Phosphorescence and optically detected magnetic resonance studies of echinomycin-DNA complexes

Echinomycin complexes with polymeric DNAs and model duplex oligonucleotides have been studied by low-temperature phosphorescence and optical detection of triplet-state magnetic resonance (ODMR) spectroscopy, with the quinoxaline chromophores of the drug used as intrinsic probes. Although not optically resolved, plots of ODMR transition frequencies versus monitored wavelength revealed heterogeneity in the phosphorescence emission of echinomycin, which was ascribed to the presence of two distinct quinoxaline triplet-state environments (referred to as the blue and red triplet states of echinomycin in this report). We think that a likely origin of the two triplet states of echinomycin is the occurrence of two or more distinct conformations of the drug in aqueous solutions. Spectroscopically observed perturbations of the triplet-state properties of echinomycin such as the phosphorescence emission spectrum, phosphorescence lifetime, ODMR spectrum, and zero-field splitting (zfs) energies were investigated upon drug binding to the double-stranded alternating copolymers poly(dG-dC).poly(dG-dC) [abbreviated as poly[d(G-C)2]] and poly(dA-dT).poly(dA-dT) [abbreviated as poly[d(A-T)2]], the homopolymer duplexes poly(dG).poly(dC) [abbreviated as poly(dG.dC)] and poly(dA).poly(dT) [abbreviated as poly(dA.dT)], and the natural DNAs from Escherichia coli, Micrococcus lysodeikticus, and calf thymus. Echinomycin bisintercalation complexes with the self-complementary oligonucleotides d(ACGT), d(CGTACG), and d(ACGTACGT), which are thought to model drug binding sites, were also investigated. Phosphorescence and ODMR spectroscopic results indicate that the quinoxaline chromophores of the drug are involved in aromatic stacking interactions in complexes with the natural DNAs as evidenced by red shifts in the phosphorescence 0,0 band of the drug, a small but significant reduction in the phosphorescence lifetime of the red triplet state, and reduction of the zfs D-value of both the blue and red triplet states upon drug complexation. These changes in the triplet-state properties of echinomycin are consistent with stacking interactions that increase the polarizability of the quinoxaline environment. The extent of the reduction of the D parameter for the red triplet state upon complexation with the polymeric DNAs was found to correlate with the binding affinities measured for these targets [Wakelin, L. P. G., & Waring, M. J. (1976) Biochem. J. 157, 721-740], with the single exception of the drug-poly[d(G-C)2] complex, for which an increase in the D-value was noted. In addition, upon drug binding to the natural DNAs, there is a reversal of signal polarity in the ODMR spectra of the red triplet state. Among the synthetic DNA polymers investigated, a reversal of ODMR signal polarity was found only with the echinomycin-poly[d(A-T)2] complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Interaction of topotecan,DNA topoisomerase I inhibitor,with double-stranded polydeoxyribonucleotides. 4. Topotecan binds preferably to the GC base pairs of DNA

Interaction of topotecan (TPT) with synthetic double-stranded polydeoxyribonucleotides has been studied in solutions of low ionic strength at pH = 6.8 by linear flow dichroism (LD), circular dichroism (CD), UV-Vis absorption and Raman spectroscopy. The complexes of TPT with poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dC).poly(dG-dT), poly(dA).poly(dT) and previously studied by us complexes of TPT with calf thymus DNA and coliphage T4 DNA have been shown to have negative LD in the long-wavelength absorption band of TPT, whereas the complex of TPT with poly(dA-dT).poly(dA-dT) has positive LD in this absorption band of TPT. Thus, there are two different types of TPT complexes with the polymers. TPT has been established to bind preferably to GC base pairs because its affinity to the polymers of different GC composition decreases in the following order: poly(dG-dC).poly(dG-dC) > poly(dG).poly(dC) > poly(dA-dC).poly(dG-dT) > poly(dA).poly(dT). The presence of DNA has been shown to shift monomer-dimer equilibrium in TPT solutions toward dimer formation. Several duplexes of the synthetic polynucleotides bound together by the bridges of TPT dimers may participate in the formation of the studied type of TPT-polynucleotide complexes. Molecular models of TPT complex with linear and ring supercoiled DNAs and with deoxyguanosine have been considered. TPT (and presumably all camptothecin family) proved to be a representative of a new class of DNA-specific ligands whose biological action is associated with formation of dimeric bridges between two DNA duplexes.     

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.     

DNA solution conformation via infrared circular dichroism: experimental and theoretical results for B-family polymers

Infrared (vibrational) circular dichroism (VCD) has been observed for the DNA models d(CG)5, poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dT).poly(dA-dT), and poly(dA).poly(dT) in the B-conformation in buffered, aqueous solution. The observed results are quantitatively interpreted in terms of the exciton model for coupled carbonyl stretching vibrational states.     

Sequence Specific Binding of Beta Carboline Alkaloid Harmalol with Deoxyribonucleotides: Binding Heterogeneity,Conformational, Thermodynamic and Cytotoxic Aspects

Background

Base dependent binding of the cytotoxic alkaloid harmalol to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by various photophysical and calorimetric studies, and molecular docking.

Methodology/Principal Findings

Binding data obtained from absorbance according to neighbor exclusion model indicated that the binding constant decreased in the order poly(dG-dC).poly(dG-dC)>poly(dA-dT).poly(dA-dT)>poly(dA).poly(dT)>poly(dG).poly(dC). The same trend was shown by the competition dialysis, change in fluorescence steady state intensity, stabilization against thermal denaturation, increase in the specific viscosity and perturbations in circular dichroism spectra. Among the polynucleotides, poly(dA).poly(dT) and poly(dG).poly(dC) showed positive cooperativity where as poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT) showed non cooperative binding. Isothermal calorimetric data on the other hand showed enthalpy driven exothermic binding with a hydrophobic contribution to the binding Gibbs energy with poly(dG-dC).poly(dG-dC), and poly(dA-dT).poly(dA-dT) where as harmalol with poly(dA).poly(dT) showed entropy driven endothermic binding and with poly(dG).poly(dC) it was reported to be entropy driven exothermic binding. The study also tested the in vitro chemotherapeutic potential of harmalol in HeLa, MDA-MB-231, A549, and HepG2 cell line by MTT assay.

Conclusions/Significance

Studies unequivocally established that harmalol binds strongly with hetero GC polymer by mechanism of intercalation where the alkaloid resists complete overlap to the DNA base pairs inside the intercalation cavity and showed maximum cytotoxicity on HepG2 with IC₅₀ value of 14 µM. The results contribute to the understanding of binding, specificity, energetic, cytotoxicity and docking of harmalol-DNA complexation that will guide synthetic efforts of medicinal chemists for developing better therapeutic agents.     

Hexammineruthenium (III) chloride: a highly efficient promoter of the B-DNA to Z-DNA transition of poly-(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC)

The effects of Ru(NH3)(3+)6 on the conformation of poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC) were studied by circular dichroism (CD) spectroscopy. Ru(NH3)(3+)6 at very low concentrations provokes the Z-DNA conformation in both polynucleotides. In the presence of 50 mM NaCl, the concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) is 4 microM compared to 5 microM for Co(NH3)(3+)6. The half-lives of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of 10 microM Ru(NH3)(3+)6 and Co(NHG3)(3+)6 are at 23 and 30 min, respectively. The concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-dC).poly(dG-dC) is 50 microM. These results demonstrate that Ru(NH3)(3+)6 is a highly efficient trivalent cation for the induction of B to Z transition in poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC). In contrast, Ru(NH3)(3+)6 has no significant effect on the conformation of calf thymus DNA, poly(dA-dT).poly(dA-dT) and poly(dA-dC).poly(dG-dT).     

B----Z DNA conformational changes induced by a family of dinuclear bis(platinum) complexes.

The reactions of bis(platinum) complexes of general formula [(PtClm(NH3)3-m)2(NH2(CH2)nNH2)]2(2-m)+ were studied with poly(dG-dC).poly(dG-dC), poly(dG-m5dC).poly(dG-m5dC) and poly(dG).poly(dC). When m = 0 (Complexes II, n = 2,4) the complexes are saturated 4+ cations capable only of electrostatic interactions with the polynucleotide. Where m = 1 the complexes contain two monodentate platinum coordination spheres with the chloride trans to the diamine bridge (Complexes I, n = 2,4, 1,1/t,t). Complexes I give CD spectra characteristic of a 'Z-like' conformation upon reaction with poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) but not poly(dG).poly(dC). The B----Z transition appears independent of interplatinum diamine chain length. As little as 1 bis(platinum) complex per 25-30 base pairs is sufficient to observe the Z-like spectrum. Covalent binding is however not a prerequisite for Z-DNA formation because the polyvalent cations II are also very effective in inducing the B----Z transition in either poly(dG-dC).poly(dG-dC) or poly (dG-m5dC).poly(dG-m5dC). In these cases, the concentrations of II required are significantly lower than analogous monomeric agents such as [Co(NH3)6]3+. The possible biological consequences of the Z-DNA induction by bis(platinum) complexes are discussed.     

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.     

Conformational peculiarities of polynucleotides with a nonrandom base sequence according to the 1H----3H exchange rate in C8H groups of purinic residues

We have determined the 1H----3H exchange rate constants between water and C8H groups of purinic residues of alternating polynucleotides poly(dA-dT).poly(dA-dT), poly(dG-dC).poly(dG-dC) and poly(dA-dC).poly(dG-dT) as well as homopolynucleotides poly(dA).poly(dT) and poly(dG).poly(dC) in aqueous solutions with high-salt concentrations (3 M NaCl and 4-6 M CsF), in water-ethanol (60%) solution and in 0.15 M NaCl at 25 degrees C. The rate constants for adenine (kA) and guanine (kG) of polynucleotides were compared with corresponding constants for E. coli DNA. dGMP nd dAMP at the same conditions. The relation between exchange rates and conformations of polynucleotides permits the study of their conformational peculiarities in solution. Of three alternating polynucleotides examined in 0.15 M NaCl the exchange retardation was observed only for poly(dA-dT).poly(dA-dT) as compared with that in B-DNA, which is in good agreement with the B-alternating "wrinkled" DNA model. The conformations of poly(dG-dC).poly(dG-dC) and poly(dA-dC).poly(dG-dT), according to the exchange data obtained are within the B form. For homopolynucleotides in 0.15 M NaCl, the KA value for poly(dA).poly(dT) is nearly the same as kA for B-DNA, which indicates the similarity of their conformations, whereas the kG value for poly(dG).poly(dC) is 1.7-fold lower in comparison with the kG value in B-DNA. This seems to be connected with the existence of B = A conformation equilibrium for poly(dG).poly(dC) in solution. The increase of NaCl concentration to 3 M results in a B----Z transition in the case of poly(dG-dC).poly(dG-dC) and in the shift of B = A equilibrium towards the A-form in the case of poly(dG).poly(dC) as is evidenced by alterations of their KG values. Poly(dA-dT).poly(dA-dT) in 6 M CsF and poly(dA-dC).poly(dG-dT) in 4.3 M CsF maintain their inherent conformations in 0.15 M NaCl in spite of the fact that they are characterised by the "X-type" CD-spectrum at these conditions. According to the exchange data the conformation of poly(dA).poly(dT) in 6 M CsF corresponds to the "heteronomous" DNA model or some other structure with lower accessibility of C8H groups of adenylic residues.     

Conformational transitions of polynucleotides in the presence of rhodium complexes

We studied the effects of hexammine and tris(ethylene diamine) complexes of rhodium on the conformation of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) using spectroscopic techniques and an enzyme immunoassay. Circular dichroism spectroscopic measurements showed that Rh(NH3)6(3+) provoked a B-DNA----Z-DNA----psi-DNA conformational transition in poly(dG-dC).poly(dG-dC). Using the enzyme immunoassay technique with a monoclonal anti-Z-DNA antibody, we found that the left-handedness of the polynucleotide was maintained in the psi-DNA form. In addition, we compared the efficacy of Rh(NH3)6(3+) and Rh(en)3(3+) to provoke the Z-DNA conformation in poly(dG-dC).poly(dG-dC) and poly(dG-m5dC.poly(dG-m5dC). The concentrations of Rh(NH3)6(3+) and Rh(en)3(3+) at the midpoint B-DNA----Z-DNA transition of poly(dG-dC).poly(dG-dC) were 48 +/- 2 and 238 +/- 2 microM, respectively. The psi-DNA form of poly(dG-dC).poly(dG-dC) was stabilized at 500 microM Rh(NH3)6(3+). With poly(dG-m5dC).poly(dg-m5dC), both counterions provoked the Z-DNA form at approximately 5 microM and stabilized the polynucleotide in this form up to 1000 microM concentration. These results show that trivalent complexes of Rh have a profound influence on the conformation of poly(dG-dC).poly(dG-dC) and its methylated derivative. Furthermore, the Rh complexes are capable of maintaining the Z-DNA form at concentration ranges far higher than that of other trivalent complexes. Our results also demonstrate that the efficacy of trivalent inorganic complexes to induce the B-DNA to Z-DNA transition of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) is dependent on the nature of the ligand as well as the polynucleotide modification. Differences in charge density and hydration levels of counterions or base sequence- and counterion-dependent specific interactions between DNA and metal complexes might be possible mechanisms for the observed effects.     

Optically detected magnetic resonance of Escherichia coli glutamic acid specific transfer ribonucleic acid and its anticodon-anticodon complex with yeast phenylalanine-specific transfer ribonucleic acid

The low-temperature phosphorescence and the optically detected magnetic resonance (ODMR) spectra of Escherichia coli tRNA2Glu and its anticodon-anticodon complex with yeast tRNAPhe are reported. The ODMR signals are assigned to the modified base 5-[(methylamino)-methyl]-2-thiouracil (mnm5S2U) located at the "wobble" position of the anticodon. The zero-field splittings (zfs) are larger than those found for 1-methyl-2-thiouracil previously studied in a 1-methyluracil host system by ODMR. They are comparable, however, to those found for neat, polycrystalline 1-methyl-2-thiouracil and for the latter dissolved in ethyl acetate solvent. In the polycrystalline sample, five traps with widely varying zfs are assigned. A very large (15-25%) reduction in the D parameter of mnm5S2U is found to occur on formation of the anticodon-anticodon complex with yeast tRNAPhe. Additional ODMR signals found in the complex are assigned to the wybutine base of yeast tRNAPhe. The extreme sensitivity of the zfs parameters of S2U to environmental perturbations is ascribed to the variable involvement of the heavy atom containing chromophore, C = S, in the triplet-state wave function, which is largely localized on the C = C - C = O portion of the molecule.     

Interaction of drugs with Z-DNA: cooperative binding of actinomycin D or actinomine to the left-handed forms of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) reverses the conformation of the helix

The interaction of actinomycin D and actinomine with poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) under B- and Z-form conditions has been investigated by optical and phase partition techniques. Circular dichroism data show that the conformation at the binding site is right-handed, even though adjacent regions of the polymer have a left-handed conformation. Actinomycin D binds in a cooperative manner to poly(dG-dC).poly(dG-dC) under both B-form and Z-form conditions. Analysis of the circular dichroism data shows that 5 +/- 1 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl switch to a right-handed conformation for each bound actinomycin D. When the left-handed form of poly(dG-dC).poly(dG-dC) is stabilized by the presence of 40 microM [Co(NH3)6]Cl3, 25 +/- 5 base pairs switch from a left-handed to a right-handed conformation for each bound actinomycin D. Actinomine binds cooperatively to left-handed poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and to left-handed poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2. Actinomine does not bind to left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl at concentrations as high as 100 microM. Each bound actinomine converts 11 +/- 3 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and 7 +/- 2 base pairs of left-handed poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2. The binding isotherm data also indicate that the binding site has a right-handed conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adduct formation between the carcinogen N-acetoxy-2-acetylaminofluorene and synthetic polydeoxyribonucleotides.

The chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF) was reacted with poly(dG-dC) - poly(dG-dC); poly dG - poly dC; poly(dA-dT) - poly (dA-dT); and poly dA - poly dT under a variety of conditions. Poly (dG-homo GC polymer and 10--20 more reactive the A + T polymers. Lowering the ionic strength increased the extent of reaction, while pH change (8.9 vs. 5.5) had only a small effect. If ionic strength was adjusted so that the two guanine-containing polymers showed equal thermal stability (as judged by Tm) then the alternating copolymer was 7 times as reactive as the homopolymer. In aggreement with previous investigators, the major product was found to be 8-(N-2-fluorenylacetamido) deoxyguanosine.     

Conformational variability of poly(dA-dT).poly(dA-dT) and some other deoxyribonucleic acids includes a novel type of double helix

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.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular recognition of DNA by small molecules: AT base pair specific intercalative binding of cytotoxic plant alkaloid palmatine

The base dependent binding of the cytotoxic alkaloid palmatine to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by competition dialysis, spectrophotometric, spectrofluorimetric, thermal melting, circular dichroic, viscometric and isothermal titration calorimetric (ITC) studies. Binding of the alkaloid to various polynucleotides was dependent upon sequences of base pairs. Binding data obtained from absorbance measurements according to neighbour exclusion model indicated that the intrinsic binding constants decreased in the order poly(dA).poly(dT)>poly(dA-dT).poly(dA-dT)>poly(dG-dC).poly(dG-dC)>poly(dG).poly(dC). This affinity was also revealed by the competition dialysis, increase of steady state fluorescence intensity, increase in fluorescence quantum yield, stabilization against thermal denaturation and perturbations in circular dichroic spectrum. Among the polynucleotides, poly(dA).poly(dT) showed positive cooperativity at binding values lower than r=0.05. Viscosity studies revealed that in the strong binding region, the increase of contour length of DNA depended strongly on the sequence of base pairs being higher for AT polymers and induction of unwinding-rewinding process of covalently closed superhelical DNA. Isothermal titration calorimetric data showed a single entropy driven binding event in the AT homo polymer while that with the hetero polymer involved two binding modes, an entropy driven strong binding followed by an enthalpy driven weak binding. These results unequivocally established that the alkaloid palmatine binds strongly to AT homo and hetero polymers by mechanism of intercalation.     

Dependence of the Raman signature of genomic B-DNA on nucleotide base sequence.

The vibrational spectra of four genomic and two synthetic DNAs, encompassing a wide range in base composition [poly(dA-dT). poly(dA-dT), 0% G + C; Clostridium perfringens DNA, 27% G + C; calf thymus DNA, 42% G + C; Escherichia coli DNA, 50% G + C; Micrococcus luteus DNA, 72% G + C; poly(dG-dC).poly(dG-dC), 100% G + C] (dA: deoxyadenosine; dG: deoxyguanosine; dC: deoxycytidine; dT: thymidine), have been analyzed using Raman difference methods of high sensitivity. The results show that the Raman signature of B DNA depends in detail upon both genomic base composition and sequence. Raman bands assigned to vibrational modes of the deoxyribose-phosphate backbone are among the most sensitive to base sequence, indicating that within the B family of conformations major differences occur in the backbone geometry of AT- and GC-rich domains. Raman bands assigned to in-plane vibrations of the purine and pyrimidine bases-particularly of A and T-exhibit large deviations from the patterns expected for random base distributions, establishing that Raman hypochromic effects in genomic DNA are also highly sequence dependent. The present study provides a basis for future use of Raman spectroscopy to analyze sequence-specific DNA-ligand interactions. The demonstration of sequence dependency in the Raman spectrum of genomic B DNA also implies the capability to distinguish genomic DNAs by means of their characteristic Raman signatures.