Vibrational circular dichroism studies of the A-to-B conformational transition in DNA.

The vibrational circular dichroism (VCD) spectra of several natural DNAs as well as tRNA, poly(dG-dC).poly(dG-dC), and poly(dA-dT).poly(dA-dT) are reported for the base deformation modes in the IR region from 1700 to 1550 cm-1 for the polymers in D2O as well as in high alcohol dehydrating conditions. Spectra of both the B- and A-forms were identified. The A-form DNA VCD, not previously reported, has characteristics that can be found in the VCD spectra of RNAs as would be expected from the similarity of their structures. The VCD is sequence-dependent. Under the dehydrating conditions studied, poly(dA-dT)poly(dA-dT),poly(dA).poly(dT), and a high-A-T fraction natural DNA had a different bandshape from the other DNAs, which was similar to that of poly(rA).poly(rU). Poly(dG-dC).poly-(dG-dC) did not form an A-form in high-alcohol conditions but instead had a VCD spectrum much like that of its high-salt-induced Z-form. Qualitative differences seen experimentally between A- and B-form DNA VCD were suggested by the differences in the coupled oscillator VCD calculated for the two forms.     

The different binding modes of Hoechst 33258 to DNA studied by electric linear dichroism.

The binding mode of the bisbenzimidazole derivative Hoechst 33258 to a series of DNAs and polynucleotides has been investigated by electric linear dichroism. Positive reduced dichroisms were measured for the poly(dA-dT).poly(dA-dT)- and poly(dA).poly(dT)-Hoechst complexes in agreement with a deep penetration of the drug into the minor groove. Similarly, the drug displays positive reduced dichroism in the presence of the DNAs from calf thymus, Clostridium perfringens and Coliphage T4. Conversely, negative reduced dichroisms were obtained when Hoechst 33258 was bound to poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT) and poly(dG).poly(dC) as well as with the GC-rich DNA from Micrococcus lysodeikticus indicating that in this case minor groove binding cannot occur. Substitution of guanosines for inosines induces a reversal of the reduced dichroism from negative to positive. Therefore, as anticipated it is the 2-amino group of guanines protruding in this groove which prevents Hoechst 33258 from getting access to the minor groove of GC sequences. The ELD data obtained with the GC-rich biopolymers are consistent with an intercalative binding. Competition experiments performed with the intercalating drug proflavine lend credence to the involvement of an intercalative binding rather than to an external or major groove binding of Hoechst 33258 at GC sequences.     

Interaction of nucleic acids with a non-intercalative anti-leukemic compound containing bisquarternary heterocycles

The binding of an antitumour drug with bisquarternary ammonium heterocyclic structure, NSC-101327, to nucleic acids has been examined by using ultraviolet absorption and CD measurements. Like the minor groove-binding oligopeptides, netropsin and distamycin A, the optically inactive chromophoric system of NSC-101327 shows induced Cotton effects in the CD spectra of complexes with various DNAs, RNA and single-stranded polynucleotides. This property directly reflects interaction of NSC-101327 with different types of nucleic acids at moderate ionic strength, which contrasts with previous findings of a higher selective binding of netropsin to B-DNA. However, an efficient interactin of NSC-101327 with dA·dT basepair sequences is demonstrated by a large melting temperature increase of dA·dT-rich DNAs. NSC-101327 also reacts with dG·dC base pairs of B-DNA and forms a complex with Z-DNA of poly(br⁸dG-dC)·poly(br⁸DG-dC). The affinity of NSC-101327 to poly(dG-dC)·poly(dG-dC) is, however, lower, and the CD spectral binding effect depends on the ionic strength. The CD results of the complex with poly(dA-dT)·poly(dA-dT) suggests at least two binding modes, in accordance with previous conclusions. This is indicated by a clear-cut initial increase of the CD signal and a subsequent large decrease to negative CD signals. Competition experiments with netropsin suggest that binding of NSC-101327 occurs preferentially in the minor groove without intercalation. NSC-101327 also tends to interact with lower binding affinity to dG-dC pairs in B-DNA, with rA·rU pairs of RNA and with single-stranded polynucleotides. Thus our results suggest that NSC-101327 represents a DNA groove-binding ligand of lower basepair specificity and lower conformational selectivity compared to the B-specific netropsin probe.     

Preferential binding of DNA methyltransferase and increased de novo methylation of deoxyinosine containing DNA

Mammalian DNA-cytosine 5-methyltransferases methylate cytosines in deoxyinosine containing DNA polymers more rapidly than in other synthetic or naturally occurring DNAs. The initial methylation rate of poly(dI-dC) X poly(dI-dC) is about 10-times higher than that of poly-(dG-dC) X poly(dG-dC) or of the native Micrococcus luteus DNA. In competitive binding experiments, DNA methyltransferase has about 10-fold higher affinity for the dI-containing alternating DNA polymer than for poly(dG-dC) X poly(dG-dC). The observed high methyl accepting capacity of poly(dI-dC) X poly(dI-dC) may be a useful methodological advance to determine de novo DNA methyltransferase activity in extracts of mammalian cells.     

Binding of tetrakis(pyrazoliumyl)porphyrin and its copper(II) and zinc(II) complexes to poly(dG-dC)2 and poly(dA-dT)2

Interactions of cationic porphyrins bearing five-membered rings at the meso position, meso-tetrakis(1,2-dimethylpyrazolium-4-yl)porphyrin (MPzP; M is H₂, Cu^II or Zn^II), with synthetic polynucleotides poly(dG-dC)₂ and poly(dA-dT)₂ have been characterized by viscometric, visible absorption, circular dichroisim and magnetic circular dichroism spectroscopic and melting temperature measurements. Both H₂PzP and CuPzP are intercalated into poly(dG-dC)₂ and are outside-bound to the major groove of poly(dA-dT)₂, while ZnPzP is outside-bound to the minor groove of poly(dA-dT)₂ and surprisingly is intercalated into poly(dG-dC)₂. The binding constants of the porphyrin and poly(dG-dC)₂ and poly(dA-dT)₂ are on the order of 10⁶ M⁻¹ and are comparable to those of other cationic porphyrins so far reported. The process of the binding of the porphyrin to poly(dG-dC)₂ and poly(dA-dT)₂ is exothermic and enthalpically driven for H₂PzP, whereas it is endothermic and entropically driven for CuPzP and ZnPzP. These results have revealed that the kind of the central metal ion of metalloporphyrins influences the characteristics of the binding of the porphyrins to DNA.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

The antitumor agent mitoxantrone binds cooperatively to DNA: evidence for heterogeneity in DNA conformation

The equilibrium binding of the antitumor compound DHAQ, or mitoxantrone [1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10- anthracenedione], to various DNAs has been examined by optical titration and equilibrium dialysis methods. At low r (bound drug/DNA base pair) values, r less than 0.03, DHAQ binds, in a highly cooperative manner, to calf thymus and Micrococcus lysodeikticus DNAs. The binding isotherms for the interaction of DHAQ with Clostridium perfringens DNA and poly(dA-dT).poly(dA-dT) exhibit a small positive slope at low r values, suggestive of cooperative binding. In contrast, the binding of DHAQ to poly(dG-dC).poly(dG-dC) shows no evidence of cooperative binding even at very low r values. At higher r values (r greater than 0.05), the binding of DHAQ to all the DNAs studied is characterized by a neighbor-exclusion process. A model is proposed to account for the two modes of binding exhibited in the cooperative binding isotherms. The main feature of the proposed model is that local sequence and structural heterogeneity of the DNA give rise to sets of binding sites to which DHAQ binds in a highly cooperative manner, while the majority of the DNA sites bind DHAQ via a neighbor-exclusion process. This two-site model reproduces the observed binding isotherms and leads to the conclusion that DHAQ binds in clusters to selected regions of DNA. It is suggested that clustering may play a role in the physiological activity of drugs.     

Berberine-DNA complexation: new insights into the cooperative binding and energetic aspects

The equilibrium binding of the cytotoxic plant alkaloid berberine to various DNAs and energetics of the interaction have been studied. At low ratios of bound alkaloid to base pair, the binding exhibited cooperativity to natural DNAs having almost equal proportions of AT and GC sequences. In contrast, the binding was non-cooperative to DNAs with predominantly high AT or GC sequences. Among the synthetic DNAs, cooperative binding was observed with poly(dA).poly(dT) and poly(dG).poly(dC) while non-cooperative binding was seen with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Both cooperative and non-cooperative bindings were remarkably dependent on the salt concentration of the media. Linear plots of ln K(a) versus [Na(+)] for poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT) showed the release of 0.56 and 0.75 sodium ions respectively per bound alkaloid. Isothermal titration calorimetry results revealed the binding to be exothermic and favoured by both enthalpy and entropy changes in all DNAs except the two AT polymers and AT rich DNA, where the same was predominantly entropy driven. Heat capacity values (DeltaCp(o)) of berberine binding to poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), Clostridium perfringens and calf thymus DNA were -98, -140, -120 and -110 cal/mol K respectively. This study presents new insights into the binding dependent base pair heterogeneity in DNA conformation and the first complete thermodynamic profile of berberine binding to DNAs.     

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.     

Binding of nonintercalative antitumor drugs to DNA-polymers: structural effects of bisquaternary ammonium heterocycles

The binding of the antitumor agents SN-16814 nd SN-13232 to various DNA's in solution was monitored by CD and UV absorption measurements. In addition comparative studies with dA.dT containing duplex DNA of the related ligands SN-6136 and SN-6324 were included with respect to effects of structural variations. In general all four ligands show a dA.dT preference in their binding affinity to DNA. Differences were observed for the reaction of SN-16814 which contains bicyclic ring system: it has a lower base pair selectivity, shows some affinity to poly(dG-dC).poly(dG-dC), poly(rA).poly(rU) and poly(rU). The binding mechanism of SN-16814 is associated with a significant time dependent binding effect in CD spectra and UV absorption in case of reaction with poly(dA).poly(dT) and poly(dI).poly(dC) indicating a slow kinetics. The preferred binding to dA.dT base pairs in DNA decreases in the order from SN-61367 greater than SN-13232 greater than SN-6324,SN-16814 as judged from CD titration studies, salt dissociation and melting temperature data. Competitive binding experiments with netropsin (Nt) or distamycin-5 revealed that SN-16814 and SN-13232 are displaced from poly(dA.dT).poly(dA-dT) suggesting that both ligands are less strongly bound than Nt and Dst-5 within the minor groove of B-DNA. These studies are consistent with results of the DNAse I cleavage of poly(dA-dT).poly(dA-dT) which show the same relative order of inhibition of the cleavage reaction due to ligand binding. The results suggest that the variability of the DNA binding and dA.dT sequence specificity may reside in the adaptability of benzamide-type ligands in the helical groove which is influenced by distinct structural modifications of the ligand conformation.     

The CD of ligand-DNA systems. 2. Poly(dA-dT) B-DNA.

A systematic theoretical study of the CD of [poly(dA-dT)]2 and its complexes with achiral small molecules is presented. The CD spectra of [poly(dA-dT)]2 and of poly(dA):poly(dT) are calculated for various DNA structures using the matrix method. The calculated and experimental spectra agree reasonably well for [poly(dA-dT)]2 but less well for poly(dA):poly(dT). The calculated CD spectrum of [poly(dA-dT)]2 fails to reproduce the wavelength region of 205-245 nm of the experimental spectrum. This discrepancy can be explained by a magnetic dipole allowed transition contributing significantly to the CD spectrum in this region. The induced CD of a transition moment of a molecule bound to [poly(dA-dT)]2 is also calculated. As was the case for [poly(dG-dC)]2, the induced CD of a groove bound molecule is one order of magnitude stronger than that of an intercalated molecule. The calculations also show considerable differences between pyrimidine-purine sites and purine-pyrimidine sites. Both signs and magnitudes of the CD induced into ligands bound in the minor groove agree with experimental observations.     

Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA

The activity of eukaryotic DNA methyltransferase diminishes with time when the enzyme is incubated with high concentrations (200-300 micrograms/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S-adenosyl-L-methionine. The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT), but not poly(dI-dC).poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the 'de novo' activity of the enzyme.     

Conformation dependent binding of netropsin and distamycin to DNA and DNA model polymers

The binding of the antibiotics netropsin and distamycin A to DNA has been studied by thermal melting, CD and sedimentation analysis. Netropsin binds strongly at antibiotic/nucleotide ratios up to at least 0.05. CD spectra obtained using DNA model polymers reveal that netropsin binds tightly to poly (dA) · poly (dT), poly (dA-dT) · poly(dA-dT) and poly (dI-dC) · poly (dI-dC) but poorly, if at all, to poly (dG) · poly (dC). Binding curves obtained with calf thymus DNA reveal one netropsin-binding site per 6.0 nucleotides (K_a=2.9 · 10⁵ M⁻¹); corresponding values for distamycin A are one site per 6.1 nucleotides with K_a= 11.6 · 10⁵ M⁻¹. Binding sites apparently involve predominantly A·T-rich sequences whose specific conformation determines their high affinity for the two antibiotics. It is suggested that the binding is stabilized primarily by hydrogen bonding and electrostatic interactions probably in the narrow groove of the DNA helix, but without intercalation. Any local structural deformation of the helix does not involve unwinding greater than approximately 3° per bound antibiotic molecule.     

Conformational transitions of alternating purine-pyrimidine DNAs in perchlorate ethanol solutions.

Conformational transitions of poly(dA-dC).poly(dG-dT), poly(dA-dT).poly(dA-dT), and other alternating purine-pyrimidine DNAs were studied in aqueous ethanol solutions containing molar concentrations of sodium perchlorate, which is a novel solvent stabilizing non-B duplexes of DNA. Using CD and UV absorption spectroscopies, we show that this solvent unstacks bases and unwinds the B-forms of the DNAs to transform them into the A-form or Z-form. In the absence of divalent cations poly(dA-dC).poly(dG-dT) can adopt both of these conformations. Its transition into the Z-form is induced at higher salt and lower ethanol concentrations, and at higher temperatures than the transition into the A-form. Submillimolar concentrations of NiCl2 induce a highly cooperative and slow A-Z transition or Z-Z' transition, which is fast and displays low cooperativity. Poly(dA-dT).poly(dA-dT) easily isomerizes into the A-form in perchlorate-ethanol solutions, whereas high perchlorate concentrations denature the polynucleotide, which then cannot adopt the Z-form. At low temperatures, however, NiCl2 also cooperatively induces the Z'-form in poly(dA-dT).poly(dA-dT). Poly(dI-dC).poly(dI-dC) is known to adopt an unusual B-form in low-salt aqueous solution, which is transformed into a standard B-form by the combination of perchlorate and ethanol. NiCl2 then transforms poly(dI-dC).poly(dI-dC) into the Z'-form, which is also adopted by poly(dI-br5dC).poly(dI-br5dC).     

Comparison of the conformation of poly(dI-dC) with poly(dI-dbr5C) and the B and Z forms of poly(dG-dC). One- and two-dimensional NMR studies

One- and two-dimensional nuclear Overhauser effects (2D NOE) have been used to compare the conformational properties of 60-80 base pair long duplexes of the synthetic DNA polymer poly(dI-dC) with those of poly(dI-dbr5C) and poly(dG-dC) in the B and Z conformations. Cross peaks in the 2D NOE spectra arising from proton-proton dipolar interactions which are more or less independent of the DNA conformation are used to assign the spectra of these molecules. Other cross peaks are sensitive to the conformational details, and these are used to make deductions about the average conformation in solution. The proton-proton interactions that give rise to the cross peaks in the 2D NOE spectrum of poly(dI-dC) are indicative of a B family conformation and rule out the possibility of some alternative conformations, including A, Z, alternating B, and left-handed B-DNA. The spectra are similar to those obtained from B-form poly(dI-dbr5C) and poly(dG-dC) but different from Z-form poly(dG-dC). Taken together, these results indicate that the solution conformation of poly(dI-dC) is not unusual but more closely resembles that of other B-form DNAs.     

A pared-down version of 5,10,15,20-tetra(N-methylpyridinium-4-yl)porphyrin intercalates into B-form DNA regardless of base composition: binding studies of tri(N-methylpyridinium-4-yl)porphyrins

Positively charged N-methylpyridinium-4-yl substituents promote the binding of a porphyrin to DNA, but they also impose steric constraints. To clarify when intercalative binding is most feasible, this report describes syntheses and binding studies of two tricationic ligands: 5,10,15-tri(N-methylpyridinium-4-yl)porphyrin (H2Tri4) and 5-methyl-10,15,20-tri(N-methylpyridinium-4-yl)porphyrin (H2MeTri4). Techniques used to characterize the binding interactions include viscometry and spectroscopic studies of the absorption, emission, and circular dichroism. The striking observation is that intercalation is the only detectable binding motif when the trisubstituted porphyrin H2Tri4 combines with [poly(dA-dT)]2, [poly(dG-dC)]2, or salmon testes DNA. H2Tri4 is, however, a limiting case. Parallel studies of H2MeTri4 and the copper(II) derivative Cu(MeTri4) reveal that external binding to [poly(dA-dT)]2 becomes important when a fourth meso substituent is present, even one as small as the methyl group. Intercalation of H2Tri4 is sterically feasible because two N-methylpyridinium-4-yl substituents can reside in the major groove, though the charge alignment is not optimal. However, the presence of the fourth substituent on H2MeTri4 further destabilizes the intercalated form, and external binding becomes competitive for a flexible host like [poly(dA-dT)]2.     

Rotation of periphery methylpyridine of meso-tetrakis(n-N-methylpyridiniumyl)porphyrin (n = 2, 3, 4) and its selective binding to native and synthetic DNAs

By utilizing circular and linear dichroism, the binding mode of meso-tetrakis(n-N-methylpyridiniumyl)porphyrin (n = 2, 3, 4) to various DNAs was studied in this work. 2-N-(methylpyridiniumyl)porphyrin(o-TMPyP), in which rotation of the periphery pyridinium ring is prevented, exhibits similar spectral properties when bound to DNA, poly[d(G-C)(2)] and poly[d(A-T)(2)], suggesting a similar binding mode. Close analysis of the spectral properties led us to conclude that o-TMPyP sits in the major groove. However, both 3-N- and 4-N-(methylpyridiniumyl)porphyrin (m- and p-TMPyP), of which the periphery pyridinium ring is free to rotate, intercalate between the basepairs of DNA and poly[d(G-C)(2)]. In the presence of poly[d(A-T)(2)], m-TMPyP exhibits a typical bisignate excitonic CD spectrum in the Soret band, while p-TMPyP shows two positive CD bands. The excitonic CD spectrum of the m-TMPyP-poly[d(A-T)(2)] complex and the positive CD band of the o-TMPyP-poly[d(A-T)(2)] complex were not affected by the presence of the minor groove binding drug, 4',6-diamidino-2-phenylindole (DAPI), indicating that this porphyrin is bound in the major groove. In contrast, two positive CD bands of the p-TMPyP-poly[d(A-T)(2)] complex altered in the presence of DAPI. From the changes in CD spectrum and other spectral properties, a few possible binding modes for p-TMPyP to poly[d(A-T)(2)] are suggested.     

Interactions of DNA with a new electron-deficient tentacle porphyrin: meso-Tetrakis[2,3,5,6-tetrafluoro-4-(2-trimethylammoniumethylamine)phenyl]porphyrin

A new electron-deficient tentacle porphyrin meso-tetrakis[2,3,5,6-tetrafluoro-4-(2-trimethylammoniumethylamine)phenyl]porphyrin (TθF₄TAP) has been synthesized. The binding interactions of TθF₄TAP with DNA polymers were studied for comparison to those of an electron-deficient tentacle porphyrin and an electron-rich tentacle porphyrin; these previously studied porphyrins bind to DNA primarily by intercalative and outside-binding modes, respectively. The three tentacle porphyrins have similar size and shape. The basicity of TθF₄TAP indicated that it has electronic characteristics similar to those of the intercalating electron-deficient tentacle porphyrin. However, TθF₄TAP binds to calf thymus DNA, [poly(dA-dT)]₂, and [poly(dG-dC)]₂ in a self-stacking, outside-binding manner under all conditions. Evidence for this binding mode included a significant hypochromicity of the Soret band, a conservative induced CD spectrum, and the absence of an increase in DNA solution viscosity. As found previously for the electron-rich porphyrin, the results suggest that combinations of closely related self-stacked forms coexist. The mix of forms depended on the DNA and the solution conditions. There are probably differences in the detailed features of the self-stacking adducts for the two types of tentacle porphyrins, especially at high R (ratio of porphyrin to DNA). At low R values, the induced CD signal of TθF₄TAP/CT DNA resembled that of TθF₄TAP/[poly(dA-dT)]₂, suggesting that TθF₄TAP binds preferentially at AT regions. Competitive binding experiments gave evidence that TθF₄TAP binds preferentially to [poly(dA-dT)]₂ over [poly(dG-dC)]₂. Thus, despite the long, positively charged, flexible substituents on the porphyrin, the binding of TθF₄TAP is significantly affected by base-pair composition. Similar characteristics were found previously for the electron-rich tentacle porphyrin. Thus, significant changes in electron richness have relatively minor effects on this outside binding selectivity for AT regions. TθF₄TAP is the first porphyrin with electron deficiency and shape similar to intercalating porphyrins that does not appear to intercalate. All porphyrins reported to intercalate have had pyridinium substituents. Thus, the electronic distribution in the porphyrin ring, not just the overall electron richness, may play a role in facilitating intercalation. © 1997 John Wiley & Sons, Inc. Biopoly 42: 203–217, 1997     

Conformational Variability of Poly(dA-dT)·Poly(dA-dT) and Some Other Deoxyribonucleic Acids Includes a Novel Type of Double Helix

Abstract

The article reviews data indicating that poly(dA-dT)?poly (dA-dT) is able of adopting three distinct double helical structures in solution, of which only the A form conforms to classical notions. The other two structures have dinucleotides as double helical repeats. At low salt concentrations poly(dA-dT)?poly(dA-dT) adopts a B-type alternating conformation which is exceptionally variable. Its architecture can gradually move in the limits demarcated by the CD spectra with inverted long wavelength CD bands and the ³¹P NMR spectra with a very low and a 0.6 ppm separation of two resonances. Contrary to Z-DNA, the ³¹P NMR spectrum of the limiting alternating B conformation of poly(dA-dT)?poly(dA-dT) is characterized by an upfield shift of one resonance. We attribute the exceptional conformational flexibility of the alternating B conformation to the unequal tendency of bases in the dA-dT and dT-dA steps to stack.

However, by assuming the limiting alternating B conformation, the variability of the synthetic DNA is not exhausted. Specific agents make it isomerize into another conformation by a fast, two-state mechanism, which is reflected by a further deepening of the negative long wavelength CD band and a downfield shift of the ³¹P NMR resonance of poly (dA-dT)?poly(dA-dT) that was constant in the course of the gradual alterations of the alternating B conformation. These changes are, however, qualitatively different from the way poly(dG-dC)?poly(dG-dC) behaves in the course of the B-Z isomerization. Poly(dG-dC) ?poly(dG-dC) displays purine-pyrimidine (dGpdC) resonance in the characteristic downfield position, while the downfield resonance of poly(dA-dT)?poly(dA-dT) belongs to the pyrimidine-purine (dTpdA) phosphodiester linkages. Consequently, phosphodiester linkages in the purine-pyrimidine steps play a similar role in the appearance of the Z form to the pyrimidine-purine phosphodiesters in the course of the isomerization of poly(dA-dT)?poly(dA-dT). This excludes that the high-salt structures of poly(dA-dT)?poly(dA-dT) and poly(dG-dC)?poly(dG-dC) are members of the same conformational family. We call the high-salt conformation of poly(dA-dT)?poly(dA-dT) X-DNA.

It furthermore follows from the review that synthetic molecules of DNA with alternating purine-pyrimidine sequences of bases can adopt either the Z form or the X form, or even both, depending on the environmental conditions. This introduces a new dimension into the DNA double helix conformational variability. The possible biological relevance of the X form is suggested by experiments with linear molecules of natural DNA. These indicate that Arich regions in natural DNAs can isomerize into the X form while the bulk of the molecule remains in the B form. The coexistence of both structures in a single DNA molecule may be understood in view of the favourable kinetic and thermodynamic properties with which the X form appears.     

Binding of Nonintercalative Antitumor Drugs to DNA-Polymers: Structural Effects of Bisquaternary Ammonium Heterocycles

Abstract

The binding of the antitumor agents SN-16814 nd SN-13232 to various DNA's in solution was monitored by CD and UV absorption measurements. In addition comparative studies with dA · dT containing duplex DNA of the related ligands SN-6136 and SN-6324 were included with respect to effects of structural variations. In general all four ligands show a dA · dT preference in their binding affinity to DNA.

Differences were observed for the reaction of SN-16814 which contains bicyclic ring system: it has a lower base pair selectivity, shows some affinity to poly(dG-dC) · poly(dG-dC), poly(rA) · poly(rU) and poly(rU). The binding mechanism of SN-16814 is associated with a significant time dependent binding effect in CD spectra and UV absorption in case of reaction with poly(dA) · poly(dT) and poly(dI) · poly(dC) indicating a slow kinetics.

The preferred binding to dA · dT base pairs in DNA decreases in the order from SN-61367 > SN-13232 > SN-6324, SN-16814 as judged from CD titration studies, salt dissociation and melting temperature data. Competitive binding experiments with netropsin (Nt) or distamycin-5 revealed that SN-16814 and SN-13232 are displaced from poly(dA-dT) · poly(dA-dT) suggesting that both ligands are less strongly bound than Nt and Dst-5 within the minor groove of B-DNA. These studies are consistent with results of the DNAase I cleavage of poly(dA-dT) · poly(dA-dT) which show the same relative order of inhibition of the cleavage reaction due to ligand binding. The results suggest that the variability of the DNAbinding and dA · dT sequence specificity may reside in the adaptability of benzamide-type ligands in the helical groove which is influenced by distinct structural modifications of the ligand conformation.