A 19F-NMR study of 2-fluoro-4-demethoxydaunomycin intercalation complexes with the decanucleotides d(G-C)5 and d(A-T)5

Binding configurations and equilibria of intercalation complexes formed by the novel anthracycline drug, 2-fluoro-4-demethoxydaunomycin (2FD), with the decanucleotides d(G-C)5 and d(A-T)5 have been studied by 19F-NMR spectroscopy. The 19F chemical shift of 2FD bound to d(A-T)5 was approximately 1.5 ppm downfield of that observed for 2FD bound to d(G-C)5. By mixing equimolar amounts of aqueous d(G-C)5, d(A-T)5 and 2FD, the distribution of drug between the nucleotides was followed by observing relative peak intensities and showed no G-C or A-T binding preference at room temperature. It was shown that each decanucleotide duplex bound three 2FD molecules, giving a neighbour exclusion parameter, n, of n = 3 for this drug. The stoichiometric complexes, which we denote by [d(A-T)5][2FD]3 and [d(G-C)5][2FD]3, were also purified and isolated in this study.     

Monitoring anthracycline binding to nucleotides by two dimensional cosy NMR experiments.

The nucleotide binding ability of the novel anthracycline drug, 3-fluoro-4-demethoxydaunomycin, has been studied by two dimensional 1H NMR correlated spectroscopy (COSY). In the COSY spectrum of the nucleotide mini-helix d(CTGCAG)2 cross-peaks are observed from the spin-coupled H6 and H5 protons of the cytidine bases. Additional cytidine H6/H5 cross-peaks are observed in the COSY spectrum of the anthracycline-d(CTGCAG)2 complex. These additional cytidine cross-peaks enable the identification of the anthracycline binding sites and the determination of the relative kinetic stability of the bound drug at each binding site.     

Effects of solute conditions on the relative affinities of the oligonucleotides d(G-C)5 and d(A-T)5 for the anthracycline drug 2-fluoro-4-demethoxydaunomycin

19F NMR has been used to show that changes in NaCl concentration, as well as the presence of lysine or arginine, affect the equilibrium distribution of the synthetic anthracycline 2-fluoro-4-demethoxydaunomycin (2FD) between binding sites on d(G-C)5 and d(A-T)5 in a 1:1:1 molar aqueous system: 2FD/d(G-C)5/d(A-T)5. Varying the pH between 6.2 and 7.7 had no effect. NaCl concentrations below 0.1 M led to a d(G-C)5 preference while above 0.1 M a preference for d(A-T)5 was observed. At comparable solute concentrations, use of either lysine or arginine resulted in a significant drug preference for d(G-C)5 compared to systems containing only NaCl.     

Neither delta- nor lambda-tris(phenanthroline)ruthenium(II) binds to DNA by classical intercalation.

Equilibrium binding studies and viscosity experiments are described that characterize the interaction of delta- and lambda-[Ru(o-phen)3]2+ with calf thymus DNA. The mode of binding of these compounds to DNA is a matter of controversy. Both isomers of [Ru(o-phen)3]2+ were found to bind but weakly to DNA, with binding constants of 4.9 (+/- 0.3) x 10(4) M-1 and 2.8 (+/- 0.2) x 10(4) M-1 determined for the delta and lambda isomers, respectively, at 20 degrees C in a solution containing 5 mM Tris-HCl (pH 7.1) and 10 mM NaCl. We determined that the quantity delta log K/delta log [Na+] equals 1.37 and 1.24 for the delta and lambda isomers, respectively. Application of polyelectrolyte theory allows us to use these values to show quantitatively that both the delta and lambda isomers are essentially electrostatically bound to DNA. Viscosity experiments show that binding the lambda isomer does not alter the relative viscosity of DNA to any appreciable extent, while binding of the delta isomer decreases the relative viscosity of DNA. From these viscosity results, we conclude that neither isomer of [Ru(o-phen)3]2+ binds to DNA by classical intercalation.     

Structural investigation of the hedamycin:d(ACCGGT)2 complex by NMR and restrained molecular dynamics

Hedamycin, a member of the pluramycin family of drugs, displays a range of biological responses including antitumor and antimicrobial activity. The mechanism of action is via direct interaction with DNA through intercalation between the bases of the oligonucleotide and alkylation of a guanine residue at 5'-PyG-3' sites. There appears to be some minor structural differences between two earlier studies on the interaction of hedamycin with 5'-PyG-3' sites. In this study, a high-resolution NMR analysis of the hedamycin:d(ACCGGT)2 complex was undertaken in order to investigate the effect of replacing the thymine with a guanine at the preferred 5'-CGT-3' site. The resultant structure was compared with earlier work, with particular emphasis placed on the drug conformation. The structure of the hedamycin:d(ACCGGT)2 complex has many features in common with the two previous NMR structures of hedamycin:DNA complexes but differed in the conformation and orientation of the N,N-dimethylvancosamine saccharide of hedamycin in one of these structures. The preferential binding of hedamycin to 5'-CG-3' over 5'-TG-3' binding sites is explained in terms of the orientation and location of the N,N-dimethylvancosamine saccharide in the minor groove.     

Interactions of water-soluble porphyrins with hexadeoxyribonucleotides: resonance raman, UV-visible and 1H NMR studies

The interactions of the water-soluble porphyrins M(TMpy-P4) [M = H2, Cu(II), Ni(II), and Co(III); TMpy-P4 = tetrakis(4-N-methylpyridyl)porphyrinato ion], with the hexadeoxyribonucleotides d(CGTACG)2, d(TACGTA)2, d(GCATGC)2, d(TGTGCA)2, and d(CTATAG)2 have been investigated by resonance Raman and/or UV-visible spectroscopy. The results indicate that all hexamers containing the 5'CG3' as well as the 5'GC3' site, and also the mismatched hexamer d(TGTGCA)2, are capable of intercalating the H2, Cu(II) and Ni(II) porphyrins. 1H nuclear magnetic resonance spectra of d(CGTACG)2 mixed with Cu(TMpy-P4) have provided further evidence for the intercalation. For the other cases, outside binding by localized electrostatic interaction is suggested. There is no evidence of groove binding to any of the hexamers. Possible reasons for different binding properties of long and short helices are discussed.     

Actinomycin D induced DNase I cleavage enhancement caused by sequence specific propagation of an altered DNA structure.

Two DNA hexadecamers containing one central 5'-GC-3' base step have been examined by footprinting methodology in the presence and absence of actinomycin D. The results of these studies, coupled with imino proton NMR measurements indicate that the antitumor drug causes a change in DNA conformation at a distance from the actinomycin intercalation site in a molecule of sequence d[ATATATAGCTATATAT] that does not occur in d[AAAAAAAGCTTTTTTT]. The experiments demonstrate that DNase I rate enhancements associated with actinomycin D binding are caused by ligand alteration of equilibrium DNA structure.     

Synthesis of metallointercalator-DNA conjugates on a solid support

Metallointercalator-DNA conjugates were prepared by amide bond formation between active esters on the nonintercalating ligands of transition metal complexes and primary amines presented at the 5' or the 3' termini of oligonucleotides attached to solid supports. The conjugates were liberated from the support by aminolysis and purified by HPLC on C18 or C4 stationary phases, which separates the two diastereomeric forms of the conjugates containing either the Lambda or the Delta enantiomer of the octahedral metal complex. The coupling reaction proceeds with approximately 75% conversion of the amino-terminated oligonucleotide into the conjugate; the isolated yield is approximately 200 nmol for syntheses initiated on DNA-synthesis columns with a loading of 2 micromol. The conjugates were characterized by ultraviolet-visible and circular dichorism absorption spectroscopy, electrospray ionization mass spectrometry, enzymatic digestion, and polyacrylamide gel electrophoresis (PAGE). Oligonucleotides bearing [Rh(phi)(2)(bpy')](3+) (phi = 9, 10-phenanthrene quinone diimine; bpy' = 4-butyric acid-4'-methyl bipyridyl) form 1:1 duplexes with the complementary strand, and the electrophoretic mobility under nondenaturating PAGE of duplexes containing Delta-Rh is notably different from duplexes containing Lambda-Rh. High-resolution PAGE of DNA photocleavage reactions initiated by irradiation of the tethered Rh complexes reveal intercalation of the complex only near the tethered end of the duplex. Analogous DNA-binding properties were observed with [Rh(phi)(2)(bpy')](3+) tethered to the 3' terminus. By combining the 3' and 5' modification strategies, a mixed-metal DNA conjugate containing both [Os(phen)(bpy')(Me(2)-dppz)](2+) (Me(2)-dppz = 7, 8-dimethyldipyridophenazine) on the 3' terminus and [Rh(phi)(2)(bpy')](3+) on the 5' terminus was prepared and isolated. Taken together, these strategies for preparing metallointercalator-DNA conjugates offer a useful approach to generate chemical assemblies to probe long-range DNA-mediated charge transfer where the redox initiator is confined to and intercalated in a well-defined binding site.     

Concerted intercalation and minor groove recognition of DNA by a homodimeric thiazole orange dye

The thiazole orange dye TOTO binds to double-stranded DNA (dsDNA) by a sequence selective bis-intercalation. Each chromophore is sandwiched between two base pairs in a (5'-CpT-3'):(5'-ApG-3') site, and the linker spans two base pairs in the minor groove. We have used one- and two-dimensional NMR spectroscopy to examine the dsDNA binding of an analogue of TOTO in which the linker has been modified to contain a bipyridyl group (viologen) that has minor groove binding properties. We have investigated the binding of this analogue, called TOTOBIPY, to three different dsDNA sequences containing a 5'-CTAG-3', a 5'-CTTAG-3', and a 5'-CTATAG-3' sites, respectively, demonstrating that TOTOBIPY prefers to span three base pairs. The many intermolecular NOE connectivities between TOTOBIPY and the d(CGCTTAGCG):d(CGCTAAGCG) oligonucleotide in the complex shows that the bipyridyl-containing linker is positioned in the minor groove and spans three base pairs. Consequently, we have succeeded in designing and synthesizing a ligand that recognizes an extended recognition sequence of dsDNA as the result of a concerted intercalation and minor groove binding mode.     

Biological activities of phosphodiester linkage isomers of 2-5A

To determine the relative importance of the 2',5'-phosphodiester bond of 2-5A in its binding to and activation of the 2-5A-dependent ribonuclease (RNase L, RNase F), a number of phosphodiester linkage isomers of 2-5A were prepared. These isomers were obtained either by lead ion-catalyzed polymerization of adenosine 5'-phosphorimidazolidate or by T4 polynucleotide kinase-catalyzed 5'-phosphorylation of adenylyl(3' leads to 5')adenylyl(3' leads to 5')adenosine followed by reaction of the corresponding phosphorimidazolidates with tri(n-butylammonium)pyrophosphate. The following 2-5A isomers thus were prepared: ppp5'A2'p5'A3'p5'A, ppp5'A3'p5'A2'p5'A, ppp5'A3'p5'A3'p5'A("3-5A"), ppp5'A2'p5'A3'p5'A2'p5'A,and ppp5'A3'p5'A2'p5'-A2'p5'A. The ability of these isomeric 2-5As to interact with the 2-5A-dependent endonuclease was ascertained by three different criteria: (i) ability to prevent the protein synthesis inhibitory effects of 2-5A, (ii) activity as an inhibitor of translation in encephalomyocarditis RNA-programmed L cell extracts, and (iii) ability to prevent binding of the radiolabeled probe, ppp5'A2'p5'A2'p5'A2'p5'A3'[32P]p5'Cp, to the endonuclease of L cell extracts. In certain experiments, degradation of oligonucleotide was minimized or eliminated by altering assay conditions, providing alternate phosphodiesterase substrates, or by using purified endoribonuclease of Ehrlich ascites cells. By all criteria, replacement of 2',5'-bond by a 3',5'-bond led to a substantial decrease in biological activity. Generally, replacement of just one 2',5'-phosphodiester bond with a 3',5'-linkage led to at least a one order of magnitude loss of activity. In accord with this trend, ppp5'A3'p5'A3'p5'A(3-5A) was greater than 10,000 less active than 2-5A in binding to the endonuclease or as an inhibitor of protein synthesis.     

A theoretical investigation on the sequence selective binding of mitoxantrone to double-stranded tetranucleotides.

Theoretical computations are performed on the comparative binding energetics of mitoxantrone (MX), a newly synthesized intercalating anthraquinone antitumor drug, to six representative double-stranded tetranucleotides: d(GCGC)2, d(CGCG)2, d(ATAT)2, d(TATA)2, d(GTGT), d(ACAC), and d(CCGG)2. The computations are performed with the SIBFA procedure, which uses empirical formulas based on ab initio SCF computations. The best binding configuration of mitoxantrone locates its two side chains in the major groove. A considerable preference is elicited for intercalation of the chromophore ring in a pyrimidine (3'-5') purine sequence rather than the isomeric purine (3'-5') pyrimidine sequence. Contrary to the situation encountered with "simple" intercalators, in which this preference is generally attributed solely to differences in the energies of unstacking necessary to generate the intercalation sites, the preference is dictated in MX to a large extent by the intermolecular interaction energy term. This result is imposed by the interactions of the side chains of MX with the oligonucleotide.     

Mode of binding of the cytotoxic alkaloid berberine with the double helix oligonucleotide d(AAGAATTCTT)(2)

Berberine, an isoquinoline plant alkaloid, belongs to the structural class of protoberberines. Recently, the ability of these compounds to act as Topoisomerase I or II poisons, was related to the antitumor activity. The binding of protoberberins to DNA has been studied and the partial intercalation into the double helix has been considered responsible for their activity. We have studied the interaction of berberine with the double helix oligonucleotides d(AAGAATTCTT)(2), d(GCGATCGC)(2), d(CGTATACG)(2), d(CGTACG)(2), 5'-d(ACCTTTTTGATGT)-3'/5(ACATCAAAAAGGT)-3' and with the single strand 5'-d(ACATCAAAAAGGT)-3', by 1H, 31P NMR and UV spectroscopy. Phosphorus resonance experiments were performed to detect small conformational changes of the phosphoribose backbone, in the case that an intercalation process occurs. Our data reveal that berberine does not intercalate into the duplexes studied, and binds preferentially to AT rich sequences. The structure of the complex with d(AAGAATTCTT)(2) was determined by using proton 2D NOESY spectra, which allowed to obtain several NOE contacts between the drug and the nucleotide. Structural models were built up by Molecular Mechanics (MM) and Molecular Dynamics (MD) calculations, by using the inter-proton distances derived from the NOE values. Berberine results to be located in the minor groove, lying with the convex side on the helix groove and presenting the positively charged nitrogen atom close to the negative ionic surface of the oligomer. The large 1H chemical shifts variation, observed for the drug when it is added to the above duplexes, as well as to the single strand oligomer, was interpreted with non-specific ionic interactions. The binding constants were measured by UV and NMR spectroscopy. They are strongly affected by the ionic strength and by the self-association process, which commonly occurs with this type of drugs. A dimerisation constant was measured and the value was included in the calculations of the binding constants. The results obtained show that the non-specific ionic interactions represent the major contribution to the values of the binding constants. These parameters, as well as the protons chemical shift variation of the ligand, are thus not diagnostic for the identification of a drug/DNA complex.     

DNA binding and cleavage properties of certain tetrammine ruthenium(II) complexes of modified 1,10-phenanthrolines--effect of hydrogen-bonding on DNA-binding affinity

A series of ruthenium(II) mixed ligand complexes of the type [Ru(NH(3))(4)(L)](2+), where L=imidazo[4,5-f][1,10]phenanthroline (ip), 2-phenylimidazo[4,5-f][1,10]phenanthroline (pip), 2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline (hpip), 4,7-diphenyl-1,10-phenanthroline (dip), naphtha[2,3-a]dipyrido[3,2-h:2',3'-f]phenazine-5,18-dione (qdppz), 5,18-dihydroxynaphtho[2,3-a]dipyrido[3,2-H:2',3'-f]phenazine (hqdppz), have been isolated and characterized. The interaction of these complexes with calf thymus DNA (CT DNA) has been explored by using absorption, emission, and circular dichroic spectral methods, thermal denaturation studies and viscometry. All these studies suggest the involvement of the modified phenanthroline 'face' rather than the ammonia 'face' of the complexes in DNA binding. An intercalative mode of DNA binding, which involves the insertion of the modified phenanthroline ligands in between the base pairs, is suggested. The results from absorption spectral titration and circular dichroism (CD), thermal denaturation and viscosity experiments indicate that the qdppz and hqdppz complexes (K(b) approximately 10(6) and Delta T(m)=11-13 degrees C) bind more avidly than the ip, pip and hpip complexes (K(b) approximately 10(5), Delta T(m)=6-8 degrees C). Intramolecular hydrogen bonding in the hpip and hqdppz complexes increases the surface area of the intercalating diimines and enhances the DNA binding affinity substantially. The ammonia co-ligands of the complexes are possibly involved in hydrogen bonding with the intrastrand nucleobases to favour intercalation of the extended aromatic ligands. Circular dichroism spectral studies reveal that all the complexes effect certain structural changes on DNA duplex; [Ru(NH(3))(4)(ip)](2+) induces a B to A transition while [Ru(NH(3))(4)(qdppz)](2+) a B to Psi conformational change on CT DNA. Cleavage efficiency of the complexes were determined using pBR322 supercoiled plasmid DNA. All the complexes, except hqdppz complex, promote the cleavage of supercoiled plasmid (form I) to relaxed circular form (form II).     

Ethidium probing of the parallel double- and four-stranded structures formed by the telomeric DNA sequences dG(GT)4G and d(GT)5

Oligonucleotides 3'-d(GT)(5)-(CH(2)CH(2)O)(3)-d(GT)(5)-3' (parGT), containing GT repeats present in the telomeric DNA from Saccharomyces cerevisiae, had been demonstrated to form bimolecular structure, GT-quadruplex (qGT) [O. F. Borisova et al. FEBS Letters 306, 140-142 (1992)]. Four d(GT)(5) strands of the GT-quadruplex are parallel and form five G-quartets while thymines are bulged out. The four GT repeats when flanked by guanines, 3'-dG(TG)(4)G-(CH(2)CH(2)O)(3)-dG(GT)(4)G-3' (hp-GT), had been shown to form a novel parallel-stranded (ps) double helix with G.G and T.T base pairs (hp-GT ps-DNA) [A. K. Shchyolkina et al. J. Biomol. Struct. Dyn. 18, 493-503 (2001)]. In the present study the intercalator ethidium bromide (Et) was used for probing the two structures. The mode of Et binding and its effect on thermostability of qGT and hp-GT were compared. The quantum yield (q) and the fluorescence lifetime (tau) of Et:qGT (q = 0.15 +/- 0.01 and tau = 24 +/- 1 ns) and Et:hp-GT (q = 0.10 +/- 0.01 and tau = 16.5 +/- 1 ns) indicative of intercalation mode of Et binding were determined. Et binding to qGT was found to be cooperative with corresponding coefficient omega = 3.9 +/- 0.1 and the binding constant Kappa = (6.4 +/- 0.1).10(4) M(-1). The maximum number of Et molecules intercalating into GT-quadruplex is as high as twice the number of innerspaces between G-quartets (eight in our case). The data conform to the model of Et association with GT-quadruplex suggested earlier [O. F. Borisova et al. Mol. Biol. (Russ) 35, 732-739 (2001)]. The anticooperative type of Et binding was observed in case of hp-GT ps-DNA, with the maximum number of bound Et molecules, N = 4 / 5, and the association constant Kappa = (1.5 +/- 0.1).10(5) M(-1). Thermodynamic parameters of formation of Et:qGT and EtBr:hp-GT complexes were calculated from UV thermal denaturation profiles.     

Aryl substituted ruthenium bis-terpyridine complexes: intercalation and groove binding with DNA

The non-covalent interaction of five novel ruthenium(II) bis-terpyridine complexes with calf thymus DNA and, where appropriate, with poly[d(G-C)](2) and poly[d(A-T)](2) is described. Each complex is functionalised with aryl tail groups in the 4' position of the terpyridine ligands ((i) 9-anthracenyl, (ii) 4,4'-biphenyl, (iii) beta-naphthyl, (iv) 9-phenanthrenyl, and (v) 1-pyrenyl). Circular dichroism and linear dichroism show that the binding of three of the complexes (phenanthrenyl, anthracenyl and pyrenyl) at low metal complex concentration is dominated by intercalation of the aryl tail groups between the DNA bases. The complex with the biphenyl tail predominantly exhibits groove binding with no significant tail intercalation. The naphthyl derivative binds both by intercalation and a non-intercalative mode even at low metal complex concentrations. At high metal complex concentrations, aggregation of the complexes on the DNA is observed. Resonance light scattering indicates that the aggregates are of low nuclearity along the groove.     

Daunomycin intercalation stabilizes distinct backbone conformations of DNA

Daunomycin is a widely used antibiotic of the anthracycline family. In the present study we reveal the structural properties and important intercalator-DNA interactions by means of molecular dynamics. As most of the X-ray structures of DNA-daunomycin intercalated complexes are short hexamers or octamers of DNA with two drug molecules per doublehelix we calculated a self complementary 14-mer oligodeoxyribonucleotide duplex d(CGCGCGATCGCGCG)2 in the B-form with two putative intercalation sites at the 5'-CGA-3' step on both strands. Consequently we are able to look at the structure of a 1:1 complex and exclude crystal packing effects normally encountered in most of the X-ray crystallographic studies conducted so far. We performed different 10 to 20 ns long molecular dynamics simulations of the uncomplexed DNA structure, the DNA-daunomycin complex and a 1:2 complex of DNA-daunomycin where the two intercalator molecules are stacked into the two opposing 5'-CGA-3' steps. Thereby--in contrast to X-ray structures--a comparison of a complex of only one with a complex of two intercalators per doublehelix is possible. The chromophore of daunomycin is intercalated between the 5'-CG-3' bases while the daunosamine sugar moiety is placed in the minor groove. We observe a flexibility of the dihedral angle at the glycosidic bond, leading to three different positions of the ammonium group responsible for important contacts in the minor groove. Furthermore a distinct pattern of BI and BII around the intercalation site is induced and stabilized. This indicates a transfer of changes in the DNA geometry caused by intercalation to the DNA backbone.     

A tentative model of the intercalative binding of the neocarzinostatin chromophore to double-stranded tetranucleotides.

Theoretical computations are performed of the intercalative binding of the neocarzinostatin chromophore (NCS) with the double-stranded oligonucleotides d(CGCG)2, d(GCGC)2, d(TATA)2 and d(ATAT)2. Minor groove binding is preferred over major groove binding. It is found that the long axis of the stacked naphtoate ring lies approximately parallel to the long axis of the base pairs of the intercalation site. The galactosamine ammonium group interacts with specific sites of the groove (O2/N3 of bases 2 and O1' of sugar S3), whereas the dodecadyine ring system wraps around the groove towards the backbone. An overall AT versus GC preference is derived. Intercalation in a central purine-(3', 5')-pyrimidine sequence appears to be preferred over that in a central pyrimidine-(3', 5')-purine sequence.     

Drug recognition and stabilisation of the parallel-stranded DNA quadruplex d(TTAGGGT)4 containing the human telomeric repeat

The NMR structure of the parallel-stranded DNA quadruplex d(TTAGGGT)(4), containing the human telomeric repeat, has been determined in solution in complex with a fluorinated pentacyclic quino[4,3,2-kl]acridinium cation (RHPS4). RHPS4 has been identified as a potent inhibitor of telomerase at submicromolar levels (IC(50) value of 0.33(+/-0.13)microM), exhibiting a wide differential between telomerase inhibition and acute cellular toxicity. All of the data point to RHPS4 exerting its chemotherapeutic potency through interaction with, and stabilisation of, four-stranded G-quadruplex structures. RHPS4 forms a dynamic interaction with d(TTAGGGT)(4), as evident from 1H and 19F linewidths, with fast exchange between binding sites induced at 318 K. Perturbations to DNA chemical shifts and 24 intermolecular nuclear Overhauser effects (NOEs) identify the 5'-ApG and 5'-GpT steps as the principle intercalation sites; a structural model has been refined using NOE-restrained molecular dynamics. The central G-tetrad core remains intact, with drug molecules stacking at the ends of the G-quadruplex. The partial positive charge on position 13-N of the acridine ring appears to act as a "pseudo" potassium ion and is positioned above the centre of the G-tetrad in the region of high negative charge density. In both ApG and GpT intercalation sites, the drug is seen to converge to the same orientation in which the pi-system of the drug overlaps primarily with two bases of each G-tetrad. The drug is held in place by stacking interactions with the G-tetrads; however, there is some evidence for a more dynamic, weakly stabilised A-tetrad that stacks partially on top of the drug at the 5'-end of the sequence. Together, the interactions of RHPS4 increase the t(m) of the quadruplex by approximately 20 degrees C. There is no evidence for drug intercalation within the G-quadruplex; however, the structural model strongly supports end-stacking interactions with the terminal G-tetrads.     

Enantiomeric ruthenium(II) complexes binding to DNA: binding modes and enantioselectivity

A series of enantiomerically pure polypyridyl ruthenium(II) complexes, delta- and lambda-[Ru(bpy)2 (HPIP)](PF6)2 (delta-1 and lambda-1; bpy=2,2'-bipyridine, HPIP = 2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline), delta and lambda-[Ru(bpy)2(HNAIP)](PF6)2 (delta-2 and lambda-2; HNAIP = 2-(2-hydroxy-1-naphthyl)imidazo[4,5-f][1,10]phenanthroline), delta- and lambda-[Ru(bpy)2 (HNOIP)](PF6)2 (delta-3 and lambda-3; HNOIP = 2-(2-hydroxy-5-nitrophenyl)imidazo[4,5-f][1,10]phenanthroline), and delta- and lambda-[Ru(bpy)2(DPPZ)](PF6)2 (delta-4 and lambda-4; DPPZ= dipyridophenazine), have been synthesized. Binding behavior of these chiral complexes to calf thymus DNA (CT-DNA) has been investigated by electronic absorption, steady-state emission, and circular dichroism spectroscopies, as well as by viscosity measurements and equilibrium dialysis binding studies. Several points came from the results. (1) The DNA-binding properties were distinctly different for the [Ru(bpy)2L]2+ (L=HPIP, HNAIP, HNOIP) series of ruthenium(II) complexes, which indicates that the photophysical behavior of the complexes on binding to DNA can be modulated through ligand design. (2) Different binding rates of individual enantiomers of complexes 1 and 4 to DNA were observed through dialysis experiments. The lambda enantiomer bound more rapidly than the lambda enantiomer and their different intercalative binding geometries were suggested to be responsible. (3) Both delta-2 and lambda-2 bound weakly to CT-DNA; delta-2 may bind through a partial intercalation mode, whereas lambda-2 may bind in the DNA groove. (4) There was no noticeable enantioselectivity for complexes 1, 3, and 4 on binding to CT-DNA. Both of their enantiomers can intercalate into DNA base pairs. It is noted that delta-3 and lambda-3 exhibited almost identical spectral changes on addition of CT-DNA, and a similar binding manner of the isomers to the double helix was proposed.     

Probing site specificity of DNA binding metallointercalators by NMR spectroscopy and molecular modeling

The molecular recognition of oligonucleotides by chiral ruthenium complexes has been probed by NMR spectroscopy using the template Delta-cis-alpha- and Delta-cis-beta-[Ru(RR-picchxnMe(2)) (bidentate)](2+), where the bidentate ligand is one of phen (1,10-phenanthroline), dpq (dipyrido[3,2-f:2',3'-h]quinoxaline), or phi (9,10-phenanthrenequinone diimine) and picchxnMe(2)() is N,N'-dimethyl-N,N'-di(2-picolyl)-1,2-diaminocyclohexane. By varying only the bidentate ligand in a series of complexes, it was shown that the bidentate alone can alter binding modes. DNA binding studies of the Delta-cis-alpha-[Ru(RR-picchxnMe(2))(phen)](2+) complex indicate fast exchange kinetics on the chemical shift time scale and a "partial intercalation" mode of binding. This complex binds to [d(CGCGATCGCG)](2) and [d(ATATCGATAT)](2) at AT, TA, and GA sites from the minor groove, as well as to the ends of the oligonucleotide at low temperature. Studies of the Delta-cis-beta-[Ru(RR-picchxnMe(2))(phen)](2+) complex with [d(CGCGATCGCG)](2) showed that the complex binds only weakly to the ends of the oligonucleotide. The interaction of Delta-cis-alpha-[Ru(RR-picchxnMe(2))(dpq)](2+) with [d(CGCGATCGCG)](2) showed intermediate exchange kinetics and evidence of minor groove intercalation at the GA base step. In contrast to the phen and dpq complexes, Delta-cis-alpha- and Delta-cis-beta-[Ru(RR-picchxnMe(2))(phi)](2+) showed evidence of major groove binding independent of the metal ion configuration. DNA stabilization induced by complex binding to [d(CGCGATCGCG)](2) (measured as DeltaT(m)) increases in the order phen < dpq and DNA affinity in the order phen < dpq < phi. The groove binding preferences exhibited by the different bidentate ligands is explained with the aid of molecular modeling experiments.