Interaction of porphyrin with G-quadruplex structures

Isothermal titration calorimetry (ITC) is a sensitive technique for probing bimolecular processes and can provide direct information about the binding affinity and stoichiometry and the key thermodynamic parameters involved. ITC has been used to investigate the interaction of the ligand H2TMPyP to the two DNA quadruplexes, [d(AGGGT)]4 and [d(TGGGGT)]. Analysis of the ITC data reveals that porphyrin/quadruplex binding stoichiometry under saturating conditions is 1:2 for [d(AGGGT)]4 and 2:1 for [d(TGGGGT)], respectively.     

Thermodynamic analysis of quadruplex DNA-drug interaction

This work studies the binding properties of distamycin and its carbamoyl analog, containing four pyrrole units, with the [d(TGGGGT)](4) quadruplex by means of isothermal titration calorimetry (ITC). Analysis of the ITC data reveals that drug/quadruplex binding stoichiometry is 1:1 for both interactions and that distamycin analog gives approximately a 10-fold increase in the quadruplex affinity.     

A more detailed picture of the interactions between virtual screening-derived hits and the DNA G-quadruplex: NMR, molecular modelling and ITC studies

The growing amount of literature about G-quadruplex DNA clearly demonstrates that such a structure is no longer viewed as just a biophysical strangeness but it is instead being considered as an important target for the treatment of various human disorders such as cancers or venous thrombosis. In this scenario, with the aim of finding brand new molecular scaffolds able to interact with the groove of the DNA quadruplex [d(TGGGGT)]₄, we recently performed a successful structure-based virtual screening (VS) campaign. As a result, six molecules were found to be somehow groove binders. Herein, we report the results of novel NMR titration experiments of these VS-derived ligands with modified quadruplexes, namely [d(TGG^BrGGT)]₄ and [d(TGGGG^BrT)]₄. The novel NMR spectroscopy experiments combined with molecular modelling studies, allow for a more detailed picture of the interaction between each binder and the quadruplex DNA. Noteworthy, isothermal titration calorimetry (ITC) measurements on the above-mentioned compounds revealed that 2, 4, and 6 besides their relatively small dimensions bind the DNA quadruplex [d(TGGGGT)]₄ with higher affinity than distamycin A, to the best of our knowledge, the most potent groove binder identified thus far.     

Targeting DNA quadruplexes with distamycin A and its derivatives: an ITC and NMR study

The use of small molecules that bind and stabilize G-quadruplex structures is emerging as a promising way to inhibit telomerase activity in tumor cells. In this paper, isothermal titration calorimetry (ITC) and (1)H NMR studies have been conducted to examine the binding of distamycin A and its two carbamoyl derivatives (compounds 1 and 2) to the target [d(TGGGGT)](4) and d[AG(3)(T(2)AG(3))(3)] quadruplexes from the Tetrahymena and human telomeres, respectively. The interactions were examined using two different buffered solutions containing either K(+) or Na(+) at a fixed ionic strength, to evaluate any influence of the ions present in solution on the binding behaviour. Experiments reveal that distamycin A and compound 1 bind the investigated quadruplexes in both solution conditions; conversely, compound 2 appears to have a poor affinity in any case. Moreover, these studies indicate that the presence of different cations in solution affects the stoichiometry and thermodynamics of the interactions.     

INTERACTION OF DISTAMYCIN A AND NETROPSIN WITH QUADRUPLEX AND DUPLEX STRUCTURES: A COMPARATIVE 1H-NMR STUDY

ABSTRACT

Homonuclear NMR techniques have been used to investigate the interactions of the minor groove binding agents distamycin A (Dist-A) and the related drug netropsin (Net) with three quadruplexes characterized by different groove widths: [d(TGGGGT)]₄ (Q1), [d(GGGGTTTTGGGG)]₂ (Q2), and d(GGGGTTGGGGTGTGGGGTTGGGG) (Q3). Netropsin has been found to be in a fast chemical exchange with all three kinds of quadruplexes, whereas Dist-A interacts tightly with Q1 and, at a less extent, with Q2. In order to determine the degree of selectivity of Dist-A for two- rather than four-stranded DNA, we titrated with Dist-A an equimolar solution of Q1 and the duplex d(CGCAAATTTGCG)₂ (D). This comparative ¹H-NMR study allowed us to conclude that Dist-A and, consequently, Net possess higher affinity for duplex DNA.     

Shape readout of AT‐rich DNA by carbohydrates

Gene expression can be altered by small molecules that target DNA; sequence as well as shape selectivities are both extremely important for DNA recognition by intercalating and groove‐binding ligands. We have characterized a carbohydrate scaffold (1) exhibiting DNA “shape readout” properties. Thermodynamic studies with 1 and model duplex DNAs demonstrate the molecule's high affinity and selectivity towards B* form (continuous AT‐rich) DNA. Isothermal titration calorimetry (ITC), circular dichroism (CD) titration, ultraviolet (UV) thermal denaturation, and Differential Scanning Calorimetry were used to characterize the binding of 1 with a B* form AT‐rich DNA duplex d[5′‐G₂A₆T₆C₂‐3′]. The binding constant was determined using ITC at various temperatures, salt concentrations, and pH. ITC titrations were fit using a two‐binding site model. The first binding event was shown to have a 1:1 binding stoichiometry and was predominantly entropy‐driven with a binding constant of approximately 10⁸ M^?1. ITC‐derived binding enthalpies were used to obtain the binding‐induced change in heat capacity (ΔC_p) of ?225 ± 19 cal/mol·K. The ionic strength dependence of the binding constant indicated a significant electrolytic contribution in ligand:DNA binding, with approximately four to five ion pairs involved in binding. Ligand 1 displayed a significantly higher affinity towards AT‐tract DNA over sequences containing GC inserts, and binding experiments revealed the order of binding affinity for 1 with DNA duplexes: contiguous B* form AT‐rich DNA (d[5′‐G₂A₆T₆C₂‐3′]) >B form alternate AT‐rich DNA (d[5′‐G₂(AT)₆C_2‐3′]) > A form GC‐rich DNA (d[5′‐A₂G₆C₆T₂‐3′]), demonstrating the preference of ligand 1 for B* form DNA. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 720–732, 2014.     

The role of positive charges on G-quadruplex binding small molecules: Learning from bisaryldiketene derivatives

Background

G-quadruplexes are promising therapeutic targets for small molecules. In general, the introduction of steady positive charges through the in situ alkylation of nitrogen atoms within potential G-quadruplex ligands can significantly improve their quadruplex binding and stabilization abilities. However, our previous studies on bisaryldiketene derivatives showed that the derivative M4, whose central piperidone moiety is quaternized, exhibits a poor G-quadruplex stabilization ability.

Methods

To clarify this unusual finding, CD, ITC, UV and NMR analyses were performed to determine the binding behaviors of M4 and its non-quaternized analog M2 to G-quadruplex DNA [d(TGGGT)]₄. Molecular modeling approaches were also employed to help illustrate ligand–quadruplex DNA interactions.

Results

The CD melting and ITC analyses revealed that M2 exhibited much stronger stabilization and binding abilities to [d(TGGGT)]₄ compared to M4. Moreover, the CD and ITC analyses in combination with UV, NMR and MD simulations revealed that M2 tended to be end-stacked on the G-quartet, whereas M4 tended to be bound in the groove region. Analysis of the electrostatic potential showed that the charged surface of M4 was more positive than that of M2 and other reported ligands that bind to the G-quadruplex via end-stacking interactions.

Conclusions

The results indicated that the different positively charged surfaces of M2 and M4 might be the key reason for their different binding modes. These different binding modes also lead to different binding affinities and stabilization abilities for [d(TGGGT)]₄.

General significance

These results provide new clues for the rational design of G-quadruplex-binding small molecules with steady positive charges.     

Effect of the incorporation of 2'-deoxy-8-(hydroxyl)adenosine on the stability of quadruplexes formed by modified human telomeric DNA

Differential scanning calorimetry (DSC) and circular dichroism (CD) techniques were used to investigate the physico-chemical properties of the quadruplexes formed by the two different truncations of human telomeric sequence d(TAGGGT) and d(AGGGT), where the adenines were substituted by 2'-deoxy-8-(hydroxyl)adenosine (A --> A OH). CD spectra show that the modified sequences are able to form parallel-stranded quadruplex structure. Analysis of the thermodynamic parameters reveals that the introduction of the modified adenine affects in different way the thermal stability of the [d(TAGGGT)]4 and [d(AGGGT)]4 quadruplexes.     

Interaction of distamycin A and netropsin with quadruplex and duplex structures: a comparative 1H-NMR study

Homonuclear NMR techniques have been used to investigate the interactions of the minor groove binding agents distamycin A (Dist-A) and the related drug netropsin (Net) with three quadruplexes characterized by different groove widths: [d(TGGGGT)]4 (Q1), [d(GGGGTTTTGGGG)]2 (Q2), and d(GGGGTTGGGGTGTGGGGTTGGGG) (Q3). Netropsin has been found to be in a fast chemical exchange with all three kinds of quadruplexes, whereas Dist-A interacts tightly with Q1 and, at a less extent, with Q2. In order to determine the degree of selectivity of Dist-A for two- rather than four-stranded DNA, we titrated with Dist-A an equimolar solution of Ql and the duplex d(CGCAAATTTGCG)2 (D). This comparative 1H-NMR study allowed us to conclude that Dist-A and, consequently, Net possess higher affinity for duplex DNA.     

The triazatruxene derivative azatrux binds to the parallel form of the human telomeric G-quadruplex under molecular crowding conditions: biophysical and molecular modeling studies

The present study has employed a combination of spectroscopic, calorimetric and computational methods to explore the binding of the three side-chained triazatruxene derivative, termed azatrux, to a human telomeric G-quadruplex sequence, under conditions of molecular crowding. The binding of azatrux to the tetramolecular parallel [d(TGGGGT)]₄ quadruplex in the presence and absence of crowding conditions, was also characterized. The data indicate that azatrux binds in an end-stacking mode to the parallel G-quadruplex scaffold and highlights the key structural elements involved in the binding. The selectivity of azatrux for the human telomeric G-quadruplex relative to another biologically relevant G-quadruplex (c-Kit87up) and to duplex DNA was also investigated under molecular crowding conditions, showing that azatrux has good selectivity for the human telomeric G-quadruplex over the other investigated DNA structures.     

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

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

Bcl-2 Promoter Sequence G-Quadruplex Interactions with Three Planar and Non-Planar Cationic Porphyrins: TMPyP4, TMPyP3, and TMPyP2

The interactions of three related cationic porphyrins, TMPyP4, TMPyP3 and TMPyP2, with a WT 39-mer Bcl-2 promoter sequence G-quadruplex were studied using Circular Dichroism, ESI mass spectrometry, Isothermal Titration Calorimetry, and Fluorescence spectroscopy. The planar cationic porphyrin TMPyP4 (5, 10, 15, 20-meso-tetra (N-methyl-4-pyridyl) porphine) is shown to bind to a WT Bcl-2 G-quadruplex via two different binding modes, an end binding mode and a weaker mode attributed to intercalation. The related non-planar ligands, TMPyP3 and TMPyP2, are shown to bind to the Bcl-2 G-quadruplex by a single mode. ESI mass spectrometry experiments confirmed that the saturation stoichiometry is 4:1 for the TMPyP4 complex and 2:1 for the TMPyP2 and TMPyP3 complexes. ITC experiments determined that the equilibrium constant for formation of the (TMPyP4)₁/DNA complex (K₁ = 3.7 × 10⁶) is approximately two orders of magnitude greater than the equilibrium constant for the formation of the (TMPyP2)₁/DNA complex, (K₁ = 7.0 × 10⁴). Porphyrin fluorescence is consistent with intercalation in the case of the (TMPyP4)₃/DNA and (TMPyP4)₄/DNA complexes. The non-planar shape of the TMPyP2 and TMPyP3 molecules results in both a reduced affinity for the end binding interaction and the elimination of the intercalation binding mode.     

Spectroscopic studies of 9-hydroxyellipticine binding to DNA

The binding of 9-hydroxyellipticine to calf thymus DNA, poly[d(A-T)]₂, and poly-[d(G-C)]₂ has been studied in detail by means of CD, linear dichroism, resonance light scattering, and molecular dynamics. The transition moment polarizations of 9-hydroxyelliptiycine were determined in polyvinyl alcohol stretched film. Spectroscopic solution studies of the DNA/drug complex are combined with theoretical CD calculations using the final 50 ps of a series of molecular dynamics simulations as input. The spectroscopic data shows 9-hydroxyellipticine to adopt two main binding modes, one intercalative and the other a stacked binding mode involving the formation of drug oligomers in the DNA major groove. Analysis of the intercalated binding mode in poly[d(A-T)]₂ suggests the 9-hydroxyellipticine hydroxyl group lies in the minor groove and hydrogen bonds to water with the pyridine ring protruding into the major groove. The stacked binding mode was examined using resonance light scattering and it was concluded that the drug was forming small oligomer stacks rather than extended aggregates. Reduced linear dichroism measurements suggested a binding geometry that precluded a minor groove binding mode where the plane of the drug makes a 45° angle with the plane of the bases. Thus it was concluded that the drug stacks in the major groove. No obvious differences in the mode of binding of 9-hydroxyellipticine were observed between different DNA sequences; however, the stacked binding mode appeared to be more favorable for calf thymus DNA and poly[d(G-C)]₂ than for poly[d(A-T)]₂, an observation that could be explained by the slightly greater steric hindrance of the poly[d(A-T)]₂ major groove. A strong concentration dependence was observed for the two binding modes where intercalation is favored at very low drug load, with stacking interactions becoming more prominent as the drug concentration is increased. Even at DNA : drug mixing ratios of 70:1 the stacked binding mode was still important for GC-rich DNAs. © 1998 John Wiley & Sons, Inc. Biopoly 46: 127–143, 1998     

Qualitative and quantitative characterization of protein-phosphoinositide interactions with liposome-based methods

We characterized phosphoinositide binding of the S. cerevisiae PROPPIN Hsv2 qualitatively with density flotation assays and quantitatively through isothermal titration calorimetry (ITC) measurements using liposomes. We discuss the design of these experiments and show with liposome flotation assays that Hsv2 binds with high specificity to both PtdIns3P and PtdIns(3,5)P₂. We propose liposome flotation assays as a more accurate alternative to the commonly used PIP strips for the characterization of phosphoinositide-binding specificities of proteins. We further quantitatively characterized PtdIns3P binding of Hsv2 with ITC measurements and determined a dissociation constant of 0.67 µM and a stoichiometry of 2:1 for PtdIns3P binding to Hsv2. PtdIns3P is crucial for the biogenesis of autophagosomes and their precursors. Besides the PROPPINs there are other PtdIns3P binding proteins with a link to autophagy, which includes the FYVE-domain containing proteins ZFYVE1/DFCP1 and WDFY3/ALFY and the PX-domain containing proteins Atg20 and Snx4/Atg24. The methods described could be useful tools for the characterization of these and other phosphoinositide-binding proteins.     

Induction of G-quadruplex DNA structure by Zn(II) 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin

G-quadruplexes (GQ) are formed by the association of guanine-rich stretches of DNA. Certain small molecules can influence kinetics and thermodynamics of this association. Understanding the mechanism of ligand-assisted GQ folding is necessary for the design of more efficient cancer therapeutics. The oligonucleotide d(TAGGG)₂ forms parallel bimolecular GQ in the presence of ≥66 mM K⁺; GQs are not formed under Na⁺, Li⁺ or low K⁺ conditions. The thermodynamic parameters for GQ folding at 60 μM oligonucleotide and 100 mM KCl are ΔH = −35 ± 2 kcal mol⁻¹ and ΔG₃₁₀ = −1.4 kcal mol⁻¹. Quadruplex [d(TAGGG)₂]₂ binds 2-3 K⁺ ions with K_d of 0.5 ± 0.2 mM. Our work addresses the question of whether metal free 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) and its Zn(II), Cu(II), and Pt(II) derivatives are capable of facilitating GQ folding of d(TAGGG)₂ from single stranded, or binding to preformed GQ, using UV-vis and circular dichroism (CD) spectroscopies. ZnTMPyP4 is unique among other porphyrins in its ability to induce GQ structure of d(TAGGG)₂, which also requires at least a low amount of potassium. ZnTMPyP4 binds with 2:1 stoichiometry possibly in an end-stacking mode with a ∼10⁶ M⁻¹ binding constant, determined through UV-vis and ITC titrations. This process is entropically driven and has ΔG₂₉₈ of −8.0 kcal mol⁻¹. TMPyP4 binds with 3:1 stoichiometry and K_a of ∼10⁶ M⁻¹. ZnTMPyP4 and TMPyP4 are efficient stabilizers of [d(TAGGG)₂]₂ displaying ΔT_1/2 of 13.5 and 13.8 °C, respectively, at 1:2 GQ to porphyrin ratio; CuTMPyP4 shows a much weaker effect (ΔT_1/2 = 4.7 °C) and PtTMPyP4 is weakly destabilizing (ΔT_1/2 = −2.9 °C). The selectivity of ZnTMPyP4 for GQ versus dsDNA is comparable to that of TMPyP4. The ability of ZnTMPyP4 to bind and stabilize GQ, to induce GQ formation, and speed up its folding may suggest an important biological activity for this molecule.     

Designing isothermal titration calorimetry experiments for the study of 1:1 binding: problems with the "standard protocol"

Literature recommendations for designing isothermal titration calorimetry (ITC) experiments to study 1:1 binding, M+X -->/<-- MX, are not consistent and have persisted through time with little quantitative justification. In particular, the "standard protocol" employed by most workers involves 20 to 30 injections of titrant to a final titrant/titrand mole ratio (R(m)) of ~ 2-a scheme that can be far from optimal and can needlessly limit applicability of the ITC technique. These deficiencies are discussed here along with other misconceptions. Whether a specific binding process can be studied by ITC is determined less by c (the product of binding constant K and titrand concentration [M](0)) than by the total detectable heat q(tot) and the extent to which M can be converted to MX. As guidelines, with 90% conversion to MX, K can be estimated within 5% over the range 10 to 10(8)M(-1) when q(tot)/σ(q)≈700, where σ(q) is the standard deviation for estimation of q. This ratio drops to ~150 when the stoichiometry parameter n is treated as known. A computer application for modeling 1:1 binding yields realistic estimates of parameter standard errors for use in protocol design and feasibility assessment.     

Thermodynamic characterization of cytosolic phospholipase A2 alpha inhibitors

Cytosolic phospholipase A₂ alpha (cPLA₂α, type IVA phospholipase) acts at the membrane surface to release free arachidonic acid, which is metabolized into inflammatory mediators, including leukotrienes and prostaglandins. Thus, specific cPLA₂α inhibitors are predicted to have antiinflammatory properties. However, a key criterion in the identification and development of such inhibitors is to distinguish between compounds that bind stoichiometrically to cPLA₂α and nonspecific membrane perturbants. In the current study, we developed a method employing isothermal titration calorimetry (ITC) to characterize the binding of several distinct classes of cPLA₂α inhibitors. Thermodynamic parameters and the binding constants were obtained following titration of the inhibitor to the protein at 30 °C and pH 7.4. The compounds tested bound cPLA₂α with a 1:1 stoichiometry, and the dissociation constant K_d of the inhibitors calculated from the ITC experiments correlated well with the IC₅₀ values obtained from enzymatic assays. Interestingly, binding was observed only in the presence of a micellar surface, even for soluble compounds. The site of binding of these inhibitors within cPLA₂α was analyzed by testing for binding in the presence of methyl arachidonyl fluorophosphonate (MAFP), an irreversible active site inhibitor of cPLA₂α. Lack of binding of inhibitors in the presence of MAFP suggested that the compounds tested bound specifically at or near the active site of the protein. Furthermore, the effect of various detergents on the binding of certain inhibitors to cPLA₂α was also tested. The results are discussed with reference to thermodynamic parameters such as changes in enthalpy (ΔH), entropy (ΔS), and free energy (ΔG). The data obtained from these studies provide not only structure-activity relationships for compounds but also important information regarding mechanism of binding. This is the first example of ITC used for studying inhibitors of enzymes with interfacial kinetics.     

Oxidation of copper(II)-coordinated diethylenetriamine by hexacyanoferrate(III) ion. A kinetic investigation

The stoichiometry and kinetics of the reaction between [Cu(dien)(OH)]⁺ and [Fe(CN)₆]³⁻ in aqueous alkaline medium are described. The rate equation − (d[Fe(III)]/dt = {k₁[OH⁻]²[[Cu(dien)(OH)]⁺] + k₂[OH⁻] × [[Cu(dien)(OH)]⁺]²}([Fe(III)]/[Fe(II)]) (Fe(III) = [Fe(CN)₆]³⁻; Fe(II) = [Fe(CN)₆]⁴⁻, the 4:4:1 OH⁻/Fe(III)/[Cu(dien)(OH)]⁺ stoichiometric ratio and the nature of the ultimate products identified in the reaction solution suggest the fast formation of a doubly deprotonated Cu(III)-diamido complex which slowly undergoes an internal redox process where the ligand is oxidised to the Schiff base H₂NCH₂CH₂NCHCHNH.The [[Cu(dien)(OH)]⁺]² term in the rate equation is explained with the formation of a transient μ-hydroxo mixed-valence Cu dimer. A two-electron internal reduction of the Cu(III) complex yielding a Cu(I) intermediate is suggested to account for the presence of monovalent copper in a precipitate which forms at relatively high reactant concentrations and in the absence of dioxygen.     

Selectivity at a three-base bulge site in the DNA binding of ΔΔ-[{Ru(phen)2}2(μ-dppm)]4+ [dppm is 4,6-bis(2-pyridyl)pyrimidine; phen is 1,10-phenanthroline]

The binding of the stereoisomers of [{Ru(phen)₂}₂(μ-bpm)]⁴⁺, [{Ru(phen)₂}₂(μ-dppm)]⁴⁺ and [{Ru(phen)₂}₂(μ-bb)]⁴⁺ {phen is 1,10-phenanthroline; bpm is 2,2′-bipyrimidine, dppm is 4,6-bis(2-pyridyl)pyrimidine, bb is 1,2-bis[4-(4′-methyl-2,2′-bipyridyl)]ethane} to an oligonucleotide duplex [d(GCATCGAAAGCTACG)•d(CGTAGCCGATGC)] containing a three-base bulge has been studied using a fluorescence intercalator displacement assay. Of the dinuclear ruthenium complexes, the dppm-linked species showed the strongest binding to the oligonucleotide, with the ΔΔ isomer binding slightly more strongly than the meso isomer and the ΛΛ isomer exhibiting the weakest binding. In order to determine whether the ΔΔ-[{Ru(phen)₂}₂(μ-dppm)]⁴⁺ metal complex specifically bound at the three-base bulge site, a ¹H NMR study of the binding of the metal complex to the oligonucleotide duplex d(GCATCGAAAGCTACG)•d(CGTAGCCGATGC) was carried out. Although a detailed picture of the metal complex–oligonucleotide association could not be determined from the NMR results owing to the broadening of the resonances from the metal complex and nucleotide residues at the bulge site, the NMR results do indicate that the metal complex specifically binds at the three-base bulge site. The combined results of this study suggest that the dppm-bridged dinuclear ruthenium complexes have considerable potential as probes for the unusual secondary structure obtained by the insertion of a three-base bulge within duplex DNA.     

Spectroscopic and thermodynamic evidence for antimicrobial peptide membrane selectivity

In our laboratory we developed a series of antimicrobial peptides that exhibit selectivity and potency for prokaryotic over eukaryotic cells (Hicks et al., 2007). Circular dichroism (CD), isothermal calorimetry (ITC) and calcein leakage assays were conducted to determine the mechanism of lipid binding of a representative peptide 1 (Ac-GF-Tic-Oic-GK-Tic-Oic-GF-Tic-Oic-GK-Tic-KKKK-CONH₂) to model membranes. POPC liposomes were used as a simple model for eukaryotic membranes and 4:1 POPC:POPG liposomes were used as a simple model for prokaryotic membranes. CD, ITC and calcein leakage data clearly indicate that compound 1 interacts via very different mechanisms with the two different liposome membranes. Compound 1 exhibits weaker binding and induces less calcein leakage in POPC liposomes than POPC:POPG (4:1 mole ratio) liposomes. The predominant binding mechanism to POPC appears to be limited to surface interactions while the mechanism of binding to 4:1 POPC:POPG most likely involves some type of pore formation.