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PubMed1983

Fragile X chromosome and chromosome condensation.

The effect of chromosome condensation on the frequency of expression of the fragile X chromosome was examined. Chromosome decondensation substances were tested for their ability to elicit expression or improve frequencies of expression of the fragile X chromosome in five patients. The substances tested included the AT specific DNA ligands ethidium bromide, Hoechst 33258, and netropsin, and the GC specific substances actinomycin D and olivomycin. Under culture conditions appropriate for eliciting fragile X expression none of the decondensation compounds studied significantly altered frequencies of expression, nor did any of the substances elicit fragile X expression under conditions that normally suppress fragile X expression. The fragile X was found to be more frequently evident in less condensed chromosome preparations from fibroblasts. The implications of these findings with respect to the nature of fragile sites are discussed.

Aged↗
PubMed1995

Influence of the methyl substituents of a thiazole-containing lexitropsin on the mode of binding to DNA.

We have studied the DNA-binding properties of two thiazole-containing analogues of netropsin which differ by the absence (Thia-Nt) or presence (Methia-Nt) of methyl groups on the thiazole rings. The mode of binding to DNA of the two lexitropsins was investigated by circular dichroism, electric linear dichroism and viscosity measurements. The spectroscopic and hydrodynamic results indicate that the non-methylated lexitropsin binds to the minor groove of DNA, whereas the methylated analogue behaves as an intercalator. Our findings led to the notion that the methyl substituents on the thiazole rings might play a significant part in the intercalation process.

Animals↗
PubMed1994

DNA-binding properties of nitroarene oligopeptides designed as hypoxia-selective agents.

The DNA-binding properties of six candidate nitroarene-functionalized oligopeptides 8-13 (i.e. 5-nitrofuran, 2-nitroimidazole and 4-nitrobenzene derivatives) with potential hypoxia-selective activity, developed as analogues of the archetypal minor groove-binding ligands, netropsin 1 and distamycin 2, have been examined using an extended molecular mechanics and dynamics (MM/MD) modelling approach. The energies calculated for interaction with the d(CGCGAATTCGCG) duplex, or d(CGCAAATTTGCG)2 in the case of the elongated nitrobenzene 13, correlate with fluorimetric data obtained by indirect competitive displacement of ethidium bromide from double-stranded calf thymus DNA. The results suggest that the mode of interaction for these agents resembles the behavior of structurally unrelated bis(amidine) derivatives that also bind to AT-rich stretches of duplex B-DNA via the minor groove. Analysis of the binding behaviour indicates that the interactions are determined by enthalpic rather than entropic energy terms, suggesting that specific or differential hydration effects for the DNA and ligand may be unimportant. A rational structure-activity relationship is established for the interaction of this family of oligopeptides with DNA that also encompass the bis(amidine) class of ligands.

Animals↗
PubMed1994

Psoralen--lexitropsin hybrids: DNA sequence selectivity of photoinduced cross-linking from MPE footprinting and exonuclease III stop assay, and mode of binding from electric linear dichroism.

The properties of certain hybrids 3 and 5 bearing a photoactivatable psoralen group attached to DNA sequence recognizing lexitropsin carriers have been examined. The hybrids bind to poly(dA-dT) with Kapp of 2.8 and 0.9 x 10(7) M-1, i.e. greater than or equal to that of netropsin (Kapp = 1.0 x 10(7) M-1), indicating that the psoralen moiety may contribute to binding in the case of 5. Photoinduced cross-linking of DNA by 3 and 5, while efficient, is less so than that of individual psoralens and reaches a maximum at a ligand to DNA base pair ratio (r) of 0.2. Complementary strand methidium-propyl-EDTA (MPE).Fe(II) footprinting demonstrated that, in the dark, the sequence preferential recognition of hybrids 3 and 5 is dominated by the lexitropsin moiety. Examination of 360 nm photoinduced DNA cross-linking by the hybrids 3 and 5 was carried out using an exonuclease III stop assay. This revealed that > 95% of the DNA remained double stranded, indicating that 3 and 5 generate primarily biadducts at AT-rich sequences. This assay also located individual monoadduct sites, some of which are remote from the dominant cross-linked sites. When the samples were exposed to 254 nm UV light before loading onto the gel to reverse the photoproducts, the pattern of the exonuclease III stop bands was not altered significantly compared with the experiment without 254 nm irradiation. It is concluded that these termination sites include both mono- and biadducts. Electric linear dichroism examination of the DNA complexes of hybrids 3 and 5 (without light activation) provides evidence that the lexitropsin portion binds in the minor groove, while the psoralen portion intercalates in a suitably located site for subsequent photoinduced cross-linking.

Antineoplastic Agents↗
PubMedDate not supplied

[Circular dichroism of DNA complexes with dyes. III. Effect of latent optical activity and the structure of the complexes].

Circular dichroism anisotropy was studied both theoretically and experimentally for the complexes of DNA and dsRNA with dyes (proflavine, 2,7-di-t-butyl proflavine, "Hoechst-33258") and antibiotics (distamycin A, netropsin and olivomycin). Theoretical analysis showed that general features of CD anisotropy, revealed in the previous studies (CD components--delta epsilon parallel to and delta epsilon perpendicular--are ten times or more bigger than the CD-effect without orientation, and delta epsilon parallel to approximately 2 delta epsilon perpendicular) are due to the existence of a specific effect named "latent" optical activity (LOA). This effect can be observed in many cases of non-chiral symmetrical chromophores if they are oriented. The effect is due to the excitation of an electrical dipole transition and a perpendicular magnetic dipole transition (or quadrupole transition) of a molecule. The amplitude and the sign of the LOA-effect depends on the orientation of the chromophores with respect to the light beam; with a random orientation the mutual compensation of LOA-effects of different chromophores happens and no LOA-effect appears. The analitycal expressions relating the value of LOA-effect of the system with electronical characteristics of the chromophores and the geometrical parametra of their arrangement was obtained. The experimental data obtained for the oriented complexes of DNA and dsRNA with proflavine made it possible to determine an angle between the chromophore and the plane perpendicular to the DNA axis--gamma. For the calf thymus DNA gamma = = + 1.8 +/- 0.4 degrees, for the phage T2 DNA gamma = + 2.2 +/- 0.4 degrees, and for phage f2 dsRNA gamma=--3.5 +/- +/- 0.5 degrees. These results, obtained at relatively low concentrations of the bound proflavine (r approximately 0.01), are in accordance with the intercalating mode of the dye binding. A study of CD anisotropy of DNA complexes with other ligands showed that many different chromophores possess LOA-effect. This phenomenon can be used to obtain both spectroscopic and structural information about the systems similar to those reported here.

Acridines↗
PubMed1996

Characterizing the DNA binding modes of a topoisomerase I-poisoning terbenzimidazole: evidence for both intercalative and minor groove binding properties.

We have used a broad range of spectroscopic and viscometric techniques to demonstrate that the complexation of a cytotoxic, topoisomerase I-poisoning terbenzimidazole (5PTB) with the poly(dA).poly(dT) duplex exhibits properties characteristic of both intercalation and minor groove binding. Our results reveal the following features: (i) Optical melting profiles reveal that 5PTB binding enhances the thermal stability of the poly(dA).poly(dT) duplex; (ii) Fluorescence-detected 5PTB binding to the poly(dA).poly(dT) duplex reveals four apparent "site sizes," ranging from 1 to 13 base pairs (bp) per bound drug; (iii) Flow linear dichroism data suggest conformational heterogeneity among the poly(dA).poly(dT)-bound 5PTB molecules, with substantial contributions from drug molecules bound in the minor groove; (iv) Fluorescence resonance energy transfer data reveal properties characteristic of a significant contribution from an intercalative mode of binding; (v) Viscometric, fluorescence quenching, and netropsin competition data are consistent with 5PTB binding to poly(dA).poly(dT) by "mixed" modes, which are operationally defined as single or multiple binding populations that individually and/or collectively express both intercalative and minor groove binding properties. We comment on a potential correlation between drugs that exhibit such "mixed" mode binding motifs and those that express antineoplastic activity through inhibition of topoisomerase I.

Benzimidazoles↗
PubMed1997

Photoreaction of furocoumarins with DNA is similarly inhibited by minor and major groove-interacting ligands.

It was found that methyl green, a major groove binding ligand and the minor groove binding ligands, netropsin and 2,7-di-tert-butylproflavine inhibit, to a similar extend a monoadduct forming benzopsoralen and monoadduct and diadduct forming derivatives of psoralen (8-methoxypsoralen and 3,4'-dimethyl-8-methoxypsoralen). Caffeine exhibits an inhibitory effect on furocoumarin photobinding to DNA at 10(3) fold higher concentration. Together with the previously published results it is concluded that both occupancy of the major and minor groove as well as intercalation hinder photobinding of furocoumarins to DNA.

Animals↗
PubMed1998

Effect of DNA-interacting drugs on phage T7 RNA polymerase.

9-Aminoacridine carboxamide derivatives studied here form with DNA intercalative complexes which differ in the kinetics of dissociation. Inhibition of total RNA synthesis catalyzed by phage T7 and Escherichia coli DNA-dependent RNA polymerases correlates with the formation of slowly dissociating acridine-DNA complex of time constant of 0.4-2.3 s. Their effect on RNA synthesis is compared with other ligands which form with DNA stable complexes of different steric properties. T7 RNA polymerase is more sensitive to distamycin A and netropsin than the E. coli enzyme while less sensitive to actinomycin D. Actinomycin induces terminations in the transcript synthesized by T7 RNA polymerase. Despite low dissociation rates of DNA complexes with acridines and pyrrole antibiotics no drug dependent terminations are observed with these ligands.

Amides↗
PubMed1978

Spore coat protein of Bacillus subtilis. Structure and precursor synthesis.

The coat protein of Bacillus subtilis spores comprises about 10% of the total dry weight of spores and 25% of the total spore protein. One protein with a molecular weight of 13,000 to 15,000 comprises a major portion of the spore coat. This mature spore coat protein has histidine at its NH2 terminus and is relatively rich in hydrophobic amino acids. Netropsin, and antibiotic which binds to A-T-rich regions of DNA and inhibits sporulation, but not growth, decreased the synthesis of this spore coat protein by 75%. A precursor spore coat protein with a molecular weight of 25,000 is made initially at t1 of sporulation and is converted to the mature spore coat protein with a molecular weight of 13,500 at t2 - t3. These data indicate that the spore coat protein gene is expressed very early in sporulation prior to the modifications of RNA polymerase which have been noted.

Amino Acids↗
PubMed1989

Interactions of nucleic acids with distamycins. The drug monomeric chromophore.

A study of the monomeric chromophore of the oligopeptides netropsin (1), distamycin III (2), and distamycin V (3) by polarization spectroscopy techniques and molecular orbital calculations is reported. Linear dichroism spectra of the monomeric model compounds 1-methyl-2(ethylcarbamoyl)-4-acetamido-pyrrole (4) and 1-methyl-2(ethylcarbamoyl)-pyrrole (5) dissolved and oriented in lyotropic and thermotropic liquid crystals provide, together with the magnetic CD spectra, experimental checks of the theoretical calculations. The polarization directions of the investigated transition obtained by these means in this study allow us to build up in the following paper the exciton states of (1)-(3) and these provide a stereochemical interpretation of the flow linear dichroism spectra of the complexes of DNA with (2) and (3).

DNA↗
PubMed1980

Interactions between pairs of DNA-binding dyes: results and implications of chromosome analysis.

A number of DNA-binding dyes, with spectral properties making them suitable as components of energy donor-acceptor pairs, are described. If such pairs are used to stain metaphase chromosomes, and if the energy acceptor (e.g., actinomycin D or methyl green) has a binding specificity opposite to the binding or fluorescence specificity of the donor (e.g., 33258 Hoechst, quinacrine or chromomycin A3), contrast in donor fluorescence can be enhanced, leading to patterns selectively highlighting standard or reverse chromosome bands or particular polymorphic regions. Such results presumably reflect chromosomal regions enriched in 10-20 base pair clusters to which the donor binds and fluoresces but to which the acceptor cannot bind. For other pairs, involving counterstains such as netropsin or echinomycin, which are not suitable as energy acceptors, specific changes observed in polymorphic region fluorescence are most likely due to binding competition between dyes. Dye pairs producing contrast by either method can be used to differentiate between homologous chromosomes or to facilitate detection of specific chromosomal rearrangements. Preliminary data indicate that contrast enhancement generated in fixed metaphase chromosomes spread on microscopic slides can also be observed in suspensions of unfixed metaphase chromosomes, reinforcing the expectation that the methodology described will be of use in flow cytometry.

Animals↗
PubMed1984

Counterstaining human chromosomes for flow karyology.

Isolated human metaphase chromosomes stained with the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) and chromomycin A3(CA3), and counterstained with nonfluorescent netropsin (NTR), have been analyzed by dual-laser flow cytometry. Counterstaining with NTR reduces DAPI fluorescence except at regions on chromosomes 1,9,15,16, and Y, corresponding to C-band heterochromatin. Bivariate flow karyology of human chromosomes treated with this triple-stain combination resolves chromosomes 1,9, and Y distinctly from the remaining chromosomes and resolves variations between chromosome homologues not detected by staining with propidium iodide (PI) or with the double stain combination Hoechst 33258(HO) and CA3.

Cells, Cultured↗
PubMed1985

Identification of inverted duplicated #15 chromosomes using bivariate flow cytometric analysis.

A dual laser FACS IV cell sorter has been used to obtain bivariate flow histograms of human metaphase chromosomes stained with the DNA-specific dyes, 33258 Hoechst and chromomycin A3. Approximately twenty distinct chromosomal fluorescence populations can be resolved using this double staining technique and the flow cytometer which has been modified only by the substitution of a specially designed air-spaced achromat for the standard focusing lens. Metaphase chromosomes from two different cell lines bearing inverted duplicated #15 autosomes have been subjected to bivariate chromosome analysis. In both cases, the inverted duplicated #15 chromosomes have been identified in the bivariate flow histogram. This identification was supported by experiments in which doubly stained chromosomes were counterstained with either netropsin or distamycin A, resulting in a relative increase in the 33258 Hoechst fluorescence intensity of the structurally abnormal #15 chromosomes, compared with the other chromosomes, as predicted by cytological studies. The possibility of identifying and separating small abnormal autosomes using commercially available instrumentation should facilitate the use of recombinant DNA techniques for the construction of libraries which are highly enriched for DNA sequences from limited autosomal subregions important in the study of chromosomal abnormalities such as deletions, translocations and inversion duplications.

Bisbenzimidazole↗
PubMed1989

SPKK, a new nucleic acid-binding unit of protein found in histone.

A new DNA-binding unit of a protein different from the alpha-helix, the beta-sheet and the Zn-finger is proposed based on the analysis of the structure of the N-terminus of sea urchin spermatogenous histone H1. DNA-binding arms of the sea urchin spermatogenous histones, H1 and H2B, are composed of repeats of Ser-Pro-Lys(Arg)-Lys(Arg) (SPKK) residues. A six-times repeat of SPKK (S6 peptide) was isolated from H1 and the competition of S6 for DNA binding with a DNA-binding dye, Hoechst 33258, was analysed. The S6 peptide is shown to be a competitive inhibitor of Hoechst 33258, and it is concluded that the SPKK repeat binds to DNA in its minor groove with a binding constant, KS6 = 1.67 X 10(10) M-1. The circular dichroism (CD) spectrum of a synthetic peptide, SPRKSPRK (S2 peptide), is quite different from those of both the alpha-helix and the beta-sheet and resembles that of a random coil. From statistical consideration of protein structures it is proposed that SPKK forms a compact beta-turn stabilized by an additional hydrogen bond. Since a repeated chain of such turn of SPKK offers a repeat of amides of Ser residues at a distance similar to that of DNA-binding amides of the drugs, Hoechst 33258 and netropsin, and since the amides of these drugs bind to DNA replacing the spine of hydration in a minor groove, it is proposed that a repeat of SPKK binds to DNA in the minor groove using similar hydrogen bonds.

Amino Acid Sequence↗
PubMed1992

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.

Base Sequence↗
PubMed1994

DNA recognition by intercalators and hybrid molecules.

Experiments are described which probe the role of the 2-amino group of guanine as a critical determinant of the recognition of nucleotide sequences in DNA by specific ligands. Homologous samples of tyrT DNA substituted with inosine or 2,6-diaminopurine residues in place of guanosine or adenine respectively yield characteristically modified footprinting patterns when challenged with sequence-selective antibiotics such as echinomycin, actinomycin or netropsin. The capacity of small molecules to recognise particular DNA sequences is exploited in the 'combilexin' strategy to target small molecules to defined sites in DNA. A composite molecule containing a distamycin moiety linked to an intercalating ellipticine derivative has been synthesised and shown to bind tightly to DNA but without much sequence-selectivity. Refinement of this molecule based on predictions from molecular modelling has led to the synthesis of a second generation derivative bearing an additional positive charge: this new hybrid molecule is strongly selective for binding to AT-rich tracts in DNA.

Antineoplastic Agents↗
PubMed2006

Stability analysis for double-stranded DNA oligomers and their noncovalent complexes with drugs by laser spray.

Laser spray, which is a newly developed ionization technique, can characterize the stability of noncovalent complexes in the solution phase. By using this advantage, laser spray has been applied to probe the intrinsic stability of double-stranded DNA (dsDNA) sequences and their binding affinities with various drugs in the solution phase. Systematic experiments were carried out using six 16-mer and three 22-mer dsDNA oligomers, together with the complexes of the 16-mer dsDNA with minor groove binders: berenil, Hoechst 33342, DAPI, and netropsin. Dissociation curves for each dsDNA or each complex were plotted as a function of laser power. The laser power (E50%), where 50% of each dsDNA or each complex was dissociated, was compared with its melting temperature (Tm) determined by UV spectroscopy. Linear correlations between E50% and Tm were obtained not only for the dsDNA oligomers (correlation factor r = 0.9835) but also for the 16-mer dsDNA complexes with minor groove binders (r = 0.9966). In addition, laser spray has successfully clarified the binding affinities of a 16-mer dsDNA with two intercalators: daunomycin and nogalamycin. In the case of the dsDNA-daunomycin complex, by changing the molar ratio of dsDNA : drug from 1 : 1 to 1 : 5, the concentration-dependent stability of the complex was confirmed by laser spray. The present results demonstrate that laser spray mass spectrometry can be a powerful and convenient method to investigate the relative binding affinities of dsDNA-ligand complexes in the solution phase, which could be applied to the early stage of high-throughput screening of drugs targeting for dsDNA.

DNA↗
PubMed2005

Combination of solid-phase affinity capture on magnetic beads and mass spectrometry to study non-covalent interactions: example of minor groove binding drugs.

A simple and novel approach was developed to detect non-covalent interactions. It is based on combination of solid-phase affinity capture with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). One of the interacting molecules is bound to magnetic beads and is incubated with the target molecules in solution. The complex bound on the solid support is removed from the solution and transferred for MALDI analysis. Mass spectrometry is used only to detect the target compound, which is far more straightforward than detecting the intact non-covalent complex. To demonstrate the applicability of the method, an AT-rich oligonucleotide (5'-CCCCCAATTCCCCC-3') and its complementary biotinylated sequence (5'-biotin-GGGGGAATTGGGGG-3') were hybridized and immobilized to paramagnetic particles by streptavidin-biotin interaction. The immobilized duplex oligonucleotide was reacted with minor groove binding drugs, Netropsin, Distamycin A, Hoechst 33258 and 4',6-diamidino-2-phenylindole. The resulting DNA-drug complex bound to the particles was separated and analyzed by linear MALDI-TOFMS after washing. Drugs were selectively detected in the spectra. Relative binding strengths were also estimated using competitive complexation.

Biotin↗