1H NMR study of the binding of bis(acridines) to d(AT)5.d(AT)5. 2. Dynamic aspects

Measurements of the 1H NMR spectra and relaxation rates were used to study the dynamic properties of 9-aminoacridine (9AA) and four bis(acridine) complexes with d(AT)5.d(AT)5. The behavior of the 9AA (monointercalator) and that of C8 (bisintercalator containing an eight-carbon atom linker chain) are entirely similar. For both compounds, the lifetime of the drug in a particular binding site is 2-3 ms at approximately 20 degrees C, and neither affects the A.T base pair opening rates. The complex with C10 (bisintercalator containing a 10-carbon atom linker chain) is slightly more stable than the C8 complex since its estimated binding site lifetime is 5-10 ms at 29 degrees C. Base pairs adjacent to the bound C10 are destabilized, relative to free d(AT)5.d(AT)5, but other base pairs in the C10 complex are little affected. Bis(acridine) pyrazole (BAPY) and bis(acridine) spermine (BAS) considerably stabilize those base pairs that are sandwiched between the two acridine chromophores, but in the BAS complex proton exchange from the two flanking base pairs appears to be accelerated, relative to free d(AT)5.d(AT)5. The lifetime of these drugs in specific binding sites is too long (>10 ms) to be manifested in increased line widths, at least up to 41 degrees C. An important conclusion from this study is that certain bisintercalators rapidly migrate along DNA, despite having large binding constants (K>10(6) M-1). For C8 and C10 complexes, migration rates are little different from those deduced for 9AA. The rigid linker chain in BAPY and the charge interactions in BAS retard migration of these two bisintercalators. These results provide new parameters that are useful in understanding the biochemical and biological properties of these and other bisintercalating drugs.     

Removal of DNA curving by DNA ligands: gel electrophoresis study.

The removal of inherent curving in Crithidia fasciculata kinetoplast DNA by various small DNA ligands, groove binders and mono- and bisintercalators, has been studied by gel retardation and electron microscopy. The migration of the kinetoplast DNA fragment is highly retarded during gel electrophoresis. We demonstrate that this retardation is suppressed by DNA ligands such as distamycin and ditercalinium, which have different modes of binding and sequence specificities. Observation by electron microscopy confirms that the effect of ditercalinium on gel migration of curved DNA is linked to DNA uncurving. As the drug is progressively added to DNA, a large broadening of the retarded band is observed during gel electrophoresis for distamycin and ditercalinium. In the case of distamycin, the retarded DNA band splits into two broad bands, whereas the noncurved DNA bands remain homogeneous. This indicates that the drug-DNA exchange is extremely slow in the gel and that a limited number of specific sites on DNA are critical for the removal of bending. GC-specific quinomycin, monointercalators, and bisintercalators act in a manner similar to that of AT-specific distamycin. This indicates that direct drug binding at the dAn tracts is not required for DNA uncurving. We propose that the uncurving of kinetoplast DNA by drugs is caused by a global alteration of DNA structure; subsequent increased flexibility leads to the suppression of rigid bending at the AT tract junctions.     

The mutagenic effects of diacridines and diquinolines in microbial systems

Two series of difunctional DNA-intercalating agents (diacridines and diquinolines) were tested for mutagenic properties in Salmonella typhimurium strain TA1537, and for 'petite' mutagenesis activity in Saccharomyces cerevisiae, and also compared in terms of their structural, lipophilic and DNA-binding properties. Diacridines with only a short chain length were monointercalators, while those with an alkyl linker chain longer than C6 were bisintercalators. Although the bisintercalators especially bound very tightly to DNA, none of these compounds was as effective a frameshift mutagen in TA1537 as the parent chromophore 9-aminoacridine. However, the two (monointercalating) diacridines of shortest chain length were still able to cause frameshifts, and this ability returned (albeit weakly) in the bisintercalators of longest chain length. Although 9-aminoacridine showed no ability for 'petite' mutagenesis, the diacridines of longer chain length were very effective in causing this mitochondrial event. In the quinoline series, both the parent chromophore (4-aminoquinoline) and all the diquinolines were weak monointercalators. None of these compounds showed any ability for frameshift mutagenesis, although some were very weak mitochondrial mutagens. It is concluded that linking two acridines produces compounds whose mutagenic properties might have been predicted from our current knowledge of the parent molecules. However, despite a similar ability to intercalate DNA, the diquinolines show no resemblance to acridines in their mutagenic properties.     

Proton NMR study of the [d(ACGTATACGT)]2-2echinomycin complex: conformational changes between echinomycin binding sites.

The interactions of echinomycin and the DNA decamer [d(ACGTATACGT)]2 were studied by proton NMR. Echinomycin binds cooperatively as a bisintercalator at the CpG steps. The terminal A.T base pairs are Hoogsteen base paired, but none of the four central A.T base pairs are Hoogsteen base paired. However, binding of the drug induces unwinding of the DNA which is propagated to the central ApT step. All four central A.T base pairs are destabilized relative to those in the free DNA. Furthermore, based on these and other results from our laboratory, we conclude that the formation of stable Hoogsteen base pairs may not be the relevant structural change in vivo. The structural changes propagated between adjacent ACGT binding sites are the unwinding of the duplex and destabilization of the base pairing between binding sites.     

Genotoxicity of non-covalent interactions: DNA intercalators

This review provides an update on the mutagenicity of intercalating chemicals, as carried out over the last 17 years. The most extensively studied DNA intercalating agents are acridine and its derivatives, that bind reversibly but non-covalently to DNA. These are frameshift mutagens, especially in bacteria and bacteriophage, but do not otherwise show a wide range of mutagenic properties. Di-acridines or di-quinolines may be either mono- or bis-intercalators, depending upon the length of the alkyl chain separating the chromophores. Those which monointercalate appear as either weak frameshift mutagens in bacteria, or as non-mutagens. However, some of the bisintercalators act as "petite" mutagens in Saccharomyces cerevisiae, suggesting that they may be more likely to target mitochondrial as compared with nuclear DNA. Some of the new methodologies for detecting intercalation suggest this may be a property of a wider range of chemicals than previously recognised. For example, quite a number of flavonoids appear to intercalate into DNA. However, their mutagenic properties may be dominated by the fact that many of them are also able to inhibit topoisomerase II enzymes, and this property implies that they will be potent recombinogens and clastogens. DNA intercalation may serve to position other, chemically reactive molecules, in specific ways on the DNA, leading to a distinctive (and wider) range of mutagenic properties, and possible carcinogenic potential.     

New findings in the study on the intercalation of bisdaunorubicin and its monomeric analogues with naked and nucleus DNA

DNA is a target molecule for anthracycline anticancer drugs. We have used new anthracycline derivatives, bisdaunorubicin (WP631) and its monomeric analogues (WP700 serie), and look if there was a relation between the drug binding affinity to naked DNA and to cell nucleus in the cell with its cytotoxicity. Circular dichroism (CD) and fluorescence were used to follow the interaction of anthracycline derivatives with naked DNA and cell nuclei. WP631 interacts with DNA at two distinct stoichiometries, 6:1 and 3:1 base pair (bp)/WP631 molecule (3:1 and 1.5:1 per anthracycline rings). Monomeric daunorubicin (DNR) with its amino sugar N-bound to amino- and nitro-substituted benzyl moiety, representing p-xylenyl linker present in WP631 bisintercalator, is much more binding to DNA than DNR or WP631. These findings are supported by the study of drug binding by nuclei of K562 cells. Around 70% of WP700 intercalate to nucleus DNA in the steady-state, while only 45% of DNR intercalate DNA in the cell. The binding of WP631 by K562 cells is even less effective ( approximately 20%). WP 700 compounds, which are very similar to each other in their binding to DNA, self-association and cell accumulation, differ very distinctly in their cytotoxicity power. The most effective compounds are amino-benzyl derivatives of WP 700 series. The nitro-benzyl compounds have very low toxicity, even if they bind to DNA with similar power with that of the amino derivatives. The comparison of the all data clearly indicates no relation between cytotoxicity of the drug and its ability to intercalate DNA.     

Molecular mechanisms and kinetics between DNA and DNA binding ligands

Mechanical properties of single double-stranded DNA (dsDNA) in the presence of different binding ligands were analyzed in optical-tweezers experiments with subpiconewton force resolution. The binding of ligands to DNA changes the overall mechanic response of the dsDNA molecule. This fundamental property can be used for discrimination and identification of different binding modes and, furthermore, may be relevant for various processes like nucleosome packing or applications like cancer therapy. We compared the effects of the minor groove binder distamycin-A, a major groove binding alpha-helical peptide, the intercalators ethidium bromide, YO-1, and daunomycin as well as the bisintercalator YOYO-1 on lambda-DNA. Binding of molecules to the minor and major groove of dsDNA induces distinct changes in the molecular elasticity compared to the free dsDNA detectable as a shift of the overstretching transition to higher forces. Intercalating molecules affect the molecular mechanics by a complete disappearance of the B-S transition and an associated increase in molecular contour length. Significant force hysteresis effects occurring during stretching/relaxation cycles with velocities >10 nm/s for YOYO-1 and >1000 nm/s for daunomycin. These indicate structural changes in the timescale of minutes for the YOYO-DNA and of seconds for the daunomycin-DNA complexes, respectively.     

Characterization of TioF, a tryptophan 2,3-dioxygenase involved in 3-hydroxyquinaldic acid formation during thiocoraline biosynthesis

Thiocoraline is a thiodepsipeptide antitumor agent that belongs to the family of bisintercalator natural products that bind duplex DNA through their two planar intercalating moieties. In thiocoraline, the 3-hydroxyquinaldic acid (3HQA) chromophores required for intercalation are derived from L-Trp. We have expressed the Micromonospora sp. ML1 tryptophan 2,3-dioxygenase(TDO) TioF, purified it from E. coli, and confirmed its role in the irreversible oxidation of L-Trp to N-formylkynurenine, the proposed first step during 3HQA biosynthesis. We have established that TioF is a catalyst with broader specificity than other TDOs, but that is less promiscuous than indoleamine 2,3-dioxygenases. TioF was found to display activity with various L-Trp analogs (serotonin, D-Trp, and indole). The TioF reaction products generated during this study will be used as substrates for subsequent analysis of the other enzymes involved in 3HQA biosynthesis.     

Towards echinomycin mimetics by grafting quinoxaline residues on glycophane scaffolds

Echinomycin is a natural depsipeptide, which is a bisintercalator, inserting quinoxaline units preferentially adjacent to CG base pairs of DNA. Herein the design and synthesis of echinomycin mimetics based on grafting of two quinoxaline residues onto a macrocyclic scaffold (glycophane) is addressed. Binding of the compounds to calf-thymus DNA was studied using UV-vis and steady state fluorescence spectroscopy, as well as thermal denaturation. An interesting observation was enhancement of fluorescence emission for the peptidomimetics on binding to DNA, which contrasted with observations for echinomycin. Molecular dynamics simulations were exploited to explore in more detail if bis-intercalation to DNA was possible for one of the glycophanes. Bis-intercalating echinomycin complexes with DNA were found to be stable during 20ns simulations at 298K. However, the MD simulations of a glycophane complexed with a DNA octamer displayed very different behaviour to echinomycin and its quinoxaline units were found to rapidly migrate out from the intercalation site. Release of bis-intercalation strain occurred with only one of the quinoxaline chromophores remaining intercalated throughout the simulation. The distance between the quinoxaline residues in the glycophane at the end of the MD simulation was 7.3-7.5?, whereas in echinomycin, the distance between the residues was ～11?, suggesting that longer glycophane scaffolds would be required to generate bis-intercalating echinomycin mimetics.     

Structure and dynamics of condensed DNA probed by 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)bis[4-[[3- methylbenz-1,3-oxazol-2-yl]methylidine]-1,4-dihydroquinolinium] tetraiodide fluorescence

Information on the structure and dynamics of condensed forms of DNA is important in understanding both natural situations such as DNA packaging and artificial systems such as gene delivery complexes. We have established the fluorescence of bisintercalator 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)bis[4-[[3-methylbenz-1,3-oxazol-2-yl]methylidine]-1,4-dihydroquinolinium] tetraiodide (YOYO-1) as a novel probe for DNA condensation. When the level of DNA-bound YOYO-1 is sufficiently large, condensation by either polyethylenimine (PEI) or the cationic detergent cetyltrimethylammonium bromide (CTAB) leads to electronic interaction among YOYO-1 molecules bound on the same DNA molecule. This interaction results in an excitonic blue shift of the absorption spectra of YOYO-1 and dramatic decrease in the fluorescence quantum yield. These observations constitute a signature of the condensation of DNA. We further examined the comparative properties of DNA condensed by PEI, CTAB, or Co(NH(3))(6)(3+) through the steady-state and dynamic fluorescence of YOYO-1. Condensation by either PEI or CTAB was associated with a blue shift in the absorption spectra of YOYO-1, although the magnitude of the shift was larger in the case of PEI when compared to that of CTAB. In contrast, condensation by Co(NH(3))(6)(3+) was not associated with a measurable shift in the absorption spectra. These results were interpreted as signifying the varying level of compactness of the DNA condensates. Quenching of fluorescence by acrylamide showed that condensation by all three agents led to an increase in the level of solvent exposure of the base pairs. Observation of the decay of fluorescence intensity and anisotropy of DNA-bound YOYO-1 showed that while condensation by either PEI or CTAB froze the segmental mobility of the helix, condensation by Co(NH(3))(6)(3+) enhanced the flexibility of DNA. The relevance of our findings to functions such as efficiency of gene delivery is discussed.     

Noncovalent drug-DNA binding interactions that inhibit and stimulate (A)BC excinuclease.

(A)BC excinuclease from Escherichia coli catalyzes the initial step of nucleotide excision repair. It recognizes and binds to many types of covalent modifications in DNA and incises the damaged strand on both sides of the lesion. We employed a variety of noncovalent DNA binding drugs to examine in vitro the mechanisms and the nature of the DNA-drug interactions responsible for two phenomena: inhibition of excision repair by caffeine and other noncovalent DNA binding compounds; incision of undamaged DNA produced by (A)BC excinuclease in the presence of the bisintercalating drug ditercalinium. All of the chemicals examined (e.g., actinomycin D, caffeine, ethidium bromide, and Hoechst 33258) inhibited incision of a covalent adduct by (A)BC excinuclease, and direct evidence is given for a common mechanism in which UvrA is depleted by binding to drug-undamaged DNA complexes. In the absence of significant amounts of undamaged DNA, another mechanism of inhibition was observed, in which enzyme bound to noncovalent drug-DNA complexes in the vicinity of the lesion prevents formation of preincision complexes at the lesion. Ditercalinium and unexpectedly all of the other drugs examined promoted the incision of undamaged DNA when the enzyme was present at high concentration. Thus, this activity contrary to previous assumptions is not unique to bisintercalators. Another unexpected finding was stimulation of incision at certain sites of photodamage in DNA produced by low concentrations of noncovalent DNA binding chemicals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acridine dimers: influence of the intercalating ring and of the linking-chain nature on the equilibrium and kinetic DNA-binding parameters

The rigidity of the linking chain of bifunctional intercalators in the ditercalinium series was shown to be critical for antitumor activity. In order to study the influence of the rigidity of the linking chain on the DNA-binding properties of DNA bifunctional intercalators, fluorescent 9-aminoacridine and 2-methoxy-6-chloro-9-aminoacridine analogues with chains of variable rigidity were synthesized. 1H-NMR studies show that the conformation of 9-aminoacridine dimers is almost independent of the nature of the linking chain. A strong self-stacking of the aromatic rings of the 2-methoxy-6-chloro-9-aminoacridine is observed for dimers with flexible chains but not for those with rigid chains. All the dimers having a linking chain long enough to bisintercalate in DNA according to the excluded site model are indeed bisintercalators. The kinetic association constant of all monomers and dimers for poly[d(A-T)].poly[d(A-T)] are in the same range (2-4 x 10(7) M-1 s-1). The large increase of DNA binding affinity observed for the dimers is always associated with the expected decrease of the dissociation rate constant. The effect of chain rigidity and pH on the calf thymus DNA binding of 9-aminoacridine and 2-methoxy-6-chloro-9-aminoacridine dimers is quite different. In the series of 9-aminoacridine the pKa of the dimers remains high and therefore no difference of DNA-binding affinity is observed between pH 5 and 7.4. The rigidity of the linking chain does not significantly alter the DNA-binding affinity. In the 2-methoxy-6-chloro-9-aminoacridine series, the pKa of all dimers became smaller than the physiological pH and a dramatic decrease of DNA-binding affinity is observed when the pH is increased from pH 5 to 7.4. This decrease appears significantly smaller for dimers with rigid chains. A similar dramatic decrease of binding affinity at pH 7.4 is not observed for poly[d(A-T)].poly[d(A-T)]. This factor makes these dimers strongly specific for the alternating polymer at pH 7.4.     

Binding Kinetics of Bisintercalator Triostin A with Optical Tweezers Force Mechanics

The binding kinetics of the intercalative binding of Triostin A to λ-DNA was investigated by measuring the force extension response of the DNA-ligand complexes with an optical tweezers system. These force response curves, containing the information about different binding properties, were analyzed based on a recent method (put forth by another research group) for monointercalators that was extended to bisintercalators. Our binding analysis reveals an exponential dependence of the association constant on the applied external force as well as a decreasing binding site size. In general, our results are in agreement with those for the monointercalator ethidium. However, to explain the high-force binding site size, a new model for bisintercalation of Triostin A at high forces is proposed.     

Optimization of Cross-Linked Lexitropsins

Abstract

In attempts to optimize the cross-linked lexitropsin design, a number of cross-linked dimers composed of two tris(N-methylpyrrolecarboxamide) strands were synthesized and their binding interactions with poly d(A)? poly d(T) and poly d(A-T)?poly d(A-T) were characterized by circular dichroism and ethidium fluorometry. While all alkanediyl-linked dimers showed a similar binding behavior to the homo AT polymer, particularly at low ligand concentrations, the decanediyl linker was found to be the optimal linker permitting the bidentate anti parallel side-by-side binding of the corresponding dimer to the alternating AT polymer. Thus, in comparison with the monomer, the decanediyl-linked dimer has a binding strength enhancement of about 1400 times in the I: I binding mode. Moreover, the hydrophilicity of the linker has a significant effect on the bidentate binding strength. The (3,6)-dioxaoctanediyl-linked dimer has a further binding strength enhancement of 10 times over the decanediyl-linked dimer. Overall, the best optimized dimer has a binding strength enhancement of over 14,000 times in comparison with the monomer in the I: I binding mode. This binding enhancement parallels that observed in the best optimized bisintercalators. Distance-restrained molecular modeling provides support for the experimental results. Dimers of longer linkers can readily accommodate a bidentate anti parallel side-by-side binding mode but those of shorter linkers necessitate marked structural distortions in the bound ligand molecules. It is further observed that the binding strength enhancement to the alternating AT polymer is not always accompanied by the binding specificity improvement. Our analysis suggests that the non-specific appendage-DNA backbone interaction is a key factor that controls the specificity improvement.     

Transport of a carcinogen, benzo[a]pyrene, from particulates to lipid bilayers: a model for the fate of particle-adsorbed polynuclear aromatic hydrocarbons which are retained in the lungs

DNA from Ehrlich ascites tumor cells is nicked or gapped by a reaction which is induced by proteases such as autodigested pronase, proteinase K, trypsin, chymotrypsin and subtilisin. The cleavage of the protease-sensitive sites is inhibited by protease inhibitors. The nicks or gaps induced by proteases can be demonstrated by nuclease S1 sensitivity of native DNA and by a change of the sedimentation rate of alkali-denatured DNA. The limit size of denatured DNA released after optimal protease treatment is 8.5 x 10(6) daltons (27 kilo bases). The molecular weight of the native DNA pieces released after nuclease S1 degradation of DNA containing the protease-induced nicks or gaps is in the same order indicating that the protease-sensitive sites are alternatively arranged on the opposite DNA strands at an average distance of 13.5 kilo base pairs. Since the protease-induced nicks or gaps in phosphatase-treated DNA are not attacked by Escherichia coli polymerase I, one or both ends liberated by the protease treatment must be blocked by a material other than phosphate groups. The results are most compatible with peptide/protein linkers joining adjacent single-strand DNA subunits. Alternative explanations such as alkali-stable RNA linkers, protein-protected RNA linkers, site-specific nuclease contaminations in the protease preparations or cellular nucleases activated by the protease treatment are eliminated by the results presented in this paper.     

The relationship between DNA strand-scission and DNA synthesis inhibition in HeLa cells treated with neocarzinostatin.

Neocarzinostatin inhibits DNA synthesis in HeLa S3 cells and induces the rapid limited breakage of cellular DNA. The fragmentation of cellular DNA appears to precede the inhibition of DNA synthesis. Cells treated with drug at 37 degrees C for 10 min and then washed free of drug show similar levels of inhibition of DNA synthesis or cell growth, or of strand-scission of DNA as when cells were not washed. If cells are preincubated with neocarzinostatin at 0 degrees C before washing, the subsequent incubation of 37 degrees C results in no inhibition of DNA synthesis or cell growth, or cutting of DNA. Isolated nuclei or cell lysates derived from neocarzinostatin-treated HeLa S3 cells are inhibited in DNA synthesis but this can be overcome in cell lysates by adding activated DNA. A cytoplasmic fraction from drug-treated cells can stimulate DNA synthesis by nuclei isolated from untreated cells, whereas nuclei from drug-treated cells are not stimulated by the cytoplasmic fraction from untreated cells. By contrast, neocarzinostatin does not inhibit DNA synthesis when incubated with isolated nuclei, but it can be shown that under these conditions the DNA is already degraded and is not further fragmented by the drug. These data suggest that the drug's ability to induce breakage of cellular DNA in HeLa S3 cells is an essential aspect of its inhibition of DNA replication and may be responsible for the cytotoxic and growth-inhibiting actions of neocarzinostatin.     

Construction of linear functional expression elements with DNA and RNA hybrid primers: a flexible and fast method for proteomics

Two methods of constructing linear functional expression elements (LFEE) using hybrid DNA and RNA primers in DNA amplification for rapid gene expression are described. In both methods, it is not necessary to have additional transformation or bacterial propagation. The promoter, open reading frame (ORF) and terminator are amplified using Pfu or Taq DNA polymerase. Three elements containing DNA or RNA overhang are covalently ligated by T4 DNA ligase. The recombinant molecule is amplified with element-specific primers. The LFEE can be generated by both methods in a few hours and can be expressed in mammalian cells.