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.     

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.     

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.     

Solution structural studies of the Ag(I)-DNA complex.

We report equilibrium dialysis and electric dichroism studies of the two strong complexes (I and II) of silver ion with DNA. Cooperative conversion of DNA to the stronger type I complex results in a 9% length decrease, and a structure in which intercalated ethidium is perpendicular to the helix axis. Upon addition of more Ag+ to form the type II complex, the DNA length reverts to its original value and bound ethidium once again becomes tilted from the plane perpendicular to the helix axis. In both type I and type II Ag (I) - DNA complexes, ethidium binding is mildly cooperative. We interpret the results in terms of a sequence of silver-induced cooperative switches of DNA from its B-form structure with propeller twisted base pairs to a structure with flat base pairs in the type I complex, and back again to propellered base pairs in the type II complex.     

Retention of helical structure of pBR322 covalently closed circular duplex DNA at high alkaline pH

Denaturation of covalently closed circular duplex replicative form (RF) I at high pH yields a form with high sedimentation coefficient even after neutralization. This form allowed less ethidium bromide to be intercalated but yielded a circular dichroic spectrum which had reduced magnitude of both positive circular dichroism at 273 nm and negative circular dichroism at 245 nm. The circular dichroic spectrum of this form is similar to that of RFV DNA. Gel electrophoretic analysis of this DNA revealed that, although part is retained in the groove, another part appeared as a faster-moving band, which we designated as RF I_d. This faster-moving form is cleaved by the restriction endonuclease BamHI at a single site giving a single RF III, comigrating with the RF III obtained from RF I by BamHI cleavage. This signifies that the two strands of RF I did not slide over one another during the formation of RF I_d as suggested previously.     

Quinacrine and 9-amino acridine inhibit B-Z and B-H(l) form DNA conformational transitions

The interaction of quinacrine and 9-amino acridine with right-handed B-form, left-handed Z-form, and left-handed protonated (H(L))-form structures of polydG-me(5)dC was investigated by circular dichroism and absorption spectral analysis. Both the compounds bind strongly to the B-form structure and convert the Z-form and H(L)-form back to the bound right-handed form. Circular dichroic data revealed that the conformation at the binding site is right-handed even though adjacent regions of the polynucleotide may have left-handed conformation. The rate and extent of B-form-to-Z-form transition were decreased in the presence of these compounds. Scatchard analysis revealed that both quinacrine and 9-amino acridine bind strongly to the polynucleotide in the B-form in a noncooperative manner, in sharp contrast to the highly cooperative binding to the Z-form and H(L)-form. Results indicated that the cooperative binding of these drugs with the Z-form and the H(L)-forms was associated with a sequential conversion of the polynucleotide from a left-handed to a bound right-handed conformation. Experimental data enabled the calculation of the number of base pairs of Z-form (7-8 with quinacrine and 9-amino acridine) and H(L)-form (4 and 25, respectively, with quinacrine and 9-amino acridine) that adopt a right-handed conformation for each bound ligand. As these compounds are known to bind preferentially to alternating guanine--cytosine sequences, which are capable of easily undergoing the B-to-Z or B-to-H(L) transition, these effects may be important in understanding their biological activities.     

Effects of the substitution positions of Br group in intercalative ligand on the DNA-binding behaviors of Ru(II) polypyridyl complexes

Two new polypyridyl ligands containing substituent Br at different positions in the phenyl ring, PBIP [PBIP=2-(4-bromophenyl)imidazo[4,5-f]1,10-phenanthroline], OBIP [OBIP=2-(2-bromophenyl)imidazo[4,5-f]1,10-phenanthroline] and their Ru(II) complexes, [Ru(phen)2PBIP]2+ 1, [Ru(phen)2OBIP]2+ 2 (phen=1,10-phenanthroline), have been synthesized and characterized. The binding strength of the two complexes to calf thymus DNA (CT DNA) was investigated with spectrophotometric methods, viscosity measurements, as well as equilibrium dialysis and circular dichroism spectroscopy. The theoretical calculations for these two complexes were also carried out applying the density functional theory (DFT) method. The experimental results show that the Br group substituting H at different positions of the phenyl ring in the intercalated ligand has significant effects on the spectral properties and the DNA-binding behaviors of Ru(II) complexes. Both the complexes can bind to CT DNA in intercalative mode and interact with CT DNA enantioselectively. Moreover, complex 1 can bind to CT DNA more strongly than complex 2, and complex 2 can become a much better candidate as an enantioselective binder to CT DNA than complex 1. The theoretical calculations show that both intercalative ligands, PBIP and OBIP, in these two complexes are essentially planar, and the obtained electronic structures of the complexes can be used to explain reasonably some of their experimental regularities or trends. Such experimental and theoretical information will be useful in design of novel probes of nucleic acid structures.     

Site-site interactions in EF-hand calcium-binding proteins. Laser-excited europium luminescence studies of 9-kDa calbindin, the pig intestinal calcium-binding protein

Europium(III) binding to 9-kDa calbindin from pig intestines was studied by direct excitation of the 7Fo----5Do transition of the ion and by near-ultraviolet circular dichroic spectroscopy. Europium(III) binding is clearly biphasic. As with other lanthanides the C-terminal metal-binding site (site II) is filled first. The europium ion in this site gives an excitation spectrum with a single peak at 579.1 nm (peak 2). The occupation of the N-terminal site (site I) by europium gives excitation spectra that are pH-dependent and show a peak at 579.4 nm (peak 1a) at pH 5 which shifts to 578.7 nm (peak 1b) over the pH range 5-7. At pH 8.07 the fluorescence from europium in site I largely disappears because of weak binding, whereas that from site II is quenched by about 75% in spite of full occupancy of the site as shown by circular dichroic titration. There is a strong interaction between the two sites in spite of the very different affinities. The fluorescence from site II increases stoichiometrically with the addition not only of the first equivalent of europium, but also concomitantly with the fluorescence from site I upon addition of the second equivalent. Furthermore, when Eu1-calbindin is titrated with calcium the fluorescence at 579.1 nm is quenched by about 30% during the addition of one equivalent of calcium which fills site I. Subsequent titration with large excesses of calcium displaces europium from site II. The affinity of site II for europium is about 100 times that of calcium under these conditions.     

Molecular aspects on the interaction of sanguinarine with B-form, Z-form and HL-form DNA structures

The interaction of sanguinarine with right-handed (B-form), left-handed (Z-form) and left-handed (HL-form) structures of poly(dG-dC).poly(dG-dC) has been investigated by measuring the circular dichroism (CD) and UV-absorption spectral analysis. Sanguinarine binds strongly to the B-form DNA and does not bind to Z-form or HL-form, but it converts the Z-form and the HL-form back to the bound right handed form as evidenced from CD spectroscopy. Sanguinarine inhibits the rate of B to Z transition under ionic conditions that otherwise favour the left-handed conformation of the polynucleotides. UV absorption kinetic studies show that the Z-form reverses back to B-form to B-form on binding to sanguinarine. Binding isotherms obtained from spectrophotometric data show that sanguinarine binds strongly to the B-form polymer in a non-cooperative manner, in sharp contrast to the highly cooperative interaction under Z-form and HL-form polynucleotides. These studies reveal that the alternating GC sequence undergoes defined conformational changes and interacts with sanguinarine which may be an important aspect in understanding its extensive biological activities.     

Fluorescence-detected circular dichroism of ethidium bound to poly(dG-dC) and poly(dG-m5dC) under B- and Z-form conditions

The equilibrium binding of ethidium to poly(dG-dC) and poly(dG-m5dC) under conditions favoring B and Z forms was investigated with fluorescence-detected circular dichroism (FDCD) and optical titration methods. FDCD spectra indicate a similar geometry for the intercalated ethidium under both B- and Z-form conditions, even at low levels of bound ethidium. The magnitude of the 310-330-nm FDCD band as a function of the bound drug to base pair ratio (r) indicates ethidium binds to poly(dG-dC) in 4.4 M NaCl and to poly(dG-m5dC) in 25 mM MgCl2 by clustering. Under these conditions, circular dichroism spectra indicate the polymer is largely Z form. Thus, it appears ethidium clusters into regions it has induced into a right-handed form. For all conditions studied, the FDCD spectra provided no evidence for a left-handed binding site. Under B-form conditions, binding is random.     

Interactions of berberine with poly(A) and tRNA.

The interaction of berberine chloride with poly(A) and tRNA has been studied by various spectroscopic techniques. Binding parameters determined from spectrophotometric and spectrofluorimetric measurements by Scatchard analysis indicate a very high effective binding capacity of berberine to poly(A) as compared to DNA or tRNA. The circular dichroism studies show that binding of berberine to poly(A) causes a significant change in the circular dichroic spectrum of poly(A) itself, as manifested by (i) a decrease of both positive and negative bands and (ii) appearance of a conservative type of extrinsic circular dichroic spectrum in the wavelength range of 300-400 nm, while it does not cause any significant alteration to the A form structure of tRNA. It is concluded that berberine interacts stronger with poly(A) than DNA or tRNA. The results are interpreted in terms of its reported biological activities.     

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.     

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.     

Renaturation of denatured, covalently closed circular DNA

The rate of renaturation of denatured, covalently closed, circular DNA (form Id DNA) of the phi X174 replicative form has been investigated as a function of pH, temperature, and ionic strength. The rate at a constant temperature is a sharply peaked function of pH in the range of pH 9 to 12. The position on the pH scale of the maximum rate decreases as the temperature is increased and as the ionic strength is increased. The kinetic course of renaturation is pseudo-first order: it is independent of DNA concentration, but falls off in rate from a first order relationship as the reaction proceeds. The rate of renaturation depends critically on the temperature at which the denaturation is carried out. Form Id, prepared at an alkaline pH at 0 degrees C, renatures from 5 to more than 100 times more rapidly than that similarly prepared at 50 degrees C. Both the heterogeneity in rate and the effect of the temperature of denaturation depend, in part, on the degree of supercoiling of the form I DNA from which the form Id is prepared. However, it is concluded that a much larger contribution to both arises from a configurational heterogeneity introduced in the denaturation reaction. The renaturation rate was determined by neutralization of the alkaline reaction and analytical ultracentrifugal analysis of the amounts of forms I and Id. The nature of the proximate renatured species at the temperature and alkaline pH of renaturation was investigated by spectrophotometric titration and analytical ultracentrifugation. It is concluded that the proximate species are the same as the intermediate species defined by an alkaline sedimentation titration of the kind first done by Vinograd et al. ((1965) Proc. Natl. Acad. Sci. U. S. A. 53, 1104-1111). Observations are included on the buoyant density of form Id and on depurination of DNA at alkaline pH values and high temperatures.     

Thermodynamic and structural study of phenanthroline derivative ruthenium complex/DNA interactions: probing partial intercalation and binding properties

The binding of [Ru(PDTA-H₂)(phen)]Cl (PDTA = propylene-1,2-diaminetetra-acetic acid; phen = 1,10 phenanthroline) with ctDNA (=calf thymus DNA) has been investigated through intrinsic and induced circular dichroism, UV-visible absorption and fluorescence spectroscopies, steady-state fluorescence, thermal denaturation technique, viscosity and electrochemical measurements. The latter indicate that the cathodic and anodic peak potentials of the ruthenium complex shift to more positive values on increasing the DNA concentration, this behavior being a direct consequence of the interaction of both the reduced and oxidized form with DNA binding. From spectrophotometric titration experiments, the equilibrium binding constant and the number of monomer units of the polymer involved in the binding of one ruthenium molecule (site size) have been quantified. The intrinsic circular dichroism (CD) spectra show an unwinding and a conformational change of the DNA helix upon interaction of the ruthenium complex. Quenching process, thermal denaturation experiments and induced circular dichroism (ICD) are consistent with a partial intercalative binding mode.     

On the various types of circular dichroism induced on acridine orange bound to poly(S-carboxymethyl-L-cysteine).

Circular dichroism and absorption spectra are measured on mixed solutions of acridine orange and poly(S-carboxymethyl-L -cysteine) at different pH and P/D mixing ratios. The observed circular dichroism spectra are classified into several types, mainly based on the number and sign of circular dichroic bands in the visible region. Three of them are associated with the absorption spectra characteristic of dimeric dye or higher aggregates of dye. Type I is observed with solutions, of which the pH is acid and P/D is higher than 4, and it has an unsymmetrical pair of positive and negative dichroic bands at 470 and 430 nm. This type is induced on the dye bound to the polymer in the β-conformation. Types II and III are considered to be characteristic of randomly coiled polymers. Type II is exhibited by solutions of P/D higher than 1 at pH 5–7 and has two dichroic bands around the same wavelengths as Type I but with opposite signs and an additional positive band at 560 nm. Type III, shown by solutions of P/D 2–0.6 at pH 6–10.5, has three dichroic bands around the same wavelengths as Type II but with signs opposite to it. The other two types of circular dichroism, induced for the solutions of P/D less than 1 at slightly acid pH, are associated with the absorption spectra of monomeric dye and are observed with disordered or randomly coiled polymer. They have a pair of dichroic bands at 540 and 425 nm, and the signs of these bands are opposite to each other in these two types.     

Role of the 20-hydroxyl group in camptothecin binding by the topoisomerase I-DNA binary complex

Recent findings concerning the structure of the covalent binary complex formed by DNA topisomerase I and its DNA substrate, as well as the nature of interactions with inhibitors that bind reversibly to this binary complex, have led to two proposed models for the binding of the prototype inhibitor camptothecin to the DNA-topisomerase I binary complex. While these models differ in many regards, they both suggest the involvement of the 20-OH group of camptothecin in a donor hydrogen bond with an enzyme side chain functional group. Presently, five analogues of camptothecin that differ only at C-20 have been evaluated for their ability to bind to the topoisomerase I-DNA binary complex and thereby inhibit enzyme function. Both 20-chloro- and 20-bromocamptothecin bound as well to the enzyme-DNA binary complex as 20-aminoCPT despite the absence of a substituent at C-20 capable of contributing a donor hydrogen bond.     

Synthesis, DNA binding, topoisomerases inhibition and cytotoxic properties of 4-arylcarboxamidopyrrolo-2-carboxyanilides

Three 4-arylcarboxamidopyrrolo-2-carboxyanilides bearing different substituents on the pyrrole nitrogen were synthesized and evaluated for their capacities to bind to specific sequences within the minor groove of DNA and to inhibit human topoisomerases I and II in vitro. The cytotoxicity of the drugs correlates with their DNA binding affinities. The two drugs bearing a N-methyl or N-benzyl pyrrole stabilize topoisomerase I-DNA complexes.     

Effect of ionic strength of a solution on the protonation of DNA molecule

The influence of the ionic strength of solution on the DNA molecule protonation was studied by means of circular dichroism (CD), spectrophotometric and potentiometric titration methods over a wide range of the supporting electrolyte concentrations [( NaCl] = 0.0005 divided by 4 M). Consideration of the obtained CD spectra shown that the acidation of the solution induces two cooperative structural transitions in the double stranded DNA molecule in the pre-denaturation pH region. Further decrease in the solution pH results in acidic melting of the DNA molecule. Analysis of the potentiometric data shows that diluted DNA solutions exhibit marked buffer capacity at pH greater than 4.2. A concept of local pH dependent on the electrostatic potential in the vicinity of the polyion was used for interpreting the obtained results. A phase diagram, which describes the polymorphic transformations of the protonated macromolecule, was constructed in terms of pHloc and -log[Na+]. Consideration of this phase diagram allows to hypothesize that: 1) in the neutral diluted DNA solution with a very low supporting electrolyte content the macromolecule exists in a polymorphic state; 2) at [NaCl] greater than or equal to 0.001 M the acid-base equilibrium in the DNA molecule is invariant in respect to the ionic strength of the solution.     

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.