Berberine, a strong polyriboadenylic acid binding plant alkaloid: spectroscopic, viscometric, and thermodynamic study

The interaction of berberine with single stranded poly(rA) structure was investigated using a combination of spectrophotometric, spectrofluorimetric, circular dichroic, viscometric, and thermodynamic studies. The interaction process was characterized by typical hypochromic and bathochromic effects in the absorption spectrum of berberine, enhancement of fluorescence intensity of berberine, increase of viscosity, and perturbation of circular dichroic spectrum of single stranded poly(rA). Scatchard plot obtained from spectrophotometric analysis showed that berberine bound strongly to single stranded poly(rA) in a non-cooperative manner. In contrast, berberine does not show any significant effect (i) in its absorbance and fluorescence spectra on binding to double stranded poly(rA), (ii) alter the circular dichroic spectrum of double stranded poly(rA), or (iii) increase of viscosity of double stranded poly(rA) indicating that it does not bind at all to double stranded poly(rA) structure. Thermodynamic parameters indicated that the binding of the alkaloid to single stranded poly(rA) is an endothermic process and entropy driven. All these findings, taken together clearly support that berberine binds strongly to single stranded poly(rA) structure by a mechanism of partial intercalation leading to its use in gene regulation in eukaryotic cells.     

Spectroscopic and thermodynamic studies on the binding of sanguinarine and berberine to triple and double helical DNA and RNA structures

A comparative study on the interaction of sanguinarine and berberine with DNA and RNA triplexes and their parent duplexes was performed, by using a combination of spectrophotometric, UV thermal melting, circular dichroic and thermodynamic techniques. Formation of the DNA and RNA triplexes was confirmed from UV-melting and circular dichroic measurements. The interaction process was characterized by increase of thermal melting temperature, perturbation in circular dichroic spectrum and the typical hypochromic and bathochromic effects in the absorption spectrum. Scatchard analysis indicated that both the alkaloids bound to the triplex and duplex structures in a non-cooperative manner and the binding was stronger to triplexes than to parent duplexes. Thermal melting studies further indicated that sanguinarine stabilized the Hoogsteen base paired third strand of both DNA and RNA triplexes more tightly compared to their Watson-Crick strands, while berberine stabilized the third strand only without affecting the Watson-Crick strand. However, sanguinarine stabilized the parent duplexes while no stabilization was observed with berberine under identical conditions. Circular dichroic studies were also consistent with the observation that perturbations of DNA and RNA triplexes were more compared to their parent duplexes in presence of the alkaloids. Thermodynamic data revealed that binding of sanguinarine and berberine to triplexes (T.AxT and U.AxU) and duplexes (A.T and A.U) showed negative enthalpy changes and positive entropy changes but that of sanguinarine to C.GxC(+) triplex and G.C duplex exhibited negative enthalpy and negative entropy changes. Taken together, these results suggest that both sanguinarine and berberine can bind and stabilize the DNA and RNA triplexes more strongly than their respective parent duplexes.     

Circular Dichroism Studies of the Structure of DNA Complex with Berberine

Abstract

The binding of the benzodioxolo-benzoquinolizine alkaloid, berberine chloride to natural and synthetic DNAs has been studied by intrinsic and extrinsic circular dichroic measurements. Binding of berberine causes changes in the circular dichroism spectrum of DNA as shown by the increase of molar ellipticity of the 270nm band, but with very little change of the 240nm band. The molar ellipticity at the saturation depends strongly on the base composition of DNA and also on salt concentration, but always larger for the AT rich DNA than the GC rich DNA The features in the circular dichroic spectral changes of berberine-synthetic DNA complexes were similar to that of native DNA but depends on the sequence of base pairs.

On binding to DNA and polynucleotides, the alkaloid becomes optically active. The extrinsic circular dichroism developed in the visible absorption region (300–500nm) for the berberine-DNA complexes shows two broad spectral bands in the regions 425–440nm and 340–360nm with the maximum varying depending on base composition and sequence of DNA While the 425nm band shows less variation on the binding ratio, the 360nm band is remarkably dependent on the DNA/alkaloid ratio. The generation of the alkaloid associated extrinsic circular dichroic bands is not dependent on the base composition or sequence of base pairs, but the nature and magnitude of the bands are very much dependent on these two factors and also on the salt concentration. The interpretation of the results with respect to the modes of the alkaloid binding to DNA are presented.     

Circular dichroism studies of the structure of DNA complex with berberine.

The binding of the benzodioxolo-benzoquinolizine alkaloid, berberine chloride to natural and synthetic DNAs has been studied by intrinsic and extrinsic circular dichroic measurements. Binding of berberine causes changes in the circular dichroism spectrum of DNA as shown by the increase of molar ellipticity of the 270nm band, but with very little change of the 240nm band. The molar ellipticity at the saturation depends strongly on the base composition of DNA and also on salt concentration, but always larger for the AT rich DNA than the GC rich DNA. The features in the circular dichroic spectral changes of berberine-synthetic DNA complexes were similar to that of native DNA, but depends on the sequence of base pairs. On binding to DNA and polynucleotides, the alkaloid becomes optically active. The extrinsic circular dichroism developed in the visible absorption region (300-500nm) for the berberine-DNA complexes shows two broad spectral bands in the regions 425-440nm and 340-360nm with the maximum varying depending on base composition and sequence of DNA. While the 425nm band shows less variation on the binding ratio, the 360nm band is remarkably dependent on the DNA/alkaloid ratio. The generation of the alkaloid associated extrinsic circular dichroic bands is not dependent on the base composition or sequence of base pairs, but the nature and magnitude of the bands are very much dependent on these two factors and also on the salt concentration. The interpretation of the results with respect to the modes of the alkaloid binding to DNA are presented.     

Spectroscopic studies on the interaction of aristololactam-beta-D-glucoside with DNA and RNA double and triple helices: A comparative study.

The interaction of aristololactam-beta-D-glucoside (ADG), a DNA intercalating alkaloid, with the DNA triplexes, poly(dT). poly(dA)xpoly(dT) and poly(dC).poly(dG)xpoly(dC+), and the RNA triplex poly(rU).poly(rA)xpoly(rU) was investigated by circular dichroic, UV melting profile, spectrophotometric, and spectrofluorimetric techniques. Comparative interaction with the corresponding Watson-Crick duplexes has also been examined under identical experimental conditions. Triplex formation has been confirmed from biphasic thermal melting profiles and analysis of temperature-dependent circular dichroic measurements. The binding of ADG to triplexes and duplexes is characterized by the typical hypochromic and bathochromic effects in the absorption spectrum, quenching of steady-state fluorescence intensity, a decrease in fluorescence quantum yield, an increase or decrease of thermal melting temperatures, and perturbation in the circular dichroic spectrum. Scatchard analysis indicates that ADG binds both to the triplexes and the duplexes in a noncooperative manner. Binding parameters obtained from spectrophotometric measurements are best fit by the neighbor exclusion model. The binding affinity of ADG to the DNA triplexes is substantially stronger than to the RNA triplex. Thermal melting study further indicates that ADG stabilizes the Hoogsteen base-paired third strand of the DNA triplexes whereas it destabilizes the same strand of RNA triplex but stabilizes its Watson-Crick strands. Comparative data reveal that ADG exhibits a stronger binding to the triple helical structures than to the respective double helical structures.     

Physical studies of the interaction of a calf thymus helix-destablizing protein with nucleic acids

UP1, a calf thymus protein that destabilizes both DNA and RNA helices, dramatically accelerates the conversion of the inactive conformers of several small RNA molecules to their biologically active forms [Karpel, R. L., Swistel, D. G., Miller, N. S., Geroch, M. E., Lu, C., & Fresco, J. R. (1974) Brookhaven Symp. Biol. 26, 165-174]. Using circular dichroic and spectrophotometric methods, we have studied the interaction of this protein with a variety of synthetic polynucleotides and yeast tRNA3Leu. As judged by perturbations in polynucleotide ellipticity or ultraviolet absorbance, the secondary structures of the single-stranded helices poly(A) and poly(C), as well as the double-stranded helices poly[d(A-T)] and poly(U.U), are largely destroyed upon interaction with UP1 at low ionic strength. This effect can be reversed by an increase in [Na+]: half the UP1-induced perturbation of the poly(A) CD spectrum is removed at 0.05 M Na+. The variation of poly(A) ellipticity and ultraviolet absorbance with [UP1]/[poly(A)]p is used to determine the length of single-stranded polynucleotide chain covered by the protein: 7 +/- 1 residues. A model is presented in which the specificity of UP1 for single strands and their concomitant distortion are a consequence of maximal binding of nucleic acid phosphates to a unique matrix of basic residues on the protein. Analogous to the effect on polynucleotides, UP1-facilitated renaturation of yeast tRNA3Leu follows the partial destruction of the inactive tRNA's secondary structure. At the tRNA absorbance maximum, UP1 effects a hyperchromic change of 10%, representing one-third of the secondary structure of the inactive conformer. This change is also clearly observable as a perturbation of the tRNA's circular dichroism spectrum.     

Protonated structures of naturally occurring deoxyribonucleic acids and their interaction with berberine

Protonation-induced conformational changes in natural DNAs of diverse base composition under the influence of low pH, low temperature, and low ionic strength have been studied using various spectroscopic techniques. At pH3.40, 10mM [Na+], and at 5 degrees C, all natural DNAs irrespective of base composition adopted an unusual and stable conformation remarkably different from the canonical B-form conformation. This protonated conformation has been characterized to have unique absorption and circular dichroic spectral characteristics and exhibited cooperative thermal melting profiles with decreased thermal melting temperatures compared to their respective B-form counterparts. The nature of this protonated structure was further investigated by monitoring the interaction of the plant alkaloid, berberine that was previously shown from our laboratory to differentially bind to B-form and H(L)-form of poly[d(G-C)] [Bioorg. Med. Chem.2003, 11, 4861]. Binding of berberine to protonated conformation of natural DNAs resulted in intrinsic circular dichroic changes as well as generation of induced circular dichroic bands for the bound berberine molecule with opposite signs and magnitude compared with B-form structures. Nevertheless, the binding of the alkaloid to both the B and protonated forms was non-linear and non-cooperative as revealed from Scatchard plots derived from spectrophotometric titration data. Steady state fluorescence studies on the other hand showed remarkable increase of the rather weak intrinsic fluorescence of berberine on binding to the protonated structure compared to the B-form structure. Taken together, these results suggest that berberine can detect the formation of significant population of H(L)-form structures under the influence of protonation irrespective of heterogeneous base compositions in natural DNAs.     

Self-structure induction in single stranded poly(A) by small molecules: Studies on DNA intercalators, partial intercalators and groove binding molecules

Self-structure induction in single stranded poly(A) has been one typical example of the various ways that could be used to modulate nucleic acid structural aspects through binding of small molecules. For the first time, the interaction between a series of small molecules and poly(A) has been investigated to understand the nature of the structural features in DNA binding small molecules that could be responsible for the formation of self-structure in single stranded poly(A) molecules. Classical intercalators like ethidium, coralyne, quinacrine and proflavine, partial intercalators like berberine and palmatine and classical minor groove binders like hoechst 33258 and DAPI have been chosen for this study. The binding of each of these molecules to poly(A) has been characterized by absorption spectral titration, job plot and isothermal titration calorimetry. Self-structure formation was monitored from circular dichroic melting, optical melting and differential scanning calorimetry. The results revealed that while all the intercalators studied induced self-structure formation, partial intercalators did not induce the same in poly(A). Of the two classical DNA minor groove binding molecules investigated, hoechst was effective in inducing self-structure while DAPI was ineffective. Self-structure induction in poly(A) was observed to be directly linked to the cooperative binding of the molecules to poly(A) in that all the molecules that bound cooperatively induced self-structure in poly(A). Structural and thermodynamic aspects of the interaction leading to self-structure formation are described.     

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.     

Protonated forms of poly[d(G-C)] and poly(dG).poly(dC) and their interaction with berberine

The pH -induced structural changes on the conformation of homo- and hetero-polymers of guanosine-citydine (G.C) sequences were investigated using spectrophotometric and circular dichroic techniques. At pH 3.40, 10 mM [Na(+)] and 10 degrees C both polynucleotides adopted a unique and stable structural conformation different from their respective B-form structures. The protonated hetero-polymer is established as left-handed structure with Hoogsteen base pairing (H(L)-form) while the homo-polymer favored Watson-Crick base pairing with different stacking arrangements from that of B-form structure as evident from thermal melting and circular dichroic studies. The interaction of berberine, a naturally occurring protoberberine group of plant alkaloid, with the protonated structures was studied using various biophysical techniques. Binding of berberine to the H(L)-form structure resulted in intrinsic circular dichroic changes and generation of extrinsic circular dichroic bands with opposite sign and magnitude compared to its B-form structure while with the homo-polymer of G.C no such reversal of extrinsic circular dichroic bands was seen indicating different stacking arrangement of berberine at the interaction site. Scatchard analysis of the binding data, however, indicated non-cooperative binding to both the protonated forms similar to that of their respective B-form structure. Fluorescence spectral studies, on the other hand, showed remarkable increase in the intrinsic fluorescence of the alkaloid in presence of the protonated forms compared to their respective B-form structure. These results suggest that berberine could be used as a probe to detect the alteration of structural handedness due to protonation and may potentiate its use in regulatory roles for biological functions.     

Interaction of berberine chloride with deoxyribonucleic acids: evidence for base and sequence specificity

The interaction of berberine chloride with natural and synthetic DNAs of differing base composition and sequences was followed by various spectroscopic and viscometric studies. The binding of berberine chloride was characterized by hypochromism and bathochromism in the absorption bands, enhancement of fluorescence intensity, stabilization against thermal denaturation, perturbations in the circular dichroic spectrum, increase in the contour length of sonicated rod-like DNA and induction of unwinding-rewinding process of covalently closed superhelical DNA, depending on the base composition and sequences of base pairs. Binding parameters determined from absorbance and fluorescence titration by Scatchard analysis, according to an excluded-site model, indicated a very high specificity of berberine to AT-rich DNAs and alternate AT polymer. Fluorescence quantum yield was maximum for the complexes with AT-rich DNAs and alternate AT polymer. Taken together, these results suggest that berberine chloride exhibits considerable specificity towards alternating AT polymer and binds to AT-rich DNAs by a mechanism of classical intercalation.     

Interaction of the antitumour alkaloid coralyne with duplex deoxyribonucleic acid structures: spectroscopic and viscometric studies.

The interaction of coralyne, an antitumour alkaloid with natural and synthetic duplex DNAs was investigated under conditions where the drug existed fully as a true monomer for the first time using spectrophotometric, spectrofluorimetric, circular dichroic and viscometric techniques. The absorption spectrum of coralyne monomer showed hypochromic and bathochromic effects on binding to duplex DNAs. This effect was used to determine the binding parameters of coralyne. The binding constants for four natural DNAs and four synthetic polynucleotides obtained from spectrophotometric titration, according to an excluded site model, using McGhee-von Hippel analysis, were all in the range of (0.38-9.8) x 10(5) M-1, and showed a relatively high specificity for the GC rich ML DNA and the alternating GC polynucleotide. The binding of coralyne decreased with increasing ionic strength, indicating that the binding affinity has a strong electrostatic component. Coralyne stabilized all the DNAs against thermal strand separation. The intense steady state fluorescence of coralyne was effectively quenched on binding to DNAs and the quantitative data on the Stern-Volmer quenching constant obtained was sequence dependent, being maximum with the GC rich DNA and alternating GC polymer. Circular dichriosm studies further evidenced for a strong perturbation of the B-conformation of DNAs consequent to coralyne binding with the concomitant development of extrinsic circular dichroic bands for the bound drug molecules suggesting their strong intercalated geometry in duplex DNAs. Further tests of intercalation using viscosity measurements on linear and covalently closed plasmid DNA conclusively proved the strong intercalation of coralyne in duplex DNA. Binding of the closely related natural alkaloid, berberine under these conditions showed considerably lower affinity to duplex DNAs in all experiments. Taken together, these results suggest that coralyne binds strongly to duplex DNAs by a mechanism of intercalation with specificity towards alternating GC duplex structure.     

Binding of protoberberine alkaloid coralyne with double stranded poly(A): a biophysical study

Recognition of double stranded ribonucleic acid is a critical event in many biological pathways such as trafficking, editing and maturation of mRNA, interferon antiviral response and RNA interference. In the context of probing double stranded RNA binding small molecules, the interaction of the antitumor protoberberine alkaloid coralyne with double stranded poly(A) has been studied by various biophysical techniques. Typical hypochromic and bathochromic shifts in the absorption spectrum and appreciable quenching of the intrinsic fluorescence of coralyne indicated the strong affinity of coralyne to poly(A). The corresponding intrinsic binding constant evaluated from Scatchard analysis was in the order of 10(5) M(-1). The strong binding was further characterized by significant polarization of the alkaloid fluorescence and stabilization of poly(A) helix against thermal strand separation. The binding process was manifested by remarkable perturbation of the intrinsic circular dichroic spectrum of poly(A) with concomitant generation of optical activity in the bound alkaloid molecules that are otherwise achiral. Job plot analysis showed the binding stoichiometry of the interaction process to be two base pairs per alkaloid molecule. The energetics of the strong interaction was studied by isothermal titration and differential scanning calorimetric techniques that suggested the binding to be exothermic and favoured by both negative enthalpy and positive entropy changes. All these results, together with the Stern-Volmer quenching experiment in fluorescence, revealed the molecular details of the intercalation of coralyne into poly(A) duplex leading to its potential use as an agent in gene regulation in eukaryotic cells.     

Interaction of sanguinarine with double stranded RNA structures

Interaction of sanguinarine with A-form RNA structures of poly(rI)poly(rC) and poly(rA).poly(rU) has been studied by spectrophotometric, spectrofluorimetric, UV melting profiles, circular dichroism and viscometric analysis. The binding of sanguinarine to A-form duplex RNA structures is characterised by the typical bathochromic and hypochromic effects in the absorption spectrum, increasing steady state fluorescence intensity, an increase in fluorescence quantum yield of sanguinarine, an increase in fluorescence polarization anisotropy, an increase of thermal transition temperature, an increase in the contour length of sonicated rod-like RNA structure and perturbation in circular dichroic spectrum. Scatchard analysis indicates that sanguinarine binds to each polymer in a non-cooperative manner. Comparative binding parameters determined from absorbance titration by Scatchard analysis, employing the excluded site model, indicate a higher binding affinity of sanguinarine to poly(rI).poly(rC) structure than to poly(rA).poly(rU) structure. On the basis of these observations, it is concluded that the alkaloid binds to both the RNA structures by a mechanism of intercalation.     

Molecular aspects on the specific interaction of cytotoxic plant alkaloid palmatine to poly(A)

The interaction of the protoberberine alkaloid palmatine with single and double stranded structures of poly(A) was studied by various biophysical techniques. Comparative binding studies were also performed with double stranded DNA, t-RNA, poly(C)·poly(G), poly(U) and poly(C). The results of competition dialysis, fluorescence, and absorption spectral studies converge to reveal the molecular aspects of the strong and specific binding of palmatine to single stranded poly(A). The binding affinity of palmatine to natural DNA, t-RNA and double stranded poly(A) was weaker while no binding was apparent with single stranded poly(U), poly(C) and double stranded poly(C)·poly(G). The strong affinity of the alkaloid to single stranded poly(A) in comparison to the double stranded structure was also revealed from circular dichroic and viscometric studies. The effect of [Na⁺] ion concentration on the binding process revealed the significant role of electrostatic forces in the complexation. The presence of bound alkaloid also remarkably affected denaturation–renaturation of stacked helical poly(A). The energetics of the strong binding to poly(A) was studied from thermodynamic estimation from van Hoff’ analysis of the temperature dependent binding constants and ultra sensitive isothermal titration calorimertry, both suggesting the binding to be exothermic and enthalpy driven. This study provides detailed insight into the binding specificity of the natural alkaloid to single stranded poly(A) over several other single and double stranded nucleic acid structures suggesting its potential as a lead compound for RNA based drug targeting.     

Modifications of circular DNA by photoalkylation

The effects of photoalkylation on superhelical PM2 DNA were examined. The chief product was 8-(2-hydroxy-2-propyl)guanine, formed exclusively in sequences of alternating purines and pyrimidines. Other purine damages included 8-(2-hydroxy-2-propyl)adenine and smaller quantities of two uncharacterized adenine products. DNA strand breaks were formed with increasing irradiation. A small quantity of thymine-containing photodimers was formed. Photoalkylation of poly(dG-dC):poly(dG-dC) reduced the concentration of salt required to effect inversion of the circular dichroic spectrum. This suggests that photoalkylation induces the transition of poly(dG-dC):poly(dG-dC) from the right-handed B form of DNA to the left-handed Z form.     

Molecular aspects on the interaction of aristololactam-beta-D-glucoside with H(L)-form deoxyribonucleic acid structures

Synthetic alternating GC-rich DNA polymers can adopt Hoogsteen base-paired structures (H(L)-form) under the influence of low pH and temperature. The interaction of aristololactam-beta-D-glucoside (ADG), a natural glucoside derivative of aristolochia group of alkaloids, with protonation-induced structures (H(L)-form) of poly(dG-dC).poly(dG-dC) and poly(dG-m(5)dC).poly(dG-m(5)dC) has been studied using different biophysical techniques. The binding of ADG to protonated DNA is characterized by typical hypochromism and bathochromism of the absorption spectrum of the alkaloid, quenching of steady state fluorescence intensity, decrease in quantum yield, increase in fluorescence polarization anisotropy values, increase in thermal transition temperature of polynucleotides following alkaloid binding and perturbation of circular dichroic spectrum of polynucleotides as a result of its interaction with the alkaloid. Scatchard analysis of the data indicates that ADG binds to protonated structures in a nonlinear noncooperative manner. The binding parameters determined from spectrophotometric titration data employing excluded site model indicate that protonated poly(dG-m(5)dC).poly(dG-m(5)dC) is more favorable for ADG binding than the corresponding nonmethylated analog. The binding of ADG to protonated structures renders a higher degree of stabilization against thermal denaturation compared to respective B-form-ADG interactions and induces a conformational switch to a bound altered form which is different from its interaction with B- and Z-form DNA structures. Thermodynamic parameters (Delta G degrees, Delta H degrees and Delta S degrees ) obtained by van't Hoff analysis of the data indicate that the binding of alkaloid to protonated structures is an exothermic process and the binding free energy arises primarily from a negative enthalpy change. Moreover, the binding leads to an increase in the contour length of protonated DNAs. These results suggest that ADG possibly binds to protonated DNAs by the mechanism of intercalation.     

Preferential binding of quinolones to DNA with alternating G,C / A,T sequences: a spectroscopic study

The binding of quinolones, nalidixic acid (Nal), oxolinic acid (Oxo) with double stranded polynucleotides was undertaken by using UV-melting, UV-Vis absorption, fluorescence and CD spectroscopic techniques. The binding of Nal or Oxo to the polynucleotides under low-salt buffer conditions were determined for poly (dA).(dT), poly [d(A-T)], poly (dG).(dC), poly [d(G-C)] and E. coli DNA. The fluorescence data were analyzed using a previously established two step mechanism with two different DNA-Drug complexes [Rajeswari et al., Biochemistry 26, 6825-31 (1987)]. The first complex [DN](1) with a binding constant K(1), is formed where the interactions are 'nonspecific' and complex [DN](2) with a binding constant K(2), is formed where the interactions are "specific" which involve (additional) hydrophobic type of interactions like 'stacking' of the drug and the overall association constant is represented as K(=K(1)K(2)). The order of binding for Nal and Oxo is: poly [d(G-C)] > poly [d(A- T)] > E. coli > poly (dG).(dC) > poly (dA).(dT). Interaction of quinolones seems to be preferential in the alternating G, C or A, T stretches of DNA than those of non-alternating. Within any alternating or non-alternating in DNA sequences the G, C rich sequences have distinctly greater binding than A, T sequences. The overall association constant data (K) reveal higher binding of Oxo to DNA compared to Nal to any given polynucleotide investigated; which also explains the higher antibacterial potency of Oxo. Changes in the absorption difference spectra and in circular dichroic spectra also manifest these results. As the melting temperatures of the polynucleotides were only marginally raised in presence of the quinolone, we rule out the possibility of 'classical intercalation' of the drug. Amino group of guanine facilitates the binding of quinolones and therefore has the greater binding with the DNA. However, poly (dG).(dC) is known to exist in 'A' conformation which is not adopted by quinolones as in the case of poly (dA).(dT). Present results suggest that Nal or Oxo bind to DNA in a non-classical fashion which is partially stacking in nature.     

Conversions of the left-handed form and the protonated form of DNA back to the bound right-handed form by sanguinarine and ethidium: a comparative study

The interaction of sanguinarine and ethidium with right-handed (B-form), left-handed (Z-form) and left-handed protonated (designated as H(L)-form) structures of poly(dG-dC).poly(dG-dC) and poly(dG-me5dC).poly(dG-me5dC) was investigated by measuring the circular dichroism and UV absorption spectral analysis. Both sanguinarine and ethidium bind strongly to the B-form DNA and convert the Z-form and the H(L)-form back to the bound right-handed form. Circular dichroic data also show that the conformation at the binding site is right-handed, even though adjacent regions of the polymer have a left-handed conformation either in Z-form or in H(L)-form. Both the rate and extent of B-form to Z-form transition were decreased by sanguinarine and ethidium under ionic conditions that otherwise favour the left-handed conformation of the polynucleotides. The rate of decrease is faster in the case of ethidium as compared to that of sanguinarine. Scatchard analysis of the spectrophotometric data shows that sanguinarine binds strongly to both the polynucleotides in a non-cooperative manner under B-form conditions, in sharp contrast to the highly-cooperative binding under Z-form and H(L)-form conditions. Correlation of binding isotherms with circular dichroism data indicates that the cooperative binding of sanguinarine under the Z-form and the H(L)-form conditions is associated with a sequential conversion of the polymer from a left-handed to a bound right-handed conformation. Determination of bound alkaloid concentration by spectroscopic titration technique and the measurement of circular dichroic spectra have enabled us to calculate the number of base pairs of Z-form and H(L)-form that adopt a right-handed conformation for each bound alkaloid. Analysis reveals that 2-3 base pairs (bp) of Z-form of poly(dG-dC).poly(dG-dC) and poly(dG-me5dC).poly(dG-me5dC) switch to the right-handed form for each bound sanguinarine, while approximately same number of base pairs switch to the bound right-handed form in complexes with H(L)-form of these polynucleotides. Comparative binding analysis shows that ethidium also converts approximately 2 bp of Z-form or H(L)-form to bound right-handed form under same experimental conditions. Since sanguinarine binds preferentially to alternating GC sequences, which are capable of undergoing the B to Z or B to H(L) transition, these effects may be an important part in understanding its extensive biological activities.