Cyanine dyes as intercalating agents: kinetic and thermodynamic studies on the DNA/Cyan40 and DNA/CCyan2 systems

The interaction of cyanines with nucleic acids is accompanied by intense changes of their optical properties. Consequently these molecules find numerous applications in biology and medicine. Since no detailed information on the binding mechanism of DNA/cyanine systems is available, a T-jump investigation of the kinetics and equilibria of binding of the cyanines Cyan40 [3-methyl-2-(1,2,6-trimethyl-4(1H)pyridinylidenmethyl)-benzothiazolium ion] and CCyan2 [3-methyl-2-[2-methyl-3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-benzothiazolium ion] with CT-DNA is performed at 25 degrees C, pH 7 and various ionic strengths. Bathochromic shifts of the dye absorption band upon DNA addition, polymer melting point displacement (DeltaT = 8-10 degrees C), site size determination (n = 2), and stepwise kinetics concur in suggesting that the investigated cyanines bind to CT-DNA primary by intercalation. Measurements with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) reveal fair selectivity of CCyan2 toward G-C basepairs. T-jump experiments show two kinetic effects for both systems. The binding process is discussed in terms of the sequence D + S left arrow over right arrow D,S left arrow over right arrow DS(I) left arrow over right arrow DS(II), which leads first to fast formation of an external complex D,S and then to a partially intercalated complex DS(I) which, in turn, converts to DS(II), a more stable intercalate. Absorption spectra reveal that both dyes tend to self-aggregate; the kinetics of CCyan2 self-aggregation is studied by T-jump relaxation and the results are interpreted in terms of dimer formation.     

A kinetic analysis of cyanine selectivity: CCyan2 and Cyan40 intercalation into poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC)

A T-jump investigation of the binding of Cyan40 [3-methyl-2-(1,2,6-trimethyl-4(1H)pyridinylidenmethyl)-benzothiazolium ion] and CCyan2 [3-methyl-2-[2-methyl-3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-benzothiazolium ion] with poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC) is performed at I = 0.1M (NaCl), 25 degrees C and pH 7. Two kinetic effects are observed for both systems. The binding process is discussed in terms of the sequence D + P <==> P,D <==> PD(I) <==> PD(II), which leads first to fast formation of a precursor complex P,D and then to a partially intercalated complex PD(I) which converts to the fully intercalate complex PD(II). Concerning CCyan2 the rate parameters depend on the polymer nature and their analysis shows that in the case of poly(dG-dC) x poly(dG-dC) the most stable bound form is the fully intercalated complex PD(II), whereas in the case of poly(dA-dT) x poly(dA-dT) the partially intercalated complex PD(I) is the most stable species. Concerning Cyan40, the rate parameters remain unchanged on going from A-T to G-C indicating that this dye is unselective.     

Relaxation kinetics of the interaction between RNA and metal-intercalators: the Poly(A).Poly(U)/platinum-proflavine system

The interactions of Poly(A).Poly(U) with the cis-platinum derivative of proflavine [{PtCl(tmen)}(2){HNC(13)H(7)(NHCH(2)CH(2))(2)}](+) (PRPt) and proflavine (PR) are investigated by spectrophotometry, spectrofluorimetry and T-jump relaxation at I=0.2M, pH 7.0, and T=25 degrees C. Base-dye interactions prevail at high RNA/dye ratio and binding isotherms analysis reveals that both dyes bind to Poly(A).Poly(U) according to the excluded site model (n=2). Only one relaxation effect is observed for the Poly(A).Poly(U)/PRPt system, whereas two effects are observed with Poly(A).Poly(U)/PR. The results agree with the sequence D+S <==> D, S <==> DS(I) <==> DS(II), where D,S is an external complex, DS(I) is a partially intercalated species, and DS(II) is the fully intercalated complex. Formation of DS(II) could be observed in the case of proflavine only. This result is interpreted by assuming that the platinum-containing residue of PRPt hinders the full intercalation of the acridine residue.     

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.     

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.     

Internal motions in B- and Z-form poly(dG-dC).poly(dG-dC): 1H NMR relaxation studies

Proton NMR relaxation measurements are used to compare the molecular dynamics of 60 base pair duplexes of B- and Z-form poly(dG-dC).poly(dG-dC). The relaxation rates of the exchangeable guanine imino protons (Gim) in H2O and in 90% D2O show that below 20 degrees C spin-lattice relaxation is exclusively from proton-proton magnetic dipolar interactions while proton-nitrogen interactions contribute about 30% to the spin-spin relaxation. The observation that the spin-lattice relaxation is nonexponential and that the initial spin-lattice relaxation rate of the Gim, G-H8 and C-H6 protons depends on the selectivity of the exciting pulse shows that spin-diffusion dominates the spin-lattice relaxation. The relaxation rates of the Gim, C-H5, and C-H6 in B- and Z-form poly(dG-dC).poly(dG-dC) cannot be explained by assuming the DNA behaves as a rigid rod. The data can be fit by assuming large-amplitude out of plane motions (+/- 30-40 degrees, tau = 1-100 ns) and fast, large-amplitude local torsional motions (+/- 25-90 degrees, tau = 0.1-1.5 ns) in addition to collective torsional motions. The results for the B and Z forms show that the rapid internal motions are similar and large in both conformations although backbone motions are slightly slower, or of lower amplitude, in Z DNA. At high temperatures (greater than 60 degrees C), imino proton exchange with solvent dominates the spin-lattice relaxation of B-form poly(dG-dC).poly(dG-dC), but in the Z form no exchange contribution (less than 2 s-1) is observed at temperatures as high as 85 degrees C. Conformational fluctuations that expose the imino protons to the solvent are strikingly different in the B and Z forms. The results obtained here are compared with those previously reported for poly(dA-dT).poly(dA-dT).     

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.     

1H two-dimensional nuclear Overhauser effect and relaxation studies of poly(dA).poly(dT)

The structure of poly(dA).poly(dT) in aqueous solution has been studied by using 1H two-dimensional nuclear Overhauser effect (2D NOE) spectroscopy and relaxation rate measurements on the imino and nonexchangeable protons. The assignments of the 1H resonances are determined from the observed cross-relaxation patterns in the 2D NOE experiments. The cross-peak intensities together with the measured relaxation rates show that the purine and pyrimidine strands in poly(dA).poly(dT) are equivalent in aqueous solution. The results are consistent with a right-handed B-form helix where the sugars on both strands are in the C2'-endo/anti configuration. These observations are inconsistent with a proposed heteronomous structure for poly(dA).poly(dT) [Arnott, S., Chandrasekaran, R., Hall, I. H., & Puigjaner, L. C. (1983) Nucleic Acids Res. 11, 4141-4155]. The measured relaxation rates also show that poly(dA).poly(dT) has fast, large-amplitude local internal motions (+/- 20-25 degrees) in solution and that the amplitudes of the base and sugar motions are similar. The motion of the bases in poly(dA).poly(dT) is also similar to that previously reported for poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) [Assa-Munt, N., Granot, J., Behling, R. W., & Kearns, D. R. (1984) Biochemistry 23, 944-955; Mirau, P. A., Behling, R. W., & Kearns, D. R. (1985) Biochemistry 24, 6200-6211].     

Intercalation of Zn(II) and Cu(II) complexes of the cyclic polyamine Neotrien into DNA: equilibria and kinetics

The equilibria and kinetics of the interaction of the Zn(II) and Cu(II) complexes of the macrocyclic polyamine 2,5,8,11-tetraaza[12]-[12](2,9)[1,10]-phenanthrolinophane (Neotrien) with calf thymus DNA have been investigated at pH=7.0 and T=25 degrees C by spectrophotometry, spectrofluorimetry and stopped-flow method. At low dye/polymer ratios both complexes bind to DNA according to the excluded site model. At high dye/polymer ratios the binding displays cooperative features. The logarithm of the binding constant depends linearly on -log[NaCl]. The kinetic results suggest the D + S <==> D, S <==> DS mechanism where the metal complexes (D) react with the DNA sites (S) leading to fast formation of an externally bound form (D,S) which, in turn, is converted into internally bound complex (DS) by intercalation. The binding constants, evaluated as ratios of rate constants, agree with those obtained from equilibrium binding experiments, thus confirming the validity of the proposed model. Fluorescence titrations, where the metal-Neotrien complexes were added to DNA previously saturated with ethidium bromide (EB), show that both complexes displace EB from the DNA cavities. The reverse process, i.e. the addition of excess ethidium to the DNA/metal Neotrien systems, leads to fluorescence recovery for DNA/ZnNeotrien but not for DNA/CuNeotrien. This observation suggests that the binding of CuNeotrien induces deep alterations in the DNA structure. Experiments with Poly(dA-dT)*Poly(dA-dT) and Poly(dG-dC)*Poly(dG-dC) reveal that CuNeotrien mainly affects the structure of the latter polynucleotide.     

23Na NMR relaxation study of the effects of conformation and base composition on the interactions of counterions with double-helical DNA

NMR relaxation rates (T1(-1) and T2(-1)) have been determined for 23Na in aqueous salt solutions containing various types of helical double-stranded deoxyribonucleic acids. These measurements were performed on three synthetic polynucleotides having different overall conformations, poly-(dA-dT).poly(dA-dT) (alternating B-DNA), poly(dG-dC).poly(dG-dC) at low salt (B-DNA), and Br-poly(dG-dC).Br-poly(dG-dC) (left-handed Z-DNA), and on four types of natural DNA differing in base composition, Clostridium perfringens (26% GC), calf thymus (40% GC), Escherichia coli (50% GC), and Micrococcus lysodeikticus (72% GC). For all types of DNA investigated, except poly(dA-dT).poly(dA-dT), the 23Na NMR spectra measured at 21 degrees C and an applied field of 4.7 T are non-Lorentzian. These non-Lorentzian spectra were analyzed on the basis of the two-state model and the standard theory of nonexponential quadrupolar relaxation processes in order to obtain estimates of the correlation times (tau c) characteristic of the sodium nuclei associated with the various nucleic acids. All of the correlation times estimated in this way are in the range of nanoseconds. The magnitudes of these correlation times show a significant dependence on the overall conformation of the nucleic acid (B vs. Z) but not on its base composition. To investigate the concentration dependence of tau c, sodium or magnesium salts were added to solutions of Br-poly(dG-dC).Br-poly(dG-dC) (Z-DNA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the proton-deuteron exchange at position H8 of adenine and guanine in DNA.

Proton-NMR has been used to determine the activation energies and pre-exponential factors for the deuterium exchange of AH8 in poly(dA-dT).poly(dA-dT), and for GH8 in poly(dG-dC).poly(dG-dC). No simple relationship between the kinetic parameters and molecular conformation was found. By addition of 4.5 M NaCl a transition from the B to the Z conformation was induced for poly(dG-dC).poly(dG-dC), and an increased exchange rate was observed. The exchange rate for poly(dA-dT).poly(dA-dT) also increased below 64 degrees C, and a significant decrease in activation energy on addition of 4.5 M NaCl was observed. The exchange rates at T = 55.8 degrees C were also measured for the AH8 and GH8 in random sequence calf thymus DNA. From the difference in exchange rates, a method of preferential labeling of either the AH8 or the GH8 in high molecular weight DNA is evaluated.     

Conformation of poly(dG-dC)·Poly(dG-dC) and Poly(dA-dT)·Poly(dA-dT) interacting with the antitumor drug cis-[Pt(NH32]Cl2

The interaction of cis-dichlorodiammine platinum(II) with poly(dG-dC)·poly(dG-dC) and poly(dA-dT) ·poly(dA-dT) was studied by circular dichroism. Significant conformational changes were induced in both alternating polymers: in the case of poly(dG-dC) ·poly(dG-dC) the spectra were not conclusive in terms of a well defined conformation, even if the presence of left-handed helices could be suggested. For poly(dA-dT)·poly(dA-dT) the data were interpreted in terms of a dimer-helix → single hairpin helix transition induced by the metal. The results obtained are discussed with reference to the antitumor activity of the drug.     

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)     

Studies on binding of toromycin, an antitumor antibiotic, to DNA

Toromycin, an antitumor, bactericidal and antiviral compound, was found to bind to DNA in such a way as to interfere with the dissociation of double helix at an elevated temperature. The antibiotic did not introduce strand scission into DNA. Single-strand-specific nuclease S1-susceptibility of negatively supercoiled DNA was not influenced by its binding. The antibiotic was shown to bind to both of the alternating purine-pyrimidine copolymers, poly(dG-dC):poly(dG-dC) and poly(dA-dT):poly(dA-dT). The unique C-glycoside molecule of toromycin interacted with single-stranded DNA, but was found to have no affinity for RNA.     

DNA-binding affinities and sequence selectivity of quaternary benzophenanthridine alkaloids sanguinarine, chelerythrine, and nitidine

A comparative study on the intercalating binding of sanguinarine, chelerythrine, and nitidine with CT DNA, poly(dG-dC).poly(dG-dC), poly(dA-dT).poly(dA-dT), and seven sequence-designed double-stranded oligodeoxynucleotides has been performed using fluorometric and spectrophotometric techniques, aiming at providing insights into their sequence selectivity for DNA-binding. The results show that both sanguinarine and nitidine bind preferentially to DNA containing alternating GC base pairs [d(TGCGCA)(2)], while chelerythrine exhibits quite distinct sequence selectivity from sanguinarine, which shows a high specificity for DNA containing contiguous GC base pairs [5'-TGGGGA-3'/3'-ACCCCT-5'].     

Studies on synthetic chromatins containing poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC)

Core histones, (H2A,H2B,H3,H4)₂, were reconstituted with the synthethic polynucleotides poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) to yield synthetic chromatins containing 200 basepairs per octamer. These synthetic chromatins displayed a 36% decrease in the circular dichroism (CD) peak ellipticity from the value of the polynucleotide free in solution; the poly(dA-dT)·poly(dA-dT)/chromatin showed an increase in the complexity of the thermal denaturation profile compared to that of the polynucleotide. Both the temperature of maximum $\text{d}\text{h}\text{d}\text{T}$ for each transition (T_m) and the relative amount of poly(dA-dT)·poly(dA-dT) in the synthetic chromatin melting in each of the four thermal transitions is a function of the ionic strength over the 0–5 mM sodium phosphate range (0.25 mM EDTA, pH 7.0); a shift of material toward higher melting transitions was observed with increasing ionic strength. The CD peak ellipticity value for both synthetic chromatins was ionic strength-independent over the 0–5 mM sodium phosphate range. These results are in contrast to those observed with $\text{H}\text{1}\text{H}\text{5}$ stripped chicken erythrocyte chromatin (Fulmer, A. and Fasman, G.D. (1979) Biopolymers 18, 2875–2891), where an ionic strength dependence was found. Differences in the CD spectra between poly(dA-dT)·poly(dA-dT)/chromatin, poly(dG-dC)·poly(dG-dC)/chromatin and $\text{H}\text{1}\text{H}\text{5}$ stripped chicken erythrocyte chromatin suggest subtle differences in assembly. Finally, the temperature dependence of the CD spectra of poly(dA-dT)·poly(dA-dT)-containing synthetic chromatin, which is similar to that for the polynucleotide, suggests the core histone bound polynucleotide has a large degree of conformational flexibility allowing it to undergo the premelt transition.     

Synthesis and DNA binding studies of a new asymmetric cyanine dye binding in the minor groove of [poly(dA-dT)]2

A new asymmetric cyanine dye has been synthesised and its interaction with different DNA has been investigated. In this dye, BEBO, the structure of the known intercalating cyanine dye BO has been extended with a benzothiazole substituent. The resulting crescent-shape of the molecule is similar to that of the well-known minor groove binder Hoechst 33258. Indeed, comparative studies of BO illustrate a considerable change in binding mode induced by this structural modification. Linear and circular dichroism studies indicate that BEBO binds in the minor groove to [poly (dA-dT)](2), but that the binding to calf thymus DNA is heterogeneous, although still with a significant contribution of minor groove binding. Similar to other DNA binding asymmetric cyanine dyes, BEBO has a large increase in fluorescence intensity upon binding and a relatively large quantum yield when bound. The minor groove binding of BEBO to [poly (dA-dT)](2) affords roughly a 180-fold increase in intensity, which is larger than to that of the commonly used minor groove binding probes DAPI and Hoechst 33258.     

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.     

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.