Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 467–472

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Studies of interaction between terbium(III)-deferasirox and double helix DNA by spectral and electrochemical methods Masoomeh Shaghaghi a, Gholamreza Dehghan b,⇑, Abolghasem Jouyban c, Parisa Sistani d, Mitra Arvin b a

Department of Chemistry, University of Payame Noor, P.O. Box. 19395-3697, Tehran, Iran Department of Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran c Drug Applied Research Center and Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran d Department of Biochemistry, University of Payame Noor, P.O. Box. 19395-3697, Tehran, Iran b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t 3+

 DNA binding studies of terbium -

deferasirox (Tb3+-DFX) complex were studied.  Several spectroscopic techniques and cyclic voltammetry and viscosity measurement have been used in this research.  The binding constants (Kb) for the complex with ctDNA were estimated to be 1.8  104 M1. 3+  Tb -DFX complex can bind to ctDNA via groove binding and/or electrostatic binding modes.

a r t i c l e

i n f o

Article history: Received 22 May 2013 Received in revised form 11 September 2013 Accepted 26 September 2013 Available online 7 October 2013 Keywords: DNA interaction Terbium complex Fluorescent probe Spectroscopic approach Binding mode

a b s t r a c t DNA binding studies of terbium(III)-deferasirox (Tb3+-DFX) complex were monitored to understand the reaction mechanism and introduce a new probe for the assay of DNA. In the present work, UV absorption spectrophotometry, fluorescence spectroscopy, circular dichroism (CD), cyclic voltammetry (CV) and viscosity measurement were employed to study the interactions of Tb3+-DFX with calf thymus DNA (ctDNA). The binding of Tb3+-DFX complex to ctDNA showed a hyperchromic effect in the absorption spectra and the increase in fluorescence quenching effect (amount) of Tb3+-DFX complex in the presence of ctDNA. The binding constants (Kb) for the complex with ctDNA were estimated to be 1.8  104 M1 through UV absorption spectrophotometry and fluorescence spectroscopy. Upon addition of the complex, clear decreases were observed in the viscosity of ctDNA. The CD spectra indicated that there are certain detectable conformational changes in the DNA double helix when the complex was added. The CV method showed that both anodic and cathodic peak potentials of Tb3+-DFX complex showed negative shifts on the addition of the ctDNA. Further, competitive methylene blue binding studies with fluorescence spectroscopy have shown that the complex can bind to ctDNA through nonintercalative mode. The experimental results suggest that Tb3+-DFX complex binds to DNA via groove binding and/or electrostatic binding mode. Ó 2013 Elsevier B.V. All rights reserved.

Introduction The interactions of small molecules with DNA are an important issue in life sciences and have attracted more attentions in recent ⇑ Corresponding author. Tel.: +98 (411) 3392739; fax: +98 (411) 3356027. E-mail addresses: [email protected], [email protected] (G. Dehghan). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.09.073

decades. These binding studies were driven partly by need to understand the action mechanisms of some antibacterial, antifungal, antiviral and antitumor drugs and to design new DNA-targeted drugs and also to screen these drugs in vitro [1–3]. Also, these interactions have great importance to provide the chemical basis for carcinogenicity of environmental pollutants and toxic chemicals [4,5], to serve as analogues in the study of protein-nucleic acid recognition and to develop novel non-radioactive probes

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of DNA structure [6]. DNA is the primary intracellular target of anticancer drugs, so the interaction between small molecules can cause DNA damage in cancer cells, blocking the division of cancer cells and resulting in cell death. Basically, metal complexes interact with double helix DNA in either a noncovalent or a covalent way. The former way includes electrostatic binding, groove binding, intercalative binding and partial intercalative binding. The intercalative binding is stronger than other binding modes because the surface of intercalative molecule is sandwiched between the aromatic and hetrocyclic base pairs of DNA [7]. Fluorescent probes including organic dyes, metal ions and metal complexes are frequently employed to investigate the interactions of nucleic acids [8–10]. In recent years, the use of coordination complexes of lanthanide ions as luminescent probes to study nucleic acids has attracted much attention [7,10,11]. These complexes have luminescence characteristics that are great utility in analysis, e.g. narrow emission bonds, a large Stokes’ shift, long luminescence decay times and strong binding with biological molecules [10] and rare earth elements have more physiological activities and lower toxicities after coordinating with a ligand [7]. Among lanthanide ions, terbium(III) ion has been widely used as a probe of nucleic acid structure [10,11]. It is known to interact with guanine and xanthosine to produce high fluorescence enhancement [12]. The sensitisation of lanthanide ions luminescence, especially terbium ion, by organic ligands, has been widely employed in various applications, including the investigation of biological systems, immunoassays, DNA and RNA hybridization assays, quantification of organic compounds and chromatographic detections [13–15]. Manzoori et al. [16,17] reported that Tb3+ ion can form fluorescent complex with deferasirox (DFX). The DFX is a newly developed iron chelator that can be orally administered once a day, and is now used worldwide for the treatment of patients with iron overload, with high efficiency and was approved by the US Food and Drug Administration (FDA) in November 2005 [18]. DFX has the ability to form complexes with various cations like Tb3+. Tb3+-DFX complex possess high fluorescence intensity and was very stable and has been used as a fluorescent probe in various studies [16,17]. Many techniques have been used to investigate the interactions of drugs with DNA, including fluorescence spectroscopy, UV–Vis spectrophotometry, voltammetry, circular dichroism spectroscopy (CD), dynamic viscosity measurements and high performance liquid chromatography [19–22]. Among these techniques, spectroscopic methods and dynamic viscosity measurements have been preferred because they are sensitive, rapid and simple and small molecule-DNA interactions may be experimentally monitored by changes in the intensity and position of the spectroscopic peak responses or changes in dynamic viscosity of DNA [7,19]. In the present study we try to introduce Tb3+-DFX complex as a new probe to DNA. Thus, the techniques viz. UV–Vis spectrophotometry, fluorescence spectroscopy, CD, cyclic voltammetry (CV) and viscosity measurement have been employed for analyzing the in vitro interaction of Tb3+-DFX with calf thymus DNA (ctDNA). The major binding mode and binding constant of Tb3+-DFX complex to ctDNA was also estimated.

Experimental Reagents and chemicals

absorption at 260 nm using a molar absorption coefficient (e260 = 6600 L mol1 cm1) [8]. The purity of the DNA was checked by monitoring the ratio of the absorbance at 260 nm to that at 280 nm. The solution gave a ratio of >1.8 at A260/A280, which indicates that DNA was adequately free from protein [7,11]. A 1.0  102 M terbium(III) solution was prepared by dissolving the appropriate amount of terbium(III) chloride hexahydrate (TbCl36H2O) (Acros Organics, USA) in double distilled water and stored in a polyethylene container to avoid memory effects of terbium adsorbed on glass vessels. A stock solution (1.0  103 M) of DFX (Osvah Pharmaceutical Company, Tehran, Iran) was prepared in ethanol and double-distilled water and for experiments freshly diluted in water in order to have less than 2% ethanol. A stock solution (1.0  103 M) of methylene blue (MB) (Sigma Chem. Co., USA) was prepared by dissolving its crystals in double distilled water. A stock solution (5.0  104 M) of Tb3+-DFX complex was prepared by mixing and gentle stirring of 1 ml of 1.0  103 M terbium(III) solution and 1 ml 1.0  103 M DFX solution. The pH of all solutions was adjusted with the Tris–HCl buffer solution (0.01 M, pH 7.4). Apparatus and methods The absorption spectra were measured with a UV–visible spectrophotometer (T60, PG Instruments Ltd., Leicestershire, UK) using a 1.0 cm cell. The interaction between the Tb3+-DFX and ctDNA was carried out as follows: (I) keep the concentration of the Tb3+-DFX complex constant (5.0  105 M) while varying the ctDNA concentrations (ri = [ctDNA]/[complex] = 0.0–2). (II) Fixed amounts of the ctDNA (5.0  105 M) were titrated with increasing amounts of Tb3+-DFX solution (ri = [complex]/[ctDNA] = 0.0–0.7). The data were then fitted to Eq. (1) to obtain intrinsic binding constant, Kb [23]. 3þ

ð1Þ

where [Tb3+-DFX] is the concentration of complex, ef, eb and ea correspond to the extinction coefficients, respectively, for free ctDNA, for fully bound ctDNA and for each addition of complex to ctDNA. In particular, ef was determined by a calibration curve of the isolated ctDNA in aqueous solution, following the Beer’s law, ea was determined as the ratio between the measured absorbance and the ctDNA concentration, Aobs/[DNA]. A plot of [Tb3+-DFX]/(ea  ef) versus [Tb3+-DFX] gives a slope of 1/(eb  ef) and an intercept equal to 1/Kb (eb  ef); Kb (intrinsic binding constant) is the ratio of the slope to the intercept. To avoid from the spectral overlap of ctDNA and Tb3+-DFX complex and vice versa, after addition of ctDNA or Tb3+-DFX in each step, absorption intensity of ctDNA-Tb3+-DFX was corrected against the absorption intensity of added compound. All fluorescence measurements were carried out with a Jasco FP-750 spectrofluorimeter (Kyoto, Japan) equipped with a 150 W Xenon lamp, using a quartz cell of 1.0 cm path length. Fixed amounts of Tb3+-DFX complex (5.0  105 M) were titrated with increasing amounts of ctDNA (ri = [ctDNA]/[complex] = 0.0–3). An excitation wavelength of 328 nm was used and total fluorescence emission intensity was monitored at 490, 545 and 588 nm. The titration data were fitted into the Stern–Volmer (Eq. (2)) [24]:

F 0 =F ¼ 1 þ K sv ½DNA ctDNA (Sigma Chem. Co., USA) was used without further purification and its stock solution was prepared by dissolving an appropriate amount of ctDNA in 0.01 M Tris–HCl (tris hydroxymethyl aminomethan hydrochloride, pH 7.4) and stored at 4 °C. The concentration of ctDNA in stock solution was determined by UV

3þ

½Tb -DFX=ðea  ef Þ ¼ ½Tb -DFX=ðeb  ef Þ þ 1=kb ðeb  ef Þ

ð2Þ

where F0 and F are the fluorescence intensities of the probe in the absence and presence of the quencher (ctDNA), respectively, Ksv is Stern–Volmer quenching constant which is a measure of the efficiency of fluorescence quenching by ctDNA.

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In the competitive binding studies, concentrations of ctDNA and methylene blue were constant (5.0  105), while varying the complex concentration (ri = [Tb3+-DFX]/[MB + ctDNA] = 0.0–1.2). Samples were excited at 630 nm, and emission spectra were recorded from 645 to 740 nm. CD measurements were recorded on a Jasco J-810 spectropolarimeter by keeping the concentration of ctDNA constant (5.0  105 M) while varying the Tb3+-DFX complex concentration (ri = [complex]/[ctDNA] = 0.0–0.4). Measurements were taken at wavelengths between 220 and 320 nm. The optical chamber of the CD spectrometer was deoxygenated with dry nitrogen before use and kept in a nitrogen atmosphere during experiments. All CD experiments were performed in 0.01 M Tris–HCl buffer (pH 7.4) at room temperature. The CV measurements were performed using an Autolab (PGSTAT 30), with a three-electrode system: a 0.10-cm-diameter Glassy carbon (GC) disc as working electrode, a calomel electrode as reference electrode, and a Pt wire as counter electrode. The supporting electrolyte was 0.01 M of Tris–HCl buffer solution (pH 7.4). The Tb3+-DFX concentration was constant at 5.0  105 M, while varying the ctDNA concentration. Viscosity experiments were carried out using an Ubbelohdetype viscometer (Julobo, MD-18V, Germany) maintained at a constant temperature at 25 ± 0.1 °C in a thermostatic water-bath. Flow time was measured with a digital stopwatch; the mean values of three replicated measurements were used to evaluate the viscosity (g) of the samples. ctDNA concentration was constant at 5.0  105 M while varying the Tb3+-DFX complex concentration (ri = [complex]/[ctDNA] = 0.0–1.8). The data were presented as (g/ g0)1/3 versus the ratio of the concentration of Tb3+-DFX to that of ctDNA (ri), where g and g0 are the viscosity of DNA in the presence and absence of Tb3+-DFX complex, respectively. Viscosity values were calculated from the observed flow time of ctDNA containing solutions (t) and corrected for buffer solution (t0), g = (t – t0)/t0 [20].

Results and discussion UV–Vis absorption spectroscopy Electronic absorption spectroscopy is an effective method to ascertain the bonding of drug-metal complexes with DNA. In general, observations of hypochromism and red shift are associated with binding of small molecules to the DNA helix due to the intercalative mode as it involves a strong stacking interaction between an aromatic chromophore of the molecule and the base pairs of DNA [25]. Fig. 1a shows the absorption spectra of Tb3+-DFX complex after addition of different amounts of ctDNA. Addition of increasing amounts of ctDNA to a Tb3+-DFX solution results in the obvious hyperchromism tendency of the absorption bands, suggesting a strong interaction between the complex and ctDNA which is different from the classical interaction binding. The spectral changes of the Tb3+-DFX complex, observed in the presence of ctDNA, may occur via a non-intercalative binding mode. Amino and hydroxyl groups could form hydrogen bonds with the DNA base pairs, contributing to the overall hyperchromism [26,27]. The band at 260 nm of DNA arises due to the p–p⁄ transitions of DNA bases [28]. The absorption spectra of ctDNA and its UV–Vis titration with Tb3+-DFX complex are recorded in Fig. 1b. Generally hypochromism arises from the contraction of ctDNA in the helix axis, as well as its conformational changes; in contrast, hyperchromism is due to a damage of the ctDNA double-helix structure [11]. Absorption spectra of DNA increased upon increasing the Tb3+-DFX concentration, this is a typical hyperchromic effect and also a blue shift of the kmax from 260 to 251 nm observed. Therefore, these re-

Fig. 1. Absorption spectra: (a) Tb3+-DFX complex (5.0  105 M) in the absence and presence of increasing amounts of ctDNA (ri = [ctDNA]/[complex] = 0.0, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 and 2.0); (b) ctDNA (5.0  105 M) in the absence and presence of increasing amounts of Tb3+-DFX complex (ri = [complex]/[ctDNA] = 0.0, 0.1, 0.2, 0.3, 0.6, 0.7); (c) [Tb3+-DFX]/(ea  ef) versus [Tb3+-DFX] plot. The arrows show the absorbance changes upon increasing ctDNA (a) and Tb3+-DFX complex (b).

sults probably indicate damage to the DNA double-helical structure after Tb3+-DFX binding and binding mode is non-intercalative. Moreover, in order to further illustrate the binding strength of the Tb3+-DFX with ctDNA, the binding constant (Kb) was determined based on Eq. (1), (y = 9.85  105x + 5.60  109, R2 = 0.993). The mean values of three replicated measurements were used to evaluate the Kb value. The Kb for Tb3+-DFX complex with ctDNA was estimated to be 1.759 (±0.001)  104 M1, this value of Kb was lower than that of classical intercalators, whose binding constants are on the order of 106  107 [29,30]. Luminescence studies: characteristics of fluorescence spectra interaction between Tb3+-DFX complex and ctDNA Fluorescence spectroscopy is the most suitable technique to investigate the interaction of a drug with a biomolecule, so that the fluorescence measurements can give some information of binding of small molecule substances to biomolecule, such as the

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binding mechanism, binding mode, binding constants and binding sites [7,19]. The maximal excitation wavelength of Tb3+-DFX complex occurred at 328 nm and the maximum emission wavelength occurred at 490, 545 and 588 nm. Fig. 2a shows the characteristic changes in fluorescence emission spectra during the titration of Tb3+-DFX complex with ctDNA. The maximum emission intensities of Tb3+-DFX complex gradually decreased with increasing concentration of ctDNA, indicated that DNA could quench the intrinsic fluorescence of Tb3+-DFX complex and the binding of Tb3+-DFX complex to DNA indeed exists. Plotting F0/F vs [Q] (based on Eq. (2)) shows an upward curvature. Typically, the linearity of the Stern–Volmer plot suggests the (i) existence of one binding site for the ligand in the proximity of the fluorophore, or (ii) more than one binding site equally accessible to the ligand [31]. Thus, the gradual divergence from linearity of the Stern–Volmer plots on the continuous addition of the ctDNA reveals the presence of more than one binding site with different accessibilities and/or of the occurrence of combined static/dynamic quenching [32]. Binding constant and number of binding sites Fluorescence data were analyzed to calculate the binding constant and binding stoichiometry (n) according to the method of Lehrer and Fasman [33] and Chipman et al. [34] as described by Sahoo et al. [35]. The binding constant (Ka) and number of binding sites (n) have been estimated, considering a 1:1 complex between ctDNA and the ligand can be represented by the following equation:

ðF0  FÞ=ðF  F1 Þ ¼ K a  ½DNA

ð3Þ

The binding constant Ka is obtained by plotting log (F0  F)/(F  F1) versus log [Q], where F0 and F1 are the relative fluorescence intensities of Tb3+-DFX complex alone and Tb3+-DFX complex saturated with ctDNA, respectively. The slope of the double-logarithmic plot (Fig. 2b), (y = 3.26 x + 13.85, R2 = 0.942), obtained from the experimental data, is the number of equivalent binding sites (n), whereas the value of log [Q or ctDNA] at log (F0  F)/(F  F1) = 0 equals to the negative logarithm (pKa) of the binding constant (Ka) (log (F0  F)/(F  F1) = log Ka + n log [Q]). The values of Ka and n for the complex for the mean values of three replicated measurements, were found to be 1.767 (±0.006)  104 M1 and 3.5, respectively. The presence of multiple binding sites can also explain the upward curvature of the Stern–Volmer plot as discussed earlier [32,35].

Competitive binding studies To further clarify the nature of the interaction between Tb3+DFX complex and ctDNA, competitive methylene blue (MB) displacement assay was investigated. Interestingly, the emission intensity of MB is quenched on adding ctDNA. This emission quenching phenomenon reflects the change in the excited state structure as a consequence of the electronic interaction in the MB-ctDNA complex [36]. The emission-quenching phenomenon and the hypochromic and red shift effects in the absorption spectra attribute to the intercalative mode of MB to DNA. The emission spectra of the MB-ctDNA solutions in the presence of the increasing Tb3+-DFX complex concentrations is shown in Fig. 3, which clearly reveals an increase in the fluorescence intensity of the probe molecule on adding the Tb3+-DFX complex. In the case of

Fig. 2. (a) Emission spectra of Tb3+-DFX complex (5  105 M) in the absence and presence of increasing amounts of ctDNA (ri = [ctDNA]/[complex] = 0.0, 0.5, 1.0, 1.3, 1.4, 1.5, 2.0, 2.4, 2.5 and 3) and kex = 328 nm. The arrow shows the fluorescence intensity changes upon increasing ctDNA. Inset: Plot of the Stern–Volmer for the quenching of Tb3+DFX complex by ctDNA. (b) The double-logarithmic plot of complex–ctDNA interaction to determine the binding constant (Ka) and number of binding sites (n).

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the highest concentration of Tb3+-DFX complex, the emission intensity of the MB-ctDNA complex could not approach that of pure MB. The increase in fluorescence intensity should be due to a greater amount of free MB molecules in solution. Considering prior knowledge on MB molecules, intercalated into the DNA helix, these results indicate that some MB molecules are released from the ctDNA nucleobases after addition of the Tb3+-DFX complex. That is, the formation of Tb3+-DFX-ctDNA complex prevents the binding of MB. The extent of MB release and following increase in fluorescence intensity is applied to compare the strength of binding. Fig. 3 shows, that our complex could not release MB completely. Therefore, this experiment confirms our previous findings and reconfirms our prior evidence based conclusions. CD spectroscopy The CD technique is indeed very sensitive to detect minor conformational changes of the DNA conformation produced by ligand binding. The observed CD spectrum of ctDNA consists of a positive band at 275 nm due to base stacking and a negative band at 245 nm due to helicity, which is characteristic of DNA in the right-handed B form [37]. While groove binding and electrostatic interaction of small molecules with DNA show little or no perturbations on the base stacking and helicity bands, intercalation enhances the intensities of both the bands, stabilizing the righthanded B conformation of ctDNA [38]. Fig. 4 shows that the intensities of both the negative and positive bands decrease significantly (shifting to zero levels). This suggests that the interaction of the mentioned complex with DNA induces certain conformational changes, such as the conversion from a more B-like to a more C-like structure within the DNA molecule [39]. These changes are indicative of a non-intercalative mode of binding of this complex. The complex interaction effectively screens the negative charge on N(7) base sites as well as phosphate oxygen atoms simultaneously, both along the deoxyribophosphate backbone and in the groove of the helix to promote a transconformational change from a more B-like to a more C-like structure. Further transformation of DNA structure proceeds by removal of water from the base sites and the grooves of the helix [40]. Electrochemical studies

Fig. 4. CD spectra of ctDNA (5.0  105 M) in 0.01 M Tris–HCl buffer, in the presence of increasing amounts of Tb3+-DFX complex (ri = [Complex]/[DNA] = 0.0, 0.1, 0.2, and 0.3). The arrows show the CD spectra changes upon increasing Tb3+DFX complex.

useful complement to the above spectral and viscometric studies. A cyclic voltammogram of the Tb3+-DFX (Fig. 5) at a GC electrode exhibited cathodic and anodic peaks. The peak at approximately +0.28 V corresponds to the reduction of Tb3+-DFX, and the anodic peak at +0.77 V corresponds to the oxidation of Tb3+-DFX. When ctDNA is added to a solution of Tb3+-DFX, cathodic peak current height of the Tb3+-DFX increased and cathodic peak potential showed negative shifts (DEp = Ep(b)  Ep(a) = 0.22–0.28 = 0.06 V). Also the anodic peak current of Tb3+-DFX disappeared and two new anodic peaks (+0.55 and +0.16 V) in more negative potentials were formed. These negative shifts are considered as evidences that Tb3+-DFX may binds electrostatically to the negatively charged deoxyribose-phosphate backbone of DNA. From the other point of view, if a molecule intercalated into the DNA, positive peak potential shifts should be detected, because this kind of interaction is due to hydrophobic interaction [43].

Electrochemical techniques extensively were used as a simple and rapid method to study DNA interaction with different compounds [41,42]. The application of these methods to the study of interaction of redox-active metal complexes with DNA provides a

Fig. 3. Emission spectra of MB–ctDNA complex in the presence of increasing amounts of Tb3+-DFX complex (ri = [complex]/[MB + ctDNA] = 0.0, 0.2, 0.6, 0.8,1 and 1.2) and kex = 630 nm. The arrow shows the fluorescence intensity changes upon increasing Tb3+-DFX complex.

Fig. 5. Cyclic voltammograms of the Tb3+-DFX complex in Tris–HCl buffer in the absence (a) and presence (b) of ctDNA. [Tb3+-DFX] = 5  105 M; [ctDNA]: (a) 0, (b) 5  105 M. The arrows show the both anodic and cathodic peak potentials changes upon increasing ctDNA.

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binding mode. Information obtained from the present work is helpful to the development of nucleic acids molecular probes. Acknowledgements The authors would like to acknowledge the research council of the University of Payame Noor for financial support of this work. References

Fig. 6. Effect of increasing amounts of complex on the viscosity of ctDNA (5  10– M) in 0.1 M Tris–HCl buffer. (ri = [Complex]/[ctDNA] = 0.6, 0.8, 1.2, 1.6, and 1.8).

5

Viscosity measurements Spectroscopic data are necessary but inadequate to support binding mode. Viscosity measurement, which is sensitive to the changes in the length of the DNA molecule, is regarded as the least ambiguous and the most critical means studying the binding mode of small molecules with DNA in solution. Intercalating agents are expected to elongate the double helix to accommodate the ligands in between the base pairs, leading to an increase in the viscosity of DNA. In contrast, complexes that binds exclusively in the DNA grooves by partial and/or non-classical intercalation, under the same conditions, typically cause less-pronounced (positive or negative) or no change in the DNA solution viscosity [44]. Fig. 6 shows the effect of increasing amounts of the Tb3+-DFX complex on the relative viscosity of the ctDNA. The results revealed that the Tb3-DFX complex decreases the relative viscosity of ctDNA. The decreased relative viscosity of DNA may be explained by a non-intercalative binding mode, which produced bends or kinks in the DNA and thus reduced its effective length and concomitantly its viscosity [8]. The results suggest that non-intercalative binding interaction could be suggested for the Tb3+-DFX complex.

Conclusions In this work, the binding interactions of Tb3+-DFX complex with ctDNA in physiological buffer by UV–Vis spectrophotometry, fluorescence spectroscopy, CD spectral studies, CV and viscosity measurement were investigated. The absorption spectrum of ctDNA showed that as the concentration of Tb3+-DFX complex increases, a hyperchromism effect develops in the spectrum and a blue shift (10 nm) in the kmax was observed. The binding strength of Tb3+-DFX complex with CT-DNA calculated with UV and fluorescence spectroscopic titrations, have shown that Tb3+-DFX complex exhibits lower Kb value (1.8  104 M1) than that of classical intercalator. Competitive binding study with MB has revealed that Tb3+-DFX complex could not release MB completely, indicating that non-intercalation as a possible mode of its interaction with ctDNA. DNA viscosity measurements showed the clear decrease in viscosity of DNA. CD measurements in combination with CV studies revealed that a non classic intercalation between ctDNA and Tb3+-DFX complex. The results suggest that the complex bind to DNA mainly through a groove binding and/or electrostatic

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