Nonintercalative DNA-binding antitumour compounds

Summary A family of compounds which appear to bind reversibly to double stranded DNA without intercalation between DNA base pairs has been defined. Methods are described by which this non-intercalative binding can be characterised using ultraviolet spectrometry, fluorimetry with ethidium as a probe, viscometry and other hydrodynamic techniques, circular dichroism and nuclear magnetic resonance spectrometry. Antibiotics which fall into this family include the antibiotics distamycin A, netropsin, mithramycin, chromomycin and olivomycin. Synthetic antitumour agents include diarylamidines such as berenil, phthalanilides, aromatic bisguanylhydrazones and bisquaternary ammonium heterocycles. A survey has been made of the general requirements of this family of compounds for DNA binding and biological activity. Binding of drugs to the minor groove of the DNA double helix appears to be the most likely mechanism for the antitumour action of these compounds.     

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.     

Investigation of the dark interaction between furocoumarins and DNA

The complexes between some furocoumarins and DNA have been studied using various physicochemical techniques. Flow-dichroism measurements data strongly support the intercalation of the planar furocoumarin molecules between two base pairs of duplex DNA. The equilibrium dialysis and spectrophotometric data show relatively low values of the association constants of the complexes and a small number of molecules able to intercalate in DNA, thus indicating that furocoumarins have a relatively low affinity for DNA in the complex formation. The biological and photobiological consequences connected with these results are discussed.The binding curves obtained using some polynucleotides and various DNA samples having different composition with regard to base pairs, have shown that the regions of the macromolecule having alternate sequences of purine and pyrimidine represent sites useful for intercalation. No preference has been observed for A-T or G-C.     

Targeting DNA with novel diphenylcarbazoles

Double-stranded DNA is a therapeutic target for a variety of anticancer and antimicrobial drugs. Noncovalent interactions of small molecules with DNA usually occur via intercalation of planar compounds between adjacent base pairs or minor-groove recognition by extended crescent-shaped ligands. However, the dynamic and flexibility of the DNA platform provide a variety of conformations that can be targeted by structurally diverse compounds. Here, we propose a novel DNA-binding template for construction of new therapeutic candidates. Four bisphenylcarbazole derivatives, derived from the combined molecular architectures of known antitumor bisphenylbenzimidazoles and anti-infectious dicationic carbazoles, have been designed, and their interaction with DNA has been studied by a combination of biochemical and biophysical methods. The substitutions of the bisphenylcarbazole core with two terminal dimethylaminoalkoxy side chains strongly promote the interaction with DNA, to prevent the heat denaturation of the double helix. The deletion or the replacement of the dimethylamino-terminal groups with hydroxyl groups strongly decreased DNA interaction, and the addition of a third cationic side chain on the carbazole nitrogen reinforced the affinity of the compound for DNA. Although the bi- and tridentate molecules both derive from well-characterized DNA minor-groove binders, the analysis of their binding mode by means of circular and linear dichroism methods suggests that these compounds form intercalation complexes with DNA. Negative-reduced dichroism signals were recorded in the presence of natural DNA and synthetic AT and GC polynucleotides. The intercalation hypothesis was validated by unwinding experiments using topoisomerase I. Prominent gel shifts were observed with the di- and trisubstituted bisphenylcarbazoles but not with the uncharged analogues. These observations, together with the documented stacking properties of such molecules (components for liquid crystals), prompted us to investigate their binding to the human telomeric DNA sequence by means of biosensor surface plasmon resonance. Under conditions favorable to G4 formation, the title compounds showed only a modest interaction with the telomeric quadruplex sequence, comparable to that measured with a double-stranded oligonucleotide. Their sequence preference was explored by DNase I footprinting experiments from which we identified a composite set of binding sequences comprising short AT stretches and a few other mixed AT/GC blocks with no special AT character. The variety of the binding sequences possibly reflects the coexistence of distinct positioning of the chromophore in the intercalation sites. The bisphenylcarbazole unit represents an original pharmacophore for DNA recognition. Its branched structure, with two or three arms suitable to introduce a structural diversity, provides an interesting scaffold to built molecules susceptible to discriminate between the different conformations of nucleic acids.     

Binding of protoberberine alkaloid coralyne with double stranded poly(A): a biophysical study

Recognition of double stranded ribonucleic acid is a critical event in many biological pathways such as trafficking, editing and maturation of mRNA, interferon antiviral response and RNA interference. In the context of probing double stranded RNA binding small molecules, the interaction of the antitumor protoberberine alkaloid coralyne with double stranded poly(A) has been studied by various biophysical techniques. Typical hypochromic and bathochromic shifts in the absorption spectrum and appreciable quenching of the intrinsic fluorescence of coralyne indicated the strong affinity of coralyne to poly(A). The corresponding intrinsic binding constant evaluated from Scatchard analysis was in the order of 10(5) M(-1). The strong binding was further characterized by significant polarization of the alkaloid fluorescence and stabilization of poly(A) helix against thermal strand separation. The binding process was manifested by remarkable perturbation of the intrinsic circular dichroic spectrum of poly(A) with concomitant generation of optical activity in the bound alkaloid molecules that are otherwise achiral. Job plot analysis showed the binding stoichiometry of the interaction process to be two base pairs per alkaloid molecule. The energetics of the strong interaction was studied by isothermal titration and differential scanning calorimetric techniques that suggested the binding to be exothermic and favoured by both negative enthalpy and positive entropy changes. All these results, together with the Stern-Volmer quenching experiment in fluorescence, revealed the molecular details of the intercalation of coralyne into poly(A) duplex leading to its potential use as an agent in gene regulation in eukaryotic cells.     

Discriminating small molecule DNA binding modes by single molecule force spectroscopy

Drugs may interact with double stranded DNA via a variety of binding modes, each mode giving rise to a specific pharmacological function. Here we demonstrate the ability of single molecule force spectroscopy to discriminate between different interaction modes by measuring the mechanical properties of DNA and their modulation upon the binding of small molecules. Due to the unique topology of double stranded DNA and due to its base pair stacking pattern, DNA undergoes several well-characterised structural transitions upon stretching. We show that small molecule binding markedly affects these transitions in ways characteristic to the binding mode and that these effects can be detected at the level of an individual molecule. The minor groove binder berenil, the crosslinker cisplatin and the intercalator ethidium bromide are compared.     

Cloning of chicken embryo tRNA genes using single stranded nucleosomal DNA highly enriched for tRNA complementary sequences

DNA from chicken embryo nucleosome tetramers (about 760 base pairs in size) was enriched for tRNA genes by RPC-5 chromatography. The enriched DNA was hybridized with chicken embryo total tRNA and the hybridized DNA isolated utilizing a) avidinbiotin interaction, b) diazobenzyloxymethyl paper, and c) high temperature RPC-5 chromatography. The obtained single stranded DNA highly enriched for tRNA complementary sequences was hybridized with total DNA from nucleosome monomers (140--190 base pairs in size) and the excess of non hybridized monomer nucleosome DNA removed by Sepharose 4B chromatography. The hybrid molecules obtained were made fully double stranded by incubation with E. coli DNA polymerase I, DNA ligase, and exonuclease III. DNA was inserted into plasmid pBR322 by G-C joining procedure and the recombinant DNA used to transform the E. coli strain chi 1776. More than 70% of the transformants obtained hybridize to chicken embryo total tRNA.     

How much pi-stacking do DNA termini seek? Solution structure of a self-complementary DNA hexamer with trimethoxystilbenes capping the terminal base pairs

The exposed terminal base pairs of DNA duplexes are nonclassical binding sites for small molecules. Instead, small molecules usually prefer intercalation or minor groove binding. Here we report the solution structure of the DNA duplex (TMS-TGCGCA)(2), where TMS denotes trimethoxystilbene carboxamides that are 5'-tethered to the DNA. The stilbenes, for which intercalation is conformationally accessible, stack on the terminal T:A base pairs of an undisturbed B-form duplex. Two conformations, differing by the orientation of the stilbene relative to the terminal base pair, are observed, indicating that the flip rate is slow for the pi-stacked aromatic ring system. The trimethoxystilbene is known to greatly increase base pairing fidelity at the terminus. Here we show that it gauges the size of the T:A base pair by embracing the 2'-methylene group of the terminal dA residue of the unmodified terminus with its methoxy "arms", but that it does not engage the entire base pair in pi-stacking. Mismatched base pairs with their altered geometry will not allow for the same embracing interaction. On the basis of the current structure, a trimethoxychrysene carboxamide is proposed as a ligand with increased pi-stacking surface and possible applications as improved fidelity-enhancing element.     

A spectroscopic and surface plasmon resonance study of oleic acid/DNA complexes

The interaction of synthetic polynucleotide double strands with a natural lipid, oleic acid, was examined in diluted aqueous solutions by circular dichroism spectra, UV-absorption measurements, and surface plasmon resonance biosensor investigations. The investigations were performed with defined double and triple stranded oligo- and polydeoxyribonucleotides. Whereas duplexes are influenced by oleic acid ligandation, which could not be removed by ethanol dialysis procedure, no binding occurs to triple stranded DNA. The spectroscopic results indicate that oleic acid shows molecular recognition to AT b.p. motifs by groove binding. GC tracts - in particular alternating d[G-C] motifs - are strongly influenced by ligand interaction up to a ratio of one molecule per two base pairs. Likewise, the spectroscopic and morphologic changes in the supramolecular association of the complexes after treatment occur even after dialysis procedure. This was monitored with scanning force microscopy (SFM) as well. Additionally, monolayers of biotinylated DNA duplexes were immobilized on a streptavidin sensor-layer for surface plasmon resonance (SPR) observations. Small portions of the ligand were injected in continuous flow. Loosely bound molecules were removed by washing procedure. Injections of sodium hydroxide denature the DNA, releasing the tightly bound effectors. The amount of tightly bound oleic acid molecules was determined at one molecule per 2-3 base pairs. As consequence, a new mechanism of regulation of gene expression at nuclear membrane or by lipids inside DNA double helix has to be discussed.     

Ru(phen)2DPPZ]2+ is in contact with DNA bases when it forms a luminescent complex with single-stranded oligonucleotides

The luminescence intensity of the Delta- and Lambda-enantiomer of [Ru(phen)2DPPZ]2+ ([Ru(phenanthroline)2 dipyrido[3,2-a:2',3'-c]phenazine]2+) complex enhanced upon binding to double stranded DNA, which has been known as "light switch effect". The enhancement of the luminescence required the intercalation of the large ligand between DNA base pairs. In this study, we report the enhancement in the luminescence intensity when the metal complexes bind to single stranded oligonucleotides, indicating that the "light switch effect" does not require intercalation of the large DPPZ ligand. Oligonucleotides may provide a hydrophobic cavity for the [Ru(phen)2DPPZ]2+ complex to prevent the quenching by the water molecule. In the cavity, the metal complex is in contact with DNA bases as is evidenced by the observation that the excited energy of the DNA bases transfer to the bound metal complex. However, the contact of the metal complex with DNA bases is different from the stacking of DPPZ in the intercalation pocket. In addition to the normal two luminescence lifetimes, a short lifetime in the range of 1-2 ns was found for both the delta- and lambda-enantiomer of [Ru(phen)2DPPZ]2+ when complexed with single stranded oligonucleotides, which may be assigned to the metal complex that is outside of the cavity, interacting with phosphate groups of DNA.     

Spatial translational motions of base pairs in DNA molecules: Application of the extended matrix generator method

We have used the elementary generator matrices outlined in the preceding paper to examine the conformational plasticity of the nucleic acid double helix. Here we investigate kinked DNA structures made up of alternating B- and A-type helices and intrinsically curved duplexes perturbed by the intercalation of ligands. We model the B-to-A transition by the lateral translation of adjacent base pairs, and the intercalation of ligands by the vertical displacement of neighboring residues. We report a complete set of average configuration-dependent parameters, ranging from scalars (i.e., persistence lengths) to first- and second-order tensor parameters (i.e., average second moments of inertia), as well as approximations of the associated spatial distributions of the DNA and their angular correlations. The average structures of short chains (of lengths less than 100 base pairs) with local kinks or intrinsically curved sequences are essentially rigid rods. At the smallest chain lengths (10 base pairs), the kinked and curved chains exhibit similar average properties, although they are structurally perturbed compared to the standard B-DNA duplex. In contrast, at lengths of 200 base pairs, the curved and kinked chains are more compact on average and are located in a different space from the standard B- or A-DNA helix. While A-DNA is shorter and thicker than B-DNA in x-ray models, the long flexible A-DNA helix is thinner and more extended on average than its B-DNA counterpart because of more limited fluctuations in local structure. Curved polymers of 50 base pairs or longer also show significantly greater asymmetry than other DNAs (in terms of the distribution of base pairs with respect to the center of gravity of the chain). The intercalation of drugs in the curved DNA straightens and extends the smoothly deformed template. The dimensions of the average ellipsoidal boundaries defining the configurations of the intercalated polymers are roughly double those of the intrinsically curved chain. The altered proportions and orientations of these density functions reflect the changing shape and flexibility of the double helix. The calculations shed new light on the possible structural role of short A-DNA fragments in long B-type duplexes and also offer a model for understanding how GC-specific intercalative ligands can straighten naturally curved DNA. The mechanism is not immediately obvious from current models of DNA curvature, which attribute the bending of the chain to a perturbed structure in repeating tracts of A · T base pairs. © 1994 John Wiley & Sons, Inc.     

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.     

Combinatorial determination of sequence specificity for nanomolar DNA-binding hairpin polyamides

Development of sequence-specific DNA-binding drugs is an important pharmacological goal, given the fact that numerous existing DNA-directed chemotherapeutic drugs rely on the strength and selectivity of their DNA interactions for therapeutic activity. Among the DNA-binding antibiotics, hairpin polyamides represent the only class of small molecules that can practically bind any predetermined DNA sequence. DNA recognition by these ligands depends on their side-by-side amino acid pairings in the DNA minor groove. Extensive studies have revealed that these molecules show extremely high affinity for sequence-directed, minor groove interaction. However, the specificity of such interactions in the presence of a large selection of sequences such as the human genome is not known. We used the combinatorial selection method restriction endonuclease protection, selection, and amplification (REPSA) to determine the DNA binding specificity of two hairpin polyamides, ImPyPyPy-gamma-PyPyPyPy-beta-Dp and ImPyPyPy-gamma-ImPyPyPy-beta-Dp, in the presence of more than 134 million different sequences. These were verified by restriction endonuclease protection assays and DNase I footprinting analysis. Our data showed that both hairpin polyamides preferentially selected DNA sequences having consensus recognition sites as defined by the Dervan pairing rules. These consensus sequences were rather degenerate, as expected, given that the stacked pyrrole-pyrrole amino acid pairs present in both polyamides are unable to discriminate between A.T and T.A base pairs. However, no individual sequence within these degenerate consensus sequences was preferentially selected by REPSA, indicating that these hairpin polyamides are truly consensus-specific DNA-binding ligands. We also discovered a preference for overlapping consensus binding sites among the sequences selected by the hairpin polyamide ImPyPyPy-gamma-PyPyPyPy-beta-Dp, and confirmed by DNase I footprinting that these complex sites provide higher binding affinity. These data suggest that multiple hairpin polyamides can cooperatively bind to their highest-affinity sites.     

DNA Intercalation and Photoinduced Cleavage by 4-Nitro(N-Hexylamine)1,8-Naphthalimide

Abstract

A novel intercalator, 4-nitro(N-hexylamine)1,8-naphthalimide, was synthesised and its DNA binding and photoinduced DNA cleavage properties were studied. The DNA unwinding results show that it binds through intercalation. Absorption and fluorescence spectroscopy reveal the preference for A/T base pairs as compared to G/C base pairs for the binding. The intercalator produces photoinduced single strand scissions in double helical DNA.     

Binding of 4'-aminomethyl 4,5',8-trimethyl psoralen to DNA, RNA and protein in HeLa cells and Drosophila cells.

In Drosophila cells and HeLa cells treated with 4'-aminomethyl trioxsalen and ultraviolet light, this compound binds covalently to DNA and RNA. The maximum number of molecules bound to 10(3) base pairs in DNA is 60 and in RNA it is 20. In nuclei treated likewise the number of molecules bound to 10(3) base pairs in DNA can be as high as 376. When cells are irradiated in the frozen state the number of 4'-aminomethyl trioxsalen molecules bound per 10(3) base pairs in DNA is about 40 and in RNA about 20. DNA molecules from cells or nuclei treated with 4'-aminomethyl trioxsalen and ultraviolet light are highly crosslinked and appear as loops interspersed by double stranded regions when analyzed in the electron microscope under denaturing conditions. The loop sizes are heterogeneous and the fraction of double stranded regions increases to almost complete double-strandedness at high degrees of reaction. No secondary structures could be found in ribosomal RNA from Drosophila cells or HeLa cells after treatment with 4'-aminomethyl trioxsalen and ultraviolet light. In cells treated with 4'-aminomethyl trioxsalen and ultraviolet light the RNAase activity is increased considerably suggesting a release of lysosomal enzymes. 4'-aminomethyl trioxsalen and its photodecomposition products bind strongly to cellular proteins.     

A full-automatic sequence design algorithm for branched DNA structures

Production of various structures by self-assembling single stranded DNA molecules is a widely used technology in the filed of DNA nanotechnology. Base sequences of single strands do predict the shape of the resulting nanostructure. Therefore, sequence design is crucial for the successful structure fabrication. This paper presents a sequence design algorithm based on mismatch minimization that can be applied to every desired DNA structure. With this algorithm, junctions, loops, single as well as double stranded regions, and very large structures up to several thousand base pairs can be handled. Thereby, the algorithm is fast for the most structures. Algorithm is Java-implemented. Its implementation is called Seed and is available publicly. As an example for a successful sequence generation, this paper presents the fabrication of DNA chain molecules consisting of double-crossover (DX) tiles as well.     

A Full-automatic Sequence Design Algorithm for Branched DNA Structures

Abstract

Production of various structures by self-assembling single stranded DNA molecules is a widely used technology in the filed of DNA nanotechnology. Base sequences of single strands do predict the shape of the resulting nanostructure. Therefore, sequence design is crucial for the successful structure fabrication. This paper presents a sequence design algorithm based on mismatch minimization that can be applied to every desired DNA structure. With this algorithm, junctions, loops, single as well as double stranded regions, and very large structures up to several thousand base pairs can be handled. Thereby, the algorithm is fast for the most structures. Algorithm is Java-implemented. Its implementation is called Seed and is available publicly. As an example for a successful sequence generation, this paper presents the fabrication of DNA chain molecules consisting of double-crossover (DX) tiles as well.     

Zn2+ selectively stabilizes FdU-substituted DNA through a unique major groove binding motif

We report, based on semi-empirical calculations, that Zn(2+) binds duplex DNA containing consecutive FdU-dA base pairs in the major groove with distorted trigonal bipyramidal geometry. In this previously uncharacterized binding motif, O4 and F5 on consecutive FdU are axial ligands while three water molecules complete the coordination sphere. NMR spectroscopy confirmed Zn(2+) complexation occurred with maintenance of base pairing while a slight hypsochromic shift in circular dichroism (CD) spectra indicated moderate structural distortion relative to B-form DNA. Zn(2+) complexation inhibited ethidium bromide (EtBr) intercalation and stabilized FdU-substituted duplex DNA (ΔT(m) > 15 °C). Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect. DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+). A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.