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

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

Investigation of DNA Binding Modes for a Symmetrical Cyanine Dye Trication: Effect of DNA Sequence and Structure

Abstract

The DNA binding behavior of a tricationic cyanine dye (DiSC₃₊(5)) was studied using the [Poly(dA-dT)]₂, [Poly(dI-dC)]₂ and Poly(dA)?Poly(dT) duplex sequences and the Poly(dA) ?2Poly(dT) triplex. Optical spectroscopy and viscometry results indicate that the dye binds to the triplex structure by intercalation, to the nonalternating Poly(dA)?Poly(dT) duplex through minor groove binding and to the alternating [Poly(dA-dT)]₂ duplex by a combination of two binding modes: intercalation at low concentration and dimerization within the minor groove at higher concentration. Dimerization occurs at lower dye concentrations for the [Poly(dI-dC)]₂ sequence, consistent with our previous investigations on an analogous monocationic cyanine dye. [Seifert, J.L., et al. (1999) J. Am. Chem. Soc. 121, 2987–2995] These studies illustrate the diversity of DNA binding modes that are available to a given ligand structure.     

Investigation of DNA binding modes for a symmetrical cyanine dye trication: effect of DNA sequence and structure.

The DNA binding behavior of a tricationic cyanine dye (DiSC3+(5)) was studied using the [Poly(dA-dT)]2, [Poly(dI-dC)]2 and Poly(dA) x Poly(dT) duplex sequences and the Poly(dA) x 2Poly(dT) triplex. Optical spectroscopy and viscometry results indicate that the dye binds to the triplex structure by intercalation, to the nonalternating Poly(dA) x Poly(dT) duplex through minor groove binding and to the alternating [Poly(dA-dT)]2 duplex by a combination of two binding modes: intercalation at low concentration and dimerization within the minor groove at higher concentration. Dimerization occurs at lower dye concentrations for the [Poly(dI-dC)]2 sequence, consistent with our previous investigations on an analogous monocationic cyanine dye. [Seifert, J.L., et al. (1999) J. Am. Chem. Soc. 121, 2987-2995] These studies illustrate the diversity of DNA binding modes that are available to a given ligand structure.     

Molecular determinants for DNA minor groove recognition: design of a bis-guanidinium derivative of ethidium that is highly selective for AT-rich DNA sequences

The phenanthridinium dye ethidium bromide is a prototypical DNA intercalating agent. For decades, this anti-trypanosomal agent has been known to intercalate into nucleic acids, with little preference for particular sequences. Only polydA-polydT tracts are relatively refractory to ethidium intercalation. In an effort to tune the sequence selectivity of known DNA binding agents, we report here the synthesis and detailed characterization of the mode of binding to DNA of a novel ethidium derivative possessing two guanidinium groups at positions 3 and 8. This compound, DB950, binds to DNA much more tightly than ethidium and exhibits distinct DNA-dependent absorption and fluorescence properties. The study of the mode of binding to DNA by means of circular and electric linear dichroism revealed that, unlike ethidium, DB950 forms minor groove complexes with AT sequences. Accurate quantification of binding affinities by surface plasmon resonance using A(n)T(n) hairpin oligomer indicated that the interaction of DB950 is over 10-50 times stronger than that of ethidium and comparable to that of the known minor groove binder furamidine. DB950 interacts weakly with GC sites by intercalation. DNase I footprinting experiments performed with different DNA fragments established that DB950 presents a pronounced selectivity for AT-rich sites, identical with that of furamidine. The replacement of the amino groups of ethidium with guanidinium groups has resulted in a marked gain of both affinity and sequence selectivity. DB950 provides protection against DNase I cleavage at AT-containing sites which frequently correspond to regions of enhanced cleavage in the presence of ethidium. Although DB950 maintains a planar phenanthridinium chromophore, the compound no longer intercalates at AT sites. The guanidinium groups of DB950, just like the amidinium group of furamidine (DB75), are the critical determinants for recognition of AT binding sites in DNA. The chemical modulation of the ethidium exocyclic amines is a profitable option to tune the nucleic acid recognition properties of phenylphenanthridinium dyes.     

Flexible docking of DNA fragments and actinocin derivatives

We have applied molecular docking methods to systems containing nucleic acids as targets and biologically active substances as ligands. The complexes of DNA fragments and actinocin derivatives with different lengths of aminoalkyl side chains were obtained by molecular docking. It was observed that actinocin derivatives could form energetically favourable complexes with DNA both as intercalators and minor groove binders. It was shown that small changes in the binding energy (～1?kcal/mol) could result in complexes with substantially different structure. The complexes of actinocin derivatives and DNA fragments were stabilized by hydrogen bonding upon intercalation and minor groove binding. It was found that the change of solvent-accessible surface area upon binding of the actinocin derivative to DNA linear increased with the growth of methylene groups' number in ligand side chains. The solvation energy change upon binding of actinocin derivatives to DNA calculated by the WSAS method was favourable in the case of small uncharged ligands and unfavourable for positively charged ligands.     

Drug-DNA sequence-dependent interactions analysed by electric linear dichroism.

The interactions between 20 drugs and a variety of synthetic DNA polymers and natural DNAs were studied by electric linear dichroism (ELD). All compounds tested, including several clinically used antitumour agents, are thought to exert their biological activities mainly by virtue of their abilities to bind to DNA. The selected drugs include intercalating agents with fused and unfused aromatic structures and several groove binders. To examine the role of base composition and base sequence in the binding of these drugs to DNA, ELD experiments were carried out with natural DNAs of widely differing base composition as well as with polynucleotides containing defined alternating and non-alternating repeating sequences, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT),poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC). Among intercalating agents, actinomycin D was found to be by far the most GC-selective. GC selectivity was also observed with an amsacrine-4-carboxamide derivative and to a lesser extent with methylene blue. In contrast, the binding of amsacrine and 9-aminoacridine was practically unaffected by varying the GC content of the DNAs. Ethidium bromide, proflavine, mitoxantrone, daunomycin and an ellipticine derivative were found to bind best to alternating purine-pyrimidine sequences regardless of their nature. ELD measurements provided evidence for non-specific intercalation of amiloride. A significant AT selectivity was observed with hycanthone and lucanthone. The triphenyl methane dye methyl green was found to exhibit positive and negative dichroism signals at AT and GC sites, respectively, showing that the mode of binding of a drug can change markedly with the DNA base composition. Among minor groove binders, the N-methylpyrrole carboxamide-containing antibiotics netropsin and distamycin bound to DNA with very pronounced AT specificity, as expected. More interestingly the dye Hoechst 33258, berenil and a thiazole-containing lexitropsin elicited negative reduced dichroism in the presence of GC-rich DNA which is totally inconsistent with a groove binding process. We postulate that these three drugs share with the trypanocide 4',6-diamidino-2-phenylindole (DAPI) the property of intercalating at GC-rich sites and binding to the minor groove of DNA at other sites. Replacement of guanines by inosines (i.e., removal of the protruding exocyclic C-2 amino group of guanine) restored minor groove binding of DAPI, Hoechst 33258 and berenil. Thus there are several cases where the mode of binding to DNA is directly dependent on the base composition of the polymer. Consequently the ELD technique appears uniquely valuable as a means of investigating the possibility of sequence-dependent recognition of DNA by drugs.     

Interaction of procarbazine with calf thymus DNA—a biophysical and molecular docking study

Interaction of procarbazine (PCZ) with calf thymus DNA was studied using biophysical and molecular docking studies. Procarbazine was to interact with DNA with a binding constant of 6.52 × 10³ M⁻¹ as calculated using ultraviolet‐visible spectroscopy. To find out the binding mode, molecular docking was performed that predicted PCZ to interact with DNA through groove binding mode with binding affinity of −6.7 kcal/mole. To confirm the groove binding nature, different experiments were performed. Dye displacement assays confirmed the non‐intercalative binding mode. Procarbazine displaced Hoechst dye from the minor groove of DNA while it was unable to displace intercalating dyes. There was no increase in the viscosity of DNA solution in presence of PCZ. Also, negligible change in the secondary structure of DNA was observed in presence of PCZ as evident by circular dichroism spectra. Procarbazine caused decrease in the melting temperature of DNA possibly because of decrease in the stability of DNA caused by groove binding interaction of PCZ with DNA.     

Anthracycline antibiotic arugomycin binds in both grooves of the DNA helix simultaneously: an NMR and molecular modelling study.

Perturbations to the 1H and 31P chemical shifts of DNA resonances together with twenty-four intermolecular nuclear Overhauser effects show that the anthracycline antibiotic arugomycin intercalates between the basepairs of the hexamer duplex d(5'-GCATGC)2 at the 5'-CpA and 5'-TpG binding sites. In the complex two drug molecules are bound per duplex with full retention of the dyad symmetry. Arugomycin adopts a threaded binding orientation with chains of sugars positioned in both the major and minor groove of the helix simultaneously. The complex is stabilized by hydrogen bonding, electrostatic and van der Waals interactions principally in the major groove and involving substituents on the rigidly oriented bicycloamino-glucose sugar of the antibiotic. A specific hydrogen bond is identified between the C2'-hydroxyl and the guanine N7 at the intercalation site. Together, interactions in the major groove appear to account for the intercalation specificity of arugomycin that requires both a guanine and thymine at the intercalation site. We are unable to identify any sequence specific interactions between the minor groove and the arugarose sugar (S1) which binds only weakly, through van der Walls contacts, over the d(GCA).d(TGC) trinucleotide sequence. The data indicate that the sugar chains of arugomycin are flexible and play little part in the interaction of the antibiotic with DNA. The intensity of sequential internucleotide NOEs identifies the intercalation site as being assymmetric. A family of conformers computed using restrained energy minimisation and molecular dynamics indicate that basepair buckling is a feature of the anthracycline intercalation site that may serve to maximise intermolecular van der Waals interactions by wrapping the basepairs around the antibiotic chromophore.     

Head-to-head bis-hairpin polyamide minor groove binders and their conjugates with triplex-forming oligonucleotides: studies of interaction with target double-stranded DNA

Two hairpin hexa(N-methylpyrrole)carboxamide DNA minor groove binders (MGB) were linked together via their N-termini in head-to-head orientation. Complex formation between these bis-MGB conjugates and target DNA has been studied using DNase I footprinting, circular dichroism, thermal dissociation, and molecular modeling. DNase I footprint revealed binding of these conjugates to all the sites of 492 b.p. DNA fragment containing (A/T)(n)X(m)(A/T)(p) sequences, where n>3, p>3; m=1,2; X = A,T,G, or C. Binding affinity depended on the sequence context of the target. CD experiments and molecular modeling showed that oligo(N-methylpyrrole)carboxamide moieties in the complex form two short antiparallel hairpins rather than a long parallel head-to-head hairpin. Binding of bis-MGB also stabilized a target duplex thermodynamically. Sequence specificity of bis-MGB/DNA binding was validated using bis-conjugates of sequence-specific hairpin (N-methylpyrrole)/(N-methylimidazole) carboxamides. In order to increase the size of recognition sequence, the conjugates of bis-MGB with triplex-forming oligonucleotides (TFO) were synthesized and compared to TFO conjugated with single MGB hairpin unit. Bis-MGB-oligonucleotide conjugates also bind to two blocks of three and more A.T/T.A pairs similarly to bis-MGB alone, independently of the oligonucleotide moiety, but with lower affinity. However, the role of TFO in DNA recognition was demonstrated for mono-MGB-TFO conjugate where the binding was detected mainly in the area of the target sequence consisting of both MGB and TFO recognition sites. Basing on the molecular modeling, three-dimensional models of both target DNA/bis-MGB and target DNA/TFO-bis-MGB complexes were built, where bis-MGB forms two antiparallel hairpins. According to the second model, one MGB hairpin is in the minor groove of 5'-adjacent A/T sequence next to the triplex-forming region, whereas the other one occupies the minor groove of the TFO binding polypurine tract. All these data together give a key information for the construction of MGB-MGB and MGB-oligonucleotide conjugates possessing high specificity and affinity for the target double-stranded DNA.     

Interaction between azo dye Acid Red 14 and pepsin by multispectral methods and docking studies

The interaction of synthetic azo dye Acid Red 14 with pepsin was studied by fluorescence spectroscopy, UV–vis spectroscopy, circular dichroism and molecular docking. Results from the fluorescence spectroscopy show that Acid Red 14 has a strong capability to quench the intrinsic fluorescence of pepsin with static quenching. Binding constant, number of the binding sites and thermodynamic parameters were measured at different temperatures. The result indicates that Acid Red 14 interact with pepsin spontaneously by hydrogen bonding and van der Waals interactions. Three‐dimensional fluorescence spectra and circular dichroism spectra reveal that Acid Red 14 could slightly change the structure of pepsin. The hydrogen bond is formed between Acid Red 14 and Tyr‐189 and Thr‐218 residues of pepsin. Furthermore, the binding between Acid Red 14 and pepsin inhibits pepsin activity. The study can provide a way to analyze the biological safety of Acid Red 14 on digestive proteases or other proteins.     

Affinity and kinetic modulation of polyamide–DNA interactions by N‐modification of the heterocycles

Synthetic N‐methyl imidazole and N‐pyrrole containing polyamides (PAs) that can form “stacked” dimers can be programmed to target and bind to specific DNA sequences and control gene expression. To accomplish this goal, the development of PAs with lower molecular mass which allows for the molecules to rapidly penetrate cells and localize in the nucleus, along with increased water solubility, while maintaining DNA binding sequence specificity and high binding affinity is key. To meet these challenges, six novel f‐ImPy*Im PA derivatives that contain different orthogonally positioned moieties were designed to target 5′‐ACGCGT‐3′. The synthesis and biophysical characterization of six f‐ImPy*Im were determined by CD, ΔT_M, DNase I footprinting, SPR, and ITC studies, and were compared with those of their parent compound, f‐ImPyIm. The results gave evidence for the minor groove binding and selectivity of PAs 1 and 6 for the cognate sequence 5′‐ACGCGT‐3′, and with strong affinity, K_eq = 2.8 × 10⁸ M^?1 and K_eq = 6.2 × 10⁷ M^?1, respectively. The six novel PAs presented in this study demonstrated increased water solubility, while maintaining low molecular mass, sequence specificity, and binding affinity, addressing key issues in therapeutic development. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 497–507, 2013.     

Interaction of an anticancer benzopyrane derivative with DNA: Biophysical,biochemical, and molecular modeling studies

BackgroundSIMR1281 is a potent anticancer lead candidate with multi- target activity against several proteins; however, its mechanism of action at the molecular level is not fully understood. Revealing the mechanism and the origin of multitarget activity is important for the rational identification and optimization of multitarget drugs.MethodsWe have used a variety of biophysical (circular dichroism, isothermal titration calorimetry, viscosity, and UV DNA melting), biochemical (topoisomerase I & II assays) and computational (molecular docking and MD simulations) methods to study the interaction of SIMR1281 with duplex DNA structures.ResultsThe biophysical results revealed that SIMR1281 binds to dsDNA via an intercalation-binding mode with an average binding constant of 3.1 × 10⁶ M⁻¹. This binding mode was confirmed by the topoisomerases' inhibition assays and molecular modeling simulations, which showed the intercalation of the benzopyrane moiety between DNA base pairs, while the remaining moieties (thiazole and phenyl rings) sit in the minor groove and interact with the flanking base pairs adjacent to the intercalation site.ConclusionsThe DNA binding characteristics of SIMR1281, which can disrupt/inhibit DNA function as confirmed by the topoisomerases' inhibition assays, indicate that the observed multi-target activity might originate from ligand intervention at nucleic acids level rather than due to direct interactions with multiple biological targets at the protein level.General significanceThe findings of this study could be helpful to guide future optimization of benzopyrane-based ligands for therapeutic purposes.     

Use of Electric Linear Dichroism and Competition Experiments with Intercalating Drugs to Investigate the Mode of Binding of Hoechst 33258, Berenil and DAPI to GC Sequences

Abstract

The drugs Hoechst 33258, berenil and DAPI bind preferentially to the minor groove of AT sequences in DNA Despite a strong selectivity for AT sites, they can interact with GC sequences by a mechanism which remains so far controversial. The 2-amino group of guanosine represents a steric hindrance to the entry of the drugs in the minor groove of GC sequences. Intercalation and major groove binding to GC sites of GC-rich DNA and polynucleotides have been proposed for these drugs. To investigate further the mode of binding of Hoechst 33258, berenil and DAPI to GC sequences, we studied by electric linear dichroism the mutual interference in the DNA binding reaction between these compounds and a classical intercalator, proflavine, or a DNA-threading intercalating drug, the amsacrine-4-carboxamide derivative SN16713. The results of the competition experiments show that the two acridine intercalators markedly affect the binding of Hoechst 33258, berenil and DAPI to GC polynucleotides but not to DNA containing AT/GC mixed sequences such as calf thymus DNA Proflavine and SN16713 exert dissimilar effects on the binding of Hoechst 33258, berenil and DAPI to GC sites. The structural changes in DNA induced upon intercalation of the acridine drugs into GC sites are not identically perceived by the test compounds. The electric linear dichroism data support the hypothesis that Hoechst 33258, berenil and DAPI interact with GC sites via a non-classical intercalation process.     

Sequence-specific interaction of the Salmonella Hin recombinase in both major and minor grooves of DNA.

The Hin recombinase of Salmonella catalyzes a site-specific recombination event which leads to flagellar phase variation. Starting with a fully symmetrical recombination site, hixC, a set of 40 recombination sites which vary by pairs of single base substitutions was constructed. This set was incorporated into the Salmonella-specific bacteriophage P22 based challenge phage selection and used to define the DNA sequence determinants for the binding of Hin to DNA in vivo. The critical sequence-specific contacts between a Hin monomer and a 13 bp hix half-site are at two T:A base pairs in the major groove of the DNA which are separated by one base pair, and two consecutive A:T contacts in the minor groove. The base substitutions in the major groove recognition portion which were defective in binding Hin still retained residual binding capability in vivo, while the base pair substitutions affecting the minor groove recognition region lost all in vivo binding. Using in vitro binding assays, Hin was found to bind to hix symmetrical sites with A:T base pairs or I:C base pairs in the minor groove recognition sequences, but not to G:C base pairs. In separate in vitro binding assays, Hin was equally defective in binding to either a G:C or a I:C contact in a major groove recognition sequence. Results from in vitro binding assays to hix sites in which 3-deazaadenine was substituted for adenine are consistent with Hin making a specific contact to either the N3 of adenine or O2 of thymine in the minor groove within the hix recombination site on each symmetric half-site. These results taken with the results of previous studies on the DNA binding domain of Hin suggest a sequence-specific minor groove DNA binding motif.     

Ethidium bromide interactions with DNA: an exploration of a classic DNA–ligand complex with unbiased molecular dynamics simulations

Visualization of double stranded DNA in gels with the binding of the fluorescent dye ethidium bromide has been a basic experimental technique in any molecular biology laboratory for >40 years. The interaction between ethidium and double stranded DNA has been observed to be an intercalation between base pairs with strong experimental evidence. This presents a unique opportunity for computational chemistry and biomolecular simulation techniques to benchmark and assess their models in order to see if the theory can reproduce experiments and ultimately provide new insights. We present molecular dynamics simulations of the interaction of ethidium with two different double stranded DNA models. The first model system is the classic sequence d(CGCGAATTCGCG)₂ also known as the Drew–Dickerson dodecamer. We found that the ethidium ligand binds mainly stacked on, or intercalated between, the terminal base pairs of the DNA with little to no interaction with the inner base pairs. As the intercalation at the terminal CpG steps is relatively rapid, the resultant DNA unwinding, rigidification, and increased stability of the internal base pair steps inhibits further intercalation. In order to reduce these interactions and to provide a larger groove space, a second 18-mer DNA duplex system with the sequence d(GCATGAACGAACGAACGC) was tested. We computed molecular dynamics simulations for 20 independent replicas with this sequence, each with ∼27 μs of sampling time. Results show several spontaneous intercalation and base-pair eversion events that are consistent with experimental observations. The present work suggests that extended MD simulations with modern DNA force fields and optimized simulation codes are allowing the ability to reproduce unbiased intercalation events that we were not able to previously reach due to limits in computing power and the lack of extensively tested force fields and analysis tools.     

Sequence specificity of the binding of 9-aminoacridine- and amsacrine-4-carboxamides to DNA studied by DNase I footprinting.

DNase I footprinting has been used to probe the sequence selectivity of binding of a series of intercalating amsacrine-4-carboxamides and a related 9-aminoacridine-4-carboxamide to three DNA restriction fragments. These ligands have good experimental antileukemic activity, and for those members of the series that gave evaluable footprints, our principal finding is that they bind preferentially to GC-rich regions in agreement with the conclusion of equilibrium and kinetic measurements. The highest affinity sites generally occur in clusters of GC base pairs with runs of AT pairs being excluded from binding. It is important to appreciate that the 9-aminoacridine- and amsacrine-4-carboxamides exhibit a very high degree of selectivity for GC sites which, to our knowledge, has not been previously matched by acridine derivatives in footprinting experiments. The principal determinant of specificity appears to be the 4-carboxamide group itself since neither variations in the terminal funtionality of the 4-carboxamide sidechain nor the presence of the 9-anilino substituent modifies sequence preferences. The molecular origins of selectivity may be discerned in terms of potential hydrogen bonding interactions between the 4-carboxamide moiety and carbonyl oxygen and amino groups of GC base pairs in the DNA minor groove at CG dinucleotide sites. The related therapeutic agent amsacrine failed to inhibit cleavage by DNase I, so no conclusion can be drawn concerning its binding selectivity, save to note that amsacrine does not possess the 4-carboxamide group which appears to be the crucial determinant of GC specificity. Whether selectivity for binding to GC-rich sequences is an important element in the antitumor activity of both the 9-aminoacridine- and amsacrine-4-carboxamides remains to be determined.     

Predicted mode of intercalation of doxorubicin with dinucleotide dimers

Intermolecular molecular mechanics energy calculations have been carried out for doxorubicin interacting with two dinucleotide dimer sequences. The preferred mode of intercalation is in the minor groove with the anthraquinone ring of the drug nearly perpendicular to the base pairs for the (CpG) sequence having alternate C3′ endo-C2′ endo sugar ring puckering. The preferred intercalation conformation of the drug is nearly identical to the N-bromacetyldaunomycin crystal structure. This prediction is qualitatively consistent with the recently reported crystal structure of a d(CpGpCpGpCpG) dimer-daunomycin complex. For the other dinucleotide sequence, (TpC-ApG), minor groove intercalation is also preferred, but the drug conformation can be changed.     

Interactions of DNA binding ligands with PNA-DNA hybrids.

The interactions of two representative mixed-sequence (one with an AT-stretch) PNA-DNA duplexes (10 or 15 base-pairs) and a PNA2/DNA triplex with the DNA binding reagents distamycin A, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, 8-methoxy-psoralen and the delta and lambda enantiomers of Ru(phen)2-dppz2+ have been investigated using optical spectroscopic methods. The behaviour of these reagents versus two PNA-PNA duplexes has also been investigated. With triple helical poly(dA)/(H-T10-Lys-NH2)2 no significant intercalative binding was detected for any of the DNA intercalators, whereas DAPI, a DNA minor groove binder, was found to exhibit a circular dichroism with a positive sign and amplitude consistent with minor groove binding. Similarly, a PNA-DNA duplex containing a central AATA motif, a typical minor groove binding site for the DNA minor groove binders distamycin A and DAPI, showed binding for both of these drugs, though with strongly reduced affinity. No important interactions were found for any of the ligands with a PNA-DNA duplex consisting of a ten base-pair mixed purine-pyrimidine sequence with only two AT base-pairs in the centre. Nor did any of the ligands show any detectable binding to the PNA-PNA duplexes (one containing an AATT motif). Various PNA derivatives with extentions of the backbone, believed to increase the flexibility of the duplex to opening of an intercalation slot, were tested for intercalation of ethidium bromide or 8-methoxypsoralen into the mixed sequence PNA-DNA duplex, however, without any observation of improved binding. The importance of the ionic contribution of the deoxyribose phosphate backbone, versus interactions with the nucleobases, for drug binding to DNA is discussed in the light of these findings.