Synthesis, characterization and DNA binding and cleavage properties of ruthenium(II) complexes with various polypyridyls

Polypyridyl chlororuthenium(II) complexes have been synthesized and characterized. The binding mode of the complexes to DNA has been evaluated from the combined results of electronic absorption spectroscopy and viscosity measurement study. The results suggest that complexes 1, 2 and 3 bind to DNA via classical intercalation, electrostatic interaction and partial intercalation mode, respectively. Complex 2 shows less affinity for DNA. Cleavage of pUC19 DNA by complexes has been checked using gel electrophoresis. The data disclose that complex 1 has the highest cleaving ability.     

1H NMR study of the complexation of the quinolone antibiotic norfloxacin with DNA

Complexation of antibiotic norfloxacin (NOR) with DNA fragments 5'-d(TpGpCpA) and 5'-d(CpGpCpG) has been studied in aqueous solution by 1H NMR spectroscopy (500 MHz). Equilibrium parameters of the complexation with single-stranded and duplex forms of DNA oligomer--equilibrium constants, enthalpy and entropy--have been obtained for the first time. Based on the analysis of the complexation parameters as well as induced chemical shifts of the antibiotic protons within different complexes, it was found that NOR binds with the tetramer duplexes mainly by intercalation. The complexation with the single-stranded form may occur either by intercalation and external binding. The site of preferential binding of the antibiotic with DNA duplex is GC site.     

The mechanism of sequence non-specific DNA binding of HMG1/2-box B in HMG1 with DNA

The DNA binding mechanism of box B in HMG1, a member of the sequence non-specific DNA binding HMG1/2-box family of proteins, has been examined by both mutation analyses and molecular modeling techniques. Substitution of the residue 102F, which is characteristically exposed to solvent, with a small hydrophobic amino acid affected its DNA binding activity. However, no additional effect was observed by the further mutation of flanking 101F. Molecular dynamics simulation and modeling studies revealed that 102F intercalates into DNA base-pairs, being supported by the flanking 101F. The mutants with a small hydrophobic residue at position 102 tolerated the substitution for 101F because the side chain at position 102 is too short to intercalate. Thus the intercalation of 102F and the positive effect of the flanking 101F residue are important for the sequence non-specific DNA binding of the HMG1/2-box. The conserved basic residues of 95K, 96R and 109R were also examined for their roles in DNA binding. These residues interacted with DNA mainly by electrostatic interaction and maintained the location of the box on the DNA, which prescribed the intercalation of 102F. The DNA intercalation by HMG1 consists of an ingenious mechanism which brings DNA conformational changes necessary for biological functions.     

Small molecule intercalation with double stranded DNA: implications for normal gene regulation and for predicting the biological efficacy and genotoxicity of drugs and other chemicals

The binding of small molecules to double stranded DNA including intercalation between base pairs has been a topic of research for over 40 years. For the most part, however, intercalation has been of marginal interest given the prevailing notion that binding of small molecules to protein receptors is largely responsible for governing biological function. This picture is now changing with the discovery of nuclear enzymes, e.g. topoisomerases that modulate intercalation of various compounds including certain antitumor drugs and genotoxins. While intercalators are classically flat, aromatic structures that can easily insert between base pairs, our laboratories reported in 1977 that a number of biologically active compounds with greater molecular thickness, e.g. steroid hormones, could fit stereospecifically between base pairs. The hypothesis was advanced that intercalation was a salient feature of the action of gene regulatory molecules. Two parallel lines of research were pursued: (1) development of technology to employ intercalation in the design of safe and effective chemicals, e.g. pharmaceuticals, nutraceuticals, agricultural chemicals; (2) exploration of intercalation in the mode of action of nuclear receptor proteins. Computer modeling demonstrated that degree of fit of certain small molecules into DNA intercalation sites correlated with degree of biological activity but not with strength of receptor binding. These findings led to computational tools including pharmacophores and search engines to design new drug candidates by predicting desirable and undesirable activities. The specific sequences in DNA into which ligands best intercalated were later found in the consensus sequences of genes activated by nuclear receptors implying intercalation was central to their mode of action. Recently, the orientation of ligands bound to nuclear receptors was found to match closely the spatial locations of ligands derived from intercalation into unwound gene sequences suggesting that nuclear receptors may be guiding ligands to DNA with remarkable precision. Based upon multiple lines of experimental evidence, we suggest that intercalation in double stranded DNA is a ubiquitous, natural process and a salient feature of the regulation of genes. If double stranded DNA is proven to be the ultimate target of genomic drug action, intercalation will emerge as a cornerstone of the future discovery of safe and effective pharmaceuticals.     

Methylene blue binding to DNA with alternating GC base sequence: continuum treatment of salt effects

Methylene blue (MB), an efficient singlet oxygen generating photoactive dye, binds to DNA and allows photosensitized reactions to be used for sequence-specific cleavage of the DNA backbone. Intercalation and groove binding are possible binding modes of the dye, depending on base sequences and environmental conditions. In a recent modeling study of methylene blue binding to a double stranded DNA decamer with an alternating GC sequence, six structural models for intercalation structures and for minor and major groove binding have been obtained. By estimating the binding energies (including electrostatic reaction field contributions of a salt-free aqueous solvent), symmetric intercalation at the 5'-CpG-3' and 5'-GpC-3' steps was found as the predominant binding mode, followed by a slightly weaker binding of the dye in the minor groove. In this study, the stability of the modeled structures has been analysed as a function of salt concentration. The results of finite difference numerical solutions of the non-linear Poisson-Boltzmann equation show that the stabilizing effect of salt is larger for free DNA than for the modeled MB-DNA complexes. Accordingly, the estimated binding energies decrease with increasing ionic strength. A slightly higher stabilization of the groove binding complexes results in comparable binding energies for symmetric intercalation and minor groove binding at high salt concentration. Both results are in qualitative agreement with experimental data.     

X-ray diffraction and infrared circular dichroism of DNA-violamycin B1 complexes

X-ray diffraction and infrared linear dichroism of oriented samples of DNA-violamycin B1 complexes have been studied at different antibiotic/DNA phosphate ratios (r) as a function of relative humidity. Violamycin B1 binds to DNA according to the intercalation as well as to the outside binding model. At low r values, where the intercalation predominates the unwinding angle of DNA helix is between 6 degrees and 12 degrees per intercalation site as followed from the dependence of the pitch of helix versus r. At r greater than or equal to 0.17 the intercalation sites are saturated and the outside binding becomes prevalent; however the violamycin B1 chromophore is still oriented in the plane of DNA bases. Conformational mobility of DNA in the violamycin B1 complexes is largely inhibited compared with pure DNA, but it is higher than that of the daunomycin complexes. At least 30% of DNA in violamycin complexes has A conformation at the medium humidities as followed by IR linear dichroism. In the case of x-ray diffraction the A conformation was not detected. The distance between DNA molecules in the complex is found to be 23.2 A, that is 2 A less than in pure DNA at the same conditions and it does not depend upon r.     

The importance of intercalation in the covalent binding of benzo(a)pyrene diol epoxide to DNA

The primary mode of non-covalent interaction of the strong carcinogen, benzo(a)pyrene diol epoxide, with DNA is through intercalation. It has variously been suggested that intercalative complexes may be prerequisite for either covalent binding or DNA-catalysed hydrolysis of the epoxide or both. Geacintov [Geacintov, N. E. (1986). Carcinogenesis 7, 589.] has recently argued that intercalation is important in covalent binding and presented theoretical constructs consistent with this proposal. A more general theoretical model is presented here which includes the possibilities that either catalysis of hydrolysis or covalent binding of benzo(a)pyrene diol epoxide DNA can occur (a) in an intercalation complex, or (b) without formation of a detectable, physically bound complex. It is shown that a variety of possible mechanisms formulated under this general theory lead to equations for overall reaction rates and covalent binding fractions which are all of the same form with respect to DNA concentration dependence. A consequence of this is that experimental studies of the dependence of hydrolysis rates and covalent binding fractions on DNA concentration do not distinguish between the various possible mechanisms. These findings are discussed in relation to the interactions of benzo(a)pyrene diol epoxide with chromatin in cells.     

Nonspecific interactions in dye binding to DNA. Influence of alcohols and amides

The binding of a few drugs (ethidium bromide, propidium diiodide, proflavine and actinomycin D) to DNA has been investigated in aqueous solutions to which cosolvents of different polarity have been added. It is found that both alcohols (less polar than water) and amides (more polar) lower the binding constant according to a linear relationship between the intercalation free energy and cosolvent concentration. The main action of cosolvents cannot be described in terms of electrostatic effects, since they predict much smaller changes in the binding constant than those observed. It appears instead that relevant solvation effects are responsible for the binding strength of the different dyes to DNA. As a general result, it is found that solvation effects largely contribute to the intercalation free energy, thereby weakening the influence of nonspecific interactions at the intercalation site.     

Synthesis, characterization, and DNA binding studies of some mixed-ligand platinum(II) complexes of 1,10-phenanthroline and amino acids

A series of complexes of the type [Pt(phen)(AA)]+ (where AA is the anion of glycine, L-alanine, L-leucine, L-phenylalanine, L-tyrosine, or L-tryptophan) has been synthesized. These complexes have been characterized by electronic absorption, infrared, and 1H NMR spectroscopy. The interaction of these complexes with calf thymus DNA has been studied using fluorescence spectroscopy. They inhibit the intercalation of ethidium bromide in DNA by intercalative binding at low concentrations and show nonintercalative binding at higher concentrations.     

Synthesis,characterization and DNA binding and cleavage properties of ruthenium(II) complexes with various polypyridyls

Polypyridyl chlororuthenium(II) complexes have been synthesized and characterized. The binding mode of the complexes to DNA has been evaluated from the combined results of electronic absorption spectroscopy and viscosity measurement study. The results suggest that complexes 1, 2 and 3 bind to DNA via classical intercalation, electrostatic interaction and partial intercalation mode, respectively. Complex 2 shows less affinity for DNA. Cleavage of pUC19 DNA by complexes has been checked using gel electrophoresis. The data disclose that complex 1 has the highest cleaving ability.     

Bifunctional intercalation of antitumor antibiotics BBM-928A and echinomycin with deoxyribonucleic acid. Effects of intercalation on deoxyribonucleic acid degradative activity of bleomycin and phleomycin

The binding of peptide antitumor antibiotics, BBM-928A and echinomycin, to superhelical PM2 DNA and the effects of the resulting conformational changes of DNA on the DNA-degradative activity of two related antitumor antibiotics, bleomycin A2 and phleomycin D1, have been studied. The bifunctional intercalative mode of the DNA binding of BBM-928A concluded previously from viscometric and fluorometric studies has been confirmed by gel electrophoretic analysis. Under the employed electrophoretic conditions, DNA-bound BBM-928A showed little dissociation whereas echinomycin and ethidium bromide showed partial and nearly complete dissociation, respectively. BBM-928A induced neither single-strand nor double-strand breaks in DNA. Competitive binding studies by fluorescence changes suggested that binding sites on DNA molecules for BBM-928A (or echinomycin) may differ from those for ethidium bromide, since binding to DNA by the two drugs was not competitive even at saturating concentrations. The lack of such a competition between the two drugs is not consistent with the neighbor-exclusion principle. The DNA-degradative activity of both bleomycin A2 and phleomycin D1 increased with the removal of the negative superhelicity of DNA by the BBM-928A intercalation and decreased with the formation of positive superhelical turns induced by high concentrations of BBM-928A. Thus the degradative activity of both bleomycin A2 and phleomycin D1 is sensitive in a similar manner to the degree of superhelicity rather than the double helicity of DNA, although there are differences between these two drugs in interaction with DNA.     

DAPI (4',6-diamidino-2-phenylindole) binds differently to DNA and RNA: minor-groove binding at AT sites and intercalation at AU sites.

The interaction of DAPI and propidium with RNA (polyA.polyU) and corresponding DNA (polydA.polydT) sequences has been compared by spectroscopic, kinetic, viscometric, Tm, and molecular modeling methods. Spectral changes of propidium are similar on binding to the AT and AU sequences but are significantly different for binding of DAPI. Spectral changes for DAPI with the DNA sequence are consistent with the expected groove-binding mode. All spectral changes for complexes of propidium with RNA and DNA and for DAPI with RNA, however, are consistent with an intercalation binding mode. When complexed with RNA, for example, DAPI aromatic protons signals shift significantly upfield, and the DAPI UV-visible spectrum shows significantly larger changes than when complexed with DNA. Slopes of log kd (dissociation rate constants) versus-log [Na+] plots are similar for complexes of propidium with RNA and DNA and for the DAPI-RNA complex and are in the range expected for an intercalation complex. The slope for the DAPI-DNA complex, however, is much larger and is in the range expected for a groove-binding complex. Association kinetics results also support an intercalation binding mode for the DAPI-RNA complex. The viscosity of polyA.polyU solutions increases significantly on addition of both propidium and DAPI, again in agreement with an intercalation binding mode for both molecules with RNA. Molecular modeling studies completely support the experimental findings and indicate that DAPI forms a very favorable intercalation complex with RNA. DAPI also forms a very stable complex in the minor groove of AT sequences of DNA, but the stabilizing interactions are considerably reduced in the wide, shallow minor groove of RNA. Modeling studies,thus,indicate that DAPI interaction energetics are more favorable for minor-groove binding in AT sequences but are more favorable for interaction in RNA.     

DNA interaction with low molecular ligands of different structure. II. Complexes of DNA with actinomine and its analogs

DNA-complexes with actinomine and its analogues containing omega-dialkylaminoalkyl groups at 1,9 positions of the phenoxazone moiety were studied by technique of spectrophotometry, viscometry and flow birefringence. In the process of spectrophotometry titration two groups of spectra corresponding to different DNA--ligand ratio in a complex were observed. According to the experimental data the investigated compounds are bounded to DNA by means of intercalation and external binding. There within a region of low degrees of binding the intercalation type of the ligand--DNA interaction prevails. In virtue of the spectrophotometry data the intercalation binding share was calculated. The intrinsic viscosity of a complex increases in the case of ligand intercalation and does not change as it joins to the DNA double-helix from outside. Optical anisotropy of DNA molecule increases linearly irrespective of the way of ligand binding. Data on the flow birefringence permits to conclude that under external binding the angle between the normal to the ligand chromophore plane and the axis of DNA double-helix is about zero. During ligand intercalation the equilibrium rigidity of DNA molecules increases.     

Methyl green and its analogues bind selectively to AT-rich regions of native DNA.

Methyl green has long been used as a DNA stain in histochemistry. The sequence selective binding of the cationic triphenylmethane dyes methyl green, crystal violet and Malachite green to DNA was investigated by DNAase 1 and micrococcal nuclease footprinting. At low concentrations the ligands showed similar footprinting patterns which centred around AT-rich regions with a mild preference for hompolymeric A and T. At higher concentrations the dyes bound to almost all available DNA sites. Models, with and without intercalation are discussed to account for the specific binding.     

Structural Analysis of HMGD-DNA Complexes Reveals Influence of Intercalation on Sequence Selectivity and DNA Bending

The ubiquitous, eukaryotic, high-mobility group box (HMGB) chromosomal proteins promote many chromatin-mediated cellular activities through their non-sequence-specific binding and bending of DNA. Minor-groove DNA binding by the HMG box results in substantial DNA bending toward the major groove owing to electrostatic interactions, shape complementarity, and DNA intercalation that occurs at two sites. Here, the structures of the complexes formed with DNA by a partially DNA intercalation-deficient mutant of Drosophila melanogaster HMGD have been determined by X-ray crystallography at a resolution of 2.85 Å. The six proteins and 50 bp of DNA in the crystal structure revealed a variety of bound conformations. All of the proteins bound in the minor groove, bridging DNA molecules, presumably because these DNA regions are easily deformed. The loss of the primary site of DNA intercalation decreased overall DNA bending and shape complementarity. However, DNA bending at the secondary site of intercalation was retained and most protein-DNA contacts were preserved. The mode of binding resembles the HMGB1 box A-cisplatin-DNA complex, which also lacks a primary intercalating residue. This study provides new insights into the binding mechanisms used by HMG boxes to recognize varied DNA structures and sequences as well as modulate DNA structure and DNA bending.     

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.     

Crystal structure of the 2:1 complex between d(GAAGCTTC) and the anticancer drug actinomycin D.

The crystal structures of the 2:1 complex of the self-complementary DNA octamer d(GAAGCTTC) with actinomycin D has been determined at 3.0 A resolution. This is the first example of a crystal structure of a DNA-drug complex in which the drug intercalates into the middle of a relatively long DNA segment. The results finally confirmed the DNA-actinomycin intercalation model proposed by Sobell & co-workers in 1971. The DNA molecule adopts a severely distorted and slightly kinked B-DNA-like structure with an actinomycin D molecule intercalated in the middle sequence, GC. The two cyclic depsipeptides, which differ from each other in overall conformation, lie in the minor groove. The complex is further stabilized by forming base-peptide and chromophore-backbone hydrogen bonds. The DNA helix appears to be unwound by rotating one of the base-pairs at the intercalation site. This single base-pair unwinding motion generates a unique asymmetrically wound helix at the binding site of the drug, i.e. the helix is loosened at one end of the intercalation site and tightened at the other end. The large unwinding of the DNA by the drug intercalation is absorbed mostly in a few residues adjacent to the intercalation site. The asymmetrical twist of the DNA helix, the overall conformation of the two cyclic depsipeptides and their interaction mode with DNA are correlated to each other and rationally explained.     

Daunomycin modifies the sequence-selective recognition of DNA by actinomycin.

The antitumour antibiotic actinomycin D normally binds to DNA by intercalation at sequences containing the CpG step, but in the presence of daunomycin it has been reported to interact with poly(dA-dT). This observation has neither been confirmed nor explained. Here we have used a photoreactive 7-azido derivative of actinomycin to study the effect of daunomycin on its binding to three DNA fragments. Daunomycin did indeed alter the binding of actinomycin to the DNA, such that the antibiotic was displaced from its primary GpC sites onto secondary sites in the DNA, though not to AT regions especially. These findings suggest a possible scientific explanation for the increased toxicity seen during combination chemotherapy with these two drugs.     

DNA binding properties of DAPI (4',6-diamidino-2-phenylindole) analogs having an imidazoline ring or a tetrahydropyrimidine ring: groove-binding and intercalation.

DAPI analogs containing an imidazoline ring or a tetrahydropyrimidine ring have been synthesized to study DNA binding properties. Spectroscopic (absorption, CD, flow dichroism and fluorescence) and viscosity measurements indicate that DAPI analogs interact with DNA both by intercalation and by groove binding. The solution structures of complexes between DAPI analog and DNA oligomers have been characterized by proton NMR spectroscopy.