Induction of self-structure in polyriboadenylic acid by the benzophenanthridine plant alkaloid chelerythrine: a spectroscopic approach

Ankur Bikash Pradhan , Lucy Haque, Sutanwi Bhuiya and Suman Das*
Department of Chemistry, Jadavpur University, Raja S. C. Mullick Road, Jadavpur, Kolkata 700 032, India. E-mail: sumandas10@yahoo.com; ankurpradhan727@gmail.com; lucy.haque@gmail.com; s.bhuiya12@gmail.com; Fax: +91 33 2414 6266; Tel: +91 94 3437 3164 Tel: +91 33 2457 2349

Received 14th July 2014 , Accepted 30th September 2014

First published on 6th October 2014


Abstract

The naturally occurring benzophenanthridine plant alkaloid chelerythrine (CHL) was found to bind strongly to single-stranded polyriboadenylic acid (poly-A) with a high association constant of the order of 107 M−1. The association was monitored by various spectroscopic and viscometric techniques. Binding of the alkaloid induced self-structure formation of a poly-A helix that showed cooperative melting transition in circular dichroism. The mode of binding of CHL to poly-A was intercalation, as revealed by fluorescence quenching, sensitization of fluorescence experiment and viscosity measurement. Transfer of fluorescence energy from RNA bases to CHL has been demonstrated from fluorimetric studies. Thermodynamic data obtained from temperature dependence of the binding constant revealed that association was driven by a negative enthalpy change and opposed by a negative entropy change. Since the interaction of naturally occurring small molecules with RNA is an active area of research, this study renders the scope of exploring chelerythrine as RNA targeted therapeutic agent.


Introduction

Interactions of small molecules with DNA have been in the focus of exploration for a long time in hopes of discovering effective therapeutic agents. Comparatively less attention has been paid to the study of interaction of small molecules with RNA, which in fact represents a great wealth of targets for the development of novel compounds with pharmacological properties. Study of RNA structures has drawn immense interest from researchers due to the emerging knowledge of their potential and critical roles in many cellular activities.1,2 The ever-growing realization of the variety of biochemical roles of RNA in all living organisms is leading to an increasing appreciation that RNAs provide fascinating targets for treatment of many infectious diseases. Furthermore, RNA is the genetic material of pathogenic viruses such as HIV or hepatitis C virus (HCV), and thus it provides numerous opportunities for the discovery of new drugs to treat the devastating illnesses caused by these agents.3–6 However, the identification of RNA-binding drugs is still in its infancy. Therefore, interaction studies with various RNA structures with biologically active small molecules are indispensable to an understanding of the basic fundamentals of RNA–ligand interaction.

Most cellular RNA molecules are single stranded, but they may form secondary structures, such as hairpins and stem-loops. Though many studies have addressed the interactions of small molecules with nucleic acids, comparatively little is known about ligand recognition of single-stranded A-rich RNAs. Among the single-stranded nucleic acids, polyriboadenylic acid [herein after, poly-A] is of particular biological significance because of its role in mRNA functioning and gene expression.7,8 All eukaryotic mRNAs have the long poly-A tail at the 3′ end and it is an important factor for the maturation and stability of mRNA, and in the initiation of translation.9–11 Since the discovery that Neo-PAP (a recently identified human poly-A polymerase) is significantly over-expressed in some human cancer cells, it has been suggested that the poly-A tails of mRNA might interfere with the full processing of mRNA by PAP and switching off protein synthesis.12–14 This suggests that poly-A tail is a potential tumour-specific target.14 The control of such over-expression of poly-A by the binding of small molecule drugs could be a potential means to RNA-targeted therapeutic intervention. Poly-A has unique characteristics: it exists in single-stranded helical and parallel double-stranded structures in vitro.15–17 The structure of the double helix was established by X-ray crystallography.15 Recently, it has been shown that a variety of small molecules bind to single-stranded poly-A and induce unique structural transformation to a self-structure stabilized by adenine–adenine base pairs.18–24 In vivo, such molecules that can bind and structurally modulate the polyadenylate tail could stop poly-A chain elongation, inhibit mRNA function and stop subsequent protein production in the cell.

Naturally occurring small molecules have been the center of attention as therapeutic agents for their high abundance and low side effects. The alkaloids represent a very extensive group of nitrogen-containing secondary metabolites with diverse structures, distribution in nature and important biological activities. The biological activity of alkaloids has been known to play important roles in medicinal chemistry due to their interaction with nucleic acids. Study of the interaction mechanism between many alkaloid molecules and DNA/RNA has encouraged the development of new drugs.25,26 Among the alkaloids, the benzophenanthridine group of plant alkaloids have been the focus of increasing attention for its wide range of biological activities.27–34 Compounds of this group exhibit strong nucleic acid binding ability, as well as inhibitory effects of different important enzymes.35 One of the remarkable structural features of the benzophenanthridine alkaloids is their ability to show a pH-dependent structural transition between the iminium and alkanolamine forms.36 Among the benzophenanthridine group of alkaloids, sanguinarine is a well-studied compound. It shows diverse biological activity as well as strong nucleic acid binding capacity.21 Sanguinarine has been reported to interact with different polymorphic forms of nucleic acid structures.37 It induces self-structure to poly-A under specific environmental conditions and also binds to the double-stranded form of poly-A.21,38 Chelerythrine (herein after, CHL, Fig. 1) (1,2-dimethoxy-12-methyl[1,3]benzodioxolo[5,6-c]phenanthridin-12-ium) is another benzophenanthridine alkaloid; it differs slightly from sanguinarine in the type and position of substituents in its structure. CHL exhibits pronounced cytotoxicity,31,39 anticancer28,34 and antitumour activity.29,34 CHL is a potent protein kinase C inhibitor and also inhibits the anti-apoptotic Bcl-2 family proteins.40 This compound has been shown to initiate the rapid mitochondrial apoptotic death of H9c2 cardiomyoblastoma cells.40 CHL shows significant anti-proliferative activities, suggesting that it can be considered a promising agent for cancer therapy.41 CHL has been reported to activate p38 MAP kinase and JNK signaling pathways and induce apoptosis in cancer cells both in vitro and in vivo.34,39,42,43 Recent studies have revealed that CHL can exist between the iminium form (charged) and alkanolamine form (uncharged), with a pKa of 8.58.44 There are few studies on the DNA binding of CHL.33,45 Very recently, a detailed study on the interaction of this compound with DNA has been reported by Basu et al.44 They reported that the iminium form of CHL binds to DNA by intercalation, whereas the alkanolamine form does not bind at all to DNA. However, their studies are limited to calf thymus (CT) DNA, and no work has been reported so far by any other group on the interaction of CHL with RNA. Keeping in view the diverse biological effects of CHL and importance of structure and function of poly-A, our focus is to study the interaction of CHL with poly-A using different spectroscopic and viscometric techniques. Overall, our aim is to elucidate and understand the structural and energetic aspects of CHL binding to poly-A and its capability to induce self-structure formation to single-stranded poly-A.


image file: c4ra07075e-f1.tif
Fig. 1 Chemical structure of CHL.

Experimental section

Materials

CHL and poly-A were purchased from Sigma Aldrich Corporation (St. Louis, MO, USA) and used without further purification. The concentration of CHL was checked spectrophotometrically using the known molar absorption coefficient value of 37[thin space (1/6-em)]060 M−1 cm−1 at 316 nm.44 The solutions of CHL were kept in the dark at all times to avoid any light-induced photochemical change. A molar absorption coefficient value of 10[thin space (1/6-em)]000 M−1 cm−1 at 257 nm was used to calculate the concentration of poly-A solution.14 No deviation from Beer's law was observed in the experimental concentration range employed in this study. A fresh solution of CHL was prepared each day for better experimental results. 10 mM citrate phosphate buffer containing 25 mM NaCl of pH 6.5 (CPB buffer) was used for all experiments. Distilled deionized water and analytical grade reagents were used for preparation of the buffers. All buffer solutions were filtered through Millipore filters of 0.45 μm pore size before use. Under our buffer conditions, CHL remained almost 100% in the iminium form. At the same time, free poly-A remained in the single-stranded form in the experimental buffer condition containing 25 mM NaCl, as evidenced from observed CD spectrum.

Methods

UV-absorption experiments. All UV-vis absorbance studies were performed on a Shimadzu model UV-1800 spectrophotometer (Shimadzu Corporation, Japan) in matched quartz cells of 1 cm path length. A thermoprogrammer was attached to maintain the temperature of this spectrometer by the Peltier effect. Initially, to a fixed concentration of CHL, small aliquots of poly-A were added to verify whether there were any interactions of CHL with RNA. In this case, maximum aliquot volume was 2 μL, and the total added volume of poly-A solution was such that the final dilution remained within 1%.

Typical hypochromism and bathochromism of CHL absorption was noted along with the appearance of the isosbestic point. This clearly indicated binding equilibrium between the alkaloid and the RNA polymer. However, in this process there is a limitation: we cannot reach a very high ratio of polymer to ligand, as saturation was reached earlier. To avoid this problem, spectrophotometric titrations were performed using a methodology described previously.37,46 Using this method, we could start with a very high ratio of polymer to ligand. Briefly, a known concentration of poly-A was kept in the sample and reference cells, and small aliquots of a known concentration of CHL were added into the sample cell. After each addition, the solution was mixed and allowed to re-equilibrate for at least 5 min before recording the data. To avoid possible aggregation and to prevent adsorption on the walls of the cuvette, the absorbance values were kept at the minimum for optical studies. This spectrophotometric data were then cast into Scatchard plots of r/Cf versus r, as described previously.37,46

Spectrofluorimetric studies. Steady state fluorescence measurements were performed on a Shimadzu RF-5301PC spectrofluorimeter (Shimadzu Corporation, Kyoto, Japan) that was attached to a highly sensitive temperature controller. Measurements were made in a fluorescence-free quartz cell of 1 cm path length. A fixed concentration of CHL was titrated by increasing the concentration of poly-A under constant stirring. All measurements were conducted while keeping an excitation and emission band pass of 5 nm.
Analysis of binding data and evaluation of binding parameters. Spectrophotometric and spectrofluorimetric titration data were used to construct Scatchard plots of r/Cf versus r.47 In the case of spectrophotometric titration, we used the data from direct titration as well as reverse titration, and we used the direct titration in spectrofluorimetry. The advantage of reverse titration is that we could get data up to a very low value of r, whereas we could not reach such a low value of r in direct titration. All the Scatchard plots were nonlinear and showed negative slopes at low r values as observed in non-cooperative binding isotherms, and hence were analyzed by the excluded site model for non-linear non-cooperative ligand binding phenomenon using the McGhee–von Hippel equation:48
 
image file: c4ra07075e-t1.tif(1)
where K′ is the intrinsic binding constant to an isolated site; n is the number of the nucleotides occluded after the binding of one single ligand molecule; r is the number of moles of CHL bound per mole of nucleotide; and Cf represents the free CHL concentration. The binding data were analyzed using Origin 7.0 software to determine the best-fit parameters of K′ and n.
Determination of binding stoichiometry. Job's continuation method49 was employed to find the binding stoichiometry of CHL with poly-A by fluorescence spectroscopy. At constant temperature, the fluorescence intensity (λmax = 564 nm) was measured for the solution where the concentration of both poly-A and ligand were varied, but the sum of their concentrations was kept constant at 10 μM. The relative difference of fluorescence intensity of CHL at 564 nm was plotted against the mole fraction of CHL. The breakpoint of the plot gave the mole fraction of CHL in the complex. The stoichiometry was obtained in terms of poly-A[thin space (1/6-em)]:[thin space (1/6-em)]CHL [(1 − χCHL)/χCHL] where χCHL denotes the mole fraction of CHL. The results reported here are average of at least three experiments.
Fluorescence polarization anisotropy. The steady state fluorescence anisotropy was also measured using the same spectrofluorimeter. Steady state anisotropy (r′) was defined by:50
 
image file: c4ra07075e-t2.tif(2)
where G is the ratio IHV/IHH used for instrumental correction. 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. The excitation and emission wavelengths were fixed at 400 nm and 564 nm, respectively. The excitation and emission slit width was fixed at 5 nm. Readings were observed 5 min after each addition to ensure stable complex formation. Each reading was an average of five measurements. Anisotropy values were plotted as a function of increasing poly-A concentration.
Mode of binding: fluorescence quenching studies. Fluorescence quenching studies were carried out with the anionic quencher potassium iodide (KI). The solution of KI was mixed with the solution of KCl in different proportions to give a fixed total ionic strength. Quenching experiments were performed at a constant P/D ratio (P/D = nucleotide/CHL molar ratio) monitoring fluorescence intensity changes at 564 nm as a function of concentration of the iodide. The data were plotted in the form of a Stern–Volmer equation:51
 
image file: c4ra07075e-t3.tif(3)
where Fo and F are the fluorescence intensities of the alkaloid-complex with poly-A (P/D = 6 where complete saturation was assured) in the absence and in the presence of the quencher (Q) KI, and KSV is the Stern–Volmer quenching constant. KSV is indicative of the accessibility of the bulky quencher (iodide) to the fluorophore CHL. The slope of the Fo/F versus [KI] plot yields the value of KSV.
Viscometric study. Viscometric measurements were carried out using a Cannon-Manning Semi-Micro Dilution Viscometer type 75 (Cannon Instruments Co., State College, PA, USA) submerged vertically in a constant temperature bath maintained at 25 ± 0.5 °C. The molecular weight of the poly-A sample was estimated to be in the order of 2–2.5 × 105 Da with an intrinsic viscosity of 2.7 dL g−1. 700 μL of 500 μM RNA solution was placed in the viscometer, and aliquots of the stock solution of CHL were added directly into the viscometer to obtain increasing D/P (CHL/nucleotide molar ratio) values. After each addition of CHL to the solution of poly-A in the viscometer, mixing was assured by passing air bubbles slowly through the solution using a narrow teflon tube. Flow times of poly-A in absence and in presence of increasing concentrations of CHL were measured after allowing an equilibration time of 15 min. Measurements were done in triplicate with an accuracy of ±0.01 s, and the relative specific viscosity was calculated using the equation:
 
image file: c4ra07075e-t4.tif(4)
where ηsp and ηsp are the specific viscosity of poly-A in presence and in absence of CHL; tcomplex and tcontrol are the time of flow of complex and control solution; and t0 is the same for buffer solution, as described previously.52
Sensitization of alkaloid emission, quantum efficiency determination and fluorescence energy transfer. The excitation spectrum of CHL was recorded in the wavelength range 220–310 nm to study the energy transfer from the RNA bases to CHL. The excitation spectrum was recorded by monitoring the emission wavelength at 564 nm. This was further confirmed by recording the nucleobase-sensitized emission spectrum of CHL in the wavelength region 500–650 nm.53 The quantum efficiency (Φ) of a ligand is a measure of the amount of energy transfer from a nucleic acid to a ligand upon binding. Φ was evaluated for different wavelengths from the ratio of the quantum efficiency of the ligand (CHL) bound to nucleic acid (ϕb) to the quantum efficiency of free CHL (ϕf) using the equation:54
 
image file: c4ra07075e-t5.tif(5)
where Ib and If are the fluorescence intensities of bound and free CHL, respectively. These values were obtained during fluorescence titration of CHL by poly-A. εf is molar absorption value of free CHL, which is already known. The calculation to determine the εb value has been described by Garbett et al.55 The ratio between the quantum efficiency of bound CHL excitation in the UV spectral region (Φλ) to that at 310 nm (Φ310) was calculated. The wavelength 310 nm was chosen for the normalization process because of insignificant absorbance of RNA at this wavelength.
Circular dichroism spectral studies. Circular dichroism (CD) measurements were carried out on a PC-driven JASCO J815 spectropolarimeter (Jasco International Co., JAPAN) with an attached temperature controller and a thermal programmer model PFD-425L/15 interfaced in a rectangular quartz cuvette of 1 cm path length. All CD spectra were recorded in the wavelength range 200–450 nm with a scan speed 100 nm min−1. Each spectrum was averaged from five readings. Final CD spectra were expressed in terms of molar ellipticity ([θ]) in units of deg cm2 dmol−l using the software provided with the spectropolarimeter. The molar ellipticity is based on the poly-A concentration.
Thermal melting studies. The denaturation profiles of poly-A in the absence and in the presence of CHL were taken in the same spectropolarimeter by monitoring the change in CD at 264 nm. The rate of heating was 0.5 °C min−1 in the temperature range 15 °C to 100 °C.
Thermodynamic studies: temperature dependent spectrophotometry. Temperature-dependent absorption spectra were recorded using a Shimadzu UV-1800 double-beam spectrophotometer with an attached thermometric cell temperature programmer and temperature controller. These measurements were performed at 15, 20, 25 and 35 °C, allowing an equilibrium period of 10 min for each addition.
Evaluation of thermodynamic parameters. The values of K′ and n were determined at different temperatures using UV-visible spectrophotometric measurements. Thermodynamic parameters were estimated by analysis of a Van't Hoff plot (ln[thin space (1/6-em)]Kversus 1/T) obtained over the temperature range of the study. The slope of the plot gives the binding enthalpy change (ΔHo) as:
 
image file: c4ra07075e-t6.tif(6)

The Gibbs free energy change (ΔGo) was determined from the binding constant at a particular temperature according to the relation:

 
ΔGo = −RT[thin space (1/6-em)]ln(K′) (7)

The entropy change (ΔSo) was then estimated from the following equation:

 
ΔSo = (ΔHo − ΔGo)/T (8)

Results and discussion

UV-vis absorption spectral studies

It is known that binding of small molecules with nucleic acids can be monitored by employing absorption spectral titration when the former has absorption characteristics in regions where nucleic acids do not absorb.44 The absorption spectral study on the interaction of CHL with poly-A in CPB buffer at 25 °C is represented in Fig. 2A. With an increase in concentration of RNA, the maximum absorption peak (∼316 nm) of the alkaloid was red shifted to about 5 nm, and at the same time a significant hypochromic effect was noted. Such finding can be rationalized as follows: the empty π*-orbital of the ligand molecule couples with the π*-orbital of the RNA bases, causing an energy decrease, and a decrease of the π–π* transition energy. As a result, a bathochromic shift is noted. Again, the empty π*-orbital is partially filled with electrons to reduce the transition probability, which causes hypochromism.56 The presence of a sharp isosbestic point at 466 nm indicated the presence of two-state systems consisting of bound and free alkaloid species, enabling application of equilibrium conditions in the complexation. A summary of the optical properties of the free and RNA bound form of CHL are given in Table S1. The data obtained from reverse spectrophotometric titration (described earlier) were cast into the form of a Scatchard plot of r/Cf versus r (Fig. 3A). It was found that the plot was nonlinear, and that there was always a negative slope for all r values. Such negative slope values for the whole range of r indicated a noncooperative type of binding. In this case, it is to be noted that we could not reach a very low value of r when we used the direct titration of CHL with increasing amounts of poly-A. The plot was fitted and analyzed by non-cooperative binding using the McGhee–von Hippel equation.48 The binding constant was found to be (2.06 ± 0.1) × 107 M−1, and the number of bases occluded (n) for a single CHL molecule was ∼2. To date, such high binding affinity of any naturally occurring small molecule with poly-A has not been reported. The maximum binding affinity of a ligand, that includes either DNA intercalator or groove binder to poly-A has been reported to be around 1 × 107 M−1.18,21,22,57 Very recently, Kumar and his group reported the detailed investigation on the interaction of CHL with calf thymus (CT) DNA, where the binding affinity was in the order of ∼105 M−1.44
image file: c4ra07075e-f2.tif
Fig. 2 Representative absorption and fluorescence emission spectral changes of CHL in presence of poly-A in CPB buffer at 25 °C. (A) Curves 1–8 denote the absorption spectrum of CHL (5.06 μM) treated with 0, 1.8, 3.3, 4.9, 6.6, 8.4, 10.1 and 13.6 μM of poly-A, respectively. (B) Curves 1–9 denote the fluorescence spectrum of CHL (3.38 μM) treated with 0, 0.88, 1.95, 3.21, 4.77, 6.75, 8.94, 10.55 and 12.50 μM of poly-A, respectively. The excitation wavelength was 400 nm at a spectral excitation and emission bandwidth of 5 and 5 nm, respectively.

image file: c4ra07075e-f3.tif
Fig. 3 Scatchard plots of the binding of CHL with poly-A monitored from (A) absorption spectrophotometry and (B) spectrofluorimetry. The points in the figures represent the data points, and the solid line represents the best fit to the McGhee–von Hippel eqn (1). The experimental points are the average of four determinations.

Fluorescence spectral studies

Binding of CHL with poly-A was further investigated by fluorescence intensity measurements. The emission spectrum of CHL was recorded in the wavelength range 430–800 nm by excitation at 400 nm. The emission maximum was found around 564 nm when excited at 400 nm. Representative excitation spectra of the iminium and alkanolamine forms of CHL (keeping emission fixed at 564 nm) are shown in Fig. S1.

In the pH range above 10.0, CHL exists almost exclusively in the alkanolamine form, and it exists almost exclusively in the iminium form when the pH of the medium is below 6.7.44 Under our experimental conditions, pH was maintained at 6.5, which ensured that CHL was exclusively in the iminium form. From the absorption spectrum of the alkanolamine form, it is observed that there is no absorption around 400 nm, whereas for the iminium form of CHL, a strong absorption band is present around 400 nm. Thus, the absorption band around this wavelength is a characteristic feature of the iminium form and not the alkanolamine form. The excitation spectrum of the alkanolamine form of CHL (keeping emission fixed at 564 nm) shows the absence of any band around 400 nm, whereas there is a strong band in the iminium form spectrum. From the absorption and excitation spectra of the two forms of CHL, it is clear that the emission at 564 nm for CHL was solely produced by the iminium form of the alkaloid when excited at 400 nm (though presence of very trace amounts of alkanolamine form at equilibrium cannot be ruled out). In other words, excitation wavelength of 400 nm instead of 316 nm or 350 nm was chosen to ensure the excitation of iminium form of CHL only and not the alkanolamine form. In our study, we are only interested with the iminium form of the alkaloid.

Complex formation was monitored by titration studies, keeping a constant concentration of the alkaloid and increasing the concentration of poly-A. With increasing concentration of poly-A, a progressive enhancement in the fluorescence intensity of the alkaloid was observed and eventually reached a saturation point with a ∼3 nm blue shift in the wavelength maximum (Fig. 2B). The results of the spectrofluorimetric titrations were analyzed by constructing Scatchard plots. The Scatchard plot (Fig. 3B) exhibited non-cooperative behaviour, as revealed by the negative slope, and hence was analyzed further by the McGhee–von Hippel methodology for non-cooperative binding. Analysis yielded a binding constant of (2.14 ± 0.07) × 107 M−1 and n value of 2.32 ± 0.04 (at 25 °C). The binding parameters obtained from spectrofluorimetry are in excellent agreement with the spectrophotometric results (Table 1).

Table 1 Binding parameters for the interaction of CHL with poly-A in CPB buffer at 25 °C obtained from spectrophotometry and spectrofluorimetrya
Parameters Methods Values
a Average of three determinations.
K′ × 10−7, the intrinsic binding constant (M−1) [A] Spectrophotometry 2.06 ± 0.10
[B] Spectrofluorimetry 2.14 ± 0.07
N, the no of base occluded [A] Spectrophotometry 1.80 ± 0.05
[B] Spectrofluorimetry 2.32 ± 0.04
KSV, the Stern–Volmer quenching constant (L mol−1) Spectrofluorimetry [i] Free: 12.1 ± 1.30
[ii] Bound: 8.5 ± 0.70
Fluorescence polarization anisotropy Spectrofluorimetry [i] Free: 0.040 ± 0.002
[ii] Bound: 0.13 ± 0.01


Evaluation of binding stoichiometry

The stoichiometry of association of CHL with poly-A was determined independently by continuous variation analysis (Job's plot) in fluorescence spectroscopy. Throughout the experiment, total concentration of poly-A and CHL was fixed at 10 μM. The mole fraction of CHL was varied from 0 to 1. The ratio of the difference in fluorescence intensity ΔF (difference in fluorescence intensity of CHL in presence and absence of poly-A) and the intensity of free CHL (Fo) was plotted against the mole fraction of CHL (Fig. S2). An inflection point was observed at χCHL = 0.34. The stoichiometry of the poly-A–CHL was found to be 2[thin space (1/6-em)]:[thin space (1/6-em)]1 in terms of poly-A[thin space (1/6-em)]:[thin space (1/6-em)]CHL. This value is in good agreement with the values calculated from spectrophotometric and spectrofluorimetric titrations (Table 1).

Fluorescence polarization anisotropy

Fluorescence anisotropy measurements provide effective information about the nature of the environment of biological probes. Any factor affecting the shape, size and flexibility of a molecule affects the observed anisotropy.51 Increase in the rigidity of the environment surrounding the fluorophore is manifested in an increase in the fluorescence anisotropy. Change in anisotropy can thus help us to find the probable location of a probe in micro-heterogeneous environments, such as proteins and nucleic acids.58 The variation of fluorescence anisotropy of CHL with increasing concentration of poly-A is shown in Fig. S3. A significant increase in the fluorescence anisotropy on binding with poly-A suggested that the dye was trapped in a motionally restricted region within the polynucleotide. The fluorescence polarization upon binding of CHL to the poly-A showed a value of ∼0.13 at saturation, against a value of ∼0.04 for free CHL under identical conditions. Our result indicates that CHL binds strongly to the poly-A structure, probably by intercalation between the bases.

Fluorescence quenching study and mode of binding

Fluorescence quenching experiments provide an effective method to address the mode of binding of small ligands to nucleic acids.51 In principle, molecules that are either free or bound on the surface of nucleic acid are easily accessible by the quencher, whereas those that are inserted between the bases of the polynucleotide may be inaccessible to the quencher. Negative charge on the phosphate groups causes an electrostatic barrier at the helix surface and limits the penetration of an anionic quencher into the interior core of the helix. Hence, very little or no quenching may be observed in the presence of such a quencher, if the binding involves strong stacking. As a result, the magnitude of the Stern–Volmer quenching constant (KSV) of the ligands that are bound inside will be lower than that of the free molecules. It is known that intercalation of small molecules into the DNA double strands protects the entrapped molecules from an ionic quencher.59–61 Electrostatic binding and groove binding, on the contrary, leave the probe molecule exposed to the bulk aqueous phase and do not appreciably obstruct the approach of the quencher.59 Compared with intercalative binding, groove binding offers much less protection for the fluorophore.61 It is observed that binding to the poly-A resulted in an increase in fluorescence intensity of CHL (Fig. 2B). Representative Stern–Volmer plots for free and RNA-bound CHL are shown in Fig. S4. KSV values for free CHL and its complex with poly-A were 12.5 and 6.2 L mol−1, respectively. This indicates that the bound CHL is less accessible to the quencher, which in turn indicates that the bound alkaloid molecules are considerably protected and sequestered away from the solvent. This observation leads us to suggest an intercalative mode of binding of CHL with poly-A. A similar observation has been reported for the binding of CHL to CT DNA.44 We have done the quenching experiments in solutions of higher ionic strength (in presence of 0.1 M NaCl), but the Stern–Volmer quenching constant was found to be almost unaffected. This also confirmed the binding mode to be intercalation and not groove binding.

Viscometric study

To investigate the mode of binding of the alkaloid to the poly-A helix, a viscosity study was undertaken (Fig. 4). The relative specific viscosity of the poly-A–alkaloid complex increased with increase in D/P. As hydrodynamic measurements are sensitive to length changes, they are regarded as the most critical for elucidating the binding mode of small molecules to nucleic acids in solution.59 Our results clearly underscore the intercalation type of binding of CHL to the poly-A helix. Nevertheless, because single-stranded poly-A has only a stacked helical structure (no base pairing), a true intercalation model59 where planar ligand molecules are fully sandwiched between hydrogen-bonded base pairs of double-stranded DNA cannot be ideated. This data, along with the hypochromism in the absorbance spectrum and fluorescence quenching results, supports an intercalating complexation of CHL with poly-A.
image file: c4ra07075e-f4.tif
Fig. 4 A plot of change of relative specific viscosity of poly-A with increasing concentration of CHL in CPB buffer at 25 °C. The concentration of poly-A was 500 μM.

Energy transfer from RNA bases to bound CHL

Strong interaction between CHL and poly-A was evidenced from spectrophotometric and spectrofluorimetric titration. Fluorescence quenching studies, along with viscometric data, clearly indicate intercalative binding of the alkaloid to the poly-A helix. The chance of excited energy transfer from the bases of poly-A to the bound CHL molecules (Fig. 5D) was explored by measuring the excitation spectrum of CHL. Fluorescence emission was monitored at 564 nm, and the excitation was varied from 220 to 550 nm. Energy transfer was studied by recording the excitation spectrum of CHL in presence of poly-A (in the concentration ratio CHL[thin space (1/6-em)]:[thin space (1/6-em)]poly-A = 1[thin space (1/6-em)]:[thin space (1/6-em)]10). In absence of poly-A, the observed excitation spectrum of CHL (curve 1, Fig. S5) matches that of its absorption spectrum in the wavelength region 300–550 nm (curve 1, panel A, Fig. S1). But in this case, there was almost an absence of any band in the excitation spectrum around 260–280 nm, though there was presence of a strong peak around this wavelength in the original alkaloid absorption spectrum (curve 1, panel A, Fig. S1). This observation indicated that the emission of CHL at 564 nm might not be due to the absorption around the 260–280 nm band. This was further confirmed by the emission spectrum of free CHL when it was excited at 270 nm. In this case, we did not get any significant emission in the wavelength range 300–600 nm. The absorption of CHL in the region of 220–300 nm is weak compared with that of RNA, which shows a strong absorption around 260 nm. In the excitation spectrum of RNA-bound CHL, we observed very high intensity in the wave length region of 220–300 nm (not shown), which was very weak in the case of free CHL. In control experiments, pure RNA solutions showed almost no excitation bands in the 220–300 nm region when monitored at 564 nm (curve 2 of Fig. S5). The ratio of excitation spectra of the CHL–poly-A complex is presented in Fig. 5A. This plot indicates direct emission from CHL, and the ratio greater than unity indicates sensitization by RNA. Appearance of a very strong excitation band around 270 nm in the bound complex can only be explained on the basis of absorption of energy and subsequently its transfer by the RNA bases to the alkaloid. Absence of any band in the excitation spectrum of RNA in the wavelength region 220–300 nm, when emission was monitored at 564 nm, is consistent with the interpretation of energy transfer from RNA to CHL. The energy transfer from RNA to the alkaloid was further confirmed by the sensitized emission of the complex, which is more intense than the direct emission of CHL (Fig. 5B). Fluorescence energy transfer from the RNA bases to the bound CHL molecules was manifested in the enhancement of the quantum efficiency of bound molecules in the wavelength range of RNA absorption. It has been demonstrated earlier that such type of energy transfer from DNA base to ligand occurs efficiently when the ligand is intercalated in a DNA structure in which the DNA bases are in close contact with the ligand.54 Similar observations have also been reported by Kumar et al.62 They have shown that such energy transfer depends on the sequence of nucleic acid polymer. As energy transfer occurs due to close contact, from such a sensitization study, both strong binding as well as the intercalative mode of binding of CHL to poly-A is further confirmed. A plot of Φλ/Φ310 against wavelength at a P/D (RNA base/CHL molar ratio) of 10 is shown in Fig. 5C. An increase in quantum efficiency in the RNA absorption region was observed. This implies energy transfer from the RNA bases to bound alkaloid molecules and also indicates an intercalative mode of binding. A representative scheme for energy transfer is shown in Fig. 5D. Such fluorescence energy transfer has been reported in the case of CHL–DNA binding.44 Thus, from our study, along with the study of Pritha et al., it can be concluded that both DNA and RNA bases can transfer energy to CHL when the alkaloid is intercalated in the DNA/RNA structure.
image file: c4ra07075e-f5.tif
Fig. 5 (A) Fluorescence excitation spectrum of CHL recorded in presence of poly-A at emission of wavelength 564 nm. (B) Sensitized fluorescence spectra of CHL in presence (curve 1) and absence of poly-A. (C) Variation of relative quantum efficiency of CHL in the presence of poly-A. (D) Energy transfer scheme illustrating the sensitization of alkaloid emission by the RNA bases. All spectra were taken in CPB buffer at 25 °C.

Spectropolarimetric results

CD spectral study on the binding of CHL to poly-A. Conformational changes in poly-A on binding of CHL were investigated from intrinsic circular dichroism studies. The intrinsic CD spectra of poly-A showed a large positive band around 262 nm and a negative band around 245 nm followed by a small positive band around 220 nm. On the other hand, the benzophenanthridine alkaloid, CHL, does not have any intrinsic CD activity. To examine the CHL-induced changes in the conformation of poly-A, the CD spectra was recorded in the region of 200–400 nm under varying D/P values. CD spectral changes of poly-A on interaction with CHL are presented in Fig. 6. The CD spectra demonstrate that the conformation of poly-A undergoes a noticeable change in presence of CHL. The decrease in intensity of CD bands around 245 and 262 nm implies an alteration of the poly-A structure upon addition of CHL. The broad band at 262 nm disappeared at saturation, whereas an increase of the negative band maximum at ∼245 nm was observed until the saturation point. A similar observation has been reported for the interaction of coralyne with poly-A.18 Four clear isoelliptic points at 210, 238, 251 and 320 nm were observed for the poly-A–CHL interaction. The appearance of the induced CD band in the region 290–370 nm clearly indicates the binding of CHL in the asymmetric environment of the poly-A helix.
image file: c4ra07075e-f6.tif
Fig. 6 Circular dichroism spectra of poly-A (50.0 μM) with varying concentrations of CHL in CPB buffer at 25 °C. The curves 1–8 represent the CHL concentrations of 0, 3.4, 6.8, 10.1, 13.5, 15.1, 16.8 and 18.5 μM, respectively.
CD melting study. The CD data we have presented previously clearly suggested the alteration of the poly-A secondary structure in presence of CHL and also a strong asymmetric environment of the bound alkaloid in the helical structure of the polynucleotide. This is presumably due to intercalative binding. Similar types of observations on the interaction of another benzophenanthridine alkaloid, sanguinarine, with poly-A have been reported by Giri and Kumar.21 More often in DNAs, the alteration from A to B-form and from B to C-like forms manifest as large decrease of ellipticity of the long wavelength band. A direct correlation of such alteration of the CD bands with the helix parameters are complex.63 However, an ordered structural transition, such as the formation of a duplex structure, can be assumed to occur, and this may be assisted by the efficient screening of charges of the phosphate backbone by the intercalated positively charged CHL. Giri and Kumar have shown the self-structure formation of poly-A by the benzophenanthridine alkaloid sanguinarine.21 Ren and colleagues reported similar self-structure formation in poly-A in presence of coralyne.18 Prior to that, Hud's laboratory had observed the ability of coralyne to dissociate the duplex poly(dA):poly(dT) into poly(dT):poly(dA):poly(dT) triplex and a coralyne-poly(dA) self-structure.64 To examine whether the binding of CHL could induce the self-structure formation in poly-A, we investigated the melting behaviour of poly-A employing CD melting. The characteristic CD spectra of single-stranded poly-A in absence and in presence of CHL are shown in Fig. 7A. The melting was monitored at 264 nm; the profiles are depicted in Fig. 7B and C. We found cooperative melting of poly-A–CHL complex in CD (Fig. 7C), whereas no such cooperative change was found in case of free poly-A (Fig. 7B). In absence of CHL, the slow change in CD is attributed to the helix-to-coil transition of poly-A.65 The melting temperature of the poly-A–CHL complex was found to be ∼62 °C. Such cooperative transition in the melting profile of the complex unequivocally confirmed the formation of a self-assembly structure. Self-assembled structure induction in poly-A by planar molecules has been supported by intercalative geometry, and the melting results confirm such helical organization induced by CHL. Induction of self-structure in poly-A by different DNA intercalators, partial intercalators and groove binders has been reported.22,57 From the observations of Kumar et al., it appears that compounds which bind cooperatively, only induce self-structure formation in poly-A. This is found to be true for many small molecules. But in our study, we have observed the induction of self-structure in poly-A by CHL although the alkaloid binds noncooperatively (as evidenced from Scatchard analysis) to the polynucleotide.
image file: c4ra07075e-f7.tif
Fig. 7 (A) CD spectra of poly-A (50.0 μM) in absence (red) and presence of (blue) 15.1 μM CHL in CPB buffer at 25 °C. (B) CD melting profile of free poly-A (50.0 μM) in CPB buffer. (C) CD melting profile of a solution containing poly-A (50.0 μM) in presence of 15.1 μM CHL in CPB buffer.

Thermodynamics of the interaction

Thermodynamic parameters for the interaction of CHL with poly-A were evaluated from the temperature dependence of binding constants using UV-vis absorption spectroscopic studies. Spectrophotometric titrations were carried out at four temperatures: 15, 20, 25 and 35 °C in CPB buffer; the data are presented in Table 2. The binding constant obtained from analysis of the Scatchard plot was found to decrease with increase in temperature, whereas the number of nucleotide-occluded sites (n) changed only marginally (Table 2). The Van't Hoff plot for binding is shown in Fig. 8; the linear Van't Hoff plot indicates a very small change in the value of heat capacity (ΔCP ≈ 0). The values of the thermodynamic parameters are presented in Table 2. The binding was characterized by both negative enthalpy and entropy changes. This indicates that binding of CHL to poly-A is predominantly enthalpy-driven.
Table 2 Binding and thermodynamic parameters for the interaction of CHL with poly-A in CPB buffer obtained from absorption spectrophotometrya
Temperature (°C) K′ × 10−7 (M−1) n ΔGo (kJ mol−1) at 25 °C ΔHo (kJ mol−1) TΔSo (kJ mol−1) at 25 °C
a Average of three determinations.
15 4.49 ± 0.20 1.75 ± 0.10 −41.75 ± 2.0 −50.66 ± 2.5 −8.91 ± 0.50
20 3.05 ± 0.15 1.79 ± 0.15
25 2.06 ± 0.10 1.80 ± 0.20
35 1.14 ± 0.08 1.90 ± 0.30



image file: c4ra07075e-f8.tif
Fig. 8 Van't Hoff plot for the complexation of CHL with poly-A in CPB buffer.

The thermodynamic parameters for the binding process are contributed by three major components: [i] contributions from hydrogen bonding and the hydrophobic part due to interactions between the bound ligand and nucleic acid binding site; [ii] contribution from the conformational changes upon binding to either the nucleic acid or the ligand or both; and [iii] contributions from coupled processes, such as proton transfer, ion release or changes in the water of hydration. Our data revealed that the binding process was favoured by negative enthalpy change and was opposed by negative entropy change. The possible contribution of negative enthalpy change for the binding process of CHL to poly-A may be attributed to the van der Waals stacking interactions, hydrophobic as well as weak electrostatic interactions. The negative entropy change can be rationalized due to the formation of ordered self-structure and the intercalation of CHL, which causes loss of translational and rotational degrees of freedom. Such behaviour has also been reported for the interaction of another similar bezophenanthridine analogue, sanguinarine, with poly-A.21

Conclusion

In summary, in this study we have reported very high binding affinity of a naturally occurring benzophenanthridine alkaloid CHL to poly-A. From the various spectroscopic studies, we have shown that CHL could induce self-structure in poly-A. The binding constant was found to be very high—of the order of 107 M−1. Viscometric data along fluorescence quenching results revealed that the mode of binding of CHL to poly-A was intercalation that leads to self-structure induction in the polymer. The sensitized fluorescence experiment suggested that there was energy transfer from RNA bases to CHL, which in turn corroborated the intercalative mode of binding of CHL to poly-A. Thermodynamic parameters for the binding process revealed both negative enthalpy and entropy changes. As the interaction of different polymorphic forms of RNA with naturally occurring small molecules is an active area of research in medicinal chemistry and chemical biology, this study renders the scope of exploring the benzophenanthridine group of alkaloids as antiviral drugs.

Acknowledgements

ABP and LH thank the University Grant Commission, Government of India, for the award of Junior Research Fellowship. SB thanks the University Grant Commission, Government of India, for the award of RGNF research Fellowship.

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Footnotes

Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra07075e
These authors contributed equally to this work.

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