pCpG)_n duplexes exhibit a B-DNA conformation in solution.     

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.     

Binding of ethidium to bacteriophage T7 and T7 deletion mutants

Equilibrium binding of ethidium, quantitated by fluorescence enhancement, to DNA packaged in bacteriophage T7 and T7 deletion mutants has been compared with the binding of this dye to DNA released from its capsid (free DNA). During achievement of apparent equilibrium binding, no change in bacteriophage T7 structure occurred, by the criterion of agarose gel electrophoresis. However, excessive incubation with ethidium bromide caused detectable changes in bacteriophage structure, a possible explanation of disagreements in similar studies previously performed with T-even bacteriophages. Scatchard plots for packaged DNA had a curvature greater than the previously demonstrated [Bresloff, J. L. & Crothers, D. M. (1981) Biochemistry 20 , 3547–3553] curvature for free DNA. By treating plots for packaged DNA as though they were biphasic, it was found that binding to most sites occurred with an apparent association constant (K_ap) 3.3–4.3 times lower than the K_ap of free DNA. The number of these sites increased significantly as the density of packaged DNA was decreased by use of the deletion mutants. Values of ΔH° for these sites were negative and equal to the ΔH° for free DNA; values of ΔS° were positive and about half the ΔS° for free DNA. A second class of sites, roughly 1.2% of the total, had a significantly higher K_ap and more negative ΔH° than those of the majority of sites.     

86 Characterization and differentiation of binding modes of water-soluble porphyrins at complexation with DNA

The influence of water-soluble cationic meso-tetra-(4?N-oxyethylpyridyl)porphyrin (H₂TOEPyP4) and it’s metallocomplexes with Ni, Cu, Co, and Zn on hydrodynamic and spectral behavior of DNA solutions has been studied by UV/Vis absorption and viscosity measurement. It was shown that the presence of planar porphyrins such as H₂TOEPyP4, NiTOEPyP4, and СuTOEPyP4 leads to an increase in viscosity at relatively small concentrations, and then decrease to stable values. Such behavior is explained by intercalation of these porphyrins in DNA structure because the intercalation mode involves the insertion of a planar molecule between DNA base pairs which results in a decrease in the DNA helical twist and lengthening of the DNA. Further decrease of viscosity is explained by the saturation intercalation sites and occurs outside the binding mode. But, in the case of porphyrins with axial ligands such as CoTOEPyP4 and ZnTOEPyP4, the hydrodynamic parameters decrease, which is explained by self-stacking of these porphyrins in DNA surface. This data are proved by spectral measurements. The results obtained from titration experiments were used for calculation of binding parameters: the binding constant K _b and the number of binding sites per base pair n. Obtained data reveal that K _b varies between 3.4 and 5.4?×?10⁶?M^?1 for a planar porphyrins, a range typical for intercalation mode interactions, and 5.6?×?10⁵?M^?1 and 1.8?×?10⁶?M^?1 for axial porphyrins. In addition, the exclusion parameter n also testifies that at intercalation, (n～2) the adjacent base pairs are removed to place the planar molecules, and for outside binders to pack on the surface needs too few places (n～0.5–1). It is apparent that the binding is somewhat stronger at intercalation. The viscometric and spectrophotometric measurements are in good agreement.     

Energetic and structural aspects of ethidium cation intercalation into DNA minihelices

The enthalpy ΔH for the intercalation of the ethidium cation (EC) into DNA minihelices can be decomposed into (1) an energy of conformational adjustment (i.e., the energy of minihelix extension and unwinding from the B-form to the intercalated form) and (2) EC minihelix intermolecular interactions. In the present study, we have focused our attention mainly on a decomposition of the energetic factors of the EC minihelix intermolecular interactions, while the essential features of the energy of conformational adjustment have been discussed in detail elsewhere by us. The structural features of the various resulting energy-minimized EC-intercalated complexes are compared with each other and the initial x-ray model structure. ΔH is estimated to be in the range of ?12.3 to ?24.0 kcal/mol. This theoretical estimate is qualitatively and quantitatively in agreement with a variety of available experimental data. The energy of conformational adjustment is an energetically unfavorable step, while the energetically favorable contribution of the EC minihelix intermolecular interactions is responsible for the overall favorable nature of the intercalation process involving the EC. On the other hand, the observed preference for intercalation into Pyr(3′–5′)Pur DNA sequences over their isomeric Pur(3′–5′)Pyr sequences is controlled by the energy of conformational adjustment and not by the EC minihelix intermolecular interaction contribution. No base-composition effect is expected at EC concentrations normally found at cellular conditions. Moreover, the structural features of the various EC-intercalated complexes are very similar regardless of minihelix base sequence or composition. These results compare favorably with available evidence. The nature of biologically preferred sites of EC binding with the minihelices is discussed.     

Relaxation kinetics of DNA-ligand binding including direct transfer

The relaxation kinetics of binding of ethidium to calf-thymus DNA as studied previously by the temperature-jump method with absorption detection [Bresloff, J. & Crothers, D. M. (1975) J. Mol. Biol. 95 , 103] is reanalyzed in terms of a series of models for DNA-ligand interactions that include cases with and without internal and bimolecular direct transfer of ligands between different binding modes or different binding sites. The experimental results are shown to be consistent with two alternative binding mechanisms. Both models include bimolecular “direct transfer” steps and a site size of two base pairs per site. In the first model, there exist two distinct modes of binding to the DNA double helix at each binding site. In the second model, sites containing at least one GC base pair constitute a different and stronger class of binding sites than those containing only AT base pairs. The existence of a direct transfer pathway between two classes of bound ligands is supported by the linear increase of reciprocal relaxation times far beyond the concentration regions, where off-rates should have become rate limiting. The second model, incorporating the notion of base selectivity, is in quantitative agreement with published work on ethidium binding to DNAs of different base compositions and with nmr measurements of bound ligand lifetimes.     

Intercalation of bulky Delta,Delta- and Lambda,Lambda-bis-Ru(II) complex between DNA base pairs

Interaction of Δ,Δ- and Λ,Λ-bis-Ru(II) complexes with native DNA was investigated by isotropic absorption and polarized spectroscopy including circular and linear dichroism (CD and LD). Despite the steric hindrance originating from its four bulky phenanthroline ligands at both ends of the molecule, this molecule rapidly intercalates between DNA base pairs. Intercalation was judged by large hypochromism and red shift in the UV-visible absorption spectra in the absorption region of the bridging moiety as well as in the metal-to-ligand-charge transfer absorption region. Further support for the intercalation is found in the fact that the magnitude of negative reduced LD signal in the absorption region of the bridging moiety was comparable to that of the DNA absorption region, indicating that the bridge connecting the two Ru(II) complexes is nearly parallel to the DNA base planes. No difference in the binding mode between the two enantiomers was observed. In the presence of either bis-Ru(II) complex, ethidium bromide, a classical intercalator, can intercalate into the empty sites but was not able to replace the Ru(II) complexes. Near the saturation, ground state interaction between ethidium and bis-Ru(II) complex was evident by LD.     

Interactions of Molecules with Nucleic Acids. X. Covalent Intercalat e Binding of the Carcinogenic BPDE I(+) to Kinked DNA

Abstract

A theoretical model is proposed for the covalent binding of (+) 7 β,8α-dihydroxy-9α, 10α- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene denoted by BPDE I(+), to N2 on guanine. The DNA must kink a minimum of 39° to allow proper hybrid configurations about the C10 and N2 atoms involved in bond formation and to allow stacking of the pyrene moiety with the non-bonded adjacent base pair. Conservative (same sugar puckers and glycosidic angles as in B-DNA) and non-conservative (alternating sugar puckers as in intercalation sites) conformations are found and they are proposed structures in pathways connecting B-DNA, an intercalation site, and a kink site in the formation of a covalently intercalative bound adduct of BPDE I(+) to N2 on guanine. Stereographic projections are presented for (3′) and (5′) binding in the DNA. Experimental data for bending of DNA by BPDE, orientation of BPDE in DNA and unwinding of superhelical DNA is explained. The structure of a covalent intercalative complex is predicted to result from the reaction. Also, an anti ? syn transition of guanine results in a structure which allows the DNA to resume its overall B-form. The only change is that guanine has been rotated by 200° about its glycosidic bond so that the BPDE I(+) is bound in the major groove. The latter step may allow the DNA to be stored with an adduct which may produce an error in the genetic code.     

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.