Targeting abasic sites and single base bulges in DNA with metalloinsertors

The site-specific recognition of abasic sites and single base bulges in duplex DNA by sterically expansive rhodium metalloinsertors has been investigated. Through DNA photocleavage experiments, Rh(bpy)2(chrysi)3+ is shown to bind both abasic sites and single base bulges site-specifically and, upon irradiation, to cleave the backbone of the defect-containing DNA. Photocleavage titrations reveal that the metal complex binds DNA containing an abasic site with high affinity (2.6(5) x 106 M-1), comparably to the metalloinsertor and a CC mismatch. The complex binds single base bulge sites with lower affinity (approximately 105 M-1). Analysis of cleavage products and the correlation of affinities with helix destabilization suggest that Rh(bpy)2(chrysi)3+ binds both lesions via metalloinsertion, as observed for Rh binding at mismatched sites, a binding mode in which the mismatched or unpaired bases are extruded from the helix and replaced in the base stack by the sterically expansive ligand of the metalloinsertor.     

Recognition of DNA base pair mismatches by a cyclometalated Rh(III) intercalator

Two cyclometalated complexes of Rh(III), rac-[Rh(ppy)2chrysi]+ and rac-[Rh(ppy)2 phi]+, have been synthesized and characterized with respect to their binding to DNA. The structure of rac-[Rh(ppy)2 phi]Cl.H2O.CH2Cl2 has been determined by X-ray diffraction (monoclinic, P2(1)/c, Z = 4, a = 18.447(3) A, b = 9.770(1) A, c = 17.661(3) A, beta = 94.821(11) degrees, V = 3172.0(8) A3) and reveals that the complex is a distorted octahedron with nearly planar ligands, similar in structure to the DNA mismatch recognition agent [Rh(bpy)2chrysi]3+. The 2-phenylpyridyl nitrogen atoms are shown to be in the axial positions, as a result of trans-directing effects. This tendency simplifies the synthesis and purification of such complexes by limiting the number of possible isomers generated. The abilities of [Rh(ppy)2chrysi]+ and [Rh(ppy)2 phi]+ to bind and, with photoactivation, to cleave DNA have been demonstrated in assays on duplex DNA in the absence and presence of a single CC mismatch. [Rh(ppy)2chrysi]+ was shown upon photoactivation to cleave DNA selectively at the base pair mismatch whereas [Rh(ppy)2 phi]+ cleaves B-DNA nonspecifically. The reactivity of [Rh(ppy)2chrysi]+ was also compared to that of the known mismatch recognition agent [Rh(bpy)2chrysi]3+. Competitive photocleavage studies revealed that a 14-fold excess of [Rh(ppy)2chrysi]+ was required to achieve the same level of binding as that of [Rh(bpy)2chrysi]3+. However, the ratio of damage induced by [Rh(bpy)2chrysi]3+ to that induced by [Rh(ppy)2chrysi]+ is considerably greater than this value, indicating that decreased photoefficiency for the cyclometalated complex must contribute to its significantly attenuated photoreactivity. These cyclometalated intercalators provide the starting points for the design of a new family of metal complexes targeted to DNA.     

DNA strand cleavage near a CC mismatch directed by a metalloinsertor

Reagents for recognition and efficient cleavage of mismatched DNA without photoactivation were designed. They contain a combination of a mismatch-directing metalloinsertor, [Rh(bpy)2(chrysi)]3+ (bpy=2,2'-bipyridyl, chrysi=5,6-chrysenequinone diimine), and an oxidative cleavage functionality, [Cu(phen)2]+ (Cu). Both unconjugated (Rh+Cu) and conjugated (Rh-Cu) frameworks of the Rh insertor and Cu were prepared. Compared to Cu, both constructs Rh+Cu and Rh-Cu exhibit efficient site-specific DNA scission only with mismatched DNA, confirmed by experiments with 32P-labeled oligonucleotides. Furthermore, these studies indicate that DNA cleavage occurs near the mismatch in the minor groove and on both strands. Interestingly, the order of reactivity of the three systems with a CC mismatch is Rh+Cu>Rh-Cu>Cu. Rh binding appears to direct Cu reactivity with or without tethering. These results illustrate advantages and disadvantages in bifunctional conjugation.     

Binding of Ru(bpy)2(eilatin)2+ to matched and mismatched DNA

The DNA-binding properties of Ru(bpy)2(eilatin)(2+) have been investigated to determine if the sterically expansive eilatin ligand confers specificity for destabilized single-base mismatches in DNA. Competitive DNA photocleavage experiments employing a sequence-neutral metallointercalator, Rh(bpy)2(phi)(3+) (phi = 9,10-phenanthrenequinonediimine), and a mismatch-specific metalloinsertor, Rh(bpy)2(chrysi)(3+) (chrysi = chrysene-5,6-quinonediimine), reveal that the eilatin complex binds to a CC mismatched site with an apparent binding constant of 2.2(2) x 10(6) M(-1). Nonetheless, the selectivity in binding mismatched DNA is not high: competitive titrations with Rh(bpy)2(phi)(3+) show that the complex binds also to well-matched B-form sites. Thus, Ru(bpy)2(eilatin)(2+), despite containing the extremely expansive eilatin ligand, displays lower selectivity for the mismatch than does Rh(bpy)2(chrysi)(3+), a metalloinsertor containing the smaller, though still bulky, chrysene-5,6-quinonediimine ligand. In summary, the size and shape of the eilatin ligand allow stacking with both well-matched and mismatched DNA.     

Λ-、△-[Rh（bpy）2chrysi]^3＋对含C：T错配的DNA序列识别作用的分子模拟研究

通过分子模拟方法研究了手性金属配合物[Rh（bpy）2Chrysi]^3＋（bpy=2，2’-bipyridineChrysi=5，6-chrysenequinonediimine）对包含C：T错配碱基对的B-DNA序列的识别作用．结合类似的针对含G：A错配的和正常的B-DNA序列的识别作用研究，发现配合物I-Rh（bpy）zChrysi]^3＋可以对错配B-DNA序列进行序列特异性的识别．能量对比计算结果表明，该经典插入识别作用倾向于在错配碱基对附近进行，其中△-[Rh（bpy）2chrysi]^3＋比其手性异构体更占优势．这同Barton教授工作组的实验结果是一致的．另外插入作用倾向于在错配序列中的正常双碱基对C3A4／G3T4（错配碱基对附近）中从小沟进行．与该配合物对含G：A错配的和正常的B-DNA序列的识别作用不同的是，对包含C：T错配碱基对的B-DNA序列的识别作用倾向于从小沟进行．这一点可能源于C：T碱基对结构的不同．     

Λ-、Δ-[Rh(bpy)2chrysi]3+对含C:T错配的DNA序列识别作用的分子模拟研究

通过分子模拟方法研究了手性金属配合物[Rh(bpy)2Chrysi]3 (bpy=2,2’- bipyridine;Chrysi=5,6-chrysenequinonediimine)对包含C:T错配碱基对的B-DNA序列的识别作用。结合类似的针对含G:A错配的和正常的B-DNA序列的识别作用研究,发现配合物[Rh(bpy)2Chrysi]3 可以对错配B-DNA序列进行序列特异性的识别．能量对比计算结果表明,该经典插入识别作用倾向于在错配碱基对附近进行,其中Δ-[Rh(bpy)2charysi]3 比其手性异构体更占优势．这同Barton教授工作组的实验结果是一致的。另外插入作用倾向于在错配序列中的正常双碱基对C3A4／G374(错配碱基对附近)中从小沟进行．与该配合物对含G:A错配的和正常的B-DNA序列的识别作用不同的是,对包含C:T错配碱基对的B-DNA序列的识别作用倾向于从小沟进行．这一点可能源于C:T碱基对结构的不同．     

Detecting DNA mismatches with metallo-insertors: a molecular simulation study

Molecules that selectively recognize DNA mismatches (MMs) play a key role as nucleic acids probes and as chemotherapeutic agents. Metallo-insertors bind to the minor groove (mG) of double strand (ds) DNA, expelling the mismatched base pairs and acting as their π-stacking replacement. In contrast, metallo-intercalators bind to the major groove (MG) of ds DNA and π-stack to adjacent base pairs. In this study we focused on structural and energetic properties of Δ-[Rh(bpy)(2)(chrysi)](3+) (1), Δ-[Ru(bpy)(2)(ddpz)](2+) (2), and Δ-[Ru(bpy)(2)(eilatin)](2+) (3) as prototypical examples of metallo-insertors and intercalators. For all molecules we characterized both insertion and intercalation into a DNA dodecamer via force field based molecular dynamics (MD) and hybrid quantum-classical (QM/MM) MD simulations. A structural analysis of the 1-3/DNA noncovalent adducts reveals that the insertion provokes an untwist of the DNA, an opening of the mG and of the phosphate backbone in proximity of the mismatch, while the intercalation induces smaller changes of these structural parameters. This behavior appears to be correlated with the size of the inserting/intercalating ligand in proximity of the metal coordination site. Moreover, our simulations show that the different selectivity of 1 toward distinct MM types may be correlated with the thermodynamic stability of the MMs in the free DNA and with that of the corresponding insertion adduct. Understanding the factors which tune a specific insertion is of crucial importance for designing specific luminescent probes that selectively recognize MMs, as well as for developing more effective anticancer drugs active in MM repair of deficient cells lines.     

Dinuclear ruthenium(II) complexes as potential probes for RNA bulge sites

1H NMR spectroscopy and molecular modelling have been used to investigate the binding of the DeltaDelta-and LambdaLambda-enantiomers of the dinuclear ruthenium(II) complex [[Ru(Me2bpy)2]2(mu-bpm)]4+ [Me2bpy = 4,4'-dimethyl-2,2'-bipyridine; bpm = 2,2'-bipyrimidine] to an RNA tridecanucleotide duplex containing a single-base bulge [r(CCGAGAAUUCCGG)2]], and the corresponding control dodecanucleotide [r(CCGGAAUUCCGG)2]. Both enantiomers bound the control RNA sequence weakly. From upfield shifts of the metal complex H3 and H3' protons throughout the titration of the control dodecanucleotide with DeltaDelta-[[Ru(Me2bpy)2]2(mu-bpm)]4+, a binding constant of 1 x 10(3) M(-1) was determined. In NOESY spectra of the control sequence with added DeltaDelta-[[Ru(Me2bpy)2]2(mu-bpm)]4+, NOEs were only observed to protons from the terminal base-pair residues. No significant changes in chemical shift were observed for either the metal complex or RNA protons upon addition of the LambdaLambda-enantiomer to the control dodecanucleotide. The DeltaDelta-[[Ru(Me2bpy)2]2(mu-bpm)]4+ complex bound the bulge-containing RNA with a significantly greater affinity (6 x 10(4) M(-1)) than the non-bulge control RNA duplex. Competition binding experiments indicated that the LambdaLambda-isomer bound the tridecanucleotide with similar affinity to the DeltaDelta-enantiomer. Addition of DeltaDelta-[[Ru(Me2bpy)2]2(mu-bpm)]4+ to the bulge-containing tridecanucleotide induced selective changes in chemical shift for the base H8 and sugar H1' resonances from the adenine bulge residue, and resonances from nucleotide residues adjacent to the bulge site. Intermolecular NOEs observed in NOESY spectra of the tridecanucleotide with added DeltaDelta-[[Ru(Me2bpy)2]2(mu-bpm)]4+ confirmed the selective binding of the ruthenium complex at the bulge site. Preliminary binding models, consistent with the NMR data, showed that the ruthenium complex could effectively associate in the RNA minor groove at the bulge site.     

Inert benzothiazole functionalised ruthenium(II) complexes; potential DNA hairpin binding agents

The two enantiomers of [Ru(bpy)2(bbtb)]2+{bpy = 2,2'-bipyridine; bbtb = 4,4'-bis(benzothiazol-2-yl)-2,2'-bipyridine} have been isolated and fully characterised. Both enantiomers have been shown to have a strong association with calf thymus DNA by UV/visible absorption, emission and CD spectroscopy, with the Lambda enantiomer having the greater affinity. The binding of both enantiomeric forms of [Ru(bpy)2(Me2bpy)]2+ and [Ru(bpy)2(bbtb)]2+{Me(2)bpy = 4,4'-dimethyl-2,2'-bipyridine} to a range of oligonucleotides, including an octadecanucleotide and an icosanucleotide which contain hairpin-sequences, have been studied using a fluorescent intercalator displacement (FID) assay. The complex [Ru(bpy)2(bbtb)]2+ exhibited an interesting association with hairpin oligonucleotides, again with the Lambda enantiomer binding more strongly. A (1)H NMR spectroscopic study of the binding of both enantiomers of [Ru(bpy)2(bbtb)]2+ to the icosanucleotide d(CACTGGTCTCTCTACCAGTG) was conducted. This sequence contains a seven-base-pair duplex stem and a six-base hairpin-loop. The investigation gave an indication of the relative binding of the complexes between the two different regions (duplex and secondary structure) of the oligonucleotide. The results suggest that both enantiomers bind at the hairpin, with the ruthenium centre located at the stem-loop interface. NOE studies indicate that one of the two benzothiazole substituents of the bbtb ligand projects into the loop-region. A simple model of the metal complex/oligonucleotide adduct was obtained by means of molecular modelling simulations. The results from this study suggest that benzothiazole complexes derived from inert polypyridine ruthenium(II) complexes could lead to the development of new fluorescent DNA hairpin binding agents.     

Molecular dynamics study on the interaction of a mithramycin dimer with a decanucleotide duplex

The complex of a minor groove binding drug mithramycin (MTR) and the self-complementary d(TAGCTAGCTA) 10-mer duplex was investigated by molecular dynamics (MD) simulations using the AMBER 7.0 suite of programs. There is one disaccharide and trisaccharide segment projecting from opposite ends of an aglycone chromophore of MTR. A MTR dimer complex (MTR)2Mg2+ is formed in the presence of a coordinated ion Mg2+. A NMR solution structure of two (MTR)2Mg2+ complexes bound with one DNA duplex, namely, the 2:1 duplex complex, was taken as the starting structure for the MD simulation. The partial charge on each atom was calculated using the multiple-RESP fitting procedure, and all of the missing parameters in the Parm99 force field used were adapted comparably from the literature. The length of the MD simulation was 5 ns, and the binding free energy for the formation of a 1:1 or 2:1 duplex complex was determined from the last 4 ns of the simulation. The binding free energies were decomposed to components of the contributions from different energy types, and the changes in the helical parameters of the bound DNA duplex plus the glycosidic linkages between sugar residues of the bound MTR dimer were determined. It was found that binding of the first (MTR)2Mg2+ complex with the DNA duplex to form a 1:1 duplex complex does not cause stiffening of the duplex especially in the unoccupied site of the duplex. However, the overall flexibility of the DNA duplex is reduced substantially once the second (MTR)2Mg2+ complex is bound with the unoccupied site to form the 2:1 duplex complex. The van der Waals interactions were found to be dominant in the central part of the DNA duplex where sugar residues from each bound (MTR)2Mg2+ complex were inwardly pointing and the corresponding minor groove was widened.     

Ru(bpy)2(L)]Cl2: luminescent metal complexes that bind DNA base mismatches

Here we report the synthesis of luminescent ruthenium complexes that bind DNA base pair mismatches. [Ru(bpy)2(tpqp)]Cl2 (tpqp = 7,8,13,14-tetrahydro-6-phenylquino[8,7-k][1,8]phenanthroline), [Ru(bpy)2(pqp)]Cl2 (pqp = 6-phenylquino[8,7-k][1,8]phenanthroline), and [Ru(bpy)2(tactp)]Cl2 [tactp = 4,5,9,18-tetraazachryseno[9,10-b]triphenylene] have been synthesized, and their spectroscopic properties in the absence and presence of DNA have been examined. While [Ru(bpy)2(pqp)]2+ shows no detectable luminescence, [Ru(bpy)2(tpqp)]2+ is luminescent in the absence and presence of DNA with an excited-state lifetime of 10 ns and a quantum yield of 0.002. Although no increase in emission intensity is associated with binding to mismatch-containing DNA, luminescence quenching experiments and measurements of steady-state fluorescence polarization provide evidence for preferential binding to oligonucleotides containing a CC mismatch. Furthermore, by marking the site of binding through singlet oxygen sensitized damage, the complex has been shown to target a CC mismatch site directly with a specific binding affinity, Kb = 4 x 10(6) M(-1). [Ru(bpy)2(tactp)]2+, an analogue of [Ru(bpy)2(dppz)]2+ containing a bulky intercalating ligand, is luminescent in aqueous solution at micromolar concentrations and exhibits a 12-fold enhancement in luminescence in the presence of DNA. The complex, however, tends to aggregate in aqueous solution; we find a dimerization constant of 9.8 x 10(5) M(-1). Again, by singlet oxygen sensitization it is apparent that [Ru(bpy)2(tactp)]2+ binds preferentially to a CC mismatch; using a DNase I footprinting assay, a binding constant to a CC mismatch of 8 x 10(5) M(-1) is found. Hence results with these novel luminescent complexes support the concept of using a structurally demanding ligand to obtain selectivity in targeting single base mismatches in DNA. The challenge is coupling the differential binding we can obtain to differential luminescence.     

meso-[{Ru(phen)2}2(mu-HAT)]4+: a high-affinity DNA hairpin probe {HAT = 1,4,5,8,9,12-hexaazatriphenylene; phen = 1,10-phenanthroline}

1H NMR spectroscopy and fluorescent intercalator displacement (FID) assays have been used to investigate the DNA-binding abilities of two series of dinuclear polypyridyl ruthenium(II) complexes of the form [{Ru(L)2}2(mu-BL)]4+ {L = 2,2'-bipyridine (bpy), 4,4'-dimethyl-2,2'-bipyridine (Me2bpy), 1,10-phenanthroline (phen), or 4,7-dimethyl-1,10-phenanthroline (Me2phen); BL = 2,2'-bipyrimidine (bpm) or 1,4,5,8,9,12-hexaazatriphenylene (HAT)}. Preliminary FID surveys of these metal complexes against a variety of different oligonucleotides revealed that those complexes based upon the HAT bridging ligand induced greater fluorescence decreases in dye-bound DNA than did their bpm-bridged counterparts, suggesting a higher binding affinity by the HAT-bridged species. Furthermore, the greatest fluorescence decreases were typically observed in an oligonucleotide featuring a six-base hairpin loop. The apparent binding affinity of the metal complexes was also found to be a function of the stereochemistry and identity of the terminal ligands of the complex. The meso (DeltaLambda) stereoisomer generally induced greater fluorescence decreases than did either enantiomer (DeltaDelta or LambdaLambda), phen-based terminal ligands performed better than bpy-based terminal ligands, and those terminal ligands with methyl substituents demonstrated stronger apparent binding than did their non-methylated analogues. NMR experiments on meso-[{Ru(phen)2}2(mu-HAT)]4+ and meso-[{Ru(Me2phen)2}2(mu-HAT)]4+ demonstrated that both complexes bound with high affinity to the six-base hairpin oligonucleotide at the stem-loop interface and provided evidence to support stronger binding by the methylated species. meso-[{Ru(phen)2}2(mu-HAT)]4+ was found to bind poorly to duplex DNA and smaller four-base hairpin loops in FID and NMR experiments, whereas FID data suggest that the methylated analogue binds relatively strongly to most oligonucleotide sequences (the four- and six-base hairpins in particular). These results demonstrate that binding affinity can come at the expense of selectivity, with meso-[{Ru(phen)2}2(mu-HAT)]4+ proving to be an efficient compromise between the two as a high-affinity DNA hairpin probe.     

Carbohydrate recognition at the minor-groove of the self-complementary duplex d(CGCGAATTCGCG)2 by a synthetic glyco-oligoamide

The structure of a neutral glyco-conjugate β-Gal-Py-γ-Py-Ind (1), designed as a probe for analyzing sugar-DNA interactions, when bound to a self-complementary oligonucleotide duplex d(CGCG AATT CGCG)(2) has been deduced by employing (1)H NMR techniques. Analysis of the formed 1:1 complex demonstrated that the glycol ligand is bound in a hairpin-like conformation in which both pyrrole amino acid moieties are stacked, whereas the indole and the sugar residues are spatially close. The binding site is defined by the minor groove formed by the -AATT- stretch. In particular, the -Py-γ-Py- region of the ligand is sited near the A5-A6 oligonucleotide residues, whereas the indole and the sugar rings are next to the T7-T8 base pairs. More relevant, the existence of a variety of intermolecular NOE correlations permitted the close proximity of the sugar to the minor groove to be assessed, thus showing that the binding of the glycoconjugate at the minor groove is the origin of the specificity of the glycoconjugate-DNA interaction. The experimental NMR data have been combined with restrained and unrestrained molecular dynamics calculations, to provide the 3D structure of the complex.     

Molecular modeling on recognition of sheared and normal DNA by novel metal complex Λ- and Δ-[Co(phen)2hpip]

Recognition of sheared and normal DNA by a novel metal complex [Co(phen)₂hpip]³⁺ (phen=1,10-phenanthroline, HPIP=2-(2-hydroxyphenyl)imidazole[4,5-f][1,10]phenanethroline) is studied by molecular modeling. Calculating results indicate that, this complex can specifically recognize DNA segment of sequence –MMNNMM– (M means mismatch base pairs and N means normal base pairs). Intercalating from minor groove between the middle normal duplex into the sheared DNA with the depth of 1.2 nm is of preference and enantioselectivity is observed. Comparison on the two DNA structures of optimal conformation and analysis on the interaction between DNA and the two tail ligands of the complex show that, the effect of the two neighboring mismatch duplexes on the structure of the middle normal base pairs and the steric interaction between the mismatch duplexes and the two tail ligands of the complex are the essential reason to the segment specificity. Investigation on the detailed energy terms indicate that, in effecting enantioselectivity, the electrostatic distribution of the complex is in the majority and steric interaction is at the next place. But, steric interaction is surely the only factor determining the intercalating from minor groove.     

Solution structure and stability of a disulfide cross-linked nucleopeptide duplex

NMR methods are used to study the structure and stability of the duplex formed by the nucleopeptide [Ac-Cys-Gly-Ala-Hse(p3'dGCATGC)-Ala-OH]2[S-S], in which the oligonucleotide is self-complementary and the cysteine residues of the two peptide chains form a disulfide bridge; thermal transitions and NMR-derived structural calculations are consistent with a 3-D structure in which the oligonucleotide forms a standard B-DNA helix without significant distortions; the peptide chains are relatively disordered in solution and lie in the minor groove of the DNA helix; this nucleopeptide duplex exhibits a high melting temperature, indicating that peptide-oligonucleotide conjugates containing cysteines are suitable molecules to establish cross-links between DNA strands and stabilize the duplex.     

新型双核配合物的形成及荧光性质研究

利用光谱学方法研究了[Ru(bpy)2TPPHZ]2+(TPPHZ=四吡啶[3,2-a: 2',3'-c: 3",2"-h: 2'",3'"-j]吩嗪)和[Ru(bpy)2ODHIP]2+(ODHIP=3,4-二羟基-咪唑并[4,5-f][1,10]邻菲咯啉)与Ni2+的配位情况及配位后的荧光性质变化, 探讨了配合物与Ni2+配位形成双核配合物后与DNA的作用机制变化. 结果表明, [Ru(bpy)2TPPHZ]2+和[Ru(bpy)2ODHIP]2+均可与Ni2+配位, 形成双核配合物[Ru(bpy)2(TPPHZ)Ni]4+和[Ru(bpy)2(ODHIP)Ni]4+, 配合物的荧光强度随着Ni2+浓度的增加而减弱. 与DNA作用后, 配合物仍可与Ni2+配位形成双核配合物, [Ru(bpy)2(TPPHZ)Ni]4+的荧光几乎完全消失, 同时配合物与DNA保持插入模式作用, 而配合物[Ru(bpy)2(ODHIP)Ni]4+与DNA的作用则由沟面结合改为插入结合, 同时配合物的荧光减弱.     

Exploring the interaction of ruthenium(II) polypyridyl complexes with DNA using single-molecule techniques

Here we explore DNA binding by a family of ruthenium(II) polypyridyl complexes using an atomic force microscope (AFM) and optical tweezers. We demonstrate using AFM that Ru(bpy)2dppz2+ intercalates into DNA (K(b) = 1.5 x 10(5) M(-1)), as does its close relative Ru(bpy)2dppx2+ (K(b) = 1.5 x 10(5) M(-1)). However, intercalation by Ru(phen)3(2+) and other Ru(II) complexes with K(b) values lower than that of Ru(bpy)2dppz2+ is difficult to determine using AFM because of competing aggregation and surface-binding phenomena. At the high Ru(II) concentrations required to evaluate intercalation, most of the DNA strands acquire a twisted, curled conformation that is impossible to measure accurately. The condensation of DNA on mica in the presence of polycations is well known, but it clearly precludes the accurate assessment by AFM of DNA intercalation by most Ru(II) complexes, though not by ethidium bromide and other monovalent intercalators. When stretching individual DNA molecules using optical tweezers, the same limitation on high metal concentration does not exist. Using optical tweezers, we show that Ru(phen)2dppz2+ intercalates avidly (K(b) = 3.2 x 10(6) M(-1)) whereas Ru(bpy)3(2+) does not intercalate, even at micromolar ruthenium concentrations. Ru(phen)3(2+) is shown to intercalate weakly (i.e., at micromolar concentrations (K(b) = 8.8 x 10(3) M(-1))). The distinct differences in DNA stretching behavior between Ru(phen)3(2+) and Ru(bpy)3(2+) clearly illustrate that intercalation can be distinguished from groove binding by pulling the DNA with optical tweezers. Our results demonstrate both the benefits and challenges of two single-molecule methods of exploring DNA binding and help to elucidate the mode of binding of Ru(phen)3(2+).     

Meta-linked poly(phenylene ethynylene) conjugated polyelectrolyte featuring a chiral side group: helical folding and guest binding

A water soluble, meta-linked poly(phenylene ethynylene) featuring chiral and optically active side groups based on L-alanine (mPPE-Ala) has been studied by using absorption, fluorescence, and circular dichroism spectroscopy. Studies of mPPE-Ala in methanol/water solvent mixtures show that the polymer folds into a helical conformation, and the extent of helical folding increases with the volume % water in the solvent. The presence of the helical conformation is signaled by the appearance of a broad, excimer-like visible fluorescence band, combined with a strong bisignate circular dichroism signal in the region of the pi,pi absorption of the polymer backbone. The circular dichroism signal exhibits negative chirality, suggesting that the left-handed (M-form) of the helix is in enantiomeric excess. Binding of the metallointercalator [Ru(bpy)2(dppz)]2+ (where bpy = 2,2-bipyridine and dppz = dipyrido[3,2-a:2',3'-c]phenazine) with the helical polymer is accompanied by the appearance of the orange-red photoluminescence from the metal complex. This effect is directly analogous to that observed when [Ru(bpy)2(dppz)]2+ binds to DNA via intercalation, suggesting that the metal complex binds to mPPE-Ala by intercalating between the pi-stacked phenylene ethynylene residues. Cationic cyanine dyes also bind to the periphery of the helical polymer in a manner that is interpreted as "groove binding". A circular dichroism signal is observed that is believed to arise from exciton coupling within the chiral cyanine dye chromophore aggregate that is formed as the dye molecules are oriented by the helical mPPE-Ala "template".     

Carbohydrate-based DNA ligands: sugar-oligoamides as a tool to study carbohydrate-nucleic acid interactions

Sugar-oligoamides have been designed and synthesized as structurally simple carbohydrate-based ligands to study carbohydrate-DNA interactions. The general design of the ligands 1-3 has been done as to favor the bound conformation of Distamycin-type gamma-linked covalent dimers which is a hairpin conformation. Indeed, NMR analysis of the sugar-oligoamides in the free state has indicated the presence of a percentage of a hairpin conformation in aqueous solution. The DNA binding activity of compounds 1-3 was confirmed by calf thymus DNA (ct-DNA) NMR titration. Interestingly, the binding of the different sugar-oligoamides seems to be modulated by the sugar configuration. Semiquantitative structural information about the DNA ligand complexes has been derived from NMR data. A competition experiment with Netropsin suggested that the sugar-oligoamide 3 bind to DNA in the minor groove. The NMR titrations of 1-3 with poly(dA-dT) and poly(dG-dC) suggested preferential binding to the ATAT sequence. TR-NOE NMR experiments for the sugar-oligoamide 3-ct-DNA complex both in D(2)O and H(2)O have confirmed the complex formation and given information on the conformation of the ligand in the bound state. The data confirmed that the sugar-oligoamide ligand is a hairpin in the bound state. Even more relevant to our goal, structural information on the conformation around the N-glycosidic linkage has been accessed. Thus, the sugar asymmetric centers pointing to the NH-amide and N-methyl rims of the molecule have been characterized.