Spectroscopic, electrochemical and molecular docking study of the binding interaction of a small molecule 5H-naptho[2,1-f][1,2] oxathieaphine 2,2-dioxide with calf thymus DNA

International Journal of Biological Macromolecules 101 (2017) 527–535

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Spectroscopic, electrochemical and molecular docking study of the binding interaction of a small molecule 5H-naptho[2,1-f][1,2] oxathieaphine 2,2-dioxide with calf thymus DNA Abhijit Mukherjee a,∗ , Shovan Mondal b , Bula Singh a,∗ a b

Department of Chemistry, Siksha-Bhavana, Institute of Science, Visva-Bharati, Santiniketan-731235, W.B., India Department of Chemistry, Syamsundar College, University of Burdwan, Shyamsundar-713424, W.B., India

a r t i c l e

i n f o

Article history: Received 4 February 2017 Received in revised form 9 March 2017 Accepted 10 March 2017 Available online 14 March 2017 Keywords: Calf thymus DNA NOTD Binding interaction Emission Displacement assay Denaturation Salt effect Circular dichroism Cyclic voltammetry Molecular docking

a b s t r a c t The interaction of 5H-naptho[2,1-f][1,2]oxathieaphine2,2-dioxide (NOTD) with calf thymus DNA in TrisHCl buffer at physiological pH was investigated with the help of various spectroscopic and electrochemical methods along with molecular docking study. Studying the non-covalent binding interaction of a neutral fluorophore with ctDNA has become an active field of research at the interface between medicinal chemistry and biological science. NOTD is known for its various toxicological, skin sensitization, and antiviral properties. Still, to date, its interaction style with ctDNA is not well elucidated. UV–vis absorption, fluorescence emission and circular dichroism spectroscopy (CD) suggest the complex formation between NOTD and ctDNA with binding constant value in the order of 3.12-4.1( × 104 ) M−1 . Binding nature of NOTD with ctDNA is affirmed from the DNA helix melting experiment, comparative displacement assay using known DNA intercalator, cyclic voltammetry and finally molecular docking study. It was evident from experimental result that the probe NOTD binds with ctDNA in groove binding mode as manifested by a decrease in iodide quenching effect, spectral change in CD, a substantial increase in denaturing temperature in DNA and change in potential value. Furthermore, the molecular docking study insisted the above mentioned experimental result in a very affectionate way. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Deoxyribonucleic acid (DNA) is a significant biological macromolecule that bears crucial genetic information for the soundly functioning and exploitation of living organisms. It is engaged in many biological vital processes like gene expression, mutagenesis, transcriptions, enzyme synthesis, carcinogenesis, etc. [1–5]. DNA is conceived to be the superior intracellular object for various pharmaceutical agents because the breakdown of DNA helix has important significances in medicinal chemistry, biological science as well as in biotechnology. In recent days, developing and designing of potent, efficient and exclusive pharmaceutical agents for DNA objects with fewer side effects has become a dynamic field of research for therapeutic agents against many DNA-related diseases [2,6]. For designing a potent pharmaceutical agent in the behalf as mentioned earlier, it’s essential to realize the binding style of small dye/drug molecule explicitly with DNA. Consequently, there

∗ Corresponding authors. E-mail addresses: [email protected] (A. Mukherjee), [email protected] (B. Singh). http://dx.doi.org/10.1016/j.ijbiomac.2017.03.053 0141-8130/© 2017 Elsevier B.V. All rights reserved.

is a never-ending curiosity in exploring the supramolecular binding approach of different small molecules with double helical DNA to interpret the fundamental interaction mechanisms, excepting that such studies will be helpful in predicting the probable effect of interactions of small molecules with DNA. There are different manners by which small molecules can bind to DNA host. These binding interactions can occur either through the covalent or noncovalent way. Because of their dynamic and reversible in nature, noncovalent interactions are invariably the most trusted for small molecule-DNA interaction over covalent binding. Generally, double-helix DNA strands binds with small molecule through three different noncovalent modes which are (i) electrostatic interaction: attractive force between the positively charged end of small molecules and anionic sugar phosphate anchor of DNA; (ii) groove binding: mainly governed by van der Waals’ interactions or hydrogen bonding with small molecule and nucleic acid bases in the major groove or the shallow minor groove; and (iii) embedding in base pairs which are stacked and result of this distorting the DNA backbone [7,8]. Works of literature suggest that there are diverse reports available which consist the fundamental interaction of positive and negatively charged small molecules with ctDNA [3,9]. However, to

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2.3. Instrumentation and methods

Scheme1. Schematic and optimized structures of NOTD.

the best of our knowledge, no such spectroscopic, electrochemical and molecular docking study done so far with neutral sultones like fluorophore compound 5H-naptho [2,1-f] [1,2] oxathieaphine 2, 2dioxide (NOTD) represent in Scheme 1. In our present study, we have attained a generative undertake to clarify the binding interaction of a neutral probe NOTD with ctDNA. Besides their synthetic application, NOTD scaffolds are in huge demand in medicinal science research [10]. In biological science, it is very familiar for its toxicological, skin sensitization, and antiviral activities [11]. In the present context, the binding features of a neutral probe NOTD with ctDNA in aqueous buffer solution at a physiological pH (=7.4) were investigated by applying several spectroscopic methods including UV–vis absorption spectroscopy, steady-state and time-resolved fluorescence, comparative displacement assay by the well-known DNA intercalators ethidium bromide (EtBr), acridine orange (AO), Hoechst 33258, circular dichroism (CD) spectral study, cyclic voltammetry (CV) and helix melting temperature. These studies were carried to find out binding constant, binding style and conformational shifts related with ctDNA upon interaction with NOTD. Molecular docking analysis confirms the experimental outcome in a nice way picturing the non intercalative i.e. minor groove binding of the small molecule NOTD with ctDNA. 2. Experimental

2.3.1. UV–vis spectroscopic method For UV–vis absorption spectra measurement, Shimadzu (model UV1800) spectrophotometer was used, in the wavelength range 200–350 nm. The thermal melting study of ctDNA and NOTD bound ctDNA complex was carried out on the same spectrophotometer well fitted with the peltier operating setup. The experiment was conducted in the presence of defined concentration of NOTD (15 ␮M) in a particular volume and quantified with changing concentration of ctDNA 0–25 ␮M. Using same ctDNA concentrations without NOTD were employed as the blank to notice the UV–vis spectra especial to NOTD-ctDNA complex. 2.3.2. Steady state fluorescence Emission spectra of NOTD have recorded in Perkin-Elmer fluorescence spectrophotometer (LS 55) fitted with xenon flash lamp utilizing 1.0 cm quartz cells. Exciting the sample at 265 nm the fluorescence emission spectra have been registered after adjusting the wavelength from 300 to 500 nm and both the excitation and the emission slits of 2.5 nm. Proper blank analogous to the buffer were deduced to appropriate the background fluorescence. By keeping the constant concentration of NOTD (15 ␮M), the fluorescence titration was carried out varying ctDNA concentration from 0 to 24 ␮M. 2.3.3. Time-resolved fluorescence decay measurements Time-correlated single photon counting (TCSPC) technique was used for fluorescence lifetime measurements of NOTD [19]. The samples were excited at 280 nm employing a picoseconds diode laser (IBH-NanoLED source N-280). Using a Hamamatsu MCP Photomultiplier (Model R-3809U-50), the emission spectra was gathered at magic angle polarization. The TCSPC apparatus consist of an Ortec 9327 Pico-timing amplifier. The data was accumulated with a PCI-6602 interface card as a multi-channel analyzer. The distinctive full width at half maximum (fwhm) of the system response was about 820 ps. The emission was fixed at 356 nm. For analyzing multiexponential fluorescence decay (I(t)) the following expression was employed [14]. I (t) =



2.2. Sample preparation The stock solution of NOTD (10 mM) was made in 5% absolute methanol. The ctDNA solution was obtained by dissolving the proper quantity of solid fiber ctDNA in Tris-HCl buffer maintaining pH = 7.4 and placed at 277 K. The integrity of ctDNA solution was checked by control the ratio of absorbance at 260 nm to that at 280 nm, which was in the limit 1.8–1.9. Spectrophotometrically, the concentration of the ctDNA solution, was predicted having the molar extinction coefficient εDNA = 6600 M−1 cm−1 at 260 nm [13].



(1)

i

2.1. Materials 5H-naptho[2,1-f][1,2]oxathieaphine2,2-dioxide (NOTD) was synthesized employing the method described elsewhere [12]. The product was purified through column chromatography and purity of the compound was further assured by thin layer chromatography (TLC). The sodium salt of calf thymus DNA, ethidium bromide (EB), Hoechst 33258 and urea bought from Sigma-Aldrich, USA. Acridine orange (AO) obtained from Alfa Aesar. Solid Tris hydrochloride buffer and potassium iodide (KI) of AR grade were received from Sisco Research Laborites Pvt.Ltd., India. Throughout the experiment deionised Milli-Q water (Millipore) was used. All the experimentations were executed using 0.01 M Tris-HCl buffer at pH = 7.4.



˛i exp −t/i

In which ˛i stands for the pre-exponential factor (amplitude) regarding the ith decay time constant,  i [19]. The intensity of fluorescence of biexponential decay for a given  is given by [15]

∞ F =

F (t) dt

(2)

0

Average (mean) fluorescence lifetimes ( avg ) for biexponential iterative fittings were accounted from the decay times ( 1 , 2 ) and the pre-exponential factors (a1 and a2 ) applying the following relation avg = 1 a1 + 2 a2

(3)

2.3.4. Potassium iodide (KI) quenching method Iodide quenching experimentation was performed after exciting NOTD (30 ␮M) at 268 nm and listing the fluorescence spectral data in the presence of increasing concentration of KI (0–13 mM), both in the presence and absence of ctDNA (30 ␮M). Corresponding emission spectra was recorded from 320 to 430 nm. With the help of Stern-Volmer equation binding constant (Ksv ) was determined both in absence and presence of ctDNA [16]. F0 = 1 + KSV [Q ] F

(4)

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where F0, F are the fluorescence intensities of NOTD in absence and in the presence of quencher and [Q] is the concentration of KI. 2.3.5. Effect of urea For the further studies of the binding interaction with the help of denaturation study, we have used urea as a denaturizing agent. A certain volume of NOTD bound ctDNA complex, in which both are at concentration of 30 ␮M, was titrated with varying the urea concentration from 0 to 1 M. On exciting the NOTD bound ctDNA complex at 265 nm corresponding emission spectra was recorded from the range 300–500 nm. 2.3.6. Comparative displacement assay using known DNA intercalators for binding study Displacement assay was done as early reported by known intercalator’s ethidium bromide, acridine orange and Hoechst 33258 [17]. In the first titration monitoring the emission spectra of ctDNAEtBr (50 ␮M & 5 ␮M of each) complex in the subsequent change of NOTD concentrations from 0 to 60 ␮M, to assure the binding of NOTD with ctDNA. The exciting wavelength for the EB bound ctDNA complex was 476 nm and corresponding emission spectra were recorded between 520 and 680 nm. Similarly, second and third titration have done monitoring the emission spectra of ctDNA-AO complex (50 ␮M & 5 ␮M of each) and ctDNA-Hoechst33258 complex (50 ␮M & 5 ␮M of each) in the presence of same concentration range of NOTD as mentioned earlier, to assure the binding of NOTD with ctDNA. After exciting the AO and Hoechst 33258 bound ctDNA complex at 460 nm and 345 nm emission spectra were recorded between the wavelength region 480–630 nm and 400–600 nm. 2.3.7. Circular dichroism spectroscopy The CD spectra of free ctDNA (20 ␮M) and with raising concentrations of NOTD were recorded on a JASCO (J-815) spectropolarimeter which was connected with an extremely sensitive temperature controller unit, applying a cylindrical cuvette of 0.1 cm path-length at 298 K. The described CD profiles were recorded in a range from 200 to 350 nm with a mean of four consecutive scans obtained at 20 nm min−1 scan rate with properly rectified baseline. The background spectrum of 0.01 M Tris-HCl buffer solution was subtracted from the spectra of ctDNA and the NOTD-ctDNA complex. 2.3.8. Effect of ionic strength The role of ionic strength on the binding interaction between the NOTD and ctDNA was studied by changing the NaCl concentration (0–25 mM) in a constant volume which contains an equal concentration of NOTD and ctDNA. The excitation wavelength was 265 nm and the emission spectra were recorded between 360 and 560 nm in 0.01 M Tris-HCl at pH 7.4.

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implemented therein. From the Protein Data Bank the endemic 3D structure of B-DNA (for docking analysis) has consented bearing PDB ID: 1BNA [16]. The necessary file of the NOTD, for docking of NOTD with DNA, was produced through coupled usage of Gaussian 03W [19] and Auto Dock 4.2 [20] software packages. Appling Gaussian 03W suite of programs the geometry of NOTD was first optimized at DFT//B3LYP/6-31G (d, p) level of theory and the resultant geometry was read in Auto Dock 4.2 software incompatible file format, from which the necessary file was created in Auto Dock 4.2. After adjusting the grid size to 60, 60, and 120 Å along X-, Y-, and Z-axis with a spacing of grid 0.375 Å. For the auto docking the following parameters are used. These are as follows: GA population size = 150; maximum number of energy evaluations = 2500000; GA crossover mode = two points. For each and every docking simulation the conformer which has lowest binding energy was searched out of ten different conformations, and for further analysis, consequent minimum energy structure was applied. The PyMOL software package was employed for visualizations of the docked conformation [21]. 3. Results and discussion 3.1. UV–vis absorption spectroscopy Examining the binding interaction of the small molecule with ctDNA is a significant step to perceive their mechanism of action. Absorption spectroscopy is one of the most fruitful techniques in detecting the primal interaction between NOTD with ctDNA and complex formation. Fig. 1 shows the absorption spectra of NOTD in the absence and presence of a variable ctDNA concentration. With the increase of ctDNA concentration, the absorption spectra of NOTD were continuous increases unchanging the position of the band. Generally, hyperchromism has been connected with the presence of diverse non-covalent interactions which take place outside the DNA helix [17]. These non-covalent type interactions may cover electrostatic force of binding, groove binding, and hydrogen bonding interaction. Also, absence of clear isosbestic points and enhancement of NOTD absorbance on the addition of ctDNA to NOTD buffer solution confirms the ctDNA and NOTD binding interaction. To know about the binding strength between the ctDNA and NOTD the equation was implemented [17]: 1 εNOTD εNOTD 1 = + × A − A0 εB εB C

(5)

where the terms have usual meaning A0 and A are absorbance of NOTD in absence and presence of ctDNA, εB and εNOTD are the molar extinction coefficient of NOTD bound ctDNA complex and NOTD,

2.3.9. Voltammetric study Cyclic voltammetry (CV) experiment was carried out using a potentiostat/galvanostat; model Versa Stat-IITM (Princeton Applied Research). A Pt wire (1 cm2 ), a saturated calomel and the Pt disk were used as a counter, reference and working electrode. Electrochemical measurements were conducted in 0.005 M K3 [Fe (CN)6 ] and 0.1 M KCl in 0.01 M Tris-Hcl buffer solution (pH 7.4) containing 21.5 ␮M NOTD. Fixed volume of NOTD solution with concentration 21.5 ␮M was titrated with successive addition of ctDNA solutions. In each and every CV measurement was taken after attaining equilibrium with electrodes [18]. 2.3.10. Molecular docking The molecular docking analysis for the binding interaction of NOTD with ctDNA, was carried out with Auto Dock 4.2 set of programs which utilizes the Lamarckian Genetic Algorithm (LGA)

Fig. 1. Interaction of NOTD with ctDNA using UV–vis spectroscopy.UV–vis absorption spectra of NOTD (20 ␮M) in presence of increasing concentrations of ctDNA (␮M) (i) → (vi): 0, 5, 10, 15, 20, 25 in 0.01 M Tris-HCl buffer (pH 7.4). Spectra were recorded in the range of 200–280 nm.

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Fig. 2. (a) Emission profiles of NOTD (ex = 265 nm) in the presence of (i) → (ix): 0, 3,6,9,12,15,18,21,24 ␮M ctDNA, respectively, in aqueous Tris-Hcl buffer (pH 7.4). (b) Double reciprocal plot for the binding of NOTD and ctDNA from fluorescence data.

Fig. 3. (a) Time resolved fluorescence decays of NOTD in the absence and presence of ctDNA. The sharp black profile is the lamp profile. Concentrations of ctDNA are labeled in the legends ␭exc = 280 nm, ␭em fixed at 365 nm. [NOTD] = 20 ␮M. (b) Graphical plot of average lifetime of NOTD with increasing ctDNA concentration.

concentration of ctDNA indicated by C and binding constant was denoted by K. From the graphical plot shown in Fig.S1 we get the binding constant value. Here we found the value 4.01 × 104 M−1 which was obtained from the ratio of the intercept (4.73) to the slope (1.177 × 10−4 M). Further experiments were performed to details study of the binding mode of ctDNA with small molecule NOTD.

3.2. Steady state fluorescence In Tris-HCl buffer, upon excitation at 265 nm NOTD exhibit an unstructured and single emission spectrum band peaking at around 370 nm [22]. A remarkable enhancement of the emission intensity along with a considerable hypsochromic shift near about 12 nm is observed with the continuous addition of ctDNA to the buffered solution of NOTD. Plotting the change of the fluorescence intensity and maximum emission of NOTD against ctDNA concentration we have achieve a clear image of NOTD-ctDNA binding interaction shown in Fig. 2a and 2b. The figure brings out that emission intensity of NOTD increases with the simultaneous addition of ctDNA. The maximum of the NOTD emission band also exhibits a gradual blue shift and then remains unchanged after a particular concentration of ctDNA. The variation in the fluorometric response intimates binding of NOTD with the ctDNA. In the emission spectra of the NOTD in ctDNA environment the noticeable blue shift reveals that the probe is feeling a lesser extent of polar environment than the aqueous buffer. Since the value of binding constant supplies an idea about the binding strength between NOTD and the macromolecule, we have used fluorescence titration data to determine the binding constant using the modified Benesi-Hildebrand equation [23] as given below [24].

1 1 1 1 = + × K.Fmax F Fmax [L]

(6)

where F = FX − F0 and Fmax = F∞ − F0 and F0, FX, F∞ are the fluorescence intensities of NOTD in absent of ctDNA, at an intermediate ctDNA concentration, and at a concentration for complete interaction respectively; the binding constant value is denoted by K and [L] represent the ctDNA concentration. After fluorescence measurement the plot of 1/F vs.1/[ctDNA] shown in Fig. 2b gives a straight line with R = 0.99164 and suggesting the 1:1 interaction between the ctDNA and NOTD. For the present system K value is calculated from the ratio of the intercept (3.08 × 10−7 ) to the slope (9.88 × 10−12 ) of the plot, the determined value comes out to be 3.12 × 104 M−1 at 298 K. The calculated binding constant value (normal range of binding constant for non-intercalating ∼103 –104 M−1 ) indicates groove binding nature [13] of the NOTD in the ctDNA environment since for intercalative binding, the binding constants are known to be much higher (∼105 M−1 ) [13,25].

3.3. Time-correlated fluorescence decay study The fluorescence lifetime measurement often serves as a sensorial exponent of the immediate environment of a fluorophore and for the excited state processes it is too much respondent [24,26]. To get an explicit insight into the small molecule-ctDNA interaction; the time-resolved decays of NOTD have been recorded in absence and presence of different concentration of ctDNA exciting at 265 nm. In the presence of ctDNA, an aqueous buffer solution of NOTD shows biexponential fluorescence decay. The biexponential decay of probe is very common in biomacromolecular environments [27]. Typical decay profiles of NOTD in the ctDNA

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Table 1 Time-resolved fluorescence parameters of NOTD with increasing concentration ctDNA. DNA(␮M)

␶1 (ns)

␶2 (ns)

␣1

␣2

␶(ns)

␹2

0 3 6 12 18 27 42

0.17 0.18 0.18 0.18 0.19 0.20 0.20

4.40 3.50 4.32 3.91 3.92 3.84 3.73

94.59 92.91 92.95 91.83 90.07 88.01 85.65

5.41 7.09 7.05 8.17 9.93 11.99 14.35

0.39 0.41 0.47 0.49 0.56 0.63 0.71

1.00 1.07 1.02 1.05 1.07 1.06 1.08

environment as mentioned earlier are depicted in Fig. 3a, and the deconvoluted data are assembled in Table 1. A quick look on Table.1 reflects that the littlest constituent ( 1 ) of NOTD remains unchanged in ctDNA surroundings to that of aqueous Tris-HCl buffer. So, this element ( 1 ) may be ascribed to the free NOTD molecule. The other element ( 2 ) is assigned to the ctDNA bound NOTD and changes continuously with changing ctDNA concentration. As a result, in ctDNA environment the average lifetime value ( avg ) of NOTD increases significantly shown in Fig. 3b. This enhancement of the fluorescence lifetime value of NOTD in ctDNA environment suggests the binding interaction between them [28,29]. From Table.1 it is also clear that with increasing ctDNA concentration, more and more number of NOTD molecules interact with ctDNA leading to reduction of the quantity of free probe (˛1 ) with a consequent enhance amount of ctDNA bound NOTD (˛2 ). 3.4. KI quenching studies To demonstrate the binding mode of NOTD with ctDNA, its quenching of fluorescence in presence and absence of ctDNA was well-read using popular anionic quencher potassium iodide [30]. Negatively charged iodide ions can efficaciously quench the fluorescence of NOTD like small probe molecule in aqueous Tris-HCl buffer medium. It is very well known that ctDNA hold a phosphate anchor which was negatively charged, so the anionic quenchers are readily repelled by it. Thus the emission intensity is intimately preserved of an intercalating drug or small molecule from being quenched as the neighborhood of anionic quenchers towards the fluorophore is confined [31]. Nevertheless, non-intercalative binding provides a trivial defense for the fluorophore as the fluorophore is manifested to the outer environment and iodide ions can immediately quench their fluorescence emission even in the presence of ctDNA [32]. Fluorescence quenching of NOTD by KI in the presence and absence of ctDNA was shown in Fig. 4. Using Stern-Volmer equation (4) availability of small molecule to anionic quenchers (here iodide ion) in the presence of ctDNA as well as in free medium was studied and the Ksv value was calculated. Ksv value of free NOTD by the iodide ions was 0.54 × 102 M−1 and in presence of ctDNA was 0.43 × 102 M−1 and the result is summarized in Table.S1. An ignorable decrease in Ksv ruled out the chance of intercalation between the NOTD and ctDNA base pairs. Some report contrary of groove binding of a small molecule by DNA, but this protection have no great importance as compared to intercalation [33]. Thus, we guessed that the interaction occurs between NOTD and ctDNA in a groove binding mode. 3.5. Effect of urea The double helical DNA strands were destabilized by the addition of urea and result of this double helix strands separated into single strands [24]. To analyze the mode of binding between NOTD and ctDNA the denaturizing feature of urea is utilized. With addition of urea, the appreciated (intercalate) small molecule is

Fig. 4. KI quenching experiment. Stern-Volmer plot F0 /F vs. [KI] for fluorescence quenching of NOTD (50 ␮M) by successive both the cases. Difference in Ksv values was further used to correlate the binding mode of NOTD with ctDNA addition of KI (0–13 mM) in the absence and presence of 50 ␮M ctDNA. Quenching constants were calculated in.

exempted into buffer solution result of this switch on the intensity of fluorescence whereas in non-intercalators i.e. groove binders causes very negligible effect on fluorescence behavior [34]. The successive addition of urea to NOTD-ctDNA complex have not seen any noticeable change on the fluorescence intensity shown in Fig. 5 (A) indicating the non-intercalative mode of binding. 3.6. Competitive displacement assay using known DNA intercalators To examine the intercalative or non-intercalative binding nature of NOTD with ctDNA, we have performed a correlative binding study with different well known DNA intercalators such as EB, AO and Hoechst 33258 whose mode of binding with ctDNA are already demonstrated [35]. In comparative displacement exertion, any drug or small molecule which may exchange the known intercalator antecedently adhered to the DNA, will interact with DNA in the similar way as they replaced the intercalator. Any noticeable alteration in the emission intensity of probe-DNA adduct on simultaneous addition of small molecule affords a valuable information about the mode of binding [32]. A sensible fluorescent probe EB contains a planar structure. In general EB appears as a poor fluorescent molecule. But due intercalation within the DNA base pairs the emission intensity of EB drastically increased. To determine the mode of binding between NOTD and ctDNA this property of EB is used. Fig. 6(A) represents that with subsequent change of NOTD to the EB-ctDNA complex, the emission of EB (emis max = 600.5 nm) alters vividly with an invisible change in the NOTD emission intensity (emis max = 600 nm). The negligible variation in the fluorescence intensity of EB-ctDNA complex with the simultaneous addition of NOTD implies that the intercalative mode of binding of EtBr does not disturb the binding of NOTD with ctDNA. Thus, the experiments clearly told that NOTD binds to ctDNA through non-intercalative binding mode since intercalation of EtBr with ctDNA base pair is very well known [35]. To further eliminate the chance of intercalation of NOTD we have execute the AO displacement assay. AO contains a planar aromatic structure; normally it shows weak fluorescent property. But after intercalation with DNA base pair the fluorescence intensity of AO is changed vigorously. A little alteration in the emission intensity (emis max = 523 nm) with subsequent addition of NOTD to AO-ctDNA complex, closed out the probability of intercalative binding mode. Thus, to establish the groove binding mode we have use Hoechst33258, a popular groove binder which binds to the minor

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Fig. 5. (A) Effect of ionic strength on the fluorescence emission spectra of NOTD bound ctDNA complex with increasing NaCl concentration from 0 to 1 M. (B) Effect of urea on the fluorescence emission spectra of NOTD-ctDNA interaction.

Fig. 6. (A) Fluorescence emission spectra of ctDNA bound EtBr with the increasing addition of NOTD. exc = 476 nm and emission spectra recorded in the wavelength range 500 nm to700 nm (B) Fluorescence emission spectra of ctDNA bound AO with the increasing addition NOTD. ␭exc = 460 nm and emission spectra recorded in the wavelength range 475 nm–630 nm (C) Fluorescence emission spectra of ctDNA bound Hoechst33258 with the increasing addition of NOTD. exc = 345 nm and emission spectra recorded in the wavelength range 380 nm to 600 nm. (D) Stern Volmer quenching plot of different fluorescent dye bound ctDNA complex systems by increasing NOTD addition. In each titration concentration of ctDNA and dyes was fixed and variation of NOTD 0–60 ␮M; [ctDNA] = 50 ␮M.

groove of ctDNA. In Tris-HCl buffer Hoechst33258 shows a weak emission spectra. But after addition of ctDNA to Hoechst33258 solution the emission intensity rose violently indicate the minor groove binding nature of dye. Fig. 6(C) depicts the emission intensity of Hoechst33258-ctDNA complex upon addition of NOTD, an observable fluorescence quenching (emis max = 491 nm) signifies the non-intercalative binding mode. The binding constant value for the EB, AO and Hoechst33258 bound ctDNA complex was determined using equation (4) shown in Fig. 6(D) and the result is summarized in Table 2. The tabulated result confirms the non-intercalation of NOTD with ctDNA after replacement of Hoechst33258. 3.7. DNA melting studies The double-helical ctDNA structure is unusually stable due to the presence of base stacking interactions and hydrogen bond. With simultaneous temperature increase various binding forces are debilitated and the result of this the double helical strands dis-

Table 2 Comparison of Stern-Volmer quenching constant for fluorescence quenching by displacement of different fluorescent dyes from ctDNA by NOTD. Dye

Ksv (M−1 )

Hoechst AO EB

10.0 × 10 1.08 × 103 0.40 × 103

a b

3

Ra 0.9991 0.9908 0.9987

S.Db 0.01904 0.00393 0.00129

R = Correlation coefficient. S.D = standard deviation.

sociates to single strand [36]. The DNA melting temperature (Tm ) was defined as a temperature at which half of the double-helical DNA structure is unfolded to two individual strands [37]. Interactions between ctDNA with small molecules are very familiar to predominate on Tm . The intercalative binding mode of small probe molecule can stagnate the double helical DNA form and raise the Tm by near about 5–8 ◦ C. However, non-intercalative type binding like electrostatic binding or groove binding brings out less or no

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Fig. 7. Representative CD spectra of ctDNA with varying concentrations of NOTD. Concentrations of NOTD are shown in the legends. [ctDNA] = 20 ␮M.

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Fig. 9. Cyclic voltammograms of 20 ␮M NOTD in the absence and presence of varying concentration of ctDNA (0–25 ␮M) obtained at 0.01 Vs−1 scan rate and 298 K temperature in a Tris-HCl buffer (pH 7.4).

and a negative peak at 245 nm, characteristic of the right-handed B form [39]. These bands are caused by the stacking interactions between the bases and the helical supra-structure of the polynucleotide that provides an asymmetric environment for the bases. As seen in Fig. 8, with increasing concentration of NOTD, there is no perturbation or negligible change of the helicity bands and base stacking in the CD spectra of ctDNA. Though, intercalation can cause switch in both the intensities of the peak location and the bands, composing the right-handed B-conformation of ctDNA. These observations forcibly eliminate the possibleness of intercalation of NOTD in the ctDNA helix and thereby suggest that the non-intercalative type binding between NOTD and ctDNA. 3.9. Effect of ionic strength

Fig. 8. Effect of NOTD on the melting temperature of ctDNA. Thermal melting curves of 20 ␮M ctDNA in the absence (red dots) and presence (blue dots) of NOTD (20 ␮M). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

indicative increase in Tm [38]. The melting temperature value for ctDNA in the presence and absence of NOTD was determined from the plot of normalized absorbance at 260 nm with temperature by regulating the absorbance as the alteration midpoint of the melting curve as shown in Fig.S2. From Fig. 7 it is perceivable that under the experimental conditions, the value of Tm for ctDNA alone was ◦ ◦ 69.5 C, while in the presence of NOTD, it was found to be 72.5 C. The observed change in Tm was very small, confirming the nonintercalative mode of binding. Also this outcome definitely notifies missing of intercalation binding mode even though revealing for exterior mode of binding between the NOTD and ctDNA. The slight raise in Tm is presumably owing to altering in the conformation of ctDNA as a result of destabilization ctDNA duplex in presence NOTD or due to groove binding of NOTD with ctDNA. 3.8. Circular dichroism study Adopting circular dichroism (CD) experiment the mode of binding of ctDNA with NOTD is verified, a negative result of which plucks up the chance of the intercalative mode of binding. It is well known that intercalating molecules distinctly agitated the secondary structure of ctDNA and gave up its touch through shifts in the inherent CD spectrum of the ctDNA. On the other hand, nonintercalative type binding does not alter CD spectrum of ctDNA. Fig. 8 shows the intrinsic CD spectrum of ctDNA in Tris-HCl buffer at pH = 7.4, have a positive peak at 275 nm due to base stacking pair

To recognize the mode of binding between the small molecule and ctDNA, study the affect of ionic strength is an effective method. In evidence, both intercalative and non-intercalative binding are closely connected to the double helical DNA strand, but the electrostatic interaction occurs from outside the DNA helix. A small molecule is in intimate vicinity in case of intercalative and groove binding but electrostatic binding can occur with phosphate group which are outside of DNA helix. When the small molecule-DNA complex was titrated by NaCl, due to an electrostatic binding small molecule is released from small molecule-DNA adduct and resulting enhance the intensity of fluorescence [40]. From Fig. 5(B) it is clear that with subsequent addition of NaCl (0–25 mM) no significant change in the fluorescence intensity was occurred which suggest that the involvement of groove binding rather than the intercalation. 3.10. Voltammetric study The application of the electrochemical method in the study of small molecule-ctDNA interaction provides a valuable complement to the previously used methods of investigation. As because of electrical inertness of small molecule to examine the binding interaction we use potassium ferrocyanide as a redox probe. Fig. 9 shows the change of current and potential of NOTD with subsequent addition of ctDNA. In the forward scan, a single anodic peak was observed which corresponds to the oxidation of NOTD with potential value 0.287 eV, whereas single cathodic peak was noted in the way of a reverse scan i.e. reduction occur with potential value 0.134 eV, indicating the reversibility of the process. From Fig. 9, it is found that a steady decrease in the anodic peak current with a shift of peak potential took place upon increasing the ctDNA concentration. On addition of ctDNA to the NOTD solution, the peak potential of NOTD changed from 0.287 eV to 0.277 eV

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Fig. 10. Molecular docked structures of NOTD-ctDNA complex. (a), (b), (c) and (d) docked representation showing minor groove binding of NOTD with dodecamer duplex sequence d(CGCGAATTCGCG)2 (PDBID: 1BNA). (c) and (d) stereo view of the docked conformation of the NOTD-ctDNA complex showing the possibility of non intercalative mode. The relative free energy of binding the NOTD-ctDNA complex system was found to be −7.57 kcal/ mol.

summarized in TableS2. Sun et al. reported that the interaction of probe molecule with DNA resulted in a significant increase of peak current, subsequently implying a stronger interaction between DNA and probe [41]. Furthermore, according to Devi et al., with increasing ctDNA concentration, the anodic peak potentials of the compound shifted toward lower values, indicating the nonintercalative binding nature of the compound with ctDNA [42]. 3.11. Molecular docking analysis Since simulation can suggest the most useable site of the ctDNA like biomacromolecules towards the binding of a small molecule or drug molecule, molecular docking analysis has been started to assist as a proficient tool for the purpose of the innovative biophysical research [43]. To afford a usual assist regarding the binding mode between the drug or small molecule with ctDNA through disclosing the empirical binding spot, we have carried out the molecular docking study according to the early mentioned protocol depicted in the experimental section. It is necessary to keep in mind that the results of molecular docking analysis significantly figure on the chose crystallographic ctDNA structure. To fulfill our intention, we have chosen B-DNA with PDB ID: 1BNA, which is utilized by lot of research groups for observing the exact position of the small probe molecule in ctDNA helix [39,43]. The docked form shown in Fig. 10 demonstrates that NOTD is found to be best fitted in the minor groove of double helix ctDNA with a free binding energy of −7.57 kcal/mol which is in real consent with the values of docking energies for the molecules that bind in a non-intercalative mode (Fig.S3) with ctDNA [39,43]. The acquired energy from molecular docking analysis was compared with the experimental finding i.e. the free energy of binding −7.57 kcal/mol obtained from docking studies was consistent with the spectroscopic results. Impartial of the non-existence of any positive charge on NOTD, the negative binding energy pointed out the superior potential of binding between the ctDNA and NOTD. Thus, the molecular docking analysis confirms the proposition of groove binding of NOTD with ctDNA rather than the intercalative mode of binding.

study. Through the exploration of UV–vis absorbance, steady state and time resolved fluorescence spectroscopy, circular dichroism spectral analysis, voltammetric measurements, melting studies and molecular docking. UV–vis absorbance, steady state and time resolve fluorescence measurements indicate that the complex formation between NOTD and ctDNA. The estimated binding constant was in the order of 3.12–4.1(×104 ) M−1 . Iodide prompted quenching, denaturation study, effect of ionic strength and competitive displacement experiment with known intercalator EB, AO and Hoechst 33258 revealed that NOTD interacts with ctDNA through groove binding mode and does not intercalate into the ctDNA base pairs. Furthermore, helix melting temperature, voltammetric study and CD spectral change has affirmed the complex formation between NOTD and ctDNA. However, the probability of electrostatic binding interaction between NOTD and ctDNA cannot be overlooked. Molecular docking confirmed that NOTD binds to the ctDNA minor groove with a relative binding energy of −7.57 kcal/mol. Our finding gives an important message about NOTD-ctDNA interactions, which are valuable for the rational drug designing with increased or more selective activeness and much better efficaciousness. Acknowledgements Author AM acknowledging University research fellowship. Author BS sincerely thanks the Department of Chemistry, VisvaBharati, Santiniketan-731235, India for instrumental facility and research funding for UGC-SAP. Author thanks Mr. Ramakanta Mondal of IISER Bhopal, M.P, India for helping us in the molecular docking study and also thankful to Mr. S. Debnath of Department of Chemistry, Visva-Bharati. We specially appreciate the cooperation obtained from Prof. Pranab Sarkar of Department of Chemistry, Visva-Bharati, Santiniketan for computational study and Dr. Susanta Ghosh of Department of ISERC, Visva-Bharati, Santiniketan for cyclic voltammetry study. We also acknowledge the instrumental facility of IACS, Jadavpur, Kolkata-700032, India for carrying out CD and other experiments.

4. Conclusion

Appendix A. Supplementary data

In the present study, the weak binding interactions between NOTD and ctDNA have been investigated using various biophysical techniques along with electrochemical and molecular docking

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2017. 03.053.

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