Spectroscopic and Calorimetric Studies on the Binding of Alkaloids

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J. Phys. Chem. B 2009, 113, 1210–1224

Spectroscopic and Calorimetric Studies on the Binding of Alkaloids Berberine, Palmatine and Coralyne to Double Stranded RNA Polynucleotides Md. Maidul Islam, Sebanti Roy Chowdhury, and Gopinatha Suresh Kumar* Biophysical Chemistry Laboratory, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, Kolkata 700 032, India ReceiVed: July 25, 2008; ReVised Manuscript ReceiVed: NoVember 21, 2008

The interaction of two natural protoberberine plant alkaloids berberine and palmatine and a synthetic derivative coralyne to three double stranded ribonucleic acids, poly(A). poly(U), poly(I).poly(C) and poly(C).poly(G) was studied using various biophysical techniques. Absorbance and fluorescence studies showed that the alkaloids bound cooperatively to these RNAs with the binding affinities of the order 104 M-1. Circular dichroic results suggested that the conformation of poly(A). poly(U) was perturbed by all the three alkaloids, that of poly(I).poly(C) by coralyne only and that of poly(C).poly(G) by none. Fluorescence quenching studies gave evidence for partial intercalation of berberine and palmatine and complete intercalation of coralyne to these RNA duplexes. Isothermal titration calorimetric studies revealed that the binding was characterized by negative enthalpy and positive entropy changes and the affinity constants derived were in agreement with the overall binding affinity from spectral data. The binding of all the three alkaloids considerably stabilized the melting of poly(A). poly(U) and poly(I).poly(C) and the binding data evaluated from the melting data were in agreement with that obtained from other techniques. The overall binding affinity of the alkaloids to these double stranded RNAs varied in the order, berberine ) palmatine < coralyne. The temperature dependence of the enthalpy changes afforded large negative values of heat capacity changes for the binding of palmatine and coralyne to poly(A).poly(U) and of coralyne to poly(I).poly(C), suggesting substantial hydrophobic contribution in the binding process. Further, enthalpy-entropy compensation was also seen in almost all the systems that showed binding. These results further advance our understanding on the binding of small molecules that are specific binders to double stranded RNA sequences. 1. Introduction In recent years the number of members of the RNA family has grown rapidly. A burgeoning body of recent evidence has revealed a remarkably new world for RNAs in which many small RNAs that do not code for proteins exercise control over those RNAs that do. Because these small RNAs that include micro RNAs, small interfering RNAs, small nucleolar RNAs and small nuclear RNAs can control the transcription and translation processes, they represent a newly discovered level of control over the genome. A growing body of evidence also suggests that plants, animals, and yeasts share related mechanisms of specific degradation of RNAs in which double stranded (ds) forms of RNA are involved. Double stranded RNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi) and ds sections of mRNA itself are often sites of interaction of proteins and small molecules. Consequently, this has fueled significant interest in exploiting RNAs as a potential therapeutic target for small molecules.1-5 However, very little attention has been paid so far to the recognition of RNA by small molecules in contrast to the large volume of DNA binding studies. The intricate structural architectures of the RNAs in comparison to DNA and the relatively scanty high-resolution structural information available in respect of RNA molecules were to a large extent responsible for this lacuna. The emergence of deadly RNA viruses like HIV and hepatitis C, and the knowledge that many * To whom all correspondence should be addressed. Phone: +91 33 2472 4049. Fax: +91 33 2473 0284/5197. E-mail: [email protected]/gsk.iicb@ gmail.com.

serious diseases are caused by RNA viruses led to growing interest in the development of RNA binding antiviral compounds and RNA based anticancer agents.2,6,7 This has been further accelerated by the discovery of several small RNAs that encompass many different classes of noncoding RNAs each with their own properties and functions.8 A rational design of such RNA based therapeutics molecules, however, would essentially require a detailed knowledge of the structural aspects of RNA on the one hand and the molecular nature of the mode, mechanism and specificity of binding of small molecules to various conformations of RNA on the other. In the recent past, significant advancement has taken place in the structural evaluation of various RNAs through X-ray crystallography, nuclear magnetic resonance spectroscopy, computational evaluation etc.9,10 Although all cellular RNAs have single polynucleotide chain, they are highly versatile molecules that can fold into a multitude of secondary structures and conformations. These complex structural motifs could be potential binding pockets for specific drug recognition sites, and it would be interesting to take advantage of these promising recognition capabilities of RNAs to develop new RNA binding molecules that could be modulators of cellular functions. Studies in this direction have identified one class of RNA binding compounds, viz. the aminoglycoside antibiotics which were shown to interact with the functional sites on 16S rRNA.11-13 The current understanding of the fundamentals of small molecule-RNA interactions is mostly derived exclusively from studies with aminoglycosides. Apart from aminoglycosides, no other group of compounds have been seriously studied that can bind with

10.1021/jp806597w CCC: $40.75  2009 American Chemical Society Published on Web 01/08/2009

Alkaloid-RNA Interaction

J. Phys. Chem. B, Vol. 113, No. 4, 2009 1211 poly(A),44,45 we sought to further characterize the interaction of these three alkaloids with model ds RNA structures. Double stranded RNA is formed in cells through intra- and intermolecular RNA interactions and is involved in a range of biological processes.44 Molecular recognition of ds RNA is a key event for numerous pathways like trafficking, editing, and maturation of cellular RNA, the interferon antiviral response and RNA silencing. In this paper we present the results of our investigation on the binding of natural berberine and palmatine and of synthetic coralyne to poly(A).poly(U), poly(I).poly(C) and poly(C).poly(G) from multifaceted spectroscopic and calorimetric studies. 2. Materials and Methods

Figure 1. Chemical structures of berberine, palmatine and coralyne and base paring scheme in AU, IC and CG base pairs.

RNA and it is not clear as to whether the results of aminoglycosides can be directly applied to other small molecules. One of the easiest approaches in the development of RNA targeted molecules has been to study the interaction of known DNA binding compounds with fairly well characterized interaction profiles. Wilson and co-workers have performed the interaction of a variety of DNA intercalating and groove binding molecules with various ds RNA constructs.14-16 Our primary interest has been to enhance the fundamental knowledge in this area by studying the binding of some natural alkaloids with synthetic double stranded RNA structures. Studying RNA molecules with defined sequences gives the advantage that the structures of these are known and hence the interaction profile can be understood easily. Natural products in general due to their unmatched chemical diversity and biological relevance have been widely accepted as potential high quality pools in drug screening. Protoberberines, of which berberine and palmatine (Figure 1) are the two most prominent members, constitute a class of such compounds that have been studied extensively and known to be potential lead compounds in cancer therapy.10,17 Plants containing these alkaloids have been used as folk medicines for centuries world over without a clear understanding of their molecular biotargets. Berberine and palmatine have a wide range of biochemical and pharmacological effects17,20,21 and were demonstrated to possess antitumor activity in vitro and in vivo.22-24 Berberine induces apoptosis through a mitochondria/ caspase pathway in human hepatoma cells.25 Both the alkaloids have been known to bind to DNA predominantly by intercalation exhibiting remarkable adenine-thymine base pair specificity.19,26-29 Coralyne, the synthetic protoberberine derivative, has been known for its potential antileukemic activity with low toxicity and pronounced topoisomerase I poisoning property.30-32 It also exhibits strong DNA intercalating properties, but with guanine-cytosine specificity.33-35 RNA binding studies of these molecules were reported recently from our laboratory.36-43 In the light of their strong binding to tRNA36-38 as also to single stranded RNA polynucleotides39 and remarkably stronger affinity to ss poly(A) molecules39-43 in contrast to weak binding to ds

Double stranded RNAs, poly(A).poly(U), poly(I).poly(C), and poly(C).poly(G) were purchased from Sigma-Aldrich Corporation (St. Louis, MO). Concentration of these RNA duplexes was determined spectrophotometrically using molar extinction coefficients values (ε in M-1 cm-1) of 14 280 at 260 nm for poly(A).poly(U), 10 000 at 260 nm for poly(I).poly(C) and 15 400 at 259 nm for poly(C).poly(G) expressed in terms of base pairs. RNA duplexes were sonicated to an uniform size of about 280 ( 40 base pairs using a Labsonic 2000 sonicator (B. Braun) with a needle probe of 4 mm diameter as described previuosly.46 After sonication, the polymers were extensively dialyzed under sterile conditions. The molecular mass of each of the sonicated samples was estimated to be in the range (2.2-2.5) × 105 Dalton from viscosity experiments described previously.46 The nativeness of the samples of poly(A).poly(U) and poly(I).poly(C) was confirmed from optical melting and differential scanning calorimetry where cooperative transitions with sharp melting temperatures were observed. The sharpness and reversibility and reproducibility with no hysteresis of the melting profiles together with the hyperchromic data suggested the samples to be homogeneous with perfect base pair formation. Further the DSC data gave the ratio of calorimetric and van’t Hoff enthalpy to be unity typical of cooperative reversible transitions unequivocally establishing the nativeness and proper base pairing schemes in these duplexes. Poly(C).poly(G) did not melt in the temperature range 30-110 °C and hence it was characterized by absorbance and CD spectra that was in conformity with the literature report.46 Berberine chloride (BC), palmatine chloride (PC), and coralyne chloride (CC) (alkaloids in general hereafter) were obtained from Sigma-Aldrich and were used without further purification as no detectable impurities were observed by thin layer chromatography and 1H NMR spectroscopy. All the three alkaloids were fairly soluble in aqueous buffers and hence their solutions were freshly prepared each day in the buffer and kept protected in the dark to prevent any light induced photochemical changes. The molar extinction coefficients (ε) of the alkaloids for determining their concentrations by absorbance measurements and optical properties are listed in Table 1. No deviation from Beer’s law was observed in the concentration range employed in this study. All experiments were conducted in citrate-phosphate (CP) buffer, pH 7.0, containing 10 mM Na2HPO4. pH was adjusted by the addition of citric acid. Glass distilled deionized water and analytical grade reagents were used throughout. pH measurements were made on a Cyberscan 2100 high precision bench pH meter (Eutech Instruments Pte. Ltd.) with an accuracy of >(0.001 units. All buffer solutions were filtered through Millipore filters (Millipore, India Pvt. Ltd., Bangalore, India) of 0.45 µm before use. Absorbance titrations were performed on a Shimadzu Pharmaspec 1700 spectrophotometer (Shimadzu Corp., Kyoto, Japan)

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Islam et al.

TABLE 1: Summary of the Optical Properties of Free and RNA Bound Alkaloidsa parameter λmax (free) λmax (bound) λisob εf(atλmax) εb(atλmax) with poly(A).poly(U) poly(I).poly(C) poly(C).poly(G)

berberine

palmatine

coralyne

Absorbance 344 348 360, 380, 454 22 500

345 348 356, 378, 456 25 000

421 426 435 14 500

13 450 14 700 15 035

16 030 16 675 17 125

8193 9720 9365

350 533

425 472

Fluorescence λmax (excitation) λmax (emission)

350 533

Units: λ nm; ε (molar extinction coefficient) M-1 cm-1. b Wavelengths at the isosbestic points. a

at 20 ( 0.5 °C using the methodology of Chaires et al.47 described previously.29,40 For titration of berberine and palmatine quartz cells of 1 cm path length and for coralyne quartz cells of 10 cm path length were used. Briefly, a known concentration of the RNA solution was kept in the sample and reference cells and small aliquots of a known concentration of the alkaloid was titrated into the sample cell. After each addition, the solution was exhaustively mixed and allowed to re-equilibrate for at least 10 min before noting the absorbance at the wavelength maximum and the isosbestic point. The data obtained from these titrations were used for constructing Scatchard plots. Steady state fluorescence measurements were performed on a Hitachi F4010 fluorescence spectrometer (Hitachi Ltd., Tokyo, Japan) in fluorescence free quartz cells of 1 cm path length as described previously.29,40,46 The excitation wavelength for berberine and palmatine was 350 nm whereas the same for coralyne was 425 nm. Because emission spectra of the alkaloid-RNA complexes were in a region far away from the excitation wavelength, no overlap of the bands was observed. All measurements were performed keeping an excitation and emission band-pass of 5 nm. The sample temperature was maintained at 20 ( 1.0 °C using Eylea Uni Cool U55 water bath (Tokyo Rikakikai Co. Ltd., Tokyo, Japan). The amount of free and bound alkaloid was determined as follows. In UV-vis spectroscopy, following each addition of the alkaloid to the RNA solution (40 µM), from the absorbance at the respective isosbestic point (Aiso) and the total drug concentration present was calculated as Ct ) Aiso/lεiso, where l is the path length of the cell and εiso is the molar extinction coefficient at the isosbestic point. This quantity was used to calculate the expected absorbance at the wavelength maximum, Aexp ) lCtεmax, where εmax is the molar extinction coefficient at the wavelength maximum. The difference in Aexp and the observed absorbance (Aobsd) was then used to calculate the amount of bound drug as Cb ) ∆A/1∆ε ) (Aexp - Aobsd)/l(εf εb). The amount of free drug was determined by difference, Cf ) Ct - Cb. The extinction coefficient of the completely bound drug was determined by adding a known quantity of drug to a large excess of RNA and on the assumption of total binding, εb)Amax/lCt. Alternatively, the absorbance of a known quantity of drug was monitored at the wavelength maximum while adding known amounts of RNA until no further change in absorbance was observed. Both these protocols gave similar values within experimental errors. In fluorescence, Cb was calculated from the relation Cb ) Ct(I - Io)/(Vo - 1)Io, where Ct is the known total drug concentration, I is the observed fluorescence, Io is the fluorescence intensity of identical

concentration of drug in absence of RNA and Vo is the experimentally determined ratio of the fluorescence intensity of totally bound drug to that of free drug. Free drug concentrations (Cf) were obtained from the relationship Ct ) Cb + Cf. The binding ratio r is defined as r ) Cb/[RNA]total. Binding data obtained from spectrophotometric and spectrofluorometric titrations were cast into Scatchard plots of r/Cf versus r. All the Scatchard plots revealed positive slopes at low r values as observed in cooperative binding isotherms and hence were analyzed using the following McGhee-von Hippel equation.48

r (2ω - 1)(1 - nr) + (r - R) (n-1) ) Ki(1 - nr) Cf 2(ω - 1)(1 - nr) 1 - (n + 1)r + R 2(1 - nr)

(

)

(

2

)

(1)

where R ) {[1 - (n + 1)r]2 + 4ωr(1 - nr)}1/2, where Ki is the intrinsic binding constant to an isolated binding site, n is the number of base pairs excluded by the binding of a single ligand molecule and ω is the cooperativity factor. The binding data were analyzed using the Origin 7.0 software (Origin Laboratories, Northampton, MA) that determines the best-fit parameters to eq 1. Continuous variation method of Job49,50 was employed to determine the binding stoichiometry in each case from fluorescence spectroscopy. Steady state fluorescence measurements were performed on a Hitachi F4010 fluorescence spectrometer (Hitachi Ltd., Tokyo, Japan) in fluorescence free quartz cells of 1 cm path length as described previously.39,40 All the measurements were performed at 20 ( 1 °C under conditions of constant stirring. Uncorrected fluorescence spectra are recorded. At constant temperature, the fluorescence signal was recorded for solutions where the concentrations of both RNA and alkaloid were varied while the sum of their concentrations was kept constant. The difference in fluorescence intensity (∆F) of the alkaloids in the absence and presence of RNA was plotted as a function of the input mole fraction of each alkaloid. Break point in the resulting plot corresponds to the mole fraction of the bound alkaloid in the complex. The stoichiometry was obtained in terms of RNA-alkaloid [(1 - χalkaloid)/χalkaloid] where χalkaloid denotes the mole fraction of the respective alkaloid. The results reported are average of at least three experiments. Fluorescence quenching studies were carried out with the anionic quencher [Fe(CN)6J4-. The quenching experiments were performed by mixing, in different ratios, two solutions, one containing KCl, the other containing K4[Fe(CN)6], in addition to the normal buffer components, at a fixed total ionic strength. Fluorescence quenching experiments were performed at a constant P/D (RNA base pair/alkaloid molar ratio) monitoring fluorescence intensity as a function of changing concentration of the ferrocyanide as described previously.43,44 At least four measurements were taken for each set and averaged out. The data were plotted as Stern-Volmer plots of relative fluorescence intensity (Fo/F) versus [Fe(CN)6]4-. The viscosity of the RNA-alkaloid complexes was determined by measuring the time needed to flow through a CannonManning semi micro size 75 capillary viscometer (Cannon Instruments Co., State College, PA) that was submerged in a thermostatted bath maintained at 20 ( 1 °C, as reported previously.46 Flow times were measured in triplicate to an accuracy of (0.01 s with an electronic stopwatch Casio Model HS-30W (Casio Computer Co. Ltd., Tokyo, Japan). Relative viscosities for RNA either in the presence or in the absence of


J. Phys. Chem. B, Vol. 113, No. 4, 2009 1213

the alkaloids were calculated from the relation

ηsp/ηsp ) {(tcomplex - to)/to}/{(tcontrol - to)/to}

(2)

where, η′sp and ηsp are specific viscosities of the alkaloid-RNA complex and the RNA respectively; tcomplex, tcontrol, and to are the average flow times for the RNA-alkaloid complex, free RNA and buffer, respectively. The relative increase in length, L/Lo, can be obtained from a corresponding increase in relative viscosity using the following equation51

L/Lo ) (η/ηo)1/3 ) 1 + βr

(3)

where L and Lo are the contour length of RNA in presence and absence of the alkaloids and η and ηo are the corresponding values of intrinsic viscosity (approximated by the reduced viscosity η ) ηsp/C, where C is the RNA concentration) and β is the slope when L/Lo is plotted against r. A JASCO J715 spectropolarimeter (JASCO International Co. Ltd., Tokyo, Japan) equipped with a JASCO temperature controller (model PTC 343) and controlled by a PC was used for all circular dichroic measurements at 20 ( 0.5 °C, as reported earlier.40,41 A rectangular quartz cell of 1 cm path length was used. Each spectrum was averaged from five successive accumulations at a scan rate of 50 nm/min, keeping a bandwidth of 1.0 nm at a sensitivity of 100 mdeg, and was baseline corrected and smoothed within permissible limits using the inbuilt software of the unit. The molar ellipticity values [θ] are expressed in terms of either per RNA base pair (220-400 nm region) or per bound alkaloid (300-500 nm region). The CD unit was routinely calibrated using an aqueous solution of d-10 ammonium camphor sulfonate. Absorbance versus temperature profiles (melting curves) of RNA and RNA-alkaloid complexes were measured on the Shimadzu Pharmaspec 1700 unit equipped with the peltier controlled TMSPC-8 model accessory (Shimadzu Corp., Kyoto, Japan) as described earlier.40,42 In a typical experiment, the RNA sample (50 µM) was mixed with the varying concentrations of the alkaloid under study in the desired degassed buffer into the eight-cell microoptical cuvettes of 1 cm path length and the temperature of the microcell accessory was raised at a heating rate of 0.5 °C/min while continuously monitoring the absorbance change at 260 nm. The Tm was taken as the mid point of the melting transition as determined by the maxima of the first derivative plots. All isothermal titration calorimetry experiments were performed using a MicroCal VP-ITC unit (MicroCal, Inc., Northampton, MA) using protocols developed in our laboratory and described previously.39,40,42,43 Briefly, aliquots of degassed RNA solution were injected from a rotating syringe (290 rpm) into the isothermal sample chamber containing each of the alkaloid solutions (1.4235 mL). Corresponding control experiments to determine the heat of dilution of RNA were performed by injecting identical volumes of same concentration of the RNA into buffer. The area under each heat burst curve was determined by integration using the Origin 7.0 software to give the measure of the heat associated with the injection. The heat associated with each RNA-buffer mixing was subtracted from the corresponding heat of RNA-alkaloid reaction to give the heat of alkaloid-RNA binding. The heat of dilution of injecting the buffer into each of the alkaloid solution alone was observed to be negligible. The resulting corrected injection heats were plotted as a function of the P/D molar ratio and fit with a model

for one set of binding sites and analyzed using Origin 7.0 software to provide the binding affinity (Ka), the binding stoichiometry (N) and the enthalpy of binding (∆H). The binding free energy (∆G) and the entropic contribution to the binding (T∆S), where subsequently calculated from standard relationships described earlier.39,40 To investigate the helix-coil transition, excess heat capacities as a function of temperature were measured on a Microcal VPdifferential scanning calorimeter (DSC) (MicroCal, Inc., Northampton, MA) as described previously.39,40 In a series of DSC scans, both the sample and reference cells were loaded with buffer solution, equilibrated at 20 °C for 15 min and scanned from 20° to 120 °C at a scan rate of 60 °C/h. The buffer scans were repeated until reproducible, and on cooling, the sample cell was rinsed and loaded with RNA solution and then with alkaloid/RNA complexes of different molar ratios and scanned. Each experiment was repeated twice with separate fillings. The DSC thermograms of excess heat capacity versus temperature were analyzed using the Origin 7.0 software to determine the calorimetric transition enthalpy (∆Hcal), as described earlier.39,40 This calorimetrically determined enthalpy is model-independent and unrelated to the nature of the transition. The temperature at which excess heat capacity is maximum defines the transition temperature (Tm). The modeldependent van’t Hoff enthalpy (∆Hv) was obtained by shape analysis of the calorimetric data, and the cooperativity factor was obtained from the ratio (∆Hcal/∆Hv). The reversibility of the transitions were checked by allowing the sample to cool slowly to 10 °C (10 °C/h) and performing a repeat DSC scan under identical conditions on the renatured sample. 3. Results and Discussion 3.1. Binding Aspects of Alkaloids to Double Stranded RNAs. Pronounced hypochromic and bathochromic effects were observed (not shown) in the absorbance spectra of the alkaloids when mixed with increasing concentrations of the RNA duplexes, revealing strong association of these molecules on each of the ds RNAs presumably by intercalation. The presence of sharp isosbestic points enabled the assumption of a two state system consisting of bound and free alkaloids at any particular wavelength. A summary of the optical properties of the alkaloids in the free and bound state is presented in Table 1. The spectrophotometric titrations were performed by increasing the concentration of each alkaloid to a fixed concentration of the RNA sample, and observing the absorbance change at the isosbestic point and the wavelength maximum for use in the calculation of the binding parameters (vide supra). Berberine and palmatine are weak fluorescent compounds with emission spectra in the 450-650 nm range when excited at 350 nm whereas coralyne has a strong fluorescence maximum at 472 nm when excited at 425 nm. Binding to ds DNAs is known to remarkably enhance the fluorescence intensity of berberine and palmatine and quench the fluorescence of coralyne.19,26,29 Similarly, binding to ds RNAs also resulted in the enhancement of the fluorescence of the complexed berberine and palmatine and quenching of the fluorescence of coralyne (not shown) eventually leading to saturation. The extent of change of fluorescence intensity on complexation with ds RNAs was similar in both berberine and palmatine, but more pronounced in the case of coralyne. Large fluorescence change, in each case is indicative of strong association of these molecules to ds RNA structure resulting from an effective overlap of the bound molecules with the base pairs. This result also proposes


Islam et al.

Figure 2. Representative Scatchard plots of complexation of poly(A).poly(U) (A, B, C), poly(I).poly(C) (D, E, F), and poly(C).poly(G) (G, H, I) with berberine, palmatine, and coralyne obtained from spectrophotometric titration data.

the location of the bound molecules in a hydrophobic environment similar to an intercalated state. The base pairing schemes in these ds RNAs are presented in Figure 1. The data from spectrophotometric and fluorometric titrations were used to construct Scatchard plots and such plots of r/Cf versus r are presented in Figure 2. The striking result that emerges from this is that all the Scatchard plots have positive slope at low “r” values, indicating pronounced cooperativity in the ds RNA binding of these molecules. The binding constants (Ki) and the number of excluded sites (n) for the interaction in each case were estimated from the fits to the cooperative equation of McGhee-von Hippel and are collated in Table 2. The affinity values of the cooperative binding of berberine to poly(A).poly(U), and poly(I).poly(C) were (2.24 ( 0.20) -1 -1 × 104 M and (1.42 ( 0.30) × 104 M , respectively, whereas the binding affinity of berberine to poly(C).poly(G) was significantly lower at (0.14 ( 0.03) × 103 M-1. Palmatine has higher binding affinity to poly(A).poly(U), and poly(I).poly(C) with values of (4.12 ( 0.20) × 104 M-1 and (3.50 ( 0.30) × 104 M-1 respectively. The binding affinity of palmatine to poly(C).poly(G) was again higher at (1.70 ( 0.30) × 104 M-1. The binding affinity values of coralyne complexation to poly(A).poly(U), poly(I).poly(C) and poly(C).poly(G) estimated from similar analysis of the spectrophotometric data were higher compared to the corresponding values of berberine and palmatine and were (8.86 ( 0.30) × 104, (5.62 ( 0.30) × 104 and (1.72 ( 0.20) × 104 M-1 respectively. The values of excluded sites for binding of the alkaloids to poly(A).poly(U), poly(I).poly(C) and poly(C).poly(G) were 6.6, 7.8, and 10.2 base pairs for berberine, 5.3, 6.3 and 9.4 base pairs for palmatine and 3.2, 3.8 and 4.6 base pairs for coralyne (Table 2). Similarly, from the analysis of the spectrofluorometric data, values close to that obtained from spectrophotometry were obtained (Table 2). The binding affinity of berberine complexation to poly(A).poly(U), and poly(I).poly(C) were (2.13 ( 0.20) × 104 and (1.01 ( 0.10) × 104 M-1, respectively, whereas the binding affinity of berberine to poly(C).poly(G) was low at (0.16 ( 0.02) ×

103 M-1. The binding affinity values of palmatine to poly(A).poly(U), and poly(I).poly(C) were (3.68 ( 0.30) × 104 M-1 and (2.96 ( 0.20) × 104 M-1, respectively. The binding affinity of palmatine to poly(C).poly(G) was (1.68 ( 0.15) × 104 M-1. The binding affinity values of coralyne complexation to poly(A).poly(U), poly(I).poly(C) and poly(C).poly(G) were (6.80 ( 0.20) × 104, (4.69 ( 0.20) × 104 and (1.45 ( 0.15) × 104 M-1, respectively. It can be seen that a higher binding affinity for coralyne compared to berberine and palmatine to all the three RNAs was observed from both spectrophotometry and spectrofluorometry. Similar values of binding constant for berberine, palmatine and coralyne to poly(A).poly(U) and poly(I).poly(C) are indicative of more or less similar affinity for these compounds to these RNAs. From spectrofluorometry, the numbers of excluded sites on binding of a single alkaloid molecule to AU, IC and CG polymers were found to be around 7.1, 8.2 and 11.3 for berberine, 6.1, 6.8 and 8.9 for palmatine and 3.6, 4.2 and 4.9 base pairs for coralyne. A 7% deviation in the values of n and ω between the two techniques is within experimental errors. The values of the cooperativity factor from spectrophotometric and spectrofluorometric analysis for berberine, palmatine and coralyne are also presented in Table 2. Previous results on studies of berberine palmatine and coralyne to tRNA42,43 and some sequences of DNA29,52,53 have shown cooperative binding phenomena but with synthetic single stranded RNA sequences noncooperative binding was observed.41 Positive cooperativity in DNA-drug interactions has been rationalized as an allosteric effect or as some effect mediated by conformational change in the DNA structure by small molecule binding.54,55 Alternatively, the alkaloids may be clustering to some local conformational regions of the RNAs at the first instance revealing cooperative binding. It is likely that these double stranded RNAs also have some discrete heterogeneous structures to which these alkaloids initially bind cooperatively and convert to a more conventional A-form structure to which subsequent binding is noncooperative. It is pertinent to observe that Barton and

Binding constants (Ki) and the number of occluded sites (n) refer to solution conditions of CP buffer containing 10 mM Na2HPO4, pH 7.0 at 20 °C. ω is the a

5.3 6.3 9.4 4.12 ( 0.20 3.50 ( 0.30 1.70 ( 0.30 24.5 35.2 51.4 6.6 7.8 10.2 2.24 ( 0.20 1.42 ( 0.30 0.014 ( 0.003 poly(A). poly(U) poly(I). poly(C) poly(C).poly(G)

Lippard had observed cooperative binding for a platinum metalointercalator to poly(A).poly(U).56 The overall binding stoichiometry and the possible number of binding sites of the alkaloids on the RNA duplexes were determined by continuous variation analysis (Job Plot) in fluorescence. Plots of difference in fluorescence intensity at 530 nm for berberine and palmatine and at 472 nm for coralyne versus the mole fraction of the alkaloids revealed single binding mode for all the three alkaloids on the polynucleotides (figure not shown). From the inflection points the number of base pairs spanned per alkaloid molecule were evaluated to be 3.8, 4.6, 6.5 for berberine, 2.7, 3.6, 5.6 for palmatine and 2.0, 3.0, 3.8 for palmatine binding to poly(A).poly(U), poly(I).poly(C) and poly(C).poly(G) respectively. Thermal melting is an important tool to investigate the interaction of small molecules to nucleic acids. Neutralization of the phosphate charges through external binding as well as the stacking interactions of intercalated molecules together may contribute to the enhancement of the melting temperature (Tm). In the absence of alkaloid, the optical melting of RNA duplexes (not shown) was cooperative, reversible and revealed Tm values of 38.0 and 44.0 °C for poly(A).poly(U) and poly(I).poly(C), respectively, under the conditions of our experiment (Table 3). Poly(C).poly(G) did not show any melting up to a temperature of 100 °C under the conditions of this study. The differential scanning calorimetric melting also revealed Tm values very close to that obtained from optical melting (Figure 3A,B, Table 3). Further from DSC it was also revealed that the melting was fully cooperative and reversible in that the ratio of values of calorimetric enthalpy (∆Hcal) and van’t Hoff enthalpy (∆Hv) was near unity (Table 3). Strong binding of organic molecules may result in considerable stabilization of the melting temperature of RNAs. In presence of saturating concentrations of berberine, palmatine and coralyne the melting temperatures of both RNAs increased stabilizing them by ∆Tm values of 7.0, 9.0 and 12.0 °C with poly(A).poly(U) and 3.0, 6.0 and 7.0 with poly(I).poly(C), suggesting strong stabilization of RNA by berberine and palmatine and a more stronger stabilization by coralyne. Although increases in Tm cannot be immediately correlated with binding stability, a higher ∆Tm of coralyne complex compared to berberine and palmatine certainly correlates well with the stronger binding of coralyne to poly(A).poly(U) and poly(I).poly(C). Simple electrostatic neutralizations of phosphate molecules, outside the helix, are insufficient to produce duplex structure stabilizations of the magnitude observed. The melting temperature data was used to calculate the binding constants of these molecules to the RNA duplexes using the equation derived by Crothers for DNA57

o where Tm is the optical melting temperature of the RNA in the absence of the drug, Tm is the melting temperature in presence of saturating amounts of the drug, ∆Hwc is the enthalpy of RNA melting obtained from DSC experiment, R is the gas constant, (1.987 cal K-1 mol-1), KTm is the drug binding constant at Tm, R is the free alkaloid activity, which may be estimated by onehalf of the total alkaloid concentration, and n is the site size of the alkaloid binding. The calculated apparent binding constant at the melting temperature can be extrapolated to a reference temperature using the standard relationship,

a Average of four determinations. cooperativity factor.

3.6 4.2 4.9 6.80 ( 0.20 4.69 ( 0.20 1.45 ( 0.15 22.2 23.1 39.2 6.1 6.8 8.9 3.68 ( 0.30 2.96 ( 0.20 1.68 ( 0.15 25.2 34.2 49.6 7.1 8.2 11.3 2.13 ( 0.20 1.01 ( 0.10 0.016 ( 0.002 36.2 47.8 76.8 3.2 3.8 4.6 8.86 ( 0.30 5.62 ( 0.30 1.72 ( 0.20

n

berberine

10-4Ki (M-1) ω n

coralyne

10-4Ki (M-1) ω n 10-4Ki (M-1) ω n 10-4Ki (M-1) RNA

berberine

21.2 23.2 37.7

n

coralyne

10-4Ki (M-1) ω n

palmatine

10-4Ki (M-1)

palmatine

ω

fluorescence absorbance

TABLE 2: Binding Parameters for the ds RNA-Alkaloid Complexation from Absorbance and Fluorescence Dataa

32.3 41.6 69.7

J. Phys. Chem. B, Vol. 113, No. 4, 2009 1215 ω


o 1/Tm - 1/Tm ) (R/n∆Hwc) ln(1 + KTmR)

δ[ln(Kobs)]/δ(1/T) ) -(∆Hb/R)

(4)

(5)


Islam et al.

TABLE 3: Optical Thermal Melting Data and Binding Constants from Optical Melting Data at Saturating Concentrations of Berberine, Palmatine and Coralyne with ds RNAsa system RNA

alkaloid

Tm (°C) (optical melting)

Tm (°C) (DSC)

∆Hcal (kcal/mol)

∆HV (kcal/mol)

poly(A).poly(U)

no alkaloid berberine palmatine coralyne no alkaloid berberine palmatine coralyne

38.0 45.0 47.0 50.0 44.0 47.0 50.0 51.0

39.5 46.1 48.2 50.2 45.2 47.2 50.1 51.6

22.5 27.5 28.5 29.6 16.2 18.6 17.8 18.6

23.1 29.1 28.0 31.2 15.6 17.2 17.6 18.3

poly(I).poly(C)

KTm (M-1) × 10-6

Kobs (M-1) × 10-6

0.84 ( 0.09 1.44 ( 0.08 2.73 ( 0.12

1.00 ( 0.08 1.64 ( 0.10 4.39 ( 0.12

0.57 ( 0.06 0.75 ( 0.08 0.84 ( 0.08

0.61 ( 0.06 1.13 ( 0.08 2.27 ( 0.10

a Melting stabilization of RNA duplexes in the presence of saturating amounts of the alkaloids in CP buffer containing 10 mM Na2HPO4, pH 7.0. The data are averages of four determinations. KTm is the binding constant at the melting temperature and Kobs is the drug-binding constant at 20 °C determined using eqs 4 and 5 described in the text. ∆Hv has been obtained from the DSC data.

Figure 3. Differential scanning calorimetric profiles of (A) poly(A).poly(U)-alkaloid complexes and (B) poly(I).poly(C)-alkaloid complexes.

where Kobs is the drug binding constant at the reference temperature T (in Kelvin) and ∆Hb, the binding enthalpy which was directly determined from the isothermal titration calorimetry experiment (vide infra). The binding constants (Kobs) calculated from these data using the above equations were found to be (1.00 ( 0.08) × 106, (1.64 ( 0.10) × 106 and (4.39 ( 0.12) × 106 M-1, respectively for berberine, palmatine and coralyne, binding to poly(A).poly(U) and (0.61 ( 0.06) × 106, (1.13 ( 0.08) × 106 and (2.27 ( 0.10) × 106 M-1, respectively, for binding to poly(I).poly(C) at 20 °C (Table 3). These values represent the overall binding affinity of the alkaloids to these RNAs. The binding of the alkaloids to the RNAs was further probed using fluorescence quenching experiments in presence of [Fe(CN)6]4-. The anionic quencher would not be able to penetrate the negatively charged helix and if these small molecules are buried within the RNA helix by intercalation, little or no change in fluorescence is expected. Stern-Volmer plots for the quenching of the fluorescence berberine, palmatine and coralyne-RNA complexes are shown in Figure 4. Results indicate that free molecules are quenched efficiently, so is the bound berberine and palmatine molecules to a considerable extent (Figure 4A,B). No quenching or very little quenching was observed in case of coralyne (Figure 4C), indicating the binding of coralyne to be in a relatively more protected environment compared to berberine and palmatine. The quenching constants calculated for berberine and its complexes with AU, IC and CG polymers were 267, 237, 240 and 265 M-1, for palmatine and its complexes with AU, IC and CG polymers were 303, 240, 268 and 274 M-1 and for coralyne and complexes of AU, IC and CG polymers were 196, 4, 63 and 86 M-1. From these results it can be inferred that compared to berberine and palmatine, the bound coralyne is sequestered away from the solvent indicating strong intercalative binding.

Figure 4. Stern-Volmer plots for the quenching of (A) berberine (close circle) and complexes of berberine-AU (open circle), berberine-IC (open square), berberine-CG (open triangle), (B) palmatine (close circle) and complexes of palmatine-AU (open circle), palmatine-IC (open square), palmatine-CG (open triangle), and (C) coralyne (close circle) and complexes of coralyne-AU (open circle), coralyne-IC (open square), and coralyne-CG polymers (open triangle) with increasing concentration of K4[Fe(CN)6]. The concentration of K+ ion was kept constant using KCl solution.

3.2. Mode of Binding. To probe the binding mode of the alkaloids to the RNAs, the viscosity of the RNA solution was measured in the presence of increasing concentrations of the alkaloids. The original hypothesis of Lerman suggests that the viscosity of a rodlike nucleic acid solution should increase upon complexation with an intercalator.58 This is due to the fact that the axial length of the nucleic acid enhances and it becomes more rigid. The viscosity increase occurs as both the factors enhance the frictional coefficient. Viscosity results are expressed as length enhancement estimated with respect to a standard value



Figure 5. Circular dichroic spectra of poly(A).poly(U) (50 µM) treated with (A) 0.0, 2.5, 5.0, 10, 20, 25, and 30 µM berberine (curves 1-7), (B) 0.0, 2.5, 5.0, 10, 15, 20, and 35 µM palmatine (curves 1-7), and (C) 0.0, 5.0, 10, 15, 20, 25, 30, and 40 µM coralyne (curves 1-8); poly(I).poly(C) (50 µM) treated with (D) 0.0, 5.0, 10, 20, 25, and 30 µM berberine (curves 1-6), (E) of 0.0, 5.0, 10, 15, 20, and 30 µM palmatine (curves 1-6), and (F) 0.0, 5.0, 10, 15, 20, and 30 µM coralyne (curves 1-6); and poly(C).poly (G) (50 µM) treated with 0.0, 5.0, 10, 20, and 30 µM berberine (curves 1-5), (H) 0.0, 5.0, 10, 15, 20, and 30 µM palmatine (curves 1-6), and (I) 0.0, 10, 15, and 30 µM (curves 1-4) coralyne.

(β) of 2 corresponding to a length enhancement of 0.34 nm. The β values for berberine binding to AU, IC and CG polymers were 0.19, 0.17 and 0.04 nms, for palmatine to AU, IC and CG polymers were 0.41, 0.22 and 0.18 nms and for coralyne complexation to AU, IC and CG polymers were 1.88, 1.27 and 1.07 nms, respectively. For the cationic metallo intercalator studied by Barton and Lippard, a β value of 1.64 was found with poly(A).poly(U) duplex.56 Thus, a true intercalation scenario may be envisaged in coralyne binding to the AU and IC sequences whereas partial intercalation may be assigned to coralyne binding to the CG polymer. On the other hand, berberine and palmatine binding to all three polymers were through partial intercalation to varying extents. It is pertinent to observe here that a partial intercalation mode of interaction was also suggested for berberine and palmatine binding to ds DNA sequences.18,19,26-29 Berberine and palmatine have partial saturation in the ring structure resulting in tilted structure, and it is likely that the buckled structure of these molecules is preventing a true intercalation interaction into the RNA polymers, whereas the planar structure of coralyne facilitates true intercalation. 3.3. Conformational Aspects of Binding. Conformational aspect of the binding of the alkaloids with these ds RNAs was investigated from intrinsic and induced circular dichroic studies. The intrinsic CD spectra of the RNA duplexes displays more or less gross A-form conformation characterized by a large positive band in the 260-268 nm and an adjacent weak negative band at 235 nm although there are significant differences in the nature, ellipticity and wavelength maxima. These bands are caused due to the stacking interactions between the bases and the helical structure of the polymers that provide asymmetric environment for the bases and are in conformity with the literature data.46,59,60 On the other hand, the alkaloids under investigation do not have any intrinsic optical activity but may acquire optical activity (induced CD) on binding to double

helical RNA. To record the alkaloid-induced changes in the RNA conformation, the CD spectra in the 220-400 nm regions were recorded in the presence of varying D/P values. In the presence of berberine, palmatine and coralyne the ellipticity of the long wavelength positive band of poly(A).poly(U) decreased as the interaction progressed with a slight shift in the wavelength maximum till saturation was achieved at a D/P of 0.5. The change in ellipticity was higher for palmatine and coralyne interaction compared to berberine. Not much change was observed in the negative 235 nm band. A weak isoelliptic point was seen at around 287 nm in both these cases. An induced CD with strong ellipticity was observed in the 300-400 nm region for palmatine-AU polymer complexation whereas for berberine-AU polymer and coralyne -AU polymer very broad weak bands were developed in the same region. The CD spectral data are presented in Figure 5. On the other hand, neither berberine nor palmatine binding to the IC polymer produced any significant CD changes in the polymer conformation (Figure 5D,E). But, on the contrary, coralyne-IC polymer complexation (Figure 5F) revealed considerable changes in the CD of this RNA compared to the other alkaloids manifested with the appearance of large induced CD band in the 300-350 nm region. An isoelliptic point was observed at 294 nm that further indicated that the structural changes are interdependent. The interaction of the alkaloids with the CG polymer, did not produce any significant changes to the polymer CD (Figure 5G-I), suggesting the absence of conformational changes. To examine the conformational aspects in more detail, induced CD spectra in the 300-700 nm region were recorded where neither the RNAs nor these alkaloids have any CD spectra. This region monitors the CD induced in the small molecules essentially on binding asymmetrically to the chiral RNA helix. The alkaloid molecules strongly bound may acquire induced circular dichroic characteristics in the asymmetric RNA environment, which was already visible in case of poly(A).poly(U)-palmatine and


Islam et al.

Figure 6. Induced circular dichroic spectra of berberine (50 µM) in the presence of (A) 500, 400, 300, 200, 100, and 50 µM poly(A).poly(U) (curves 1-6), (B) 500, 400, 300, 200, and 100 µM poly(I).poly(C) (curves 1-5), and (C) 500, 300, 200, and 100 µM poly(C).poly(G) (curves 1-4); palmatine (50 µM) in the presence of (D) 500, 400, 300, 200, 100, and 50 µM poly(A).poly(U) (curves 1-6), (E) 500, 400, 300, 200, 100, and 50 µM poly(I).poly(C) (curves 1-6), and (F) 500, 400, 300, 200, and 100 µM poly(C).poly(G) (curves 1-5); and coralyne (10 µM) in the presence of (G) 100, 80, 60, 40, and 10 µM poly(A).poly(U) (curves 1-5), (H) 100, 80, 60, 40, 20, and 10 µM poly(I).poly(C) (curves 1-6), and (I) 100, 80, 60, 40, 20, and 10 µM poly(C).poly(G) (curves 1-6).

coralyne from intrinsic CD studies. In Figure 6A,D,G the induced CD spectra of the complexation of berberine, palmatine and coralyne with the AU RNA are depicted. Two major peaks are observed, for berberine and palmatine, one negative one around 320 and another positive around 350 nm. As the P/D decreased, the ellipticity of both the bands decreased. In berberine, additionally a broad band was observed in the region 400-500 nm. For coralyne (Figure 6G) there were two regions where positive induced CD bands were seen; a sharp band in the 300 nm region with a remarkably higher ellipticity and a broad negative band in the 500 nm region, the ellipticity of both decreased concomitantly with decreasing P/D. In the IC polymer system, berberine showed two bands, a strong positive one around 350 nm followed by a broad weak band in the 400-500 region, but the nature of the spectra was different from that of berberine-AU system (Figure 6B). On the other hand, the palmatine-IC polymer system showed a single positive band (Figure 6E) around 350 nm similar to that of the palmatine AU system. For coralyne-IC polymer binding, the regions of the induced CD bands were almost similar to that observed with AU polymer (Figure 6H). In the CG system, berberine and palmatine produced single asymmetry with a positive induced CD in the 350 nm region (Figure 6C and F). For the coralyne-CG polymer, the induced CD pattern was similar to AU and IC systems, although there were significant differences in the ellipticity of the bands (Figure 6I). The induced CD spectral patterns suggest differences in the orientation of the bound molecule inside the helical organization of the RNA duplexes. Strong induced CD in terms of higher ellipticity values was manifested in palmatine-AU, berberine-AU, coralyne-AU and coralyne-CG systems. It is pertinent to observe that the base pairing schemes and the dipole moments of the AU, IC and GC base pairs are different and their interaction with that of the alkaloids are expected to be different leading to the

different manifestation of the induced CD patterns. The two regions of the induced CD of these alkaloid interactions with DNA and RNA have been previously characterized to be due to intercalation and external binding modes.35,41,52,61 In analogy with such interpretations, the band in the long wavelength region (>400 nm) for ds RNA complexes may be inferred to arise due to intercalated alkaloid. Based on this, the intercalation of coralyne appears to be stronger with the all the three RNAs followed by berberine with AU and IC polymers. 3.4. Thermodynamics of the Interaction. Isothermal titration calorimetry (ITC) has become an effective tool to thermodynamically characterize the binding of small molecules to macromolecules and may offer key insights into the molecular forces that drive complex formation. The advantage of ITC is that it can give a complete thermodynamic profile for the binding such as Gibbs free energy change (∆G), enthalpy change (∆H) and entropy change (∆S) together with the number of binding sites (N) and the affinity constant (Ka) and these are independent of the spectroscopic changes that occur during the reactions. The ITC profiles for the binding of berberine, palmatine and coralyne to three RNAs under investigation are presented in Figure 7. All the profiles were monophasic and revealed the binding to be exothermic, resulting in negative peaks in the plot of power versus time. Each of the heat burst curves in the figure corresponds to a single injection of the respective RNA into alkaloid that was corrected by corresponding dilution heats derived from the titration of identical amounts of RNA into buffer alone. The resulting corrected heat plotted as a function of molar ratio is depicted in the lower panel. The data points reflect the experimental injection heats, and the solid lines reflect the calculated fits of the data. Except for berberine-poly(C). poly(G) titration, the data were fitted to a single set of identical sites model that yielded a fairly reasonable fitting of the experimental data. Due to very low heat change, the experi-



Figure 7. Representative ITC profiles for the titration of poly(A).poly(U) (A, B, C), poly(I).poly(C) (D, E, F) and poly(C).poly(G) (G, H, I) with berberine, palmatine and coralyne, respectively, at 20 °C. The top panels represent the raw data for the sequential injection of RNA into the alkaloid, and the bottom panels show the integrated heat data after correction of heat of dilution against the molar ratio of RNA/[alkaloid]. The data points were fitted to one site model, and the solid lines represent the best-fit data.

mental data for berberine-poly(C).poly(G) complexation (Figure 7G) could not be fitted to any model. The thermodynamic parameters for the alkaloids binding to the three RNA duplexes are depicted in Table 4. The ITC data for the binding of berberine to poly(A).poly(U) yielded an association constant (Ka) of (8.10 ( 0.70) × 105 M-1, an enthalpy change (∆H) of -2.15 kcal/mol, an entropy change (T∆S) of 5.82 kcal/mol and a binding site size (1/N) of 3.25 base pairs. The binding of palmatine to this RNA yielded a higher association constant of (1.23 ( 0.07) × 106 M-1, a ∆H of -2.62 kcal/mol, a T∆S value of 5.60 kcal/mol and a

binding site size of 2.85 base pairs. On the other hand, binding of coralyne was markedly stronger compared to berberine and palmatine with a Ka of (3.02 ( 0.12) × 106 M-1, a ∆H of -5.86 kcal/mol, a T∆S value of 2.88 kcal/mol and a lower binding site size of 2.03 base pairs. The association constant of berberine binding to poly(I).poly(C) was slightly lower than the value with the poly(A).poly(U) at (0.61 ( 0.08) × 106 M-1. A ∆H value of -0.86 kcal/mol, a T∆S value of 6.94 kcal/mol and a binding site size of 4.25 base pairs was evaluated form the ITC analysis. Similar to berberine, the binding of palmatine to the IC polymer was also


Islam et al.

TABLE 4: Temperature Dependent Isothermal Calorimetric Data for the Binding of Berberine, Palmatine and Coralyne to ds RNAsa RNAs

alkaloid

poly(A).poly(U)

berberine palmatine coralyne

poly(I).poly(C)

berberine palmatine coralyne

poly(C).poly(G)

palmatine coralyne

temp (K)

Ka × 10-6 (M-1)

∆G (kcal /mol)

∆H (kcal /mol)

T∆S (kcal/mol)

∆Cp (cal. /mol. K)

∆G hyd (kcal /mol)

283 293 303 283 293 303 283 293 303 283 293 303 283 293 303 283 293 303 283 293 303 283 293 303

1.60 ( 0.06 0.81 ( 0.07 0.09 ( 0.04 2.48 ( 0.09 1.23 ( 0.07 0.56 ( 0.06 4.84 ( 0.08 3.02 ( 0.12 1.18 ( 0.10 0.95 ( 0.07 0.61 ( 0.08 0.06 ( 0.09 1.84 ( 0.06 0.94 ( 0.07 0.07 ( 0.03 2.69 ( 0.12 1.17 ( 0.10 0.95 ( 0.04 1.30 ( 0.02 0.78 ( 0.01 0.06 ( 0.01 1.76 ( 0.01 0.87 ( 0.01 0.08 ( 0.01

-8.08 -7.97 -6.91 -8.33 -8.22 -8.02 -8.71 -8.74 -8.47 -7.79 -7.80 -6.67 -8.16 -8.06 -6.76 -8.38 -8.19 -8.34 -7.97 -7.95 -6.67 -8.14 -8.02 -6.85

-1.75 -2.15 -3.58 -1.37 -2.62 -5.25 -2.22 -5.86 -8.92 -0.32 -0.86 -1.42 -2.55 -3.54 -4.71 -3.83 -5.21 -6.58 -1.18 -2.01 -2.88 -6.11 -6.93 -7.56

6.33 5.82 3.33 6.96 5.60 2.77 6.49 2.88 -0.45 7.47 6.94 5.25 5.61 4.52 2.05 4.55 2.98 1.76 6.79 5.94 3.79 1.62 0.57 -1.01

-102

-8.2

-194

-15.5

-335

-26.8

-55

-4.4

-108

-8.6

-138

-11.0

-85

-6.8

-93

-7.4

All the data in this table are derived from ITC experiments conducted in CP buffer, 20 mM [Na+], pH 7.0 and are average of four determinations. Ka and ∆H values were determined from ITC profiles fitting to Origin 7 software as described in the text. The values of ∆G and T∆S were determined using the equations ∆G ) - RTlnKa, and T∆S ) ∆H-∆G. All the ITC profiles were fit to a model of single binding sites. a

TABLE 5: Isothermal Titration Calorimetric Data for the Binding of Berberine, Palmatine and Coralyne to ds RNAsa poly(A).poly(U) parameters -Zφ -∆G (kcal/mol) -Gt (kcal/mol) -∆G pe (kcal/mol)

poly(I).poly(C)

poly(C).poly(G)

berberine

palmatine

coralyne

berberine

palmatine

coralyne

berberine

palmatine

coralyne

0.91 7.97 5.89 2.08

0.98 8.22 5.98 2.24

0.78 8.74 7.07 1.67

0.98 7.80 5.56 2.24

0.97 8.06 5.84 2.22

0.83 8.19 6.29 1.90

nd nd nd nd

0.99 7.95 5.68 2.27

0.87 8.02 6.03 1.99

a All values refer to solution conditions of 20 °C, 20 mM [Na+], pH 7.0. Z- is the slope of the plot of lnKa versus ln [Na+]. All other parameters are as defined in the text. nd: not determined.

weak compared to its interaction with AU polymer. The value of the association constant of palmatine binding to the IC polymer was lower at (9.40 ( 0.70) × 105 M-1. The ITC data suggests the binding of coralyne to AU duplex to be of the highest affinity. Importantly, the binding affinity values evaluated from the ITC data are comparable to the overall binding affinity values (Kiω) obtained from absorption and fluorescence spectral data. Notably, with each RNA, coralyne binds with higher affinity followed by berberine and palmatine. The free energy change in each case was more or less similar around 7.0-8.0 kcal/mol. The binding of berberine and palmatine to each RNA was favored by negative enthalpy and a larger favorable entropy change whereas, in the case with coralyne, the binding was driven by a stronger enthalpy term, with a smaller positive entropy term. Thus, the entropic contribution is most favorable in binding of berberine and palmatine to all the RNAs. The energetics of berberine and palmatine interaction are significantly different from that of coralyne and reflect the differences in the mode of interaction. The thermodynamic data of interaction of these alkaloids to RNA appear to be similar to their interaction with DNA.35,52,53 3.5. Ionic Strength Dependence of the Binding and Parsing of the Free Energy of Binding. Berberine, palmatine and coralyne are polycondensate molecules with a single charge on the exocyclic nitrogen atom, and electrostatic interaction is

thought to be the driving force in their interaction with nucleic acids. Further, cations are condensed around the polyanionic RNA helix and charged ligands compete to expel the cations for phosphate neutralization; these are thermodynamically linked processes. To provide insights into such molecular details, ITC studies were performed at three different salt conditions, viz. 20, 50 and 100 mM [Na+], and the association constants were evaluated from the fits of the ITC data and analyzed in conjunction with van’t Hoff analysis. The association constant values for the binding of berberine, palmatine and coralyne to AU, IC and CG RNA duplexes are presented in the Table 5. The following relationship between Ka and sodium ion concentration has been derived previously linking the charge to the variation of binding affinity with [Na+]62

δ log Ka/δ log [Na+] ) -Zφ

(6)

where Z is the apparent charge on the bound ligand and φ is the fraction of the [Na+] bound per RNA phosphate. It can be seen that the value of the association constant decreases as the salt increases. Variation of ln Ka versus ln [Na+] reveals straight lines (Figure 8), the slope (Zφ) of which provides values of 0.91, 0.98 and 0.78 for berberine, palmatine and coralyne



Figure 8. Left panel: plot of ln Ka versus ln [Na+] of (A) poly(A).poly(U) (B) poly(I).poly(C) and (C) poly(C).poly(G) with berberine (b) palmatine (9) and coralyne (2). Right panel: nonpolyelectrolytic (∆Gt) and polyelectrolytic (∆Gpe) contribution of blinding of alkaloids with (D) poly(A).poly(U), (E) poly(I).poly(C) and (F) poly(C).poly(G) at 20 mM [Na+]. The solid parts and hatched parts indicate respectively the nonpolyelectrolytic (∆Gt) and the polyelectrolytic (∆Gpe) contribution to the binding free energy.

binding to poly(A).poly(U), 0.98, 0.97 and 0.83 for berberine, palmatine and coralyne binding to poly(I).poly(C) and 0.99 and 0.87 for palmatine and coralyne binding to poly(C).poly(G) indicating the number of ions released. The value of φ for poly(A).poly(U) was previously determined to be around 0.89.62 The binding of the alkaloids to poly(C).poly(G) could not be estimated due to the weak binding profiles in ITC that could not fitted to any model (vide supra). The values of the ions released obtained here are in good agreement with that reported for mono cationic molecules binding to double stranded DNAs and RNAs and are in agreement with that reported earlier for binding of these molecules to tRNA.40 The observed free energies of the interaction are in the range 7.80 to 8.74 kcal/ mol (Table 5). From the dependence of Ka on [Na+] the observed free energy can be partitioned between two contributions namely the nonpolyelectrolytic contribution (∆Gt) and the polyelectrolytic contribution (∆Gpe) as done in case of several intercalators by Chaires and co-workers.63,64

∆G ) -RT ln Ka ) (∆Gt) + (∆Gpe)

(7)

The polyelectrolytic contribution at any given [Na+] may be calculated from the experimentally determined quantity ((δ ln K/δ ln [Na+]) ) -Zφ). Record and co-workers65,66 have shown that ∆Gpe ) -(Zφ)RT ln [MX], where MX is the monovalent salt concentration. The magnitude of ∆Gpe is the free energy contribution arising from coupled polyelectrolytic forces particularly that from the release of condensed counterions from the RNA helix upon binding of charged ligands. The free energy of binding of these alkaloids to the RNA and the polyelectrolytic and the nonpolyelectrolytic contribution to the same were

calculated at 20 mM and presented in Table 5. A graphical representation of the same is presented in Figure 8D-F. At 20 mM where the electrostatic contribution is also predominant the values of ∆Gt and ∆Gpe for the binding of berberine to AU polymer were respectively -5.89 and -2.08 kcal/mol. The values for palmatine binding to this RNA were -5.98 and -2.24 kcal/mol, respectively. Similarly for coralyne poly(A).poly(U) complexation, these values were respectively -7.07 and -1.67 kcal /mol. As the salt concentration was increased, the electrostatic contribution to the total binding free energy decreased and at 100 mM, the ∆Gpe contribution to the free energy was -1.23, -1.32 and -1.05 kcal/mol for berberine, palmatine and coralyne, respectively. On the other hand, the values of ∆Gt and ∆Gpe for berberine, palmatine and coralyne binding to IC polymer were -5.56, -5.84, -6.29 and -2.24, -2.22, -1.90 kcal/mol, respectively. At 100 mM salt the ∆Gpe contribution to the free energy was -1.32, -1.31 and -1.20 kcal/mol, respectively. It is pertinent to observe that the ∆Gt remained invariant at all ionic strengths. For the binding of palmatine and coralyne to poly(C).poly(G), the partitioned contributions of ∆Gt values were -5.68 and -6.03 kcal/mol and the ∆Gpe values were -2.27 and -1.99 kcal/mol, respectively (Table 5). As the salt concentration increased, the ∆Gpe contribution decreased, while the ∆Gt contribution to the total free energy remained invariant. At 100 mM, the respective contributions to the observed free energy of binding were -1.33 and -1.17 kcal/mol, respectively. It can be seen from the figure and table that in both cases there is a remarkably large magnitude from the nonelectrostatic forces to the binding free energy, clearly suggesting the role of hydrophobic forces in the RNA intercalation process of these two molecules. Further, the polyelectrolytic contribution to the free energy appears to be more or less similar in all the cases at all ionic strengths, further supporting the presumption of the weak participation of the charges on the alkaloids in the interaction. It is pertinent to observe here that the low electrostatic contribution revealed here is in stark contrast to that observed with the binding of aminoglycosides to A-site RNA interaction. These results are in agreement with our previous proposal for the binding of berberine, palmatine and coralyne to tRNA molecules.40,42,43 3.6. Heat Capacity Changes. The heat capacity changes (∆Cp) of small molecule-RNA binding interactions can be determined from the temperature dependence of the binding enthalpy using the standard relationship,

∆Cp ) δ(∆H)/δT

(8)

This information provides valuable insights into the type and magnitude of forces involved in the interaction. A large ∆Cp value is usually associated with changes in hydrophobic or polar group hydration and considered an indicator of a dominant hydrophobic effect in the binding process. Figure 9 graphically presents the temperature variation of the ∆H values listed in Table 4. The data points were fit by linear regression with the slopes of the lines giving estimates of ∆Cp values for berberine, palmatine and coralyne of -102, -194 and -335 cal/(mol K) binding to poly(A).poly(U), -55, -108 and -138 cal/(mol K) for binding to poly(I).poly(C) and -85 and -93 cal/(mol K) for binding of palmatine and coralyne to poly(C).poly(G). Similar values are observed for the binding of these alkaloids to DNA also.35,53 First of all, the values of ∆Cp are nonzero, indicating temperature dependence of the enthalpy change. Second, the values of ∆Cp in almost all cases fall within the range 100-500 cal/(mol K) or are very close to the lower limit


Figure 9. Plot of variation of enthalpy of binding (∆H) with temperature for the binding of (A) poly(A).poly(U) (B) poly(I).poly(C) and (C) poly(C).poly(G) with berberine (O) palmatine (0) and coralyne (∆) in CP buffer of 20 mM [Na+], pH 7.0.

that is frequently observed for both ligand nucleic acid and ligand protein interaction.67,68 The higher values of ∆Cp for coralyne binding to AU and IC sequences and palmatine binding to AU sequences compared to others suggest substantial conformational change in the RNA structure that was reflected in the CD as well. The significant differences in the ∆Cp values may indicate differences in the release of structured water consequent to the transfer of nonpolar groups into the interior of the helix. In nucleic acids particularly, structured water like the hydrophobic hydration can be associated with large heat capacity changes and release of such water associated with the transfer of nonpolar groups into the interior of the grooves of the helix can be attributed to the negative term to the ∆Cp. Murphy and Churchill69 have discussed four types or modes of DNA recognition, which are sequence specific, nonspecific, minimal sequence specific and structure specific. In their view, slightly negative ∆Cp values are associated with a minimal sequence specific binding. Extending the argument to RNA here, the nonzero ∆Cp values observed here, but that are smaller than values associated with sequence specific binding, may appear to denote structure specific binding of these molecules. Further studies are required to clarify this point. It is known that for intercalators and groove binding molecules a large hydrophobic contribution to the binding free energy is expected due to their aromatic ring system and binding should be energetically favorable.68 From the Records70 relationship ∆Ghyd ) (80 ( 10)∆Cp, the free energy contribution from the hydrophobic transfer step of these compounds to the RNA structures may be calculated. We observe that values of ∆Ghyd for berberine, palmatine and coralyne binding to poly(A).poly(U) to be -8.16, -15.5 and -26.8 kcal/mol, respectively, whereas that to poly(I).poly(C) binding to be -4.4, -8.6 and -11.0 kcal/mol,

Islam et al.

Figure 10. Plot of ∆G (open symbols) and ∆H (closed symbols) versus T∆S for the binding of (A) berberine, (B) palmatine and (C) coralyne with poly(A).poly(U) (O, b) poly(I).poly(C) (0, 9) and poly(C).poly(G) (∆, 2) respectively.

respectively. Such values for palmatine and coralyne binding to poly(C).poly(G) were -6.8 and -7.4 kcal/mol, respectively (Table 4). All these values are in the range that was observed for intercalators or groove binding molecules binding to DNA and RNA.68 Although Chaires, Wilson and co-workers have recently71,72 advanced more rigorous analysis of contributing factors to ∆Cp, those are beyond the scope of this paper. Enthalpy-entropy compensation is more often associated with solvent reorganization accompanying drug-nucleic acid interactions.73,74 Linear relationship of enthalpy change with T∆S with slope unity is an indication of complete compensation, and this occurs in systems with ∆Cp not equal to zero and ∆Cp > ∆S. Figure 10A-C shows the variation of ∆H as a function of T∆S. The values of the slope, that is δ∆H/ δ(T∆S), were 0.93, 0.97 and 0.93 for palmatine-poly(A).poly(U), coralyne-poly(A).poly(U) and coralyne-poly(I).poly(C), respectively. This suggests almost complete compensation in these cases. On the other hand, the slopes for berberine binding to poly(A).poly(U) and poly(I).poly(C) were 0.61 and 0.46 whereas that for palmatine binding to poly(I).poly(C) and poly(C).poly(G) were 0.58 and 0.54, respectively. The slope in case of coralyne-poly(C).poly(G) was 0.58. In all these cases we can suggest that there is partial compensation leading to finite values of ∆Cp. 4. Conclusion The present work reports the binding of three isoquinoline alkaloids with three sequence specific double stranded RNAs using a variety of biophysical techniques. This study has

Alkaloid-RNA Interaction revealed that the alkaloids berberine and palmatine bind to the RNA duplexes by weak partial intercalation and coralyne binds by strong intercalation. All the alkaloids bind to the RNAs exhibiting positive cooperativity, providing evidence for heterogeneity in the RNA conformations. Parsing of the free energy of the binding showed a large nonelectrostatic contribution to it in each case. The binding was also characterized by moderate to large change in the conformation of the RNA duplexes with the bound alkaloids acquiring induced optical activity to varying extents. Thermodynamics of the interaction revealed that the binding was favored by both negative enthalpy and positive entropy changes, but to different extents, and showed enthalpyentropy compensation behavior to varying extents. Negative heat capacity changes in all the systems are correlated to the involvement of significant hydrophobic forces in the complexation. The binding of berberine and palmatine was favored by higher entropic contribution compared to coralyne. All data suggest stronger binding of coralyne compared to berberine and palmatine. This study further advance our knowledge on the interaction of small molecules to ds RNA sequences that may be useful for designing natural product based RNA binding therapeutic molecules. Acknowledgment. This work was supported by grants of the network project on “Comparative genomics and biology of noncoding RNA in human genome” (NWP0036) from the Council of Scientific and Industrial Research (CSIR), Government of India. Md.M.I. is a Senior Research Fellow of the CSIR, awarded through the national eligibility test (NET). S.R.C. is supported by Project Assistanceship from NWP0036. We thank Prof. Siddhartha Roy, Director, IICB, for his support and encouragement, Dr. Basudev Achari, CSIR Emeritus Scientist, for critical reading of the manuscript and all the colleagues of the Biophysical Chemistry Laboratory for their help and cooperation at every stage of this work. Critical and constructive comments from the reviewers are also highly appreciated. References and Notes (1) Cheng, A. C.; Calabro, V.; Frankel, A. D. Curr. Opin. Struct. Biol. 2001, 11, 478. (2) Hermann, T. Biochimie 2002, 84, 869. (3) Vicens, Q.; Westhof, E. ChemBioChem 2003, 4, 1018. (4) Tor, Y. ChemBioChem 2003, 4, 998. (5) Sall, A.; Liu, Z.; Zhang, H. M.; Yuan, J.; Lim, T.; Su, Y.; Yang, D. Curr. Drug DiscoV. Technol. 2008, 5, 49. (6) Gallego, J.; Varani, G. Acc. Chem. Res. 2001, 34, 836. (7) Foloppe, N.; Matassova, N.; Aboul-Ela, F. Drug DiscoV. Today 2006, 11, 1019. (8) Takahashi, T. T.; Austin, R. J.; Richard, R. W. Trends Biochem. Sci. 2003, 28, 159. (9) Cheong, H. K.; Hwang, E.; Lee, C.; Choi, B. S.; Cheong, C. Nucleic Acids Res. 2004, 32, e84. (10) Musselman, C.; Pitt, S. W.; Gulati, K.; Foster, L. L.; Andricioaei, I.; Al-Hashimi, H. M. J. Biomol. NMR 2006, 36, 235. (11) Walter, F.; Vicens, Q.; Westhof, E. Curr. Opin. Chem. Biol. 1999, 3, 694. (12) Kaul, M.; Pilch, D. S. Biochemistry 2002, 41, 7695. (13) Chao, P. W.; Chow, C. S. Bioorg. Med. Chem. 2007, 15, 3825. (14) Tanious, F. A.; Veal, J. M.; Buczak, H.; Ratmeyer, L. S.; Wilson, W. D. Biochemistry 1992, 31, 3103. (15) Zhao, M.; Janda, L.; Nguyen, J.; Strekowski, L.; Wilson, W. D. Biopolymers 1994, 34, 61. (16) Wilson, W. D.; Li, K. Curr. Med. Chem. 2000, 7, 73. (17) Grycova´, L.; Dosta´l, J.; Marek, R. Phytochemistry 2007, 68, 150. (18) Maiti, M.; Suresh Kumar, G. In Topics in Heterocyclic Chemistry; Khan, M. T. H., Eds.; Springer-Verlag: Berlin, Heidelberg, 2007; Vol. 10, p 155. (19) Maiti, M.; Suresh Kumar, G. Med. Res. ReV. 2007, 27, 649. (20) Creasey, W. A. Biochem. Pharmacol. 1979, 28, 1081.

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