Journal of Photochemistry and Photobiology B: Biology 134 (2014) 64–74

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Interaction of 9-O-N-aryl/arylalkyl amino carbonyl methyl berberine analogs with single stranded ribonucleotides Anirban Basu a,b, Parasuraman Jaisankar a, Gopinatha Suresh Kumar a,b,⇑ a b

Chemistry Division, CSIR-Indian Institute of Chemical Biology, Kolkata 700 032, India Biophysical Chemistry Laboratory, CSIR-Indian Institute of Chemical Biology, Kolkata 700 032, India

a r t i c l e

i n f o

Article history: Received 18 September 2013 Received in revised form 27 January 2014 Accepted 31 March 2014 Available online 12 April 2014 Keywords: 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberines Single stranded RNA binding Intercalation Energetics

a b s t r a c t Studies on the molecular aspects of alkaloid–RNA complexation are of prime importance for the development of rational RNA targeted drug design strategies. Towards this goal, the binding aspects of three novel 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs to four single stranded ribonucleotides, poly(G), poly(I), poly(C) and poly(U), were studied for the first time employing multifaceted biophysical tools. Absorbance and fluorescence studies revealed that these analogs bound non-cooperatively to poly(G) and poly(I) with binding affinities remarkably higher than berberine. The binding of these analogs to poly(U) and poly(C) was weaker in comparison to poly(G) and poly(I) but were one order higher in comparison to berberine. Quantum efficiency values revealed that energy transfer occurred from the RNA bases to the analogs upon complexation. The binding was dominated by large positive entropic contributions and small but favorable enthalpic contributions. Salt dependent studies established that the binding was dominated by hydrophobic forces that contributed around 90% of the total standard molar Gibbs energy. The chain length of the substitution at the 9-position was found to be critical in modulating the binding affinities. These results provide new insights into the binding efficacy of these novel berberine analogs to single stranded RNA sequences. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Design and development of small molecules that can selectively bind to defined sites in RNA is an emerging field of activity in the area of RNA targeted drug design [1–3]. RNA molecules are polymorphic, and can exist in a variety of structural forms that can provide unique binding sites for small molecules [4]. Compounds that stabilize the RNA constructs might effectively block its activity by locking the nucleic acid substrate into an unfavorable conformation. Such molecules have the potential to serve as important therapeutic agents. This is owing to the fact that RNA molecules are now revealed to be involved in the processing and transmission of genetic information at the cellular levels [5]. RNA based therapeutics must be able to distinguish between the desired RNA targets from other cellular targets. This selection can be achieved by studying different RNA constructs and sequences that can mediate specific recognition of RNA structural elements.

⇑ Corresponding author at: Biophysical Chemistry Laboratory, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700 032, India. Tel.: +91 33 2472 4049/2499 5723; fax: +91 33 2472 3967. E-mail addresses: [email protected], [email protected] (G. Suresh Kumar). http://dx.doi.org/10.1016/j.jphotobiol.2014.03.024 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.

Isoquinoline alkaloids, of which berberine (Fig. 1) is the prominent member, constitute a class of compounds that have been studied extensively and are regarded to be potential lead compounds in cancer therapy [6–8]. Plants containing this alkaloid have been employed as folk medicine for centuries all over the world. Berberine exhibits a wide range of biological activities [6–16]. Berberine binds to a variety of nucleic acid structures and the biological activities of berberine are thought to be manifested through its interaction with different nucleic acids [17–20]. However, the binding affinity of berberine to many nucleic acid structures are modest and hence call for appropriate structural modifications to enhance the affinity to serve as an effective therapeutic agent. The 9-position of the isoquinoline chromophore of berberine has been shown to have the most remarkable influence on its biological activity and nucleic acid binding efficacy [21–29]. Keeping this in mind, a series of novel of 9-O-N-aryl/ arylalkyl amino carbonyl methyl substituted berberine analogs (Fig. 1) were synthesized. This 9-O-substitutent was expected to improve the nucleic acid binding efficacy of these analogs by offering additional contact points/better contact geometry with the nucleic acid bases. These analogs exhibited remarkably high binding affinities to double stranded DNA, single and double stranded poly(A) and tRNAphe [26–29]. These analogs also induced

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experiments were conducted in filtered 10 mM citrate–phosphate (CP) buffer, pH 7.0, prepared in deionised triple glass distilled water. 2.2. Spectroscopic measurements Spectrophotometric and fluorimetric measurements were made in a Jasco V660 double beam dual monochromator spectrophotometer (Jasco International Co., Hachioji, Japan) and a Shimadzu RF-5301 PC fluorimeter (Shimadzu Corporation, Kyoto, Japan). Titrations were performed keeping a constant concentration of the alkaloid solution and varying the RNA polynucleotide concentration in fluorescence free quartz cuvettes of 1 cm path length following generally the methods developed by Chaires and colleagues and described by us in details earlier [33,34]. The excitation wavelength for berberine and its analogs was 345 nm. An excitation and emission band pass of 5 nm was used for all fluorescence measurements. The sample temperature was maintained at 25 ± 1.0 °C using Eyela Uni Cool U55 water bath (Tokyo Rikakikai Co., Ltd., Japan). Uncorrected fluorescence spectra are reported. The data obtained from these titrations were utilized for constructing Scatchard and Benesi–Hildebrand plots. 2.3. Elucidation of the binding parameters Fig. 1. Chemical structure of berberine and its 9-O-substituted analogs.

self-structure in poly(A) with much greater ease compared to berberine [27]. Enhanced nucleic acid binding aspect prompted us to investigate their interaction with single stranded polyguanylic acid [poly(G)], polyinosinic acid [poly(I)], polyuridylic acid [poly(U)] and polycytidylic acid [poly(C)]. The biological importance of single stranded RNA’s has been well documented in the literature based on k phage DNA mapping studies which established that the expression of phage genome is remarkably influenced by k phage genome containing ten poly(G) binding sites [30]. Besides, the antiviral activity of poly(I) and poly(C) has also been revealed [31]. Interferon response in cell cultures can be invoked by poly(I) and poly(C) which is highly sensitive to pancreatic ribonuclease treatment [31]. Hence, in this manuscript we present a detailed study on the binding of three novel 9-O-substituted analogs of berberine to four single stranded RNA’s, poly(G), poly(I), poly(U) and poly(C), employing multifaceted biophysical tools. 2. Materials and methods 2.1. Materials Single stranded polyribonucleotides, poly(G), poly(I), poly(U) and poly(C) were obtained from Sigma–Aldrich Corporation (St. Louis, MO, USA) and were sonicated on a Labsonic Sonicator (B Braun, Germany) to a uniform size of about 280 ± 40 bases. The size was estimated from the molecualr weight through viscometry measurements [32]. Concentrations of these homoribopolynucleotides in terms of nucleotide phosphates were determined spectrophotometrically using reported molar extinction coefficients [20]. Berberine chloride was obtained from Sigma–Aldrich and was used without further purification. The analogs of berberine were synthesized, purified and characterized as reported earlier [26]. The concentrations of BER, BER1, BER2 and BER3 were determined by applying molar extinction coefficient values of 22,500, 25,000, 26,000, 26,500 M1 cm1, respectively. All buffer salts and other reagents were of analytical grade. The alkaloid and analog solutions were freshly prepared in the buffer and kept protected in the dark to prevent any light induced photochemical changes. All

Amounts of free and RNA bound alkaloid were obtained employing the following methodology described in details earlier [20,33]. Concentration of free alkaloid (Cf) was estimated from the relationship Ct = Cb + Cf. The binding ratio (r) is given by the relation, r = Cb/[RNA]total. Binding data obtained from spectrophotometric and spectrofluorimetric titrations were cast into Scatchard plots of r/Cf versus r. The Scatchard plots exhibited negative slopes at low r values indicating noncooperative binding isotherms. Hence, the plots were analyzed employing the following equation of McGhee and von Hippel [35]

r=C f ¼ K i ð1  nrÞ½ð1  nrÞ=f1  ðn  1Þrgðn1Þ

ð1Þ

where Ki is the intrinsic binding affinity to an isolated binding site and n is number of nucleotides excluded by the binding of a single alkaloid/analog molecule. The binding data were analyzed using the Origin 7.0 software (Microcal, Inc., Northampton, MA, USA) that determines the best-fit parameters of Ki and n to the above equation. The absorption spectral titration data in the case of poly(U) and poly(C) were analyzed by the Benesi–Hildebrand plot [36]. This method of analysis is applicable for the systems where the binding constants are small and equilibria can only be measured at low values of r, the ratio of bound alkaloid to total polynucleotide concentration. Under such circumstances, the apparent equilibrium constant (KBH) was obtained by plotting the variation of 1/DAbs against 1/Cs and taking the ratio of the intercept to the slope. Here, Cs represents the concentration of the polynucleotide and DAbs is the difference in absorbance of free and complexed alkaloid at kmax. 2.4. Quantum efficiency determination The quantum efficiency (Q) of a RNA polynucleotide bound alkaloid gives an estimate of the energy transferred from the polynucleotide to the alkaloid upon complexation. This is evaluated from the ratio of the quantum efficiency of the alkaloid bound to the nucleic acid (qb) to the quantum efficiency of the unbound alkaloid. Fluorescence quantum efficiency was estimated employing the following equation [37]

Q¼

qb Ib ef ¼  qf If eb

ð2Þ

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Here qb is the quantum efficiency of the alkaloid complexed to the RNA and qf is the quantum efficiency of the free alkaloid. ef and eb represent the molar extinction coefficients of the free and bound alkaloids and Ib and If are the fluorescence intensities of bound and free alkaloids, respectively. 2.5. Stoichiometry of the binding: Job plot analysis Continuous variation method of Job [19,38] was used to determine the binding stoichiometry in the case of poly(G) and poly(I) from fluorescence spectral results at 25 ± 1 °C. The stoichiometry was obtained in terms of RNA–alkaloid [(1valkaloid)/valkaloid], where, valkaloid denotes the mole fraction of the alkaloid in a plot of the difference in fluorescence intensity versus the mole fraction of the alkaloid. The results reported are average of at least three experiments. 2.6. Fluorescence quenching studies Fluorescence quenching studies were performed with the anionic quencher [Fe(CN)6]4. These experiments were carried out by mixing solutions of KCl and K4[Fe(CN)6] in different ratios at a fixed total ionic strength. Experiments were performed at a constant P/D (RNA/alkaloid molar ratio) monitoring the fluorescence intensities as a function of varying ferrocyanide ion concentration as described in details previously [18]. The data were cast into Stern–Volmer plots of relative fluorescence intensity (Fo/F) versus [Fe(CN)6]4 concentration,

F0 ¼ 1 þ K sv ½Q F

ð3Þ

where Fo and F denote the fluorescence emission intensities in the absence and in the presence of the quencher and [Q] is the quencher concentration. Ksv is the Stern–Volmer quenching constant, the magnitude of which provides an estimate of the efficiency of quenching by the quencher. 2.7. Circular dichroism studies Circular dichroism measurements were carried out on a Jasco J815 spectropolarimeter interfaced with a thermal programmer (Jasco model 425L/15) and controlled by a PC according to the procedure reported previously [17]. The CD scans were recorded in the wavelength range of 230–400 nm at a scan speed 100 nm/min and step size of 0.1 nm. The time constant was 2 s and the bandwidth was 1 nm. All measurements were carried out in a 1 cm path length cuvette. Fixed amount of the RNAs was titrated with increasing concentration of the analogs. Each reading was an average of four runs. Readings were noted 5 min after each addition to ensure complete complex formation. 2.8. Isothermal titration calorimetry All isothermal titration calorimetry (ITC) experiments were performed on a MicroCal VP-ITC unit (MicroCal, Inc.) using protocols developed in our laboratory and reported earlier [18,33]. All the ITC thermograms showed single binding event and hence were fitted to a one site binding model and analyzed using Origin 7.0 software to obtain the values of the binding affinity (Ka), the binding stoichiometry (N) and the standard molar enthalpy of binding (DHo). The standard molar Gibbs energy change (DGo) and the standard molar entropic contribution (TDSo) were subsequently deduced from standard relationships, DGo = RT ln Ka (R = 1.9872 cal mole1 K1, T = 298.15 K) and DGo = DHo–TDSo.

3. Results and discussion 3.1. Absorbance spectral studies The visible absorbance spectra of the berberine analogs underwent pronounced hypochromic and bathochromic shifts when mixed with increasing concentrations of the single stranded RNA sequences revealing strong intermolecular association. These kind of spectral changes are usually characteristic of intercalative binding, resulting from strong intimate interaction of the p electron cloud of the alkaloids and the RNA bases. The presence of three, well-defined, sharp isosbestic points in case of poly(G) and poly(I) enabled the assumption of a two state system. After verifying the existence of equilibrium, reverse spectrophotometric titrations were performed by incremental addition of aliquots of the alkaloid to a fixed concentration of the RNA samples, poly(G) and poly(I), observing the absorbance change at the wavelength maximum and the isosbestic point for calculating the binding parameters. However, for the other RNA sequences poly(U) and poly(C) the changes in the absorption spectra were significantly less pronounced and no clear isosbestic points were observed. Therefore, a Benesi–Hildebrand plot was constructed to evaluate the binding affinity values. A typical absorption spectral titration of analog BER1 with poly(G) and poly(C) polynucleotides is presented in Fig. 2. 3.2. Fluorescence studies The binding was further studied by fluorescence spectroscopy. Berberine and its analogs have inherently weak fluorescence with emission maxima in the range 450–455 nm when excited at 345 nm. However, in the presence of two polynucleotides, poly(G) and poly(I), a remarkable increase in the fluorescence was observed for all the analogs with concomitant development of a peak around 510–520 nm (Figs. 3 and S1 and S2). Again, such large enhancements may be interpreted in terms of an effective overlap of the electronic cloud of the bound alkaloids with that of the RNA bases. The ratio of the fluorescence intensity of the bound alkaloid to that of free alkaloid was found be 82.3, 4.3, 16.3 and 25.2, respectively, for the complexation of BER and its analogs BER1-3 with poly(G). Similarly, with poly(I) the ratios for the complexation of BER and its analogs were found to be 60.4, 6.1, 25.1 and 52.0, respectively. This observation, like that from the absorbance titration studies, also proposed the location of the bound alkaloid molecules to be in a hydrophobic environment similar to an intercalated state. In the case of the other two ribonucleotides, poly(U) and poly(C), only marginal alterations were observed in the peak in the 450–455 nm region upon complexation (Figs. 3 and S1 and S2). Besides, no peak developed in the 510–520 nm region upon addition of these two ribonucleic acids thereby indicating that the binding with these two RNA sequences is extremely weak. Hence, the binding affinity values could not be evaluated from fluorescence data for these two RNA sequences. Representative fluorescence spectral titration of analog BER2 with poly(G), poly(I), poly(U) and poly(C) RNA sequences are depicted in Fig. 3. 3.3. Elucidation of the binding parameters from spectroscopic experiments The binding parameters for the association of BER and its analogs with poly(G) and poly(I) ribonucleotides were estimated from Scatchard plots fitted to the McGhee-von Hippel equation for noncooperative binding [35]. The binding of BER was non-cooperative as evidenced by the negative slope at low r values in the Scatchard plot. This non-cooperative binding mode of BER was propagated in the analogs as well (negative slope at low r values). Therefore, the

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Fig. 2. Absorption spectral titration of analog (A) BER1 (4.88 lM, curve 1) with increasing concentration of poly(G) (curves 2–8) and (B) BER1 (4.56 lM, curve 1) with increasing concentration of poly(C) (curves 2–7). Inset: Plot of absorbance at 345 nm versus P/D values.

Fig. 3. Representative fluorescence spectral titration of analog BER2 (5 lM) treated with (A) 0, 50, 100, 200, 300, 400, 500, 550 lM poly(G) (curves 1–8), (B) 0, 100, 200, 400, 600, 800, 900, 1000 lM poly(I) (curves 1–8), (C) 0, 200, 400, 800, 1000 lM poly(U) (curves 1–5) and (D) 0, 200, 400, 800, 1000 lM poly(C) (curves 1–5).

Table 1 Binding parameters obtained from the analysis of the spectrophotometric and spectrofluorimetric data.a Single stranded RNA

Alkaloids

Spectrophotometry

Spectrofluorimetry

Ki  105 (M1)

n

Ki  105 (M1)

n

Poly(G)

BER BER1 BER2 BER3

2.24 ± 0.03 13.1 ± 0.03 10.7 ± 0.05 8.49 ± 0.05

8.54 ± 0.06 1.28 ± 0.05 1.41 ± 0.01 3.09 ± 0.03

2.19 ± 0.03 12.7 ± 0.03 10.4 ± 0.05 8.43 ± 0.05

8.39 ± 0.06 1.26 ± 0.05 1.45 ± 0.01 3.13 ± 0.03

Poly(I)

BER BER1 BER2 BER3

1.65 ± 0.02 10.8 ± 0.03 10.1 ± 0.02 7.07 ± 0.02

7.21 ± 0.01 3.03 ± 0.05 2.42 ± 0.01 1.62 ± 0.05

1.52 ± 0.02 10.9 ± 0.03 10.3 ± 0.02 7.24 ± 0.02

7.16 ± 0.01 3.02 ± 0.05 2.26 ± 0.01 1.71 ± 0.05

KBH  104 (M1)

n

KBH  104 (M1)

n

Poly(U)

BER BER1 BER2 BER3

0.18 ± 0.01 2.81 ± 0.04 1.70 ± 0.03 1.26 ± 0.02

nd nd nd nd

nd nd nd nd

nd nd nd nd

Poly(C)

BER BER1 BER2 BER3

0.03 ± 0.01 0.34 ± 0.02 0.31 ± 0.02 0.25 ± 0.02

nd nd nd nd

nd nd nd nd

nd nd nd nd

a All the experiments were performed in 10 mM CP buffer, pH 7.0 at 25 °C and the data in this table are average of four determinations. Ki and KBH are the intrinsic binding affinity constants derived from Scatchard and Benesi–Hildebrand analysis, respectively. n is equivalent to the number of RNA bases spanned by an alkaloid molecule. Uncertainties correspond to regression standard errors. nd is not determined.

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noncooperative binding mode of BER remained intact upon introduction of the aryl/arylalkyl amino carbonyl methyl substitution at the 9-position. The intrinsic binding affinity (Ki) values obtained from the Scatchard analysis of the spectrophotometric and spectrofluorimetric data are collated in Table 1. The binding affinity values are significantly higher for all the berberine analogs compared to BER and magnitude of the binding affinity varied as BER1 > BER2 > BER3 > BER. The binding affinity values of the analogs BER1-2 with poly(G) and poly(I) were of the order 106 M1 whereas for analogs BER3 and BER the values were of the order 105 M1. The affinity values obtained from the analysis of the spectrophotometric and spectrofluorimetric data were in excellent agreement with each other. The binding affinity values for all the analogs with poly(U) and poly(C) sequences were deduced from the Benesi–Hildebrand analysis of the spectrophotometric data. From this analysis, the affinity values for the complexation of the analogs BER1-3 with poly(U) and poly(C) ribonucleotides were found to be of the order of 104 and 103 M1, respectively, whereas for BER they were of the order 103 and 102 M1, respectively. Therefore, although weak in magnitude, the intrinsic binding affinity values were one order higher for the analogs compared to BER. The intrinsic binding affinity values were (1.31 ± 0.03)  106, (1.08 ± 0.03)  106, (2.81 ± 0.04)  104 and (3.4 ± 0.02)  103 M1, respectively, for the complexation of BER1 with sequences of poly(G), poly(I), poly(U) and poly(C).

3.4. Determination of quantum efficiency values The quantum efficiency (Q) of an alkaloid binding to the RNA polynucleotide gives a measure of the energy transferred from the RNA bases to the alkaloid upon complexation and is measured from the ratio of the quantum efficiency of alkaloid bound to the RNA (qb) to the quantum efficiency of the free alkaloid (qf). Determination of quantum efficiency values further testifies the strong binding of the analogs to poly(G) and poly(I) sequences. A plot of DAbsorbance against the inverse of the RNA concentration gave an exponential plot (not shown) from which the quantum efficiency values were determined. For the complexation of poly(G) with analogs BER1-3 the Q values were calculated to be 5.75, 24.04 and 39.57, respectively. Similarly, for the association of the analogs with poly(I) the Q values were found to be 7.67, 36.57 and 58.58, respectively. It can be observed that Q is >1.0 in each case and this denotes that all the analogs exhibit greater fluorescence when bound to the nucleic acid compared to when free in solution. Besides, Q > 1.0 also indicates greater retention of fluorescence energy by the bound alkaloids due to shielding within the RNA binding site from quenching by the solvent molecules.

3.5. Binding stoichiometry (Job Plot) To establish the binding stoichiometry of the analogs to poly(G) and poly(I) sequences, continuous variation analysis (Job plot) was performed in fluorescence. The stoichiometry of binding was determined by the molar ratio where maximal binding was observed. The Job plots of the difference in fluorescence intensity versus the mole fraction of BER and its analogs revealed exclusively a single binding mode in each case. From the inflection points, valkaloid = 0.112, 0.446, 0.406 and 0.240, the number of bases of poly(G) bound per BER and its analogs BER1-3 was estimated to be around 7.93, 1.24, 1.46 and 3.17, respectively. Again, from the inflection point, valkaloid was found to be 0.122, 0.246, 0.284 and 0.346, respectively, for the binding of poly(I) to BER and its analogs BER1-3. Thus, the number of poly(I) bases bound per BER and its analogs BER1-3 can be estimated to be around 7.20, 3.06, 2.52 and 1.89, respectively. The binding stoichiometry for poly(U) and poly(C) sequences could not be determined owing to their very weak binding affinities. 3.6. Fluorescence quenching studies and elucidation of the mode of binding Recent X-ray diffraction studies on oligonucleotide complexes have unequivocally established the intercalative binding mode of BER [39]. So it may be presumed that, like BER, its analogs may also intercalate into the RNA helix. In order to ascertain the mode of binding of the 9-substituted analogs ferrocyanide quenching experiments were performed [40]. Ferrocyanide quencher, being anionic in nature, will not be able to penetrate the negatively charged RNA helix and if these analogs are buried deep inside the helical organization of RNA strongly by intercalation then little or no change in fluorescence would occur. Stern–Volmer plots for the quenching of fluorescence of BER and its analogs clearly indicated that free molecules were quenched very efficiently with quenching constants in the range 230–200 M1. In the presence of the two ribonucleotides, poly(G) and poly(I), more quenching was observed in the case of berberine and less quenching for the analogs BER1-3 indicating that the RNA bound analogs are indeed located in a relatively more protected environment than BER. The quenching constants (Ksv) calculated for poly(G) bound BER and its analogs BER1-3 were 138, 57, 69 and 84 M1, respectively (Fig. 4). Similarly, in the presence of poly(I), the Ksv values were estimated to be 147, 67, 75 and 95 M1, respectively, for BER, BER1, BER2 and BER3 (Fig. 4). From this result it can be inferred that the bound alkaloid molecules are sequestered away from the solvent and shielded in a relatively more protected environment inside the RNA helix confirming strong intercalative binding. The

Fig. 4. Stern–Volmer plots for the quenching of the fluorescence of (A) BER1 and (B) BER3 in CP buffer, pH 7.0 in the absence of RNA (N), in the presence of poly(G) (j) and in the presence of poly(I) (d).

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Fig. 5. Circular dichroic spectra of (A) poly(G) (50 lM) treated with of 0, 5, 10, 20, 30, 40, 50 lM of BER1 (curves 1–7), (B) poly(I) (50 lM) treated with 0, 5, 10, 20, 30, 40, 50 lM of BER2 (curves 1–7), (C) poly(U) (50 lM) treated with 0, 20, 40, 50, 60 lM of BER3 (curves 1–5), and (D) poly(C) (50 lM) treated with 0, 20, 40, 50, 60 lM of BER1 (curves 1–5).

trend in the values suggested that the Ksv of poly(G) and poly(I) bound BER is higher than that of all the analogs and it varied in the order BER > BER3 > BER2 > BER1. This trend is indicative of the fact that all the analogs are better intercalated to both the RNA sequences compared to BER; the best being BER1. Besides, the Ksv values of the poly(G) bound analogs were lower than those of the poly(I) bound ones. Therefore, a better intercalation geometry may be envisaged for all the analogs binding to poly(G) compared to poly(I). 3.7. Circular dichroism studies The conformational aspects of the complexation of the analogs with the four RNA sequences were monitored by circular dichroism spectroscopy. The interaction of these analogs with the homopolynucleotides may result in changes in the intrinsic CD spectra of these polynucleotides and also may result in induction of optical activity (induced CD) for the bound alkaloid molecules. Such changes provide useful insights into the nature and mode of the association. Poly(G) is known to have a random coiled structure [41] with a positive peak at 260 nm and a negative peak around 238 nm region. The ellipticity of the long wavelength band decreased significantly in the presence of the analogs. The decrease in ellipticity was maximum for BER1 (28.4%) (Fig. 5A) followed by BER2 and then BER3. Besides, comparatively less pronounced alterations were also observed in the negative CD band at 238 nm. However, in the presence of constant concentration of the alkaloids and increasing concentration of poly(G), no induced CD with satisfactory signal to noise ratio was observed in the absorption region of the alkaloids. The CD spectrum of poly(G) exhibited only insignificant marginal changes in the presence of the parent alkaloid BER revealing the absence of any conformational changes on binding of BER. Thus, the aryl/arylalkyl amino carbonyl methyl substituent is responsible for inducing conformational changes in the poly(G) structure thereby reiterating its importance in the complexation phenomenon of the analogs.

Poly(I) has an inherent tendency to form multistranded structures at high salt conditions [42] but it exists as a single stranded helix at low salt conditions and neutral pH as employed in the present study [43]. The broad flat CD melting profile without any hyperchromic change further supports that poly(I) exists as a single stranded helix under the conditions of our study. Single stranded poly(I) structure exhibits a small characteristic negative peak at 272 nm followed by a large positive band around 252 nm and a small negative hump at 236 nm (Fig. 5B). Remarkable changes were observed in the positive CD band of poly(I) in the presence of the analogs. The ellipticity of the positive band at 252 nm decreased with increasing concentration of the analogs and crossed over to the negative side and enhanced in magnitude (Fig. 5B). The other two negative peaks of poly(I) showed comparatively smaller changes in presence of the analogs. Concomitant with these intrinsic CD changes induced CD bands were also observed in the 300– 400 nm region. The induced spectra were bisignate with both positive and negative bands. The bisignate induced CD spectra is indicative of effective interaction between the transition moments of the chirally arranged bound analogs and the RNA bases. A positive band around 362 nm and a negative band around 332 nm were observed in the induced CD spectra of BER1 and BER2. However, for analog BER3 a negative induced CD band around 338 nm was only formed indicating slight conformational differences in the binding of the different analogs and also accounting for the slightly weaker binding affinity of analog BER3 compared to BER1 and BER2. The parent alkaloid BER produced only a moderate decrease in the ellipticity of the positive band of poly(I), marginal alterations in the other two negative CD bands and no induced CD bands were observed in the visible absorption region. Poly(U) has a random structure with a very little degree of base stacking [41,44]. The CD spectrum of poly(U) exhibited a large positive band around 271 nm and a less intense negative band around 243 nm (Fig. 5C). In the presence of the analogs, moderate decrease in the ellipticity of the long wavelength positive band was observed and the negative CD band exhibited only minor changes (Fig. 5C). These conformational changes were more pronounced for

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analog BER1 at a lower D/P (alkaloid/RNA molar ratio) compared to BER2 and BER3. BER itself did not induce any conformational change in the poly(U) structure. Besides, no induced CD spectra with satisfactory signal to noise ratio was obtained for the complexation of these analogs with the uracil bases. At pH 7.0 poly(C) exists as a right-handed helix with an intermediate degree of base stacking [41]. A single stranded left handed helical structure for poly(C) was suggested by Broido and Kearns based on 2D NMR spectral studies where the sugar phosphate backbone is inside the helix and the bases are on the outside [45]. Furthermore, they also proposed that the bases outside the helix were stabilized by intramolecular H-bonding between carbonyl group of one base and the amino group of the adjacent base. Results from vibrational Raman spectra also testified for such a left handed single stranded structure of poly(C) with base tilting [46]. The CD spectra of poly(C) exhibits a positive CD band at 277 nm and a negative CD band at 234 nm which remained absolutely unaltered in presence of BER thereby indicating that BER does not induce any conformational change whatsoever upon binding to poly(C). However, the analogs induced minor conformational changes in the poly(C) structure (Fig. 5D); the long wavelength positive band ellipticity is slightly decreased and the negative CD band at 234 nm also exhibited slight alterations upon binding to the analogs. Upon complexation with the poly(C) BER and its analogs do not exhibit any induced CD spectra in the absorption region of the alkaloids. It is pertinent to mention here that the overall CD changes manifested in all the four polynucleotide sequences upon binding of these analogs can be interpreted in terms of major changes in stacking pattern as well as change in the helicity of the polynucleotides. It is also worth mentioning that the overall conformational changes include contributions from the electrostatic binding of the positively charged analogs to the negatively charged phosphates and other changes that may occur in the sugar phosphate backbone. 3.8. Energetics of the binding: isothermal titration calorimetry studies Isothermal titration calorimetry is an important and quick tool for direct and reliable measure of the thermodynamic parameters

of interaction of small molecules to biopolymers [18,33]. Since ITC measures heat exchange, it provides a reliable tool independent of the spectroscopic changes that occur for the chromophores in the reaction. ITC enables the estimation of the complete thermodynamic profile of the interaction in terms of standard molar Gibbs energy change (DGo), standard molar enthalpy (DHo), and entropy change (DSo), together with the stoichiometry (N) and binding affinity (Ka) from a single titration. Fig. 6 depicts the representative ITC thermograms for the complexation of BER1 with poly(G) and BER2 with poly(I). The upper panels represent the raw data for the sequential injection of RNA into the alkaloids in the calorimeter cell. The areas under the heat burst curves were determined by integration to yield the associated injection heats. These injection heats were corrected by subtracting the corresponding heat of dilution derived from the injection of identical amounts of the injectant (here RNA) into the buffer alone. The lower panel shows the integrated heat after correction of the heat of dilution. BER and its three analogs bound to the RNA sequences exhibiting monophasic binding events that were exothermic, resulting in negative peaks in the plot of power versus time. The heat evolved with BER was higher than with its analogs. Since the integrated heat data in the ITC profiles showed only one binding event, they were fitted to a single set of identical sites model. This was also based on the results of the Job plot analysis which revealed a single binding mode in all the cases. All the thermodynamic parameters of interaction elucidated from the single site fitting protocol are collated in Table 2. The binding affinity of the analogs BER1-3 to poly(G) were estimated to be (1.26 ± 0.05)  106 M1, (1.03 ± 0.05)  106 M1 and (8.05 ± 0.04)  105 M1, respectively, against a value of (4.21 ± 0.03)  105 M1 observed for BER. Similarly, for poly(I) the binding affinity values were found to be (2.01 ± 0.03)  105 M1, (1.11 ± 0.05)  106 M1, (1.00 ± 0.04)  106 M1 and (6.80 ± 0.04)  105 M1, respectively, for BER and its analogs BER1-3. The binding affinity values deduced from ITC clearly suggested a remarkable enhancement of the affinity from BER to BER1 and thereafter a gradual decrease from BER1 to BER2 to BER3. The site size (n), which is the reciprocal of stoichiometry (N), was found to be in the range 2.72–1.06 for all the berberine

Fig. 6. Representative ITC thermograms for the binding of (A) BER1 with poly(G) and (B) BER2 with poly(I). The top panels represent the raw data for the sequential injection of RNA into the alkaloid solutions 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 (closed rectangles) were fitted to a one site model and the solid lines represent the best fit of the experimental data.

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A. Basu et al. / Journal of Photochemistry and Photobiology B: Biology 134 (2014) 64–74 Table 2 Thermodynamic parameters for the association of alkaloids with single stranded RNA from ITC.a. Alkaloids

Single stranded RNA

Ka  105 (M1)

n

DHo (kcal/mol)

TDSo (kcal/mol)

DGo (kcal/mol)

BER

Poly(G) Poly(I) Poly(U) Poly(C)

4.21 ± 0.03 2.01 ± 0.03 nd nd

8.46 ± 0.06 7.22 ± 0.05 nd nd

4.03 ± 0.06 3.77 ± 0.06 nd nd

3.64 3.46 nd nd

7.67 ± 0.06 7.23 ± 0.06 nd nd

BER1

Poly(G) Poly(I) Poly(U) Poly(C)

12.6 ± 0.05 11.1 ± 0.05 nd nd

1.06 ± 0.01 2.72 ± 0.03 nd nd

1.37 ± 0.03 0.28 ± 0.01 nd nd

6.95 7.96 nd nd

8.32 ± 0.03 8.24 ± 0.01 nd nd

BER2

Poly(G) Poly(I) Poly(U) Poly(C)

10.3 ± 0.05 10.0 ± 0.04 nd nd

1.23 ± 0.02 2.18 ± 0.02 nd nd

2.28 ± 0.04 0.68 ± 0.02 nd nd

5.93 7.51 nd nd

8.21 ± 0.04 8.19 ± 0.02 nd nd

BER3

Poly(G) Poly(I) Poly(U) Poly(C)

8.05 ± 0.04 6.80 ± 0.04 nd nd

2.68 ± 0.03 1.30 ± 0.02 nd nd

2.71 ± 0.04 1.70 ± 0.03 nd nd

5.34 6.26 nd nd

8.05 ± 0.04 7.96 ± 0.03 nd nd

a All the data in this table are derived from ITC experiments conducted in citrate–phosphate buffer of 10 mM [Na+], pH 7.0 at 25 °C and are average of four determinations. Ka, n and DH0 values were determined from ITC profiles fitting to Origin 7.0 software as described in the text. The values of DGo and TDSo were determined using the equations DGo = RT ln Ka and TDSo = DHo–DGo. All the ITC profiles were fit to a model of single binding sites. Uncertainties correspond to regression standard errors. nd is not determined.

analogs. These values are in close agreement with the stoichiometry values deduced from Job plot analysis. The binding Gibbs energy enhanced significantly for the complexation of all the analogs with poly(G) and poly(I) in comparison to BER. Hence, we can conclude that the side chain at the 9-position of the isoquinoline chromophore offers a better contact module or additional contact points with the RNA bases thereby resulting in enhanced binding preference. However, the enthalpy driven binding of berberine switched to entropy dominated binding for all the analogs. The enthalpic contribution to the standard molar Gibbs energy was significantly reduced while the entropic contribution (TDSo) was remarkably enhanced with the introduction of 9-O-N-aryl/arylalkyl amino carbonyl methyl side chain. The decrease in enthalpic contribution suggested lesser contribution from stacking while the strong positive entropy terms are indicative of the dominant role of hydrophobic forces in the binding process, reorientation of water molecules, disordering of ion atmosphere inside the RNA helix, disruption and release of structured water molecules from the RNA helices upon intercalation of the analogs. Therefore, not only a remarkable amplification in binding affinity was observed with the introduction of the side chain at the 9-position of the isoquinoline chromophore but there was also a significant alteration in the energetics of the complexation process. The overall binding affinity values deduced from ITC analysis are in good agreement with those obtained from spectroscopic analysis. 3.9. Exploring the dependence of binding on the ionic strength of the medium and parsing of the standard molar Gibbs energy All the alkaloid analogs are monocationic owing to the presence of the quaternary nitrogen atom at the 7-position of the isoquinoline chromophore. Hence, it may be expected that their complexation to the anionic RNA sequences is thermodynamically linked to the concentration of sodium ions bound to the phosphate backbone of RNA. To explore the nature of forces governing the complexation phenomenon salt dependent isothermal titration calorimetry experiments were performed in conjunction with van’t Hoff analysis. This is critical as the binding of the positively charged alkaloids is sensitive to cation concentration because the alkaloid competes to expel the bound cations for phosphate neutralization and these are thermodynamically linked processes. ITC titrations were performed for the complexation of the most

potent analog BER1 with poly(G) and poly(I) at three different Na+ concentrations, viz. 10, 20 and 50 mM [Na+] and the binding affinity values (Table S1) were utilized for elucidating the salt dependence of the binding. The binding affinity to the two RNA polynucleotides decreased as the salt concentration increased. This is because the electrostatic repulsion between the negatively charged phosphate backbones of the adjacent ribonucleotides is reduced with increasing [Na+], and hence the binding of the alkaloid molecule is hindered. From the dependence of Ka on [Na+] the standard molar Gibbs energy can be partitioned between polyelectrolytic (DGope) and nonpolyelectrolytic (DGot) contributions as done in case of several intercalators by Chaires and co-workers [47,48]. From the polyelectrolytic theory, the slope of the best fit line for a plot of log Ka versus log [Na+] is related to the counterions release by the following relation [49]

SK ¼ @ logðK a Þ=@ logð½Naþ Þ ¼ zu

ð4Þ

where SK is equivalent to the number of counterions released upon binding of a drug, z is the apparent charge of the bound alkaloid per phosphate binding and u is the fraction of [Na+] bound per phosphate group. A plot of log Ka versus log [Na+] for the complexation of BER1 with poly(G) and poly(I) is depicted in Fig. 7A. A quantitative estimation of the polyelectrolytic contribution to the standard molar Gibbs energy was obtained employing the relationship given by Record and coworkers [50,51]

DG0pe ¼ zuRT lnð½Naþ Þ

ð5Þ 0

The enthalpic term in DG pe originates from the columbic interaction of alkaloid molecules with counter ions present in the solution and the entropic term arises from disordering of ion atmosphere inside the RNA helix upon alkaloid intercalation. As the salt concentration increased, the electrostatic contribution (DG0pe) to the total standard molar Gibbs energy decreased and at 50 mM [Na+] it was estimated to be around 7–8% of the total standard molar Gibbs energy. The difference between DG0 and DG0pe enabled the estimation of DG0t. However, the nonpolyelectrolytic contribution (DG0t) which accounted for around 90% of the total standard molar Gibbs energy remained almost invariant with changing [Na+]. Thus the RNA binding phenomenon was dominated by nonpolyelectrolytic forces at both high and low salt concentrations. The DGot contribution included all factors like hydrophobic interactions, p–p stacking interactions, H-bonding,

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Fig. 7. (A) Plot of log Ka versus log [Na+] for the binding of BER1 with poly(G) (j) and poly(I) (d). Polyelectrolytic (DG0pe) (shaded) and non-polyelectrolytic (DG0t) (white) contribution to standard molar Gibbs energy for the complexation of analog BER1 with (B) poly(G) and (C) ploy(I) at different Na+ concentrations.

and van der Waals forces. Thus, DGot corresponds to that portion of the standard molar Gibbs energy which is independent of salt concentration and contains minimal contribution from polyelectrolyte effects such as coupled ion release. A graphical representation of the partitioning of the standard molar Gibbs energy between electrostatic (DGope) and non-electrostatic (DGot) components for poly(G) and poly(I) is depicted in Fig. 7B and C. 3.10. Temperature dependence of the calorimetric data: heat capacity changes Elucidation of the thermodynamic parameters at different temperatures enable us to estimate the extent of enthalpy–entropy compensation and also determine the magnitude of heat capacity change. The constant pressure heat capacity changes (DCop) were calculated from the temperature dependent enthalpy results using the standard relationship,

DC op ¼ @ DHo =@T

ð6Þ

Heat capacity change data can provide valuable insights into the type and magnitude of forces that govern the complexation phenomenon. The thermodynamic parameters were evaluated for the complexation of BER1 (the analog with the highest binding affinity) with poly(G) and poly(I) from ITC experiments conducted in CP buffer of pH 7.0 at three temperatures, viz. 288.15, 298.15 and 308.15 K. It is pertinent to mention here that the pH of the buffer remained unchanged in the temperature range studied. The thermodynamic parameters obtained at these three temperatures are collated in Table S2. The binding affinity values decreased significantly in each case as the temperature was increased from 288.15 to 308.15 K (Table S2). With increasing temperature there was significant enthalpic and entropic alterations-while the negative enthalpy values increased, the positive entropy values decreased. DCp0 values were estimated from the variation of DH0 with temperature (Fig. 8A). The slopes of the lines gave values of 34.7 and 15.3 cal/mole K, respectively, for the binding of BER1

with poly(G) and poly(I). The negative enthalpic and positive entropic terms, both of which were strong functions of temperature, compensated each other to make the standard molar Gibbs energy change temperature independent. A plot of variation of DG0 and DH0 against TDS0 is shown in Fig. 8B. Such enthalpy– entropy compensation behavior is the characteristic of many biomolecular processes [18,52,53] and signifies dominant contribution from hydrophobic forces in the complexation process. The negative DCp0 values observed here tends to denote burial of water accessible hydrophobic surface area, resulting in the release of bound water molecules and counter ions [54,55]. Besides, negative heat capacity values also indicate destacking of the RNA bases and may also serve as a sequel to the stacking interaction between the alkaloid moiety and the RNA bases [56]. The fact that the magnitude of enthalpy increases (in absolute values) with increasing temperature suggests that stacking alone is not the dominant binding force. Other forces like hydrophobic interactions, H-bonding, van der Waals forces, and polyelectrolytic forces. are also involved in the complexation process. The differences in the DCp0 values between the two RNA sequences indicate differences in the release of structured water consequent to the transfer of nonpolar groups into the interior of the RNA helical organization. Slightly higher DCp0 value for poly(G) compared to poly(I) sequence for the binding of analog BER1 suggest conformational differences in their structure and also greater disruption of the water structure around the poly(G) helix compared to poly(I) helix. Murphy and Churchill described sequence specific, nonspecific, minimal sequence specific and structure specific modes of nucleic acid recognition by small molecules [57]. The small but negative heat capacity values observed here are the characteristic of structure specific binding. For DNA and RNA intercalators a large hydrophobic contribution to the standard molar Gibbs energy arises from their aromatic nucleus and the binding should be energetically favorable [58]. From the Records [59] relationship, DG0hyd = 80(±10)  DCp0, the standard molar Gibbs energy contribution from the hydrophobic transfer step of binding was calculated. The DG0hyd values for BER1 binding to poly(G) and poly(I) were deduced to be 2.78,

Fig. 8. (A) Plot of variation of standard molar enthalpy of binding (DH0) with temperature for the complexation of BER1 with poly(G) (j) and poly(I) (d). (B) Plot of variation of DG0 (open symbols) and DH0 (closed symbols) verses TDS0 for the binding of BER1 with poly(G) (h,j) and poly(I) (s, d).

A. Basu et al. / Journal of Photochemistry and Photobiology B: Biology 134 (2014) 64–74

and 1.22 kcal/mol, respectively. Thus, the binding of BER1 to poly(G) results in release of greater number of RNA bound water molecules as compared to poly(I) thereby accounting for higher DCp0 and DG0hyd values.

73

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jphotobiol.2014. 03.024.

4. Conclusions

References

In the present study the structural and thermodynamic characterization of the binding of three novel 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs with four sequence specific ss RNAs was performed using multifaceted biophysical techniques. This study has revealed that all the analogs are better intercalators compared to berberine and hence useful for potential biological applications. Besides, a remarkable enhancement in the binding affinity (one order higher) was observed for all the analogs binding to all the four RNA sequences; the best being BER1. Thereafter, on further increasing the chain length of the 9-substitutent the binding affinity decreased gradually indicating that the chain length is critical in modulating the RNA binding affinities. The binding to poly(G) and poly(I) RNA sequences was significantly stronger compared to poly(U) and poly(C) sequences. This is probably because the polypurine bases have a pyrimidine ring fused to an imidazole ring and thus it can offer a better/additional contact module with the analogs in comparison to the polypyrimidine bases. The non-cooperative binding mode of the parent berberine was propagated in the analogs also. All the analogs effected significant conformational changes in poly(G), remarkable conformational changes on binding to poly(I), moderate changes on complexation with poly(U) and marginal changes on association with poly(C). Besides, CD was essentially induced in the bound analog molecules on intercalating into the chiral poly(I) helix. The binding was favoured by both small negative enthalpic contribution and high positive entropic contribution. Parsing of the standard molar Gibbs energy established the involvement of large non-electrostatic contribution to the binding. Small but negative heat capacity changes in all the systems was correlated to structure specific binding and established the involvement of significant hydrophobic forces in the complexation process. The binding of the analogs was favored by higher entropic contribution in contrast to the enthalpy driven binding of berberine. Thus, the introduction of the aryl/arylalkyl amino carbonyl methyl side chain switched the enthalpy driven binding of berberine to entropy dominated binding thereby clearly establishing the role of the side chain in greater disruption and release of structured water from the RNA sequences. The overall standard molar Gibbs energy also enhanced significantly with the introduction of the substituent at the 9-position of the isoquinoline chromophore indicating that the side chain offers a more favorable contact geometry with the RNA bases. These results further advance our knowledge on the interaction of berberine analogs to single stranded RNA sequences that may be useful for the design and development of berberine based RNA binding therapeutic molecules.

[1] J.B. Chaires, Drug–DNA interactions, Curr. Opin. Struct. Biol. 8 (1998) 314–320. [2] J. Ren, J.B. Chaires, Sequence and structural selectivity of nucleic acid binding ligands, Biochemistry 38 (1999) 16067–16075. [3] L. Guan, M.D. Disney, Recent advances in developing small molecules targeting RNA, ACS Chem. Biol. 7 (2012) 73–86. [4] R.R. Sinden, DNA Structure and Function, Academic Press, San Diego, California, 1994. [5] M. Rassoulzadegan, V. Grandjean, P. Gounon, S. Vincent, I. Gillot, F. Cuzin, RNAmediated non-mendelian inheritance of an epigenetic change in the mouse, Nature 441 (2006) 469–474. [6] C.L. Kuo, C.C. Chou, B.Y. Yung, Berberine complexes with DNA in the berberineinduced apoptosis in human leukemic HL-60 cells, Cancer Lett. 93 (1995) 193– 200. [7] Y.-T. Ho, J.S. Yang, T.-C. Li, J.-J. Lin, J.-G. Lin, K.-C. Lai, C.-Y. Ma, W.G. Wood, J.-G. Chung, Berberine suppresses in vitro migration and invasion of human SCC-4 tongue squamous cancer cells through the inhibitions of FAK, IKK, NF-jB, u-PA and MMP-2 and -9, Cancer Lett. 279 (2009) 155–162. [8] Y. Sun, K. Xun, Y. Wang, X. Chen, A systematic review of the anticancer properties of berberine, a natural product from Chinese herbs, Anticancer Drugs 20 (2009) 757–769. [9] W.H. Cheuh, J.Y. Lin, Protective effect of isoquinoline alkaloid berberine on spontaneous inflammation in the spleen, liver and kidney of non-obese diabetic mice through downregulating gene expression ratios of pro-/antiinflammatory and Th1/Th2 cytokines, Food Chem. 131 (2012) 1263–1271. [10] W. Kong, J. Wei, P. Abidi, M. Lin, S. Inaba, C. Li, Y. Wang, Z. Wang, S. Si, H. Pan, S. Wang, J. Wu, Y. Wang, Z. Li, J. Liu, J.-D. Jiang, Berberine is a novel cholesterollowering drug working through a unique mechanism distinct from statins, Nat. Med. 10 (2004) 1344–1351. [11] L.-Q. Tang, W. Wei, L.-M. Chen, S. Liu, Effects of berberine on diabetes induced by alloxan and a high-fat/high-cholesterol diet in rats, J. Ethnopharmacol. 108 (2006) 109–115. [12] R. Domitrovic´, H. Jakovac, G. Blagojevic´, Hepatoprotective activity of berberine is mediated by inhibition of TNF-a, COX-2, and iNOS expression in CCl4intoxicated mice, Toxicology 280 (2011) 33–43. ˇ ern ˇ áková, D. Koštˇálová, Antimicrobial activity of berberine-a constituent [13] M. C of Mahonia aquifolium, Folia Microbiol. 47 (2002) 375–378. [14] A. Shirwaikar, A. Shirwaikar, K. Rajendran, I.S. Punitha, In vitro antioxidant studies on the benzyl tetra isoquinoline alkaloid berberine, Biol. Pharm. Bull. 29 (2006) 1906–1910. [15] W.H. Peng, K.L. Lo, Y.H. Lee, T.H. Hung, Y.C. Lin, Berberine produces antidepressant-like effects in the forced swim test and in the tail suspension test in mice, Life Sci. 81 (2007) 933–938. [16] K. Iwasa, H.-S. Kim, Y. Wataya, D.-U. Lee, Antimalarial activity and structureactivity relationships of protoberberine alkaloids, Eur. J. Med. Chem. 33 (1998) 65–69. [17] M.M. Islam, A. Basu, M. Hossain, G. Sureshkumar, S. Hotha, G. Suresh Kumar, Enhanced DNA binding of 9-x-amino alkyl ether analogs from the plant alkaloid berberine, DNA Cell Biol. 30 (2011) 123–133. [18] M.M. Islam, S. Roy Chowdhury, G. Suresh Kumar, Spectroscopic and calorimetric studies on the binding of alkaloids berberine, palmatine and coralyne to double stranded RNA polynucleotides, J. Phys. Chem. B 113 (2009) 1210–1224. [19] M.M. Islam, R. Sinha, G. Suresh Kumar, RNA binding small molecules: studies on t-RNA binding by cytotoxic plant alkaloids berberine, palmatine and the comparison to ethidium, Biophys. Chem. 125 (2007) 508–520. [20] M.M. Islam, G. Suresh Kumar, RNA targeting by small molecule alkaloids: studies on the binding of berberine and palmatine to polyribonucleotides and comparison to ethidium, J. Mol. Struct. 875 (2008) 382–391. [21] P. Krishnan, K.F. Bastow, The 9-position in berberine analogs is an important determinant of DNA topoisomerase II inhibition, Anti-cancer Drug Des. 15 (2000) 255–264. [22] J.Y. Pang, Y. Qin, W.H. Chen, G.A. Luo, Z.H. Jiang, Synthesis and DNA-binding affinities of monomodified berberines, Bioorg. Med. Chem. 13 (2005) 5835– 5840. [23] H.S. Bodiwala, S. Sabde, D. Mitra, K.K. Bhutani, I.P. Singh, Synthesis of 9substituted derivatives of berberine as anti-HIV agents, Eur. J. Med. Chem. 46 (2011) 1045–1049. [24] C.Y. Lo, L.C. Hsu, M.S. Chen, Y.J. Lin, L.G. Chen, C.D. Kuo, J.Y. Wu, Synthesis and anticancer activity of a novel series of 9-O-substituted berberine derivatives: a lipophilic substitute role, Bioorg. Med. Chem. Lett. 23 (2013) 305–309. [25] M.M. Islam, A. Basu, G. Suresh Kumar, Binding of 9-O-(x-amino) alkyl ether analogues of the plant alkaloid berberine to poly(A): insights into selfstructure induction, Med. Chem. Commun. 2 (2011) 631–637. [26] A. Basu, P. Jaisankar, G. Suresh Kumar, Synthesis of novel 9-O-N-aryl/aryl-alkyl amino carbonyl methyl substituted berberine analogs and evaluation of DNA binding aspects, Bioorg. Med. Chem. 20 (2012) 2498–2505.

Acknowledgments Financial assistance from the network project GenCODE (BSC0123) of the Council of Scientific and Industrial Research (CSIR) is gratefully acknowledged. A. Basu is a NET qualified Senior Research Fellow of the University Grants Commission, Govt. of India. The authors thank all the colleagues of the Biophysical Chemistry Laboratory for their help and cooperation during the course of this work. The critical and judicious comments of the anonymous reviewers that enabled considerable improvement of the manuscript are greatly appreciated.

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A. Basu et al. / Journal of Photochemistry and Photobiology B: Biology 134 (2014) 64–74

[27] A. Basu, P. Jaisankar, G. Suresh Kumar, 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs induce self-structure in polyadenylic acid, RSC Adv. 2 (2012) 7714–7723. [28] A. Basu, P. Jaisankar, G. Suresh Kumar, Binding of the 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs to tRNAphe, PLoS ONE 8 (2013) e58279. [29] A. Basu, P. Jaisankar, G. Suresh Kumar, Photophysical and calorimetric studies on the binding of 9-O-substituted analogs of the plant alkaloid berberine to double stranded poly(A), J. Photochem. Photobiol. B: Biol. 125 (2013) 105–114. [30] J.J. ChamPoux, D.S. Hogness, The topography of lambda DNA: polyriboguanylic acid binding sites and base composition, J. Mol. Biol. 71 (1972) 383–405. [31] E.D. Clercq, W.E. Stewart, P.D. Somer, Poly(rI) more important than poly(rC) in the interferon induction process by poly(rI)poly(rC), Virology 54 (1973) 278– 282. [32] R. Sinha, M.M. Islam, K. Bhadra, G. Suresh Kumar, A. Banerjee, M. Maiti, The binding of DNA intercalating and non-intercalating compounds to A-form and protonated form of poly(rC)poly(rG): spectroscopic and viscometric study, Bioorg. Med. Chem. 14 (2006) 800–814. [33] K. Bhadra, M. Maiti, G. Suresh Kumar, Molecular recognition of nucleic acids by small molecules: AT base pair specific intercalative binding of cytotoxic plant alkaloid palmatine, Biochim. Biophys. Acta. 1770 (2007) 1071–1080. [34] J.B. Chaires, N. Dattagupta, D.M. Crothers, Studies on interaction of anthracycline antibiotics and deoxyribonucleic acid: equilibrium binding studies on the interaction of daunomycin with deoxyribonucleic acid, Biochemistry 21 (1982) 3933–3940. [35] J.D. McGhee, P.H. von Hippel, Theoretical aspects of DNA–protein interactions: co-operative and non-co-operative binding of large ligands to a onedimensional homogeneous lattice, J. Mol. Biol. 86 (1974) 469–489. [36] H.A. Benesi, J.H. Hildebrand, A spectrophotometric investigation of the interaction of iodine with aromatic hydrocarbons, J. Am. Chem. Soc. 71 (1949) 2703–2707. [37] N.C. Garbett, N.B. Hammond, D.E. Graves, Influence of the amino substituents in the interaction of ethidium bromide with DNA, Biophys. J. 87 (2004) 3974– 3981. [38] P. Job, Formation and stability of inorganic complexes in solution, Ann. Chim. 9 (1928) 113–203. [39] M. Ferraoni, C. Bazzicalupi, A.R. Billa, P. Gratteri, X-ray diffraction analyses of the natural isoquinoline alkaloids berberine and sanguinarine complexed with double helix DNA d(CGTACG), Chem. Commun. 47 (2011) 4917–4919. [40] J.R. Lakowicz, Principles of fluorescence spectroscopy, Plenum press, New York, 1983. p. 257. [41] W. Saenger, Principles of Nucleic Acid Structure, Springer Verlag, New York, 1984. [42] S. Arnott, R. Chandrasekaran, C.M. Marttila, Structures for polyinosinic acid and polyguanylic acid, Biochem. J. 141 (1974) 537–543.

[43] D. Thiele, W. Guschlbauer, The structures of polyinosinic acid, Biophysik 9 (1973) 261–277. [44] J.C. Thrierr, M. Dourlent, M. Leng, A study of polyuridylic acid, J. Mol. Biol. 58 (1971) 815–830. [45] M.S. Broido, D.R. Kearns, Proton NMR evidence for a left-handed helical structure of poly(ribocytidylic acid) in neutral solution, J. Am. Chem. Soc. 104 (1982) 5207–5216. [46] A.F. Bell, L. Hecht, L.D. Barron, Vibrational Raman optical activity as a probe of polyribonucleotide solution stereochemistry, J. Am. Chem. Soc. 119 (1997) 6006–6013. [47] J.B. Chaires, S. Satyanarayana, D. Suh, I. Fokt, T. Przewloka, W. Priebe, Parsing the free energy of anthracycline antibiotic binding to DNA, Biochemistry 35 (1996) 2047–2053. [48] J.B. Chaires, Dissecting the free energy of drug binding to DNA, Anticancer Drug Des. 11 (1996) 569–580. [49] M.T. Record Jr., C.F. Anderson, T.M. Lohman, Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: the roles of ion association or release, screening, and ion effects on water activity, Q. Rev. Biophys. 11 (1978) 103–178. [50] M.T. Record Jr., R.S. Spolar, The biology of nonspecific DNA-protein interactions, in: A. Revzin (Ed.), CRC Press, Boca Raton, FL, 1990. pp. 33–69. [51] M.T. Record Jr., J.H. Ha, M.A. Fisher, Analysis of equilibrium and kinetic measurements to determine thermodynamic origins of stability and specificity and mechanism of formation of site-specific complexes between proteins and helical DNA, Methods Enzymol. 208 (1991) 291–343. [52] L. Jen-Jacobson, L.E. Engler, L.A. Jacobson, Structural and thermodynamic strategies for site-specific DNA binding proteins, Structure 8 (2000) 1015– 1023. [53] J.B. Chaires, A thermodynamic signature for drug–DNA binding mode, Arch. Biochem. Biophys. 453 (2006) 26–31. [54] I. Haq, Thermodynamics of drug–DNA interactions, Arch. Biochem. Biophys. 403 (2002) 1–15. [55] J. Ren, T.C. Jenkins, J.B. Chaires, Energetics of DNA intercalation reactions, Biochemistry 39 (2000) 8439–8447. [56] D.S. Pilch, C.M. Barbieri, S.G. Rzuczek, E.J. LaVoie, J.E. Rice, Targeting human telomeric G-quadruplex DNA with oxazole-containing macrocyclic compounds, Biochimie 90 (2008) 1233–1249. [57] F.V. Murphy, M.E. Churchill, Nonsequence-specific DNA recognition: a structural perspective, Structure 8 (2000) R83–R89. [58] M. Guthrie, A.D. Parenty, L.V. Smith, L. Cronin, A. Cooper, Microcalorimetry of interaction of dihydro-imidazo-phenanthridinium (DIP)-based compounds with duplex DNA, Biophys. Chem. 126 (2007) 117–123. [59] J.H. Ha, R.S. Spolar, M.T. Record Jr., Role of the hydrophobic effect in stability of site-specific protein–DNA complexes, J. Mol. Biol. 209 (1989) 801–816.