Interaction of Isoquinoline Alkaloids with an RNA Triplex: Structural

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Interaction of Isoquinoline Alkaloids with an RNA Triplex: Structural and Thermodynamic Studies of Berberine, Palmatine, and Coralyne Binding to Poly(U).Poly(A)*Poly(U) Rangana Sinha and Gopinatha Suresh Kumar* Biophysical Chemistry Laboratory, Indian Institute of Chemical Biology, CSIR, Kolkata 700032, India ReceiVed: July 22, 2009; ReVised Manuscript ReceiVed: August 24, 2009

The interaction of two natural protoberberine alkaloids berberine and palmatine and the synthetic derivative coralyne with the RNA triplex poly(U).poly(A)*poly(U) was studied using various biophysical and calorimetric techniques. All the three alkaloids bind noncooperatively to the triplex. The affinity of berberine and palmatine was in the order of 105 M-1, while that of coralyne was one order higher as inferred from spectroscopic studies. The alkaloids stabilized the Hoogsteen base-paired third strand of the triplex without affecting the stability of the duplex. Fluorescence quenching and viscosity studies gave convincing evidence for the partial intercalation of berberine and palmatine and a true intercalative binding of coralyne to the triplex. This was further supported from the significant polarization of the emission spectra of the complex and the energy transfer from the base triplets to the alkaloids. Circular dichroic studies suggested that the conformation of the triplex was perturbed significantly by the binding of the alkaloids, being more by coralyne compared to berberine and palmatine and also evidenced by the generation of strong induced optical activity in the bound coralyne molecules. Isothermal titration calorimetric studies revealed that the binding to the triplex was favored by a predominantly large negative enthalpy change (∆H° ) -5.42 kcal/mol) with small favorable entropy contribution (T∆S° ) 2.02 kcal/mol) in berberine, favored by almost equal negative enthalpy (∆H° ) -3.93 kcal/mol) and entropy changes (T∆S° ) 3.89 kcal/mol) in palmatine and driven by predominant entropy contributions (∆H° ) -1.84 and T∆S° ) 7.44 kcal/mol) in coralyne. These results advance our knowledge on the binding of small molecule isoquinoline alkaloids that are specific binders of RNA structures, particularly triplexes. 1. Introduction Triple helical nucleic acid structures have created the resurgence of interest in nucleic acid structures next to the discovery of the double helical structure for their potential application in controlling a particular gene expression via triplex formation, commonly known as the antigene strategy. The formation of triplex structures was first reported in RNA in 1957 by Felsenfeld and colleagues.1,2 There are several biological implications for triplex formation, and hence investigations on the structure and stability3-8 of triplex structures sparked much renewed interest.9-13 A triplex forming oligonucleotide can easily penetrate the cell and modify the double-stranded DNA and inhibit transcription. A triplex is formed by the binding of a third strand nucleic acid in the major groove of a duplex nucleic acid that must generally have a homopurine-homopyrimidine sequence. Two types of triplexes, namely, the parallel motif (YRY, Y ) pyrimidine and R ) purine) having a homopyrimidine third strand that binds parallel to the homopurine strand of the duplex and the antiparallel motif (YRR) with a homopurine third strand that binds antiparallel to the homopurine strand of the duplex, were characterized.11,14-18 In the parallel motif, the canonical triplexes formed are the poly(dC).poly(dG)*poly(dC+) and poly(dT).poly(dA)*poly(dT) triplexes of the DNA and the poly(U).poly(A)*poly(U) of the RNA (. and * represents the Watson-Crick and Hoogsteen base pairing, respectively). In all these, the binding of the Hoogsten base* Corresponding author. Scientist Biophysical Chemistry Laboratory, Indian Institute of Chemical Biology 4, Raja S.C. Mullick Road, Kolkata 700032, India. Phone: +91 33 2472 4049/2499 5723. Fax: +91 33 2473 5197/2472 3967. E-mail: [email protected]/[email protected].

paired third strand is weak compared to the Watson-Crick pairing resulting in low stability of the triplex and critically limiting their application in vivo. Several strategies were adopted to enhance the stability including the use of intercalators and tethers, but mismatches may also be stabilized and hence some specificity may be lost.19-22 Several intercalators conjugated at the end of the triplex enhanced the stability by anchoring the oligonucleotide through intercalation.23-31 Studies on the interaction of unbound intercalators to RNA triplexes have been scarce32-37 and with the recent discovery of the involvement of RNA in several cellular activities,38-43 RNA triplexes are gaining prominence. Further, a more recent discovery of micro RNAs and unraveling of their cellular functions led to a paradigm shift from DNA binding to RNA binding agents as potential gene regulators.44,45 It is likely that RNA triplex formation may also be an important structural motif of these small RNAs that the therapeutic agents may target for gene regulation. Understanding of the interaction of small molecules to RNA triplexes may thus be an important area of significance in the contemporary RNA targeted small molecule interaction studies. Alkaloids are important natural products with unmatched chemical diversity and biological relevance with potential highquality pools in drug screening. Protoberberines constitute an important group of alkaloids with remarkable biological relevance including potential anticancer properties. The use of plants containing protoberberines in folk medicine dates back several thousand years. Berberine, palmatine, and synthetic coralyne (Figure 1) are especially studied extensively for their utility in cancer therapy, and a large number of data are available in the literature. More recent studies reveal their potential ability

10.1021/jp9069515 CCC: $40.75  2009 American Chemical Society Published on Web 09/15/2009

Isoquinoline Alkaloids with an RNA Triplex

J. Phys. Chem. B, Vol. 113, No. 40, 2009 13411 TABLE 1: Summary of the Optical Properties of Free and Poly(U).Poly(A)*Poly(U) Bound Alkaloids parametera

b

Figure 1. Chemical structures of berberine (A), palmatine (B), and coralyne (C) and the base pairing scheme in poly(U).poly(A)*poly(U) (D).

to bind to diverse RNA structures with exceptionally high affinity and specificity some time even higher than many aminoglycosides.46,47 For example, berberine and coralyne bind to poly(A) with affinity in the order close to 107 M-1 and induce self-structure formation.47,48 In light of their strong affinity to various RNA structures, we here investigated the structure and thermodynamics of their interaction with the RNA triplex poly(U).poly(A)*poly(U) to understand the interaction specificity and energetics of binding. 2. Materials and Methods Polynucleotides poly(U) and poly(A).poly(U) required for the formation of the triplex were obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA) as sodium salts, and their nativeness was checked using absorption and circular dichroic spectroscopy. Concentrations of the polynucleotides were determined using molar extinction coefficients, ε (M-1 cm-1) reported in the literature.32 Alkaloids berberine, palmatine, and coralyne as chloride salts were obtained from Sigma-Aldrich and were used without further purification. All three alkaloids were fairly soluble in aqueous buffers, and hence their solutions were freshly prepared each day and kept protected in the dark until use. Molar extinction coefficients (ε) of the alkaloids used for determining their concentrations by absorbance measurements and other required optical properties were taken from the literature.49,50 No deviation from Beer’s law was observed in the concentration range employed in this study. All experiments were performed in 10 mM sodium cacodylate buffer containing 25 mM NaCl and 0.1 mM Na2EDTA, at pH 7.0 (total [Na+] ) 35 mM). Glass distilled deionized water and analytical grade reagents were used for the preparation of buffer. pH measurements were made on a Cyberscan 2100 high precision bench pH meter (Eutech Instruments Pte Ltd., Singapore) 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 pore size before use. The triple helix of poly(U).poly(A)*poly(U) was prepared by mixing poly(U) and poly(A).poly(U) in 1:1 molar ratio in sodium cacodylate buffer, heating to 95 °C on a peltier controlled heating device, and then cooling slowly at a programmed rate of 0.5 °C/min to 15 °C.45,47 This procedure allowed proper strand annealing. Formation of the triplex

berberine

palmatine

λmax (free) λmax (bound) λisob εf (at λmax) εb (at λmax)

Absorbance 344 345 348 348 356, 375, 440 355, 380, 440 22500 25000 13483 17715

λmax (excitation) λmax (emission)

350 517

Fluorescence 350 518

coralyne 421 424 433 14500 9710 425 470

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

structure was confirmed by its characteristic circular dichroic spectral pattern and biphasic optical melting profile, and these were in confirmity with the earlier reports.32,33 The base pairing scheme in the U.A*U triplex is depicted in Figure 1. Absorbance titrations of alkaloids with the triplex were performed on a Shimadzu Pharmaspec 1700 spectrophotometer (Shimadzu Corporation, Koyto, Japan) at 20 ( 0.5 °C using the methodology described in detail previously.51 For titration of berberine and palmatine matched quartz cells of 1 cm path length and for coralyne matched quartz cells of 10 cm path length were used. Briefly, a known concentration of the triplex solution was kept in the sample and reference cells and small aliquots of a known concentration of the alkaloid were titrated into the sample cell. After each addition, the solution was mixed and allowed to re-equilibrate for at least 5 min before recording the absorbance at the wavelength maximum (Amax) of the alkaloid and the isosbestic point (Aiso). The extinction coefficient of the alkaloid was determined at the isosbestic point (εiso), and the extinction coefficient of the bound alkaloid (εB) was obtained by addition of a known quantity to a large excess of triplex corresponding to the saturation point; εB ) Amax/lCt, where l is the path length, and Ct, the total drug concentration present, was calculated as Ct ) Aiso/lεiso. The values of Amax, Aiso, εiso, and εB etc. for each of the alkaloids are depicted in Table 1. These quantities were 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 observed absorbance was used to calculate the amount of bound drug, Cb ) (Aexp - Aobsd)/l(εf - εB). The amount of free alkaloid was determined by the difference Cf ) Ct - Cb. This Cf and the number of alkaloid molecules bound per mole of the nucleotide, r ) Cb/P, where P is the triplex concentration, were cast into Scatchard plots of r/Cf versus r. The binding isotherms with negative slopes at low r values were analyzed according to the excluded site model for the nonlinear noncooperative ligand binding system using the following equation of McGhee and von Hippel52

r/Cf ) Κi(1 - nr)[(1 - nr)/{1 - (n - 1)r}](n-1)

(1)

where Ki is the intrinsic binding constant to an isolated binding site, and n is the number of nucleotides excluded by the binding of a single alkaloid molecule. All the binding data were analyzed using Origin 7.0 software (Microcal Inc., Northampton, MA, USA) to determine the best-fit parameters of Ki and n as described in detail earlier.53 Absorbance versus temperature profiles (optical thermal melting profiles) of the triplex and triplex-alkaloid complexes

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were measured on the Shimadzu Pharmaspec 1700 unit equipped with a peltier-controlled TMSPC-8 model accessory (Shimadzu Corporation) as described earlier.54 In a typical experiment, the triplex sample (∼40 µM) was mixed with the varying concentration of the alkaloid under study in the desired degassed buffer into the eight-cell micro-optical cuvette 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 thermal melting temperature (Tm) was taken as the midpoint of the melting transition as determined by the maxima of the first derivative plot. Steady state fluorescence measurements were performed on a Shimadzu RF-5301PC specrofluorophotometer (Shimadzu Corporation) in a fluorescence free quartz cell of 1 cm path length thermoregulated at 20 ( 0.5 °C using an Eyela Uni Cool U55 water bath (Tokyo Rikakikai Co. Ltd. Tokyo, Japan) as described previously.53 The uncorrected emission spectra of the alkaloids were initially recorded and titrated with triplex solution of high concentration to prevent dilution effects. Fluorescence polarization anisotropy measurements of alkaloids and their complexes with the triplex were carried out as Larsson and colleagues55 described earlier.56 Steady state polarization anisotropy A is defined as

Α ) (Ιvv - ΙvhG)/(Ιvv + 2ΙvhG)

(2)

where Ivv, Ivh, Ihv, and Ihh represent the fluorescence signal for excitation and emission with the polarizer positions set at (0°, 0°), (0°, 90°), (90°, 0°), and (90°, 90°), respectively, and G is the ratio Ihv/Ihh used for instrumental correction. The continuous variation method of Job was employed to determine the binding stoichiometry in each case from fluorescence spectroscopy. At constant temperature, the fluorescence signal was recorded for solutions where the concentrations of both the triplex and the alkaloids 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 triplex was plotted as a function of the input mole fraction of each alkaloid. The break point in the resulting plot corresponds to the mole fraction of the bound alkaloid in the complex.ThestoichiometrywasobtainedintermsofRNA-alkaloid [(1 - χalkaloid)/χalkaloid ] where χalkaloid denotes mole fraction of the respective alkaloid. The results reported are an average of at least three experiments. Fluorescence quenching studies were carried out with the anionic quencher [Fe(CN6]4-. The quenching experiments were performed by mixing, in different ratios, two solutions, one containing KCl and the other containing K4[Fe(CN6], in addition to the normal buffer components, at a fixed total ionic strength. Fluorescence quenching experiments were performed at a constant P/D (RNA triplex/alkaloid molar ratio) monitoring fluorescence intensity as a function of changing concentration of the ferrocyanide as described previously.50 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(CN6]4- concentration according to the Stern-Volmer equation

Fo /F ) 1 + ΚSV[Q]

(3)

where Fo and F denote the fluorescence emission intensities in the absence and presence of the quencher and [Q] is the quencher concentration. KSV is the Stern-Volmer quenching

constant, which is a measure of the efficiency of quenching by the quencher. Energy transfer from the triplex to the bound alkaloid was measured from the excitation spectra of the triplex-alkaloid complex in the wavelength range 220-310 nm.25,57,58 Excitation spectra were recorded at an emission wavelength of 520 nm for berberine and palmatine and 470 nm for coralyne. The ratio Q ) qb/qf, where qb and qf are the quantum efficiencies of bound and free alkaloid, respectively, was calculated for each wavelength using the equation Q ) qb/qf ) Ibεf/Ifεb, where Ib and If are the fluorescence intensities of the alkaloids in the presence and absence of the triplex, respectively, and εb and εf are the corresponding alkaloid molar extinction coefficients. The ratio, Qλ/Q310, was then plotted against wavelength. The normalization wavelength of 310 nm was chosen because the triplex has very little absorbance at this wavelength. Circular dichroic (CD) spectra were acquired on a PC controlled spectropolarimeter JASCO J815 model (Jasco International Co. Ltd., Tokyo, Japan) equipped with a JASCO temperature programmer (model PFD 425 L/15) at 20 ( 0.5 °C as described previously.50 A rectangular strain-free quartz cell of 1 cm path length was used. Each spectrum was averaged from four successive accumulations at a scan rate of 100 nm/ min, keeping a bandwidth of 1.0 nm at a sensitivity of 100 millidegree, and was baseline corrected and smoothed within permissible limits using the inbuilt software of the unit and normalized to nucleotide concentration in the region of intrinsic CD (210-350 nm) of the triplex. The molar ellipticity (θ) is expressed in deg cm2 dmol-1. The CD unit was routinely calibrated using an aqueous solution of d-10 ammonium camphor sulfonate. The viscosity of the triplex-alkaloid complexes was determined by measuring the time needed for it to flow through a Cannon-Manning semi micro size 75 capillary viscometer (Canon Instruments Co., State College, PA, USA) that was kept submerged in a Canon thermostatted bath maintained at 20 ( 1.0 °C as described previously.51 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 triplex RNA either in the presence or absence of the alkaloids were calculated from the relation

η′sp /ηsp ) {(tcomplex - to)/to}/{(tcontrol - to)/to}

(4)

where η′sp and ηsp are the values of specific viscosity of the triplex in the presence and absence of the alkaloid and tcomplex, tcontrol, and to are the average flow times for the RNA-alkaloid complex, free triplex, and buffer, respectively. The relative increase in length, L/Lo, can be obtained from the corresponding increase in relative viscosity with the use of the following equation

L/Lo ) (η/ηo)1/3 ) 1 + βr

(5)

where L and Lo are the contour lengths of triplex in the presence and absence of the alkaloids; η and ηo are the corresponding values of intrinsic viscosity (approximated by the reduced viscosity η ) ηsp/C, where C is the triplex concentration which is kept constant throughout the experiment); and r is the number of alkaloid molecules bound per mole of the nucleotide. β is the slope when L/Lo is plotted against r and a β value of ∼2 may suggest true intercalative binding.

Isoquinoline Alkaloids with an RNA Triplex All isothermal titration calorimetry (ITC) experiments were performed using a MicroCal VP-ITC unit (MicroCal, Inc.; Northampton, MA, USA) at 20 °C using protocols developed in our laboratory and described previously in detail.46,50,53 Briefly, 10 µL aliquots of triplex RNA solution were injected from a 299 µL rotating syringe (290 rpm) into the isothermal sample chamber containing 1.4235 mL of the alkaloid solution (5 µM of berberine/palmatine or 10 µM of coralyne). Such a reverse titration with the triplex in the syringe was adopted as coralyne has a high tendency to aggregate even in very dilute solutions.59 The reverse titration protocol enabled us to keep a low concentration of the alkaloid in the calorimeter cell and still obtain good heat exchange on binding. Such protocols have been previously standardized in our laboratory for characterizing the binding of coralyne to tRNA and double-stranded RNAs.46,50,53 Corresponding control experiments to determine the heat of dilution of the RNA triplex to buffer were performed by injecting identical volumes of the same concentration of the RNA into buffer. Each injection generated a heat burst curve (microcalories per second versus time). The area under each peak was determined by integration using Origin 7.0 software to give a measure of 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 solutions 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 of one site binding site and analyzed using Origin 7.0 software to estimate the binding affinity (Kb), the binding stoichiometry (N), and the enthalpy of binding (∆H). The binding free energy and the entropic contribution to the binding were subsequently calculated from standard relationships described earlier.51,53 3. Results and Discussion 3.1. Formation of Triple Helical RNA. The RNA triplex, poly(U).poly(A)*poly(U), was prepared by the method described earlier32 (vide supra). The molar extinction coefficient value of the U.A*U triplex at the λmax of 258 nm was found to be 5900 M-1 cm-1. The formation of the triplex was confirmed from its melting profile and CD spectral pattern32,33 (Figure 2). The optical melting profile of the triplex showed a biphasic transition, the first transition representing the displacement of the Hoogsteen base-paired third strand from the triplex and the second transition representing the duplex denaturation to the singlestranded structures (Figure 2A). The first Tm (Tm1) was at 35 °C followed by the second Tm (Tm2) at 45 °C. The characteristic biphasic melting transition in this triplex with the second transition temperature corresponding to that observed for the melting of the parent duplex poly(A).poly(U) (not shown) clearly indicated the formation of a stable triple helical structure, and these were in conformity with the earlier reports.32,33 The intrinsic CD spectral pattern of the triplex (Figure 2B) was found to be significantly different from the duplex spectra (not shown) with lower ellipticity values for the peaks of the triplex32 again confirming the formation of the triplex. 3.2. Binding Aspects of Alkaloids to Triplex RNA. Progressive addition of increasing concentrations of the triplex to each of the alkaloid solutions effected pronounced hypochromic and bathochromic effects in the visible region absorbance spectra of the alkaloids (Figure 3) that essentially indicated strong intermolecular interaction involving effective overlap of the π electron cloud of the alkaloids with the base triplets that is speculative of intercalative complexation. Additionally, polarity

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Figure 2. Characteristic thermal melting profile (A) and circular dichroic spectrum (B) of poly(U).poly(A)*poly(U) in 10 mM sodium cacodylate buffer, pH 7.0. The concentration of triplex was 43.0 and 30.0 µM, respectively.

Figure 3. Representative absorption spectral changes of alkaloids in the presence of poly(U).poly(A)*poly(U) in 10 mM sodium cacodylate buffer at 20 °C. (A) Curves 1-10 denote the absorption spectrum of berberine (6.0 µM) treated with 0, 4.4, 10.2, 15.6, 20.4, 36.0, 60.0, 90.0, 150.0, and 240 µM of triplex, respectively. (B) Curves 1-10 denote the absorption spectrum of palmatine (5.0 µM) treated with 0, 3.5, 10.0, 16.5, 25.5, 40.0, 75.0, 125.0, 150.0, and 250.0 µM of triplex, respectively. (C) Curves 1-11 denote the absorption spectrum of coralyne (0.35 µM) treated with 0, 0.19, 0.38, 0.56, 0.74, 0.93, 1.11, 1.30, 1.48, 1.67, and 1.8 µM of triplex, respectively.

effects of the triplex and electron transfer from the base triplets to a minor extent may also contribute to the spectral changes. The presence of distinct isosbestic points at 356, 375, and 440 nm for berberine (Figure 3A), 355, 380, and 440 nm for palmatine (Figure 3B), and 433 nm for coralyne (Figure 3C), respectively, in the absorption spectra of alkaloid-triplex complexes revealed the existence of equilibrium between the free and bound form of the alkaloids. As described earlier,53 such titrations were performed in each case to determine the wavelength maxima, isosbestic point, and molar extinction coefficient of fully bound alkaloid, and these data are presented in Table 1. The results of absorption titration of increasing concentration of the alkaloid to a fixed concentration of triplex RNA were expressed in the form of Scatchard plots of r/Cf versus r, and these were found to be nonlinear in all the cases. In each case, a negative slope was observed at low values of bound alkaloid, and hence the plots were analyzed for noncooperative binding using the McGhee-von Hippel eq 1.52,60 The binding isotherms of the alkaloid-triplex complexations are

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Figure 4. Representative Scatchard plots of complexation of poly(U).poly(A)*poly(U) with (A) berberine (2), (B) palmatine (9), and (C) coralyne (b) obtained from spectrophotometric titration data.

TABLE 2: Binding Parameters for the Poly(U).Poly(A)*Poly(U)-Alkaloid Complexation Obtained from Scatchard Analysis of the Spectrophotometric Titration Data at 20 °Ca absorbance -1

fluorescence

alkaloids

Ki ( × 10 M )

n

Ki ( × 105 M-1)

n

berberine palmatine coralyne

1.6 ( 0.4 8.0 ( 0.3 40.0 ( 6.0

7.7 4.4 3.0

2.8 ( 0.5 7.2 ( 0.6 67.2 ( 11.2

7.0 5.0 3.5

5

a

Average of four determinations in each case. Ki is the intrinsic binding constant to an isolated binding site. n represents the number of excluded sites.

illustrated in Figure 4. The quantitative data of binding parameters obtained for the three alkaloids studied are presented in Table 2. The binding affinity values are (1.6 ( 0.4 × 105 M-1), (8.0 ( 0.3 × 105 M-1), and (4.0 ( 0.1 × 106 M-1), respectively, for berberine, palmatine, and coralyne. Thus, the binding affinity is remarkably higher for coralyne-triplex interaction compared to the corresponding values of palmatine and berberine revealing a higher affinity of coralyne with the triplex. The affinity of the alkaloids varied in the order coralyne > palmatine > berberine. Further, the numbers of excluded sites are 7.7, 4.0, and 3.0 for berberine, palmatine, and coralyne, and much lower values of n on binding of coralyne to the triplex in comparison to the other two alkaloids suggest closer binding sites for coralyne-triplex interaction indicating stronger association of coralyne to the triplex compared to the other two alkaloids. Further, the binding constant values of triplex-alkaloid interaction obtained here are found to be remarkably higher (about 10 times) in comparison to the values with the parent poly(A).poly(U) duplex-alkaloid interaction.50 Furthermore, the

Figure 5. Thermal melting profiles of poly(U).poly(A)*poly(U) (40.0 µM) (b-b) and its complexation with (A) berberine at a D/P ratio of 0.05 (O-O), 0.10 (2-2), 0.20 (∆-∆), and 0.40 (9-9); (B) palmatine at D/P ratio of 0.05 (O-O), 0.10 (2-2), 0.20 (∆-∆), 0.30 (9-9), 0.40 (0-0), and 0.50 ((-(); and (C) coralyne at D/P ratio of 0.02 (O-O), 0.03 (2-2), 0.06 (∆-∆), and 0.10 (9-9).

alkaloids bind the triplex in a noncooperative manner against the cooperative binding mode observed with the poly(A).poly(U) duplex.50 The thermal melting experiment is an important tool to investigate the interaction of small molecules to nucleic acid duplexes and triplexes. The stacking interactions of intercalated molecules as well as the neutralization of the phosphate charges through external binding together may contribute to the enhancement of the melting temperature. In particular, with triplexes, the specificity of binding of the small molecule to the Hoogsteen base-paired third strand or to the Watson-Crick base-paired duplex can be very clearly understood. It was observed that the melting profile of the triplex was identical for a heating rate of either 0.5 or 1.0 °C/min, indicating that enough time was allowed for thermal equilibrium. To examine the effect of the alkaloids on the stability of the triplex, the thermal denaturation in the presence and absence of the alkaloids was studied. The thermal melting profiles of the poly(U).poly(A)*poly(U) triplex and its complexes with various D/P (drug/nucleotide phosphate molar ratio) values of the alkaloids berberine, palmatine, and coralyne are presented in Figure 5. The quantitative data on the melting temperature of the triplex and complexes are presented in Table 3. The results show that all the three alkaloids enhanced the triplex dissociation temperature, but the extent of stabilization was more pronounced with coralyne (Figure 5C) with a ∆Tm1 ∼ 12.4 °C compared to berberine (Figure 5A) that showed a ∆Tm1 ∼ 10.2 °C and palmatine (Figure 5B) where the ∆Tm1 was ∼ 11 °C. Significantly, there was practically no effect for the alkaloids whatsoever on the duplex denaturation temperature (Tm2) as very



TABLE 3: Optical Melting Temperatures of Poly(U).Poly(A)*Poly(U) and Poly(U).Poly(A)*Poly(U)-Alkaloid Complexesa triplex/complex

D/P

[Na+] (mM)

Tm1 (°C) 3 f 2

Tm2 (°C) 2 f 1

∆Tm1 (°C) 3 f 2

∆Tm2 (°C) 2 f 1

poly(U).poly(A)*poly(U) poly(U).poly(A)*poly(U) + berberine

0 0.05 0.10 0.20 0.40 0.05 0.10 0.20 0.30 0.40 0.50 0.015 0.030 0.060 0.100

35 35 35 35 35 35 35 35 35 35 35 35 35 35 35

35.1 37.5 39.1 41.8 45.3 35.3 36.2 37.6 38.9 41.8 46.0 37.4 40.8 43.7 47.5

45.1 45.2 45.2 45.3 45.3 45.0 45.1 45.1 45.3 45.8 46.0 45.2 45.9 45.9 46.0

2.4 4.0 6.7 10.2 0.3 1.2 2.6 3.9 6.8 11.0 2.3 5.7 8.6 12.4

0.1 0.1 0.2 0.2 0.0 0.1 0.1 0.3 0.8 1.0 0.1 0.8 0.8 0.9

poly(U).poly(A)*poly(U) + palmatine

poly(U).poly(A)*poly(U) + coralyne

a Average from three experiments. Error limits for individual measurements are estimated at (0.5 °C in Tm. Tm1 (3 f 2) and Tm2 (2f1) correspond to triplex to duplex and duplex to single strand transitions, respectively. ∆Tm ) [Tm of triplex-alkaloid complex - Tm of triplex].

Figure 6. Representative fluorescence emission spectra of alkaloids in the presence of poly(U).poly(A)*poly(U) at 20 °C in 10 mM sodium cacodylate buffer. (A) Curves 1-11 denote the fluorescence spectrum of berberine (1.99 µM) treated with 0, 2.98, 5.97, 9.95, 17.91, 29.85, 49.75, 69.65, 89.55, 108.5, and 128.4 µM of triplex, respectively. (B) Curves 1-11 denote the fluorescence spectrum of palmatine (2.0 µM) treated with 0, 7.0, 12.0, 14.0, 24.0, 40.0, 60.0, 80.0, 90.0, 100.0, and 110.0 µM of triplex, respectively. (C) Curves 1-9 denote the fluorescence spectrum of coralyne (0.1 µM) treated with 0, 0.13, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, and 1.75 µM of triplex, respectively.

marginal change was observed on the second thermal transition (∆Tm2 ) 0.1-1 °C) (Table 3). In the case of berberine, saturation was reached at a D/P ratio of ∼0.4 and for palmatine at a D/P ratio of ∼0.5, while for coralyne the saturation was achieved at a much lower D/P ratio of 0.1. These results suggest specific stabilization of the triplex structure by all three alkaloids in general and a stronger stabilization of the triplex by coralyne in particular at a much lower input ratio in comparison to berberine and palmatine. This behavior is opposite to what has been reported in the case of the binding of ethidium where it was shown to have a destabilizing effect on the triple helical structure61,62 and a stabilizing effect on the duplex.36 Berberine and palmatine are weak fluorescent molecules, while coralyne is a very strong fluorophore. Berberine and palmatine have emission spectra in the 450-650 nm range when excited at 350 nm, whereas coralyne has an intense fluorescence maximum at 470 nm when excited at 425 nm. Binding to duplex DNAs and RNAs is known to remarkably enhance the fluorescence intensity of berberine and palmatine and quench the fluorescence of coralyne.63 Binding to the triplex (Figure 6) also resulted in an enhancement of the fluorescence of the complexed berberine and palmatine several fold and quenching of the fluorescence of coralyne eventually leading to saturation. The extent of change of fluorescence intensity on complexation with triplex was similar for both berberine and palmatine but more pronounced in the case of coralyne. A large fluorescence change, in each case, is indicative of strong association of these

molecules to the triplex resulting presumably from an effective overlap of the bound molecules with the base triplets. This result also proposes the location of the bound molecules in a hydrophobic environment similar to an intercalated state. The results of fluorescence titration data were also converted to Scatchard plots and analyzed according to an excluded site model for a noncooperative binding phenomenon. The values of Ki and n obtained from the analysis spectrofluorimetric data are also presented in Table 2. The values are similar to the data obtained from the spectrophotometric results. To establish the binding stoichiometry of these isoquinoline alkaloids with the RNA triplex, the continuous variation analysis procedure (Job plot) was performed in fluorescence. The Job’s plot obtained in each case is depicted in Figure 7. The plots of the difference fluorescence intensity versus alkaloid mole fraction revealed single binding in each case. The interaction of least-squares fitted lines at χ ) 0.165, 0.200, and 0.300, respectively, for berberine, palmatine, and coralyne corresponds to a site size of 5.1, 4.0, and 2.3 for one molecule of berberine, palmatine, and coralyne, respectively, which are close to the values obtained from spectrophotometric titration data. 3.3. Conformational Aspects of the Binding. Conformational changes of the triplex RNA on binding of the alkaloids was investigated from intrinsic circular dichroic studies. The intrinsic CD spectra of the U.A*U triplex showed a large positive band in the 250-285 nm region and an adjacent weak negative band in the wavelength range 230-250 followed by a small

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Figure 8. Circular dichroic spectra of poly(U).poly(A)*poly(U) (30.0 µM) treated with (A) 0.0, 3.0, 6.0, 9.0, 13.52, 18.09, and 24.0 µM of berberine (curves 1-7), (B) 0.0, 1.65, 3.28, 6.53, 9.75, 12.94, 19.22, and 25.38 µM of palmatine (curves 1-8), and (C) 0.0, 1.31, 2.60, 4.53, 5.78, 7.37, 8.93, 10.48, and 12.01 µM of coralyne (curves 1-9) in 10 mM sodium cacodylate buffer at 20 °C. Figure 7. Job plot for the binding of (A) berberine, (B) palmatine, and (C) coralyne to poly(U).poly(A)*poly(U) in 10 mM sodium cacodylate buffer at 20 °C.

positive band below the 230 nm region. These bands in triplex CD are caused most likely due to stacking interactions between the base triplets and the helical structure of the triplex strands and are in confirmity with the literature data.33 On the other hand, the alkaloids under investigation are achiral and do not have any intrinsic optical activity. To record the alkaloid induced changes in the conformation of the triplex, the CD spectra in the 210-400 nm region were recorded in the presence of varying D/P values. In the presence of these alkaloids, the conformation of the triplex was found to be perturbed (Figure 8). As the interaction progressed the characteristic positive band in the 250-285 nm region showed a red shift and enhancement in ellipticity for berberine (Figure 8A) and palmatine (Figure 8B), whereas a decrease of the same occurred with coralyne (Figure 8C) to reach saturation. This change in CD spectral pattern of the triplex was found to be most pronounced in the coralyne complex in comparison to the other two alkaloids. In all cases, there was an isoelliptic point that further indicated that the structural changes are independent. For berberine, however, no induced CD was observed, but there was the development of a weak positive induced CD band for palmatine and a strong induced CD for coralyne in the 300-400 nm region suggesting stronger interaction by coralyne compared to ber-

Figure 9. Variation of the relative fluorescence quantum yield of alkaloid berberine (2-2), palmatine (9-9), and coralyne (b-b) in the presence of poly(U).poly(A)*poly(U) in 10 mM sodium cacodylate buffer at 20 °C as a function of excitation wavelength.

berine and palmatine in consistence with that observed in spectroscopic binding and thermal melting studies. 3.4. Mode of Binding. Fluorescence energy transfer from the nucleotide to bound drugs, manifested by an increase in the fluorescence quantum yield of bound drugs in the wavelength range corresponding to DNA/RNA absorbance, can be used as evidence for intercalative binding25,54 since energy transfer can occur efficiently only if the bound drug is in close contact with, and oriented parallel to, the nucleotide base pairs/triplets. Figure 9 shows plots of the ratio Qλ/Q310 against wavelength for the



Figure 10. Stern-Volmer plots for the quenching of (A) berberine (∆) and the berberine-poly(U).poly(A)*poly(U) complex (2), (B) palmatine (0) and palmatine-U · A×U triplex complex (9), and (C) coralyne (O) and coralyne-U · A×U triplex complex (b) with increasing concentration of K4[FeCN6]. The concentration of the K+ ion was kept constant using KCl solution.

triplex at a D/P ratio of 0.1. The U.A*U triplex showed an increase in quantum yield in the region of RNA absorbance, but the increase in quantum yield was much higher for the coralyne-triplex complex in comparison to the other two alkaloids. This indicates that binding to the triplex form results in substantially higher energy transfer in case of the coralyne complex compared to berberine and palmatine complexes providing strong evidence for a true intercalative binding of coralyne, and partial intercalation of the other two alkaloids to the triplex. Fluorescence polarization anisotropy measurements also provide evidence for the binding of these three alkaloids with the triplex. It has been found that fluorescence polarization upon binding of berberine and palmatine to the triplex showed values of ∼0.16 and ∼0.17, respectively, and ∼0.23 for coralyne binding indicating coralyne to intercalate more strongly in comparison to the other two alkaloids.56 Another method of investigating the mode of binding is provided by fluorescence quenching experiments.64 In the complex, molecules that are free or bound on the surface of the triple helix are readily available to the quencher, while those that are intercalated between base triplets are not. The electrostatic barrier due to the negative charges on the phosphate groups at the helix surface limits the penetration of an anionic quencher into the interior of the helix. Hence, very little or no quenching should be observed in the presence of an anionic quencher, if the binding involves intercalation and consequently the mag-

Figure 11. Representative ITC profiles for the titration of poly(U).poly(A)*poly(U) with (A) berberine (2), (B) palmatine (9), and (C) coralyne (b), respectively, at 20 °C. The top panels represent the raw data for the sequential injection of triplex 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.

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TABLE 4: ITC Derived Thermodynamic Parameters For Binding of Berberine, Palmatine, and Coralyne to the Poly(U).Poly(A)*Poly(U) Triplex at 20 °Ca parameters -1

Ka × 10 (M ) n ∆G° (kcal mol-1) ∆H° (kcal mol-1) T∆S° (kcal mol-1) 5

berberine

palmatine

coralyne

3.5 ( 0.2 5.0 -7.44 -5.42 ( 0.24 2.02

6.8 ( 0.7 4.9 -7.82 -3.93 ( 0.14 3.89

75.9 ( 12.2 -9.28 -1.84 ( 0.05 7.44

a

All the data in this table are derived from ITC experiments and are an average of four determinations. Ka and ∆Ho values were determined from ITC profiles fitting to Origin 7 software as described in the text. The values of ∆Go and T∆So were determined using the equations ∆Go ) -RT ln Ka and ∆Go ) ∆Ho - T∆So. All the ITC profiles were fit to a model of single binding site.

nitude of the Stern-Volmer quenching constant (Ksv) of ligands that are bound inside will be lower than that of the free molecules. Figure 10 presents the effect of an anionic quencher [Fe(CN)6]4- on the fluorescence intensity of the alkaloids in the presence of the triplex represented by the Stern-Volmer plots. In Figure 10A and 10B, it is shown that binding to the triplex resulted in somewhat decreased quenching of the fluorescence intensity of berberine and palmatine. Ksv values for free berberine and its complex with the triplex were 211.77 and 113.62 L mol-1, respectively, and the same for palmatine were 210.51 and 98.94 L mol-1 which indicate that binding of these two alkaloids to the triplex decreases to some extent the accessibility of the quencher to the bound alkaloid molecules suggesting partial intercalation of berberine and palmatine inside the base triplets of the helix. Again, from Figure 10C it can be seen that while free coralyne is quenched substantially the triplex bound ones almost eliminate any sign of quenching. The Ksv value of free coralyne with [Fe(CN)6]4- was 201.68 L mol-1, while that of the bound one was 34.86 L mol-1, which demonstrates that the bound coralyne molecules are considerably protected and sequestered away from the solvent suggesting intercalative binding inside the triple helical structure. The viscometric technique is a well-established and reliable hydrodynamic method for investigating the extension of the DNA/RNA helix associated with intercalation. The original hypothesis of Lerman65 proposes that viscosity of a rodlike nucleic acid should increase upon complexation with an intercalator. 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. Hence, to further probe the binding mode of the alkaloids to the U.A*U triplex, the viscosity of the triplex solution was measured in the presence of increasing concentrations of the alkaloids, and the changes in relative viscosities with varying D/P ratio of the alkaloids were estimated (not shown). The change was found to be more rapidly pronounced for the coralyne complex compared to the other two alkaloid complexes, and the saturation was achieved at a much lower D/P ratio for coralyne again indicating tighter intercalative binding of this alkaloid. Viscosity results are expressed as length enhancement estimated with respect to a standard value (β) of 2 for a true intercalator corresponding to a length enhancement of 0.34 nm. The β values for berberine, palmatine, and coralyne binding to the U.A*U triplex were 0.84, 0.93, and 1.97, respectively. Thus, a true intercalation scenario may be envisaged for coralyne binding to the triplex, whereas partial intercalation may be assigned to berberine and palmatine binding. Berberine and palmatine have partial saturation in the ring structure resulting in tilted structure. It is likely that the buckled structure of these molecules is preventing a true intercalation into the triplex structure, whereas the planar structure of coralyne facilitates true intercalation. Similar

suggestions have been proposed for binding of these alkaloids to duplex DNA and RNA.50,66 3.5. Energetics of the Interaction. Isothermal titration calorimetry has become an effective and versatile tool for direct and reliable measurement of the thermodynamic parameters of the interaction of small molecules to nucleic acids.67 Thermodynamic characterization of the binding may offer key insights into the molecular forces that drive complex formation. Since ITC measures heat exchange, it is independent of the spectroscopic changes that occur in the reaction. Further, ITC has the great advantage in that it provides a complete thermodynamic profile for the binding such as Gibbs free energy change (∆G°), enthalpy change (∆H°), and entropy change (∆S°) along with the affinity constant (Ka) and the number of binding sites (N). The ITC profiles for the binding of berberine, palmatine, and coralyne to the U.A*U triplex are presented in Figure 11. All the profiles were monophasic and 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 triplex RNA into the alkaloid solutions that was corrected by corresponding dilution heats derived from the titration of identical amounts of RNA triplex into buffer alone. The resulting corrected heat plotted as a function of molar ratio is depicted in the lower panel of the figure. The data points reflect experimental injection points, and the solid lines reflect the calculated fits of the data. In all cases, the data were fitted to a single set of identical site models that yielded a fairly reasonable fitting of the experimental data. The thermodynamic parameters for the alkaloid binding to the triplex elucidated are depicted in Table 4. The ITC data for the binding of berberine to the triplex yielded an association constant of (3.5 ( 0.2) × 105 (M-1) and a binding site size (1/N) of 5.0. The binding of palmatine yielded a higher association constant of (6.8 ( 0.7) × 105 (M-1) and a binding site size of 4.9 nucleotides. On the other hand, binding of coralyne was markedly stronger compared to berberine and palmatine with a Ka value of (7.6 ( 1.2) × 106 (M-1) and a much lower binding site size that suggests almost 10 times stronger binding of coralyne in comparison to the other two alkaloids in conformity with the spectroscopic results. The free energy change (∆G°) was found to be similar for berberine and palmatine at ∼ -7.0 kcal/mol but higher for coralyne ∼ -9.0 kcal/mol showing the spontaneity of the interaction in all cases. Again, in all cases, negative enthalpy changes showed that the interaction was exothermic in nature. The binding of berberine to the triplex was driven by predominantly more change in enthalpy (-5.42 ( 0.24 kcal/mol) since here entropy change was relatively small (2.02 kcal/mol). This is in agreement with that previously reported by the vant Hoff analysis.33 On the other hand, for palmatine, enthalpy and entropy contributions were found to be similar ∼3.9 kcal/mol which suggests the interaction to be both enthalpy and entropy driven. For coralyne interaction, the binding was found to be

Isoquinoline Alkaloids with an RNA Triplex driven by a larger positive entropy term (7.44 kcal/mol), with a smaller enthalpy change (-1.84 ( 0.05 kcal/mol). It is pertinent to observe that the binding was found to be enthalpy driven in berberine, favored by both enthalpy and entropy changes in palmatine and entropy driven with coralyne. Previous studies on the interaction of these alkaloids to poly(A).poly(U) suggested that binding of berberine and palmatine to the RNA duplex was favored by a larger favorable entropy change, whereas in the case with coralyne, the binding was driven by a stronger enthalpy term and a smaller positive entropy term.50 Thus, these thermodynamic parameters clearly reflect the differences in the mode of interaction of these alkaloids with the RNA triplex and also show that the nature of interaction here is completely different from the mode of interaction of these alkaloids with the RNA duplex poly(A).poly(U).50 4. Conclusions This study presents the comparative binding and thermodynamics of three isoquinoline alkaloids, berberine, palmatine, and coralyne, to the RNA triplex, poly(U).poly(A)*poly(U), using a variety of biophysical and calorimetric techniques. The results of this study reveal that the alkaloids berberine and palmatine bind to the triplex by partial intercalation, while coralyne binds by true intercalation. Strong binding of all three alkaloids to the triplex was revealed from hypochromic and bathochromic effects and was noncooperative in nature as revealed from Scatchard plots. This is in contrast to the cooperative binding profiles observed previously for these alkaloids to the corresponding duplex, poly(A).poly(U). Berberine, palmatine, and coralyne stabilized the Hoogsteen base-paired third strand without affecting the stability of the Watson-Crick base paired duplex, unequivocally revealing the specificity to the triplex, being higher for coralyne at low input ratios followed by berberine and palmatine. The binding was accompanied by significant conformational changes and induction of strong optical activity in coralyne and a weak one in palmatine indicating a stronger location of coralyne in the intercalated geometry which was corroborated further from fluorescence quenching studies and energy transfer data. Further, the fluorescence of the bound alkaloid molecules was significantly polarized being higher in coralyne, and all these results together with supporting data from hydrodynamic studies in terms of length enhancement clearly propose a more favored intercalative geometry for the bound coralyne molecules compared to the partially intercalated berberine and palmatine on the triple helical RNA. Energetics of the interaction revealed that binding was favored by predominantly large negative enthalpy with small favorable entropy in berberine, favored by negative enthalpy and entropy in palmatine, and driven by predominant entropy changes in coralyne. These differences signify the importance of small structural differences and also the planarity, coralyne being planar compared to buckled berberine and palmatine in the interaction profile. All the data unequivocally suggest a stronger and tighter binding of coralyne and are similar to the trend with the parent duplex RNA. These results further advance our knowledge on the interaction of alkaloid molecules with triple helical structures and may be useful in developing RNA targeted therapeutics. Acknowledgment. Dr. Rangana Sinha is grateful to the Indian Council of Medical Research (ICMR), Government of India, for the award of Research Associateship. This work was supported by grants from the network project on “ComparatiVe genomics and biology of noncoding RNA in the human genome”

J. Phys. Chem. B, Vol. 113, No. 40, 2009 13419 (NWP0036) of the Council of Scientific and Industrial Research (CSIR), Government of India. The authors are grateful to Prof. Siddhartha Roy, Director, Indian Institute of Chemical Biology, for his patronage and all the colleagues of the Biophysical Chemistry Laboratory for their help, support, and cooperation at every stage of work. We also thank the reviewers for their comments that enabled us to improve upon the manuscript. References and Notes (1) Felsenfeld, G.; Davies, D. R.; Rich, A. J. Am. Chem. Soc. 1957, 79, 23. (2) Stevens, C. L.; Felsenfeld, G. Biopolymers 1964, 2, 293. (3) Chen, G. S.; Bhagwat, B. V.; Liao, P. Y.; Chen, H. T.; Lin, S. B.; Chern, J. W. Bioorg. Med. Chem. Lett. 2007, 17, 1769. (4) Radhakrishnan, I.; Patel, D. J. Biochemistry 1994, 33, 11405. (5) Murphy, D.; Eritza, R.; Redmond, G. Nucleic Acids Res. 2004, 32, e65. (6) Mendez, B. E.; Leumann, J. C. J. Biol. Chem. 2001, 276, 35320. (7) Leitner, D.; Schroder, W.; Weisz, K. Biochemistry 2000, 39, 5886. (8) Sugimoto, N.; Wu, P.; Hara, H.; Kawamoto, Y. Biochemistry 2001, 40, 9396. (9) Uhlmans, E.; Peymann, A. Chem. ReV. 1990, 90, 543. (10) Fox, K. R.; Dardy, R. A. J. p-360. Small molecule DNA and RNA binders; Demeunynck, M., Bailly, C., Wilson, W. D., Demeunynck, M., Eds.; Wiley VCH pub.: New York, 2004. (11) Zain, R.; Sun, J. S. Cell. Mol. Life Sci. 2003, 60, 862. (12) Frank-Kamenetskii, M. D.; Mirkin, S. M. Annu. ReV. Biochem. 1995, 64, 65. (13) Guleysee, A. L.; Praseuth, D.; Grigoriev, M.; Bellan, A. H.; Helene, C. Nucleic Acids Res. 1996, 24, 4210. (14) Mills, M.; Arimondo, P. B.; Lacrix, L.; Garestier, T.; Klump, H.; Mergny, J. L. Biochemistry 2002, 41, 357. (15) Gaffney, B. L.; Kung, P.- P.; Wang, C.; Jones, R. A. J. Am. Chem. Soc. 1995, 117, 12281. (16) Rana, V. S.; Ganesh, K. N. Nucleic Acid Res. 2000, 28, 1162. (17) Dagneaux, C.; Gousset, A. K.; Shchyolkina, A. K.; Ouali, M.; Letellier, L. J.; Florentiev, V. L.; Taillander, E. Nucleic Acids Res. 1996, 24, 4506. (18) Sun, J. S.; Helene, C. Curr. Opin. Struct. Biol. 1993, 3, 345. (19) Gianolio, D. A.; McLaughlin, L. W. Bioorg. Med. Chem. 2001, 9, 2329. (20) Kallanthottathil, R. G.; Vasant, J. R.; Ganesh, K. N. Nucleic Acids Res. 1997, 25, 4187. (21) Sollogoub, M.; Darby, R. A.; Cuenoud, B; Brown, T; Fox, K. R. Biochemistry 2002, 41, 7224. (22) Barawkar, D. A.; Rajeev, R. J.; Kumar, V. A.; Ganesh, K. N. Nucleic Acids Res. 1996, 24, 1229. (23) Sandstorm, K.; Warmlander, S.; Bergman, J.; Engqvist, R.; Leijon, M.; Graslund, J. Mol. Recognit. 2004, 17, 277. (24) Choi, S.-D.; Kim, M. S.; Kim, S. K.; Lincoln, P.; Tuite, E.; Norden, B. Biochemistry 1997, 36, 214. (25) Scaria, P. V.; Shafer, R. H. J. Biol. Chem. 1991, 266, 5417. (26) Wilson, W. D.; Mizan, S.; Tanious, F. A.; Yao, S.; Zon, G. J. Mol. Recogn. 1994, 7, 89. (27) Moraru-Allen, A. A.; Cassidy, S.; Alvarez, L. A.; Fox, K. R.; Brown, T.; Lane, A. N. Nucleic Acid Res. 1997, 25, 1890. (28) Kim, H. K.; Kim, J. M.; Kim, S. K.; Rodger, A.; Norden, B. Biochemistry 1996, 35, 1187. (29) Arya, D. P.; Micovic, L.; Charles, I.; Coffee, R. L.; Willis, B.; Xue, L. J. Am. Chem. Soc. 2003, 125, 3733. (30) Cassidy, S. A.; Strekowski, L.; Fox, K. R. Nucleic Acids Res. 1996, 24, 4133. (31) Durand, M.; Seche, E.; Maurizot, J. C. Eur. Biophys. J. 2002, 30, 625. (32) Ray, A.; Suresh Kumar, G.; Das, S.; Maiti, M. Biochemistry 1999, 38, 6239. (33) Das, S.; Suresh Kumar, G.; Ray, A.; Maiti, M. J. Biomol. Struct. Dyn. 2003, 20, 703. (34) Pilch, D. S.; Kirolos, M. A.; Breslauer, K. J. Biochemistry 1995, 34, 16107. (35) Sorokin, V. A.; Valeev, V. A.; Gladchenko, G. O.; Degtiar, M. V.; Karachevtsev, V. A.; Blagoi, Y. P Int. J. Biol. Macromol. 2003, 31, 223. (36) Garcia, B.; Leal, J. M.; Paiotta, V.; Ibeas, S.; Ruiz, R.; Seco, F.; Venturi, M. J. Phys. Chem. B 2008, 110, 16131. (37) Garcia, B.; Leal, J. M.; Paiotta, V.; Ruiz, R.; Secco, F.; Venturini, M. J. Phys. Chem. B 2008, 112, 7132. (38) Cheng, A. C.; Calabro, V.; Franket, A. D. Curr. Opin. Struct. Biol. 2001, 11, 478. (39) Hermann, T. Biochimie 2002, 84, 869. (40) Vicens, Q.; Westhof, E. ChemBioChem 2003, 4, 1018.

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JP9069515

Interaction of Isoquinoline Alkaloids with an RNA Triplex: Structural

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