Left versus right: Exploring the effects of chiral threading intercalators using optical tweezers.

Small-molecule DNA-binding drugs have shown promising results in clinical use against many types of cancer. Understanding the molecular mechanisms of DNA binding for such small molecules can be critical in advancing future drug designs. We have been exploring the interactions of ruthenium-based small molecules and their DNA-binding properties that are highly relevant in the development of novel metal-based drugs. Previously we have studied the effects of the right-handed binuclear ruthenium threading intercalator ΔΔ-[µ-bidppz(phen)4Ru2]4+, or ΔΔ-P for short, which showed extremely slow kinetics and high-affinity binding to DNA. Here we investigate the left-handed enantiomer ΛΛ-[µ-bidppz(phen)4Ru2]4+, or ΛΛ-P for short, to study the effects of chirality on DNA threading intercalation. We employ single-molecule optical trapping experiments to understand the molecular mechanisms and nanoscale structural changes that occur during DNA binding and unbinding as well as the association and dissociation rates. Despite the similar threading intercalation binding mode of the two enantiomers, our data show that the left-handed ΛΛ-P complex requires increased lengthening of the DNA to thread, and it extends the DNA more than double the length at equilibrium compared with the right-handed ΔΔ-P. We also observed that the left-handed ΛΛ-P complex unthreads three times faster than ΔΔ-P. These results, along with a weaker binding affinity estimated for ΛΛ-P, suggest a preference in DNA binding to the chiral enantiomer having the same right-handed chirality as the DNA molecule, regardless of their common intercalating moiety. This comparison provides a better understanding of how chirality affects binding to DNA and may contribute to the development of enhanced potential cancer treatment drug designs.

Hydrophobic catalysis and a potential biological role of DNA unstacking induced by environment effects.

Hydrophobic base stacking is a major contributor to DNA double-helix stability. We report the discovery of specific unstacking effects in certain semihydrophobic environments. Water-miscible ethylene glycol ethers are found to modify structure, dynamics, and reactivity of DNA by mechanisms possibly related to a biologically relevant hydrophobic catalysis. Spectroscopic data and optical tweezers experiments show that base-stacking energies are reduced while base-pair hydrogen bonds are strengthened. We propose that a modulated chemical potential of water can promote "longitudinal breathing" and the formation of unstacked holes while base unpairing is suppressed. Flow linear dichroism in 20% diglyme indicates a 20 to 30% decrease in persistence length of DNA, supported by an increased flexibility in single-molecule nanochannel experiments in poly(ethylene glycol). A limited (3 to 6%) hyperchromicity but unaffected circular dichroism is consistent with transient unstacking events while maintaining an overall average B-DNA conformation. Further information about unstacking dynamics is obtained from the binding kinetics of large thread-intercalating ruthenium complexes, indicating that the hydrophobic effect provides a 10 to 100 times increased DNA unstacking frequency and an "open hole" population on the order of 10-2 compared to 10-4 in normal aqueous solution. Spontaneous DNA strand exchange catalyzed by poly(ethylene glycol) makes us propose that hydrophobic residues in the L2 loop of recombination enzymes RecA and Rad51 may assist gene recombination via modulation of water activity near the DNA helix by hydrophobic interactions, in the manner described here. We speculate that such hydrophobic interactions may have catalytic roles also in other biological contexts, such as in polymerases.

DNA threading intercalation of enantiopure [Ru(phen)2bidppz]2+ induced by hydrophobic catalysis.

The enantiomers of a novel mononuclear ruthenium(ii) complex [Ru(phen)2bidppz]2+ with an elongated dppz moiety were synthesized. Surprisingly, the complex showed no DNA intercalating capability in an aqueous environment. However, by the addition of water-miscible polyethylene glycol ether PEG-400, self-aggregation of the hydrophobic ruthenium(ii) complexes was counter-acted, thus strongly promoting the DNA intercalation binding mode. This mild alteration of the environment surrounding the DNA polymer does not damage or alter the DNA structure but instead enables more efficient binding characterization studies of potential DNA binding drugs.

Diastereomeric Crowding Effects in the Competitive DNA Intercalation of Ru(phenanthroline)2dipyridophenazine2+ Enantiomers.

The biexponential excited-state emission decay characteristic of DNA intercalated tris-bidentate dppz-based ruthenium complexes of the general form Ru(L)2dppz2+ has previously been explained by a binding model with two distinct geometry orientations of the bound ligands, with a distinct lifetime associated with each orientation. However, it has been found that upon DNA binding of Ru(phen)2dppz2+ the fractions of short and long lifetimes are strongly dependent on environmental factors such as salt concentration and, in particular, temperature. Analyzing isothermal titration calorimetry for competitive binding of Ru(phen)2dppz2+ enantiomers to poly(dAdT)2, we find that a consistent binding model must assume that the short and long lifetimes states of intercalated complexes are in equilibrium and that this equilibrium is altered when neighboring bound ligands affect each other. The degree of intercomplex binding is found to be a subtle manifestation of several attractive and repulsive factors that are highly likely to directly reflect the strong diastereomeric difference in the binding enthalpy and entropy values. In addition, as the titration progresses and the binding sites on the DNA lattice become increasingly occupied, a general resistance for the saturation of the binding sites is observed, suggesting diastereomeric crowding of the neighboring bound ligands.

Reshaping the Energy Landscape Transforms the Mechanism and Binding Kinetics of DNA Threading Intercalation.

Molecules that bind DNA via threading intercalation show high binding affinity as well as slow dissociation kinetics, properties ideal for the development of anticancer drugs. To this end, it is critical to identify the specific molecular characteristics of threading intercalators that result in optimal DNA interactions. Using single-molecule techniques, we quantify the binding of a small metal-organic ruthenium threading intercalator (Δ,Δ-B) and compare its binding characteristics to a similar molecule with significantly larger threading moieties (Δ,Δ-P). The binding affinities of the two molecules are the same, while comparison of the binding kinetics reveals significantly faster kinetics for Δ,Δ-B. However, the kinetics is still much slower than that observed for conventional intercalators. Comparison of the two threading intercalators shows that the binding affinity is modulated independently by the intercalating section and the binding kinetics is modulated by the threading moiety. In order to thread DNA, Δ,Δ-P requires a "lock mechanism", in which a large length increase of the DNA duplex is required for both association and dissociation. In contrast, measurements of the force-dependent binding kinetics show that Δ,Δ-B requires a large DNA length increase for association but no length increase for dissociation from DNA. This contrasts strongly with conventional intercalators, for which almost no DNA length change is required for association but a large DNA length change must occur for dissociation. This result illustrates the fundamentally different mechanism of threading intercalation compared with conventional intercalation and will pave the way for the rational design of therapeutic drugs based on DNA threading intercalation.

Effects of methyl substitution on DNA binding enthalpies of enantiopure Ru(phenanthroline)2dipyridophenazine2+ complexes.

Isothermal titration calorimetry (ITC) has been utilized to investigate the effect of methyl substituents on the intercalating dppz ligand of the enantiomers of the parent complex Ru(phen)2dppz2+ (phen = 1,10-phenanthroline; dppz = dipyrido[3,2-a:2',3'-c]phenazine) on DNA binding thermodynamics. The methylated complexes (10-methyl-dppz and 11,12-dimethyl-dppz) have large, concentration-dependent, positive heats of dilution, and a strong endothermic background is also apparent in the ITC-profiles from titration of methylated complexes into poly(dAdT)2, which make direct comparison between complexes difficult. By augmenting a simple cooperative binding model with one equilibrium for complex self-aggregation in solution and one equilibrium for complex aggregation on saturated DNA, it was possible to find an excellent global fit to the experimental data with DNA affinity parameters restricted to be equal for all Δ-enantiomers as well as for all Λ-enantiomers. In general, enthalpic differences, compared to the unsubstituted complex, were small and less than 4 kJ mol-1, except for the heat of intercalation of Δ-10-methyl-dppz (-11,6 kJ mol-1) and Λ-11,12-dimethyl-dppz (+4.3 kJ mol-1).

Competitive DNA binding of Ru(bpy)2dppz2+ enantiomers studied with isothermal titration calorimetry (ITC) using a direct and general binding isotherm algorithm.

While isothermal titration calorimetry (ITC) is widely used and sometimes referred to as the "gold standard" for quantitative measurements of biomolecular interactions, its usage has so far been limited to the analysis of the binding to isolated, non-cooperative binding sites. Studies on more complicated systems, where the binding sites interact, causing either cooperativity or anti-cooperativity between neighboring bound ligands, are rare, probably due to the complexity of the methods currently available. Here we have developed a simple algorithm not limited by the complexity of a binding system, meaning that it can be implemented by anyone, from analyzing systems of simple, isolated binding sites to complicated interactive multiple-site systems. We demonstrate here that even complicated competitive binding calorimetric isotherms can be properly analyzed, provided that ligand-ligand interactions are taken into account. As a practical example, the competitive binding interactions between the two enantiomers of Ru(bpy)2dppz2+ (Ru-bpy) and poly(dAdT)2 (AT-DNA) are analyzed using our new algorithm, which provided an excellent global fit for the ITC experimental data.

Guanine Can Direct Binding Specificity of Ru-dipyridophenazine (dppz) Complexes to DNA through Steric Effects.

X-ray crystal structures of three Λ-[Ru(L)2 dppz]2+ complexes (dppz=dipyridophenazine; L=1,10-phenanthroline (phen), 2,2'-bipyridine (bpy)) bound to d((5BrC)GGC/GCCG) showed the compounds intercalated at a 5'-CG-3' step. The compounds bind through canted intercalation, with the binding angle determined by the guanine NH2 group, in contrast to symmetrical intercalation previously observed at 5'-TA-3' sites. This result suggests that canted intercalation is preferred at 5'-CG-3' sites even though the site itself is symmetrical, and we hypothesise that symmetrical intercalation in a 5'-CG-3' step could give rise to a longer luminescence lifetime than canted intercalation.

A ruthenium dimer complex with a flexible linker slowly threads between DNA bases in two distinct steps.

Several multi-component DNA intercalating small molecules have been designed around ruthenium-based intercalating monomers to optimize DNA binding properties for therapeutic use. Here we probe the DNA binding ligand [µ-C4(cpdppz)2(phen)4Ru2](4+), which consists of two Ru(phen)2dppz(2+) moieties joined by a flexible linker. To quantify ligand binding, double-stranded DNA is stretched with optical tweezers and exposed to ligand under constant applied force. In contrast to other bis-intercalators, we find that ligand association is described by a two-step process, which consists of fast bimolecular intercalation of the first dppz moiety followed by ∼10-fold slower intercalation of the second dppz moiety. The second step is rate-limited by the requirement for a DNA-ligand conformational change that allows the flexible linker to pass through the DNA duplex. Based on our measured force-dependent binding rates and ligand-induced DNA elongation measurements, we are able to map out the energy landscape and structural dynamics for both ligand binding steps. In addition, we find that at zero force the overall binding process involves fast association (∼10 s), slow dissociation (∼300 s), and very high affinity (Kd ∼10 nM). The methodology developed in this work will be useful for studying the mechanism of DNA binding by other multi-step intercalating ligands and proteins.

Dissecting the Dynamic Pathways of Stereoselective DNA Threading Intercalation.

DNA intercalators that have high affinity and slow kinetics are developed for potential DNA-targeted therapeutics. Although many natural intercalators contain multiple chiral subunits, only intercalators with a single chiral unit have been quantitatively probed. Dumbbell-shaped DNA threading intercalators represent the next order of structural complexity relative to simple intercalators, and can provide significant insights into the stereoselectivity of DNA-ligand intercalation. We investigated DNA threading intercalation by binuclear ruthenium complex [µ-dppzip(phen)4Ru2](4+) (Piz). Four Piz stereoisomers are defined by the chirality of the intercalating subunit (Ru(phen)2dppz) and the distal subunit (Ru(phen)2ip), respectively, each of which can be either right-handed (Δ) or left-handed (Λ). We used optical tweezers to measure single DNA molecule elongation due to threading intercalation, revealing force-dependent DNA intercalation rates and equilibrium dissociation constants. The force spectroscopy analysis provided the zero-force DNA binding affinity, the equilibrium DNA-ligand elongation Δxeq, and the dynamic DNA structural deformations during ligand association xon and dissociation xoff. We found that Piz stereoisomers exhibit over 20-fold differences in DNA binding affinity, from a Kd of 27 ± 3 nM for (Δ,Λ)-Piz to a Kd of 622 ± 55 nM for (Λ,Δ)-Piz. The striking affinity decrease is correlated with increasing Δxeq from 0.30 ± 0.02 to 0.48 ± 0.02 nm and xon from 0.25 ± 0.01 to 0.46 ± 0.02 nm, but limited xoff changes. Notably, the affinity and threading kinetics is 10-fold enhanced for right-handed intercalating subunits, and 2- to 5-fold enhanced for left-handed distal subunits. These findings demonstrate sterically dispersed transition pathways and robust DNA structural recognition of chiral intercalators, which are critical for optimizing DNA binding affinity and kinetics.

Diastereomeric bactericidal effect of Ru(phenanthroline)2 dipyridophenazine.

Metal susceptibility assays and spot plating were used to investigate the antimicrobial activity of enantiopure [Ru(phen)2 dppz]2+ (phen =1,10-phenanthroline and dppz = dipyrido[3,2-a:2´,3´-c]phenazine) and [µ-bidppz(phen)4 Ru2 ]4+ (bidppz =11,11´-bis(dipyrido[3,2-a:2´,3´-c]phenazinyl)), on Gram-negative Escherichia coli and Gram-positive Bacillus subtilis as bacterial models. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined for both complexes: while [µ-bidppz(phen)4 Ru2 ]4+ only showed a bactericidal effect at the highest concentrations tested, the antimicrobial activity of [Ru(phen)2 dppz]2+ against B. subtilis was comparable to that of tetracyline. In addition, the Δ-enantiomer of [Ru(phen)2 dppz]2+ showed a 2-fold higher bacteriostatic and bactericidal effect compared to the Λ-enantiomer. This was in accordance with the enantiomers relative binding affinity for DNA, thus strongly indicating DNA binding as the mode of action.

Strong DNA deformation required for extremely slow DNA threading intercalation by a binuclear ruthenium complex.

DNA intercalation by threading is expected to yield high affinity and slow dissociation, properties desirable for DNA-targeted therapeutics. To measure these properties, we utilize single molecule DNA stretching to quantify both the binding affinity and the force-dependent threading intercalation kinetics of the binuclear ruthenium complex Δ,Δ-[µ-bidppz-(phen)4Ru2]4+ (Δ,Δ-P). We measure the DNA elongation at a range of constant stretching forces using optical tweezers, allowing direct characterization of the intercalation kinetics as well as the amount intercalated at equilibrium. Higher forces exponentially facilitate the intercalative binding, leading to a profound decrease in the binding site size that results in one ligand intercalated at almost every DNA base stack. The zero force Δ,Δ-P intercalation Kd is 44 nM, 25-fold stronger than the analogous mono-nuclear ligand (Δ-P). The force-dependent kinetics analysis reveals a mechanism that requires DNA elongation of 0.33 nm for association, relaxation to an equilibrium elongation of 0.19 nm, and an additional elongation of 0.14 nm from the equilibrium state for dissociation. In cells, a molecule with binding properties similar to Δ,Δ-P may rapidly bind DNA destabilized by enzymes during replication or transcription, but upon enzyme dissociation it is predicted to remain intercalated for several hours, thereby interfering with essential biological processes.

Rate of hydrolysis in ATP synthase is fine-tuned by α-subunit motif controlling active site conformation.

Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.

Enhanced cellular uptake of antisecretory peptide AF-16 through proteoglycan binding.

Peptide AF-16, which includes the active site of Antisecretory Factor protein, has antisecretory and anti-inflammatory properties, making it a potent drug candidate for treatment of secretory and inflammatory diseases such as diarrhea, inflammatory bowel diseases, and intracranial hypertension. Despite remarkable physiological effects and great pharmaceutical need for drug discovery, very little is yet understood about AF-16 mechanism of action. In order to address interaction mechanisms, we investigated the binding of AF-16 to sulfated glycosaminoglycan, heparin, with focus on the effect of pH and ionic strength, and studied the influence of cell-surface proteoglycans on cellular uptake efficiency. Confocal laser scanning microscopy and flow cytometry experiments on wild type and proteoglycan-deficient Chinese hamster ovary cells reveal an endocytotic nature of AF-16 cellular uptake that is, however, less efficient for the cells lacking cell-surface proteoglycans. Isothermal titration calorimetry provides quantitative thermodynamic data and evidence for that the peptide affinity to heparin increases at lower pH and ionic strength. Experimental data, supported by theoretical modeling, of peptide-glycosaminoglycan interaction indicate that it has a large electrostatic contribution, which will be enhanced in diseases accompanied by decreased pH and ionic strength. These observations show that cell-surface proteoglycans are of general and crucial importance for the antisecretory and anti-inflammatory activities of AF-16.

DNA-binding properties of amidine-substituted spiropyran photoswitches.

Two amidine-substituted spiropyran derivatives have been characterized with respect to the DNA-binding properties over a broad pH interval. The two derivatives differ in the number of positive charges. By varying the pH, the protonation state of the derivatives is also changed, allowing for additional variations in the charge distribution. We show that the closed spiro isomer does not bind for either of the two derivatives, whereas the open merocyanine forms bind both in the protonated and in the nonprotonated state, but with dramatically different binding constants. Flow-oriented linear dichroism (LD) measurements also show that there are differences in the binding modes between the various forms. We rationalize these differences in terms of structure and charge distribution.

Double-lock ratchet mechanism revealing the role of alphaSER-344 in FoF1 ATP synthase.

In a majority of living organisms, FoF1 ATP synthase performs the fundamental process of ATP synthesis. Despite the simple net reaction formula, ADP+Pi→ATP+H2O, the detailed step-by-step mechanism of the reaction yet remains to be resolved owing to the complexity of this multisubunit enzyme. Based on quantum mechanical computations using recent high resolution X-ray structures, we propose that during ATP synthesis the enzyme first prepares the inorganic phosphate for the γP-OADP bond-forming step via a double-proton transfer. At this step, the highly conserved αS344 side chain plays a catalytic role. The reaction thereafter progresses through another transition state (TS) having a planar ion configuration to finally form ATP. These two TSs are concluded crucial for ATP synthesis. Using stepwise scans and several models of the nucleotide-bound active site, some of the most important conformational changes were traced toward direction of synthesis. Interestingly, as the active site geometry progresses toward the ATP-favoring tight binding site, at both of these TSs, a dramatic increase in barrier heights is observed for the reverse direction, i.e., hydrolysis of ATP. This change could indicate a "ratchet" mechanism for the enzyme to ensure efficacy of ATP synthesis by shifting residue conformation and thus locking access to the crucial TSs.

Thread insertion of a bis(dipyridophenazine) diruthenium complex into the DNA double helix by the extrusion of AT base pairs and cross-linking of DNA duplexes.

The crystal structure of the Δ,Δ enantiomer of the binuclear "light-switch" ruthenium complex [µ-(11,11'-bidppz)(1,10-phenanthroline)4 Ru2 ](4+) bound to the oligonucleotide d(CGTACG) shows that one dppz moiety of the dumbbell-like compound inserts into the DNA stack through the extrusion of an AT base pair. The second dppz moiety recruits a neighboring DNA molecule, and the complex thus cross-links two adjacent duplexes by bridging their major grooves.

Cell surface binding and uptake of arginine- and lysine-rich penetratin peptides in absence and presence of proteoglycans.

Cell surface proteoglycans (PGs) appear to promote uptake of arginine-rich cell-penetrating peptides (CPPs), but their exact functions are unclear. To address if there is specificity in the interactions of arginines and PGs leading to improved internalization, we used flow cytometry to examine uptake in relation to cell surface binding for penetratin and two arginine/lysine substituted variants (PenArg and PenLys) in wildtype CHO-K1 and PG-deficient A745 cells. All peptides were more efficiently internalized into CHO-K1 than into A745, but their cell surface binding was independent of cell type. Thus, PGs promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Uptake of each peptide was linearly dependent on its cell surface binding, and affinity is thus important for efficiency. However, the gradients of these linear dependencies varied significantly. Thus each peptide's ability to stimulate uptake once bound to the cell surface is reliant on formation of specific uptake-promoting interactions. Heparin affinity chromatography and clustering experiments showed that penetratin and PenArg binding to sulfated sugars is stabilized by hydrophobic interactions and result in clustering, whereas PenLys only interacts through electrostatic attraction. This may have implications for the molecular mechanisms behind arginine-specific uptake stimulation as penetratin and PenArg are more efficiently internalized than PenLys upon interaction with PGs. However, PenArg is also least affected by removal of PGs. This indicates that an increased arginine content not only improve PG-dependent uptake but also that PenArg is more adaptable as it can use several portals of entry into the cell.

Bridging ligand length controls at selectivity and enantioselectivity of binuclear ruthenium threading intercalators.

The slow dissociation of DNA threading intercalators makes them interesting as model compounds in the search for new DNA targeting drugs, as there appears to be a correlation between slow dissociation and biological activity. Thus, it would be of great value to understand the mechanisms controlling threading intercalation, and for this purpose we have investigated how the length of the bridging ligand of binuclear ruthenium threading intercalators affects their DNA binding properties. We have synthesised a new binuclear ruthenium threading intercalator with slower dissociation kinetics from ct-DNA than has ever been observed for any ruthenium complex with any type of DNA, a property that we attribute to the increased distance between the ruthenium centres of the new complex. By comparison with previously studied ruthenium complexes, we further conclude that elongation of the bridging ligand reduces the sensitivity of the threading interaction to DNA flexibility, resulting in a decreased AT selectivity for the new complex. We also find that the length of the bridging ligand affects the enantioselectivity with increasing preference for the ΔΔ enantiomer as the bridging ligand becomes longer.

Initial DNA interactions of the binuclear threading intercalator Λ,Λ-[µ-bidppz(bipy)4Ru2]4+: an NMR study with [d(CGCGAATTCGCG)]2.

Binuclear polypyridine ruthenium compounds have been shown to slowly intercalate into DNA, following a fast initial binding on the DNA surface. For these compounds, intercalation requires threading of a bulky substituent, containing one Ru(II), through the DNA base-pair stack, and the accompanying DNA duplex distortions are much more severe than with intercalation of mononuclear compounds. Structural understanding of the process of intercalation may greatly gain from a characterisation of the initial interactions between binuclear Ru(II) compounds and DNA. We report a structural NMR study on the binuclear Ru(II) intercalator Λ,Λ-B (Λ,Λ-[µ-bidppz(bipy)4Ru2](4+); bidppz = 11,11'-bis(dipyrido[3,2-a:2',3'-c]phenazinyl, bipy = 2,2'-bipyridine) mixed with the palindromic DNA [d(CGCGAATTCGCG)]2. Threading of Λ,Λ-B depends on the presence and length of AT stretches in the DNA. Therefore, the latter was selected to promote initial binding, but due to the short stretch of AT base pairs, final intercalation is prevented. Structural calculations provide a model for the interaction: Λ,Λ-B is trapped in a well-defined surface-bound state consisting of an eccentric minor-groove binding. Most of the interaction enthalpy originates from electrostatic and van der Waals contacts, whereas intermolecular hydrogen bonds may help to define a unique position of Λ,Λ-B. Molecular dynamics simulations show that this minor-groove binding mode is stable on a nanosecond scale. To the best of our knowledge, this is the first structural study by NMR spectroscopy on a binuclear Ru compound bound to DNA. In the calculated structure, one of the positively charged Ru(2+) moieties is near the central AATT region; this is favourable in view of potential intercalation as observed by optical methods for DNA with longer AT stretches. Circular dichroism (CD) spectroscopy suggests that a similar binding geometry is formed in mixtures of Λ,Λ-B with natural calf thymus DNA. The present minor-groove binding mode is proposed to represent the initial surface interactions of binuclear Ru(II) compounds prior to intercalation into AT-rich DNA.