Studying non-covalent drug-DNA interactions.

Archives of Biochemistry and Biophysics xxx (2015) xxx–xxx

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Review

Studying non-covalent drug–DNA interactions Sayeed Ur Rehman 1, Tarique Sarwar 1, Mohammed Amir Husain 1, Hassan Mubarak Ishqi, Mohammad Tabish ⇑ Department of Biochemistry, Faculty of Life Sciences, A.M. University, Aligarh, U.P. 202002, India

a r t i c l e

i n f o

Article history: Received 31 December 2014 and in revised form 9 March 2015 Available online xxxx Keywords: Drug–DNA binding Intercalation Groove binding Electrostatic interactions

a b s t r a c t Drug–DNA interactions have been extensively studied in the recent past. Various techniques have been employed to decipher these interactions. DNA is a major target for a wide range of drugs that may specifically or non-specifically interact with DNA and affect its functions. Interaction between small molecules and DNA are of two types, covalent interactions and non-covalent interactions. Three major modes of non-covalent interactions are electrostatic interactions, groove binding and intercalative binding. This review primarily focuses on discussing various techniques used to study non-covalent interactions that occur between drugs and DNA. Additionally, we report several techniques that may be employed to analyse the binding mode of a drug with DNA. These techniques provide data that are reliable and simple to interpret. Ó 2015 Elsevier Inc. All rights reserved.

Introduction DNA plays an important role in biological processes since it carries the hereditary information codes required for the synthesis of all proteins and enzymes. DNA directly or indirectly controls the structure and function of the cell. Ever since the discovery of the structure of DNA, it has been a prime target for various therapeutically important small molecules that belong to different classes of drugs, ranging from anticancer drugs to antibiotics. Apart from interacting with DNA associated proteins or interacting through DNA–RNA hybrids, small molecules may directly bind to the DNA helix. Such interactions result in diverse downstream processes like interfering with the activity of various important enzymes and proteins involved in maintaining the structure and functions of the cell. The interaction of small molecules with DNA has been studied extensively. These studies provide insights into the development of effective therapeutic drugs that could control gene expression. Newer and more effective DNA-targeted drugs against several diseases can be easily developed [1]. Understanding the mechanism of action of various anti-cancer drugs became possible by studying the drug–DNA interactions. The interactions between small molecules and DNA are of two types, covalent interactions and non-covalent interactions. Our article primarily focuses on studying various non-covalent interactions. The three major modes of non-covalent interactions are ⇑ Corresponding author. 1

E-mail address: [email protected] (M. Tabish). Contributed equally.

electrostatic interactions, groove binding and intercalative binding. Electrostatic binding occurs due to interaction between the negatively charged phosphate backbone of DNA and the positively charged ends of small molecules. The two different types of groove binding modes are major and minor groove binding. Groove binding involves hydrogen bonding or van der Waals interactions of the small molecule with nucleic acid bases. Intercalation occurs when the small molecules intercalate within the nucleic acid base pairs [2,3]. Many small molecules of biological importance are known to interact with DNA through non-covalent interactions [4–6]. These small molecules are known to interact with DNA by a specific binding mode. However, several studies have reported mixed or more than one binding modes for various small molecules interacting with DNA. This property of mixed binding mode can be linked to their mechanism of action and therapeutic efficiency [7]. This review article describes commonly used techniques for studying drug–DNA interactions like UV–visible spectroscopy, fluorescence spectroscopy, circular dichroism (CD)2 spectroscopy, viscosity measurement studies, isothermal titration calorimetry (ITC) and fourier transform infrared (FT-IR) spectroscopy. An overview of some other important techniques is also provided.

2 Abbreviations used: CD, circular dichroism; ITC, isothermal titration calorimetry; FT-IR, fourier transform infrared; DSMI, trans-4-[4-(dimethylamino)styryl]-1-methyl pyridinium iodide; EB, ethidium bromide; NR, neutral red; AO, acridine orange; MB, methylene blue; IR, infrared radiation; G, guanine; A, adenine, C, cytosine; AFM, atomic force microscopy; CV, cyclic voltammetry; NMR, nuclear magnetic resonance; FRET, Forster (fluorescence) resonance energy transfer; PDB, Protein Data Bank.

http://dx.doi.org/10.1016/j.abb.2015.03.024 0003-9861/Ó 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: S.U. Rehman et al., Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/j.abb.2015.03.024

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S.U. Rehman et al. / Archives of Biochemistry and Biophysics xxx (2015) xxx–xxx

Techniques employed to study drug–DNA interactions UV–visible spectroscopy UV–visible absorption spectroscopy is simple, widely used and one of the most effective methods in detecting the interaction of small molecules with DNA. The stability of DNA on interaction with small molecules is easily studied using UV–visible absorption measurement. In general, the interaction of small molecules with DNA and the formation of a new complex lead to changes in UV– visible spectra [8,9]. To elaborate, the band intensity and position of the spectra of the free small molecule as well as its complex with DNA are recorded (Fig. 1A). Any change in the band is considered to arise from formation of a complex [10–13]. Interaction of small molecules and DNA are also studied by studying the change in absorption spectra of DNA. In this case, an increasing concentration of small molecules is added to a fixed concentration of DNA (kmax is 260 nm) (Fig. 1B). On increasing the concentration of small molecules, any change in absorbance as well as any shift in the position of the peak is recorded and interpreted. Two major features of the spectra of DNA are ‘‘hyperchromic’’ effect and ‘‘hypochromic effect’’ that arise due to a change in its double helical structure [14]. Any change in the conformation and structure of DNA that occurs on binding of small molecule translates into a change in its spectral behaviour. On interaction with small molecules, any destabilisation of secondary structure of DNA leads to hyperchromism, while hypochromism originates from the stabilisation of DNA secondary structure either by electrostatic effects or intercalation of small molecules [15,16]. Generally, absorption spectra of small molecules show bathochromic shift (red shift) as well as hypochromic effect on intercalation into the DNA double helix [16]. In a typical case of intercalation, hypochromism and bathochromism in presence of DNA were >35% and >15 nm, respectively [17]. In the case of groove binding molecules, that bind on the outer surface of DNA, smaller (6–8 nm) or no bathochromism is usually observed. It is important to mention that the absorption spectra of DNA may be influenced by drug or vice versa. To remove ambiguity, baseline correction with suitable concentration of drug or DNA must be performed before studying the interaction using UV–visible spectroscopy. Thermal denaturation studies Thermal denaturation studies are usually performed to elucidate the binding mode of small molecules with DNA (Fig. 1C). On increasing the temperature of solution, the DNA double helix is denatured and single stranded regions are generated. This leads to a change in the absorbance of DNA solution at 260 nm. The temperature at which 50% of double stranded DNA is denatured to single stranded DNA is called the midpoint denaturation or melting temperature (Tm). To study the interaction of small molecules with DNA, Tm of the DNA solution in the absence and presence of small molecules or drugs are recorded. Small molecules that bind to DNA via an intercalation mode lead to stabilisation of DNA and results in an increase in the Tm of DNA by about 5–8 °C [18]. However, groove binding molecules do not alter the melting temperature of DNA duplex to such an extent [19]. Fluorescence spectroscopy One of the most widely exploited techniques to study the drug– DNA interaction is fluorescence spectroscopy. In general, compounds containing aromatic functional groups show intense fluorescence as compared to small molecules containing aliphatic, alicyclic carbonyl structure or compounds containing highly

Fig. 1. Studying the interaction of small molecules with DNA using UV–visible spectroscopy. (A) Spectra of drug in absence and presence of increasing concentration of DNA. Figure is reproduced with permission from the manuscript of Khorasani-Motlagh et al. [10]. (B) Spectra of DNA in absence and presence of increasing concentration of drug. Figure is reproduced with permission from the manuscript of Shahabadi and Hadidi [11]. (C) Thermal melting profile of DNA in presence of a groove binding and an intercalating molecule. 3-Hydroxyflavone (3HF) is a groove binding molecule that does not increase the Tm of DNA while Quercitin (Q) follows intercalating mode and increases Tm significantly. Figure is reproduced with permission from the manuscript of Jana et al. [18].

conjugated double bond structure. Since the fluorescence property of DNA is negligible, change in the intrinsic fluorescence of drug is usually studied in presence of varying DNA concentration (Fig. 2A).



The change in fluorescence intensity is interpreted through Stern– Volmer plots by using the Stern–Volmer equation:

F 0 =F ¼ 1 þ K sv ½Q where F0 is the fluorescence intensity of fluorophore in absence of DNA; F is fluorescence intensity of fluorophore in presence of DNA; Ksv is the Stern–Volmer constant and [Q] is the concentration of DNA. A linear Stern–Volmer plot indicates either one type of binding or quenching process is occurring by static or dynamic mechanism [20]. To differentiate between the quenching processes, the bimolecular quenching rate constants, Kq, is evaluated using the following equation:

K q ¼ K sv =s0 where s0 is the lifetime of the biomolecule in absence of the quencher. Since, the fluorescence lifetime of the biomolecule is 108 s [21], Kq can be easily calculated. Also, the limiting diffusion rate constant of the biomolecule is known to be around 2.0 1010, thus if the value of Kq is higher than the limiting diffusion rate constant, the quenching process is static rather than dynamic [21]. Differentiation between the static and dynamic quenching can also be done on the basis of differences in Ksv values at different temperatures. On increasing the temperature, molecules move faster and hence an increase in the collision probability occurs, resulting in an increased dynamic quenching process. However, in case of static quenching, increasing the temperature decreases the stability of the complex formed and causes a decrease in the fluorescence quenching. In short, a decrease in Ksv values on increasing the temperature occurs in case of a static quenching process, while an increase in Ksv values is seen for a dynamic quenching process [22,23]. It is also possible to calculate the intrinsic binding constant from these fluorescence quenching experiments by using the following equation [24],

log F 0 F=F ¼ log K b þ n log½Q where Kb is the binding constant; n is the number of binding sites; F0 is the fluorescence intensity of small molecule in absence of DNA, while F is the fluorescence intensity of small molecule in presence of DNA and [Q] is the concentration of quenching molecule (DNA). Kb and n are easily calculated from the double logarithm regression curve of log (F0 F)/F versus the concentration of DNA [Q]. Fluorescence spectroscopy is often used to determine the binding mode of drugs with DNA by utilising various analytical tools that are based on the fluorescence emission property of the drug. Fluorescence titration of small molecule in presence of ssDNA and dsDNA is done to elucidate the binding mode of these small molecules with DNA (Fig. 2B). If the quenching in presence of dsDNA is more efficient than in presence of ssDNA, the small molecule binds to DNA majorly in the intercalative mode [21]. However, higher quenching in presence of ssDNA suggests a groove binding mode of interaction [21]. A dsDNA releases the intercalated small molecules when strand separation occurs, leading to a reduction in the quenching property of DNA. While in case of groove binders, the small molecules interact with base pairs of DNA through hydrogen binding and van der Waals forces [21]. Fluorescence spectroscopy also finds application in deducing various thermodynamic parameters involved in the interaction of small molecules with DNA [25,26]. Hydrogen bonding, van der Waals forces, hydrophobic interactions and electrostatic interactions are the four major non-covalent interactions that play an important role in drug–DNA interaction. Complex formation between small molecules and DNA is studied at different temperatures that allow determination of various thermodynamic parameters like enthalpy (DH) and entropy (DS) by applying the Van’t Hoff equation. Intercalation of small molecules into a DNA duplex

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is stabilised by hydrophobic interactions and van der Waals forces, while minor groove binding small molecules interact mainly via hydrophobic interactions [27]. It is known that the electrostatic force is the dominant non-covalent force when DH < 0 or DH 0 and DS > 0, while hydrophobic interactions are regarded as the main driving force when DH > 0 and DS > 0. Hydrogen bonds and van der Waals interaction are associated with DH < 0 and DS < 0 [28].

KI quenching studies Iodide quenching studies are commonly used to determine the binding mode of DNA with the drugs which are fluorescent. Quenching experiments are straight forward and indicate the location of the bound molecules to be either outside or inside of the helix. Iodide ions are negatively charged quenchers that can effectively quench the fluorescence of small molecules in an aqueous medium. On interaction with DNA, the iodide ions being negatively charged are repelled by the negatively charged phosphate backbone of DNA. Any small molecule intercalated into the DNA helix is well protected from being quenched as the approach of anionic quenchers towards the fluorophore is restricted. However, electrostatically bonded molecules as well as groove binders are exposed to the surrounding solvent and are not well protected from anionic quenchers even in the DNA environment [21]. Accessibility of the flourophore to anionic quencher in free medium as well as in presence of DNA is studied using the Stern–Volmer equation:

F 0 =F ¼ 1 þ K sv ½Q where F0 and F are the fluorescence intensities in the absence and presence of the anionic quencher [Q]; Ksv is the Stern Volmer quenching constant calculated from the slope of the (F0/F) vs [Q] plot. Ksv directly indicates the accessibility of iodide quencher to the fluorophore. The relative differences in the Ksv in absence and presence of DNA environment signify the binding mode of drug. Intercalation of small molecules into the DNA helix prevents the anionic quencher from approaching the fluorophore and leads to a decrease in Ksv as compared to Ksv in absence of DNA. This is the typical case with the intercalators of DNA (Fig. 2C).While in electrostatic interaction and groove binding, molecules are well exposed to the surrounding solvent and anionic quencher, the collision probability between small molecule and iodide anions in absence and presence of DNA will be almost equal. Therefore the Ksv value in the absence and presence of DNA is expected to be same. However, there are some reports that suggest a slight protection of fluorophore by DNA even in case of groove binding [19,29], but this protection is very less as compared to intercalation. Small or no differences in relative Ksv values are reported in case of groove binding (Fig. 2D). To further confirm the binding mode of a drug with DNA, iodide quenching studies can also be done in the presence of synthetic DNA that has desired sequences. For these studies, Poly (dA dT) and poly (dG dC) sequences are commonly used. Groove binders are known to preferably bind to AT rich sequences as compared to GC rich sequences [30]. For example, binding of trans-4-[4-(dime thylamino)styryl]-1-methylpyridinium iodide (DSMI, a groove binding dye) to poly (dA dT) sequence results in a greater enhancement in fluorescence yield as compared to poly (dG dC) sequences [19]. It is expected that iodide quenching in the presence of poly (dA dT) polymer should be more efficient than in the presence of poly (dG dC). This was clearly observed by the same group, where Ksv in the presence of an AT-rich sequence was much greater than in the presence of a GC-rich sequence or CT DNA [19].


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Fig. 2. Fluorescence spectroscopy to study drug–DNA interactions. (A) Fluorescence emission spectra of drug in absence and presence of DNA. On addition of DNA, there is decrease in the fluorescence intensity due to interaction of drug with DNA. Figure is reproduced with permission from the manuscript of Shahabadi and Maghsudi [94]. (B) Quenching of fluorescence emission intensity of groove binding drug by dsDNA and ssDNA. dsDNA and ssDNA are used to distinguish the binding mode of drug with DNA. It is known that minor groove binders bind preferably to ssDNA while intercalating molecules have high affinity for dsDNA. Figure is reproduced with permission from the manuscript of Ling et al. [21]. (C) Fluorescence quenching of intercalating drug by KI in absence and presence of DNA. There is a significant difference in KI quenching in absence and presence of DNA environment for any intercalating molecules. Figure is reproduced with permission from the manuscript of Cui et al. [95]. (D) KI quenching studies in case of groove binding molecule. There is very little difference in the fluorescence quenching in absence (a) and presence (b) of DNA for groove binders. Figure is reproduced with permission from the manuscript of Ling et al. [21]. (E) Effect of denaturing agents. With increasing concentration of urea, there is release of intercalating molecule due to separation of DNA strands and hence alteration in fluorescence behaviour. Figure is reproduced with permission from the manuscript of Grueso et al. [31]. (F) Studying role of ionic strength in drug–DNA interaction. On addition of NaCl, there is no change in the fluorescence intensity of drug–DNA system suggesting for the absence of surface binding. Figure is reproduced with permission from the manuscript of Khorasani-Motlagh et al. [10].

Effect of denaturing agents

Effect of ionic strength

Chemical denaturants like guanidine HCl and urea are frequently used to destabilise the double stranded DNA helix. Such denaturants are exploited to analyse the binding mode of small molecules with DNA [31–33]. DNA accommodates the intercalating molecules in the helix. As seen in Fig. 2E, on denaturation of the DNA helix by urea, the intercalated molecules are released in the solution leading to alteration in the fluorescence behaviour [31].

Studying the effect of ionic strength on drug–DNA interaction is also a resourceful method to analyse the binding mode between small molecules and DNA (Fig. 2F). Strong electrolytes such as NaCl are used for this purpose. Addition of NaCl to the free ligand in the absence of DNA should have little or no effect on the fluorescence yield of the ligand. However, in the presence of DNA, Na+ partly neutralises the negative charges of the DNA phosphate backbone resulting in reduced electrostatic repulsion between them.



The electrostatic attraction between the small molecule and DNA surface is weakened by the addition of Na+. In case of surface binding molecules, the electrostatic binding takes place out of the groove, where fluorescent intensity is quenched on binding with DNA. A small molecule sitting in the groove of the DNA helix is much more exposed to the ionic strength in the surrounding solvent than an intercalated molecule [19]. Addition of NaCl weakens the interaction and results in the release of the drug from DNA surface resulting in enhancement of the fluorescent intensity [34]. Competitive displacement assays Various well known DNA binding dyes are used to establish the mode of drug–DNA interactions. The binding of these dyes to DNA is well studied and their binding mode has been established. In competitive displacement assays performed using these fluorescent dyes, any small molecule that displaces the dye already bound to DNA will interact with DNA in the same mode as the displaced dye. The change in fluorescent behaviour on binding of small molecules to DNA-dye system can be easily interpreted. This assay is very commonly used to differentiate between the binding modes. Ethidium bromide (EB) is extensively used as a fluorescence probe that binds to DNA in an intercalative fashion [35–39]. The fluorescence of EB increases when it gets intercalated between DNA base pairs. In competitive binding assays using EB, any molecule that binds to DNA via the same mode as EB, will displace EB from DNA helix and result in a decrease in the fluorescence intensity of DNA–EB system (Fig. 3A). The extent of fluorescence quenching of DNA–EB system can be used to determine the extent of intercalation between the molecule and DNA [37,40]. Groove binding and surface binding molecules show no effect on the DNA–EB fluorescence intensity [41,42]. In recent years, another planar phenazine dye, neutral red (NR) has been used to study the mode of interaction of small molecules with DNA. NR binds to DNA in intercalative mode and results in enhancement of fluorescence intensity of NR on binding with DNA [43]. Small molecules can displace NR from DNA helix and decrease the emission intensity of DNA-NR systems only if they intercalate into the bases of DNA [43]. Acridine Orange (AO), a well known classical intercalating dye, is also used in similar competitive displacement assays [44,45]. Any molecule that intercalates into the helix of DNA would displace AO from the intercalation sites in DNA and result in a significant decrease in the fluorescence intensity of DNA-AO system (Fig. 3B). A study also used methylene blue (MB), a phenothiazinium dye, which interacts with DNA via an intercalation mode [46]. On binding with DNA, there is a decrease in the fluorescence intensity of MB. Competitive displacement of MB from DNA helix by any other intercalator will lead to the enhancement of fluorescence intensity which was earlier quenched by DNA. However, small molecules binding to DNA in non-intercalative mode are not able to release MB from DNA helix and hence no or little change in the fluorescent intensity is observed (Fig. 3C). In case of groove binders, competitive displacement studies are done using Hoechst 33258 that binds to the minor groove of double stranded B-DNA and has high specificity for AT-rich sequences [47]. Hoechst 33258, on binding with DNA, shows enhancement in the fluorescent intensity [48–50]. Groove binding molecules are able to displace Hoechst 33258 from the minor groove of DNA helix resulting in reduced fluorescent yield of the DNA–Hoechst system (Fig. 3D). CD spectroscopy CD spectral technique is a very sensitive technique that can be utilised to detect any changes that occur in the secondary structure

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of polypeptides, proteins and DNA under the influence of interacting ligands. It has been extensively employed for the analysis of changes that occur in the DNA backbone upon binding with the drug. Changes observed in the CD signals of DNA are linked to corresponding changes in the DNA structure. Therefore, CD spectroscopy is quite often used to identify the backbone distortions in DNA affected by the binding of the drug. Various non-covalent DNA–drug interactions may affect the DNA structure leading to an altered CD spectral behaviour [5,51]. Calf thymus DNA is extensively used as representative of DNA helix to study the interaction with various small molecules. In the CD spectrum of native calf thymus DNA, two major bands are studied i.e., at 277 nm (positive), 243 nm (negative) (Fig. 4A). The positive band at 277 nm is due to base stacking, while helicity is responsible for the negative band at 243 nm, which is characteristic of DNA in right-handed B form [52]. These bands are considered to be highly sensitive towards interaction of small molecules with DNA. The CD spectra of calf thymus DNA shows no or very little alteration in case of electrostatic binding and minor groove binding (Fig. 4B). However, on intercalation (Fig. 4A), both the positive and negative bands are altered significantly [5,53]. Other inferences can also be obtained by studying the changes in position and intensity of bands or both. For example, during the transition of DNA double helix from B to A conformation, band at 277 nm shows an increase in intensity while band at 213 nm shows decrease in intensity and the position of band at 223 nm is shifted towards the higher wavelength [54]. Viscosity measurements Viscosity measurement is a hydrodynamic method of determining the binding mode of drug with DNA [55–57]. It is highly sensitive to change in the length of DNA and is considered as one of the most reliable techniques for DNA binding studies in solution. When an intercalator binds to the DNA helix, the base pairs tend to separate in order to accommodate the binding molecule into the helix leading to an increase in the overall length of the DNA and hence an increase in viscosity is observed (Fig. 4C). A decrease in relative viscosity is reported when the interaction of a ligand leads to the bending of DNA; the consequent reduction in length of DNA reduces the relative viscosity. Cisplatin covalently binds to DNA and leads to a decrease in the relative viscosity by causing a bend in the DNA structure [58]. Groove binders do not change the relative viscosity of DNA (Fig. 4D) since they do not alter the axial length of DNA upon binding [41]. However, a slight increase in the relative viscosity may also be considered as an indication of groove binding. Footprinting assay Small molecules interacting with DNA via the groove binding mode can also be identified by footprinting technique. Information such as binding affinity and sequence selectivity are often obtained using footprinting techniques [59,60]. In short, binding of small molecules to a specific region of DNA helix protects that region from cleavage by a cleaving agent. Most commonly used cleaving agents are Dnase I and hydroxyl radicals and the methods are called Dnase I footprinting and hydroxyl radical footprinting assays, respectively. Radiolabelled double stranded DNA is used in this study and the digestion products are resolved on a denaturing polyacrylamide gel. Comparison of the gel patterns obtained from the control set (without test compound) and protection set (with the test compound) is done. Missing bands in the protection set gel pattern denotes the protection of the DNA at a particular region and hence the binding of small molecules to that particular region is established (Fig. 5A).


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Fig. 3. Competitive displacement assays using dyes. (A) Ethidium bromide displacement assay. Intercalating molecules compete for the binding sites with ethidium bromide and displace it from DNA helix resulting in decreased fluorescence intensity of EB–DNA complex. Figure is reproduced with permission from the manuscript of Ozluer and Kara [39]. (B) Acridine orange displacement assay. On addition of intercalating small molecule, there is decrease in fluorescence intensity of AO–DNA complex. Figure is reproduced with permission from the manuscript of Li et al. [2]. (C) Displacement assay using methylene blue (MB). MB alone is fluorescent however, fluorescence intensity of MB decreases due to intercalation in DNA helix. In presence of intercalating molecule, MB is displaced from helix which leads to increase in the fluorescence intensity of MB. However, there is no change in the fluorescence intensity of MB-DNA system on addition of non-intercalating molecules. Figure is reproduced with permission from the manuscript of Shahabadi and Hadidi [11]. (D) Displacement of groove binding dye by groove binding molecule. Hoechst, a groove binding dye is fluorescent when complexed with DNA. However, on addition of another groove binding molecule capable of displacing Hoechst causes decrease in the fluorescence intensity of Hoechst–DNA complex. Figure is reproduced with permission from the manuscript of Shahabadi and Maghsudi [94].

FT-IR spectroscopy

Isothermal titration calorimetry (ITC)

Fourier transform infrared (FT-IR) spectroscopy finds great application in biological sciences. It provides an easy way of identifying functional groups present in a molecule and thus identity of a pure compound can be established. Infrared (IR) radiation is passed through a sample where some of the radiations are absorbed, while some are transmitted. The resulting raw data is presented in the form of a spectrum that acts like a molecular fingerprint of the compound or molecule. While studying drug–DNA interaction, spectral changes i.e. change in intensity and shift in band positions of several DNA marker bands are monitored in presence of varying concentrations of a drug [61]. Molecular fingerprint of pure DNA lies in the spectral region of 1800–700 cm1 due to in plane vibrations of various nitrogenous bases, phosphate stretching vibrations (asymmetric and symmetric) and deoxyribose stretching. Binding of small molecules to DNA bases are studied by monitoring spectral change at 1714 cm1 that is mainly attributed to guanine (G) in plane stretching vibrations, 1661 cm1 caused by thymine base vibrations (T), 1610 and 1493 cm1 caused by adenine (A) and cytosine (C) stretching [54,62–64]. Binding of small molecules to sugar phosphate backbone of DNA is studied by monitoring the changes in the bands at 1228 and 1087 cm1 that are caused by phosphate asymmetric and symmetric vibrations, respectively [64]. The transition of DNA double helix conformation from B to A form or from B to Z form can easily be detected on binding of drugs to DNA [54,64,65]. Fig. 5B depicts the FT-IR spectra of calf thymus DNA in absence and presence of an intercalating drug.

ITC is one of the most accurate methods employed to characterise the binding interaction of small molecules and DNA (Fig. 5C). Various thermodynamic parameters of the interaction are easily obtained directly or indirectly and the mechanism can be easily elucidated. ITC measures the heat absorbed or released during the interaction and calculates various parameters like binding sites (n), association constant (K), change in enthalpy (DH) and change in entropy (DS) [13,66–68], which are used to decipher the binding interaction and binding mode. For example, a large negative enthalpy of binding along with positive entropy is typical for an interaction of small molecule with DNA [69]. Strong positive entropy signifies the disruption and release of water molecules on intercalation of small molecules into the DNA helix. The magnitude and type of forces involved in interaction of small molecules with DNA can be determined by studying the change in heat capacity (DCp). For this, enthalpy of binding at different temperatures is obtained using ITC by applying the standard relationship:

DC p ¼ @ðDHÞ=@T ITC can be also employed to decipher the binding mode of small molecules to DNA using AT-rich and GC-rich sequences [7]. It is known that groove binders preferably bind to AT-rich rather than GC-rich sequences. Various parameters are obtained on interaction of small molecules with these sequences and affinity of small molecules towards these DNA sequences are easily compared [7].



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Fig. 4. CD spectroscopy and viscosity measurement studies to study drug–DNA interactions. (A) CD spectra of CT-DNA in absence and presence of an intercalating molecule. Figure is reproduced with permission from the manuscript of Husain et al. [5]. (B) CD spectra of CT-DNA in absence and presence of a groove binding molecule. Figure is reproduced with permission from the manuscript of Rehman et al. [41]. (C) Viscosity of DNA in absence and presence of increasing concentration of an intercalating molecule. There is significant increase in the viscosity of DNA. Figure is reproduced with permission from the manuscript of Husain et al. [5]. (D) Viscosity of DNA in presence of groove binding molecule. With increasing concentration of groove binding molecule, there is negligible change in the viscosity of groove binding molecules. Figure is reproduced with permission from the manuscript of Rehman et al. [41].

Atomic force microscopy Atomic force microscopy (AFM), owing to its spatial resolution, is often used to study the changes in DNA morphology on interaction with small molecules [70,71]. It provides direct evidence of DNA–drug interaction at molecular levels [72]. The two main characteristics of DNA morphology studied using AFM are the contour length (L) and persistence length (P). Contour length represents the length of completely extended DNA molecule, while the persistence length measures the DNA flexibility. On interaction of small molecules with DNA, a change in DNA contour lengths is obtained from AFM images and analysed for studying the interaction and differentiating between the intercalation and groove binding mode [71]. On interaction with intercalating molecules like doxorubicin and ethidium bromide, an increase in the contour length of DNA was observed using AFM [71]. However, no change in the contour length of DNA was observed in the presence of netropsin, a groove binding drug [71]. Fig. 6 represents the change in DNA morphology in presence of increasing concentration of doxorubicin. Many reviews have been published recently that discuss AFM in more detail [73–75]. AFM is also useful in studying the interaction of DNA with proteins [76,77] where DNA bending caused by interacting proteins is easily monitored [78]. Cyclic voltammetry Electrochemical investigation of drug–DNA interactions can complement the findings of previously used methods such as UV–visible spectroscopy and fluorescence spectroscopy among the others. The interaction of some small molecules with DNA cannot be well established because of various limitations such as weak absorption spectra, overlap of electronic transition with that of

DNA or because of weak or no fluorescence spectra. Voltammetric techniques may be employed under such circumstances. Cyclic voltammetry (CV) is overwhelmingly used for metal based compounds due to their redox states. Analysis of the changes induced in the electrochemical signal by drug–DNA interaction can provide evidences for the interaction mechanism, nature of the redox species formed, binding constant and size of binding sites [76]. A change in the peak current of a drug with increasing concentration of DNA can be used for the determination of its binding constant and the size of binding sites, whereas a shift in the peak potential due to oxidation or reduction of the bound species on the amount of added DNA can be used to ascertain the mode of interaction such as intercalative, electrostatic or hydrophobic interactions [77]. The change in the peak current with increasing concentration of DNA can be used to calculate the binging constant with the help of following equation [78],

1=½DNA ¼ ½Kð1 AÞ=1 I=I0 K where A is the proportionality constant, K is the binding constant, I0 and I are the peak currents of the drugs obtained by CV in the absence and presence of DNA. The binding constant can be easily calculated from the intercept of the plot of 1/[DNA] vs 1(1 I/I0). As seen in Fig. 7A, with increasing concentration of DNA, there was a decrease in the peak current of NBI (5-benzylideneimidazoli dine-2,4-dione), accompanied with a slight cathodic followed by an anodic shift. The initial cathodic shift indicates an electrostatic interaction of NBI with the phosphate backbone of DNA, while the anodic shift observed with increasing concentration of DNA is due to the groove binding and partial intercalation [78]. Several recent studies have implicated the use of CV to understand the drug– DNA interactions in details [79–82].


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Fig. 5. Techniques to study drug–DNA interaction. (A) DNA footprinting assay to study binding of drug with DNA. Figure is reproduced with permission from the manuscript of Hampshire et al. [59]. (B) FTIR spectra of DNA in presence of increasing concentration (a–d) of intercalating drug where (a) free calf-thymus DNA and (b–d) mitoxantrone/ DNA molar ratios of 1/50, 1/30 and 1/15 respectively. Figure is reproduced with permission from the manuscript of Agarwal et al. [54]. (C) ITC profile of thionine interaction with (a) CP DNA (b) HT DNA and (c) ML DNA. Figure is reproduced with permission from the manuscript of Paul et al. [68].

Nuclear magnetic resonance (NMR) NMR is based on the fact that certain atomic nuclei such as 1H, C, 15N and 31P carry magnetic dipoles. Every nucleus with its different characteristic spin gives rise to a signal or peak which represents a transition. 1H is the most commonly used atomic nuclei for

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DNA binding study, but to study the effects of ligand binding on the phosphate groups of DNA, 31P NMR is also used [86]. Two main features of NMR spectra studied in drug–DNA interaction are the change in line widths and chemical shifts on interaction [87]. 1H NMR signals can easily differentiate between the different types of interaction mode. Intercalation of molecules into the DNA helix



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Fig. 6. Studying interaction of small molecule with DNA using AFM. AFM images of DNA deposited on mica in 2 mM nickel and 10 mM HEPES at different doxorubicin concentrations (a = 0.1 lM, b = 0.4 lM, c = 0.7 lM, d = 2.7 lM, e = 3.7 lM, and f = 5.5 lM). There is negligible effect on the chain conformation of DNA at lower doxorubicin concentration but with increasing concentration of drug, chain loops and overlaps are clearly visible and filaments appear more and more aggregated and entangled. With further increase in the doxorubicin concentration, some aggregated and disordered coils appear with a complete collapse of DNA at the highest concentration. Figure is reproduced with permission from the manuscript of Cassina et al. [71].

results in the total line broadening of 1H NMR signal. In case of partially intercalated molecule, line broadening and upfield chemical shift of the signal is recorded. These changes are possibly due to two reasons: (i) weak restriction of molecular tumbling in the DNA-complex; and/or (ii) slow rate of exchange between various DNA binding sites and the unbound states. However, in case of groove binding molecules, no line broadening or upfield chemical shift is recorded. This indicates presence of rapid tumbling of molecules as well as fast exchange between various binding sites of DNA [88]. Fig. 7B represents the 1H NMR spectra of chlorobenzylidine in the absence and presence of CT-DNA. The spectra clearly show extensive broadening of different peaks in the presence of DNA, suggesting an intercalation mode of interaction between chlorobenzylidine and DNA [88].

acceptor [5]. Spectral overlap was calculated from the Fig. 8A, as the emission spectrum of drug–DNA complex overlaps with the absorption spectrum of EB. Also, direct interaction of EB with naproxen was negligible as seen in Fig. 8B. In another experiment, a continuous decrease in the emission intensity of naproxen–DNA complex was observed with the increasing concentration of EB (Fig. 8C). Interestingly, a new peak appearing at 600 nm was also observed (Fig. 8C inset). Since the naproxen–DNA solution was excited at 230 nm where EB has minimum absorption, the new peak appearing was possibly due to FRET between the excimer of naproxen and EB in presence of DNA. With the help of data obtained from these experiments, FRET was calculated according to Forster’s theory where the efficiency of FRET depends on the inverse sixth power of the distance between the donor and acceptor molecule (RDA):

Forster (fluorescence) resonance energy transfer (FRET)

EFRET ¼ ½1 þ ðRDA =Ro Þ6

FRET involves distance dependant radiationless transfer of energy from an excited donor molecule to an appropriate acceptor molecule. It is widely used to determine distances, orientations, proximities and dynamic properties of biomolecular structures. Due to its sensitivity to distance, FRET has been used to investigate molecular level interactions. Interaction of small molecules with the DNA has also been studied with the help of FRET [5,51]. There are certain criteria that must be fulfilled in order to study the drug–DNA interaction with the help of FRET. The first criterion is that the fluorescence emission spectrum of donor molecule must overlap the excitation spectrum of acceptor molecule. Also, the donor and acceptor molecules must be in close proximity to one another. Finally, the donor and acceptor transition dipole moment must be correctly oriented and donor should have high quantum yield [89]. In order to explain the application of FRET in studying the drug– DNA interaction, a work published earlier is discussed here [5]. Interaction of naproxen with DNA was studied using FRET where drug–DNA complex was taken as energy donor and EB as energy

1

where Ro is the Forster distance that represents the distance at which the efficiency of transfer is 50%. Ro is calculated using equation given below [20]: 2

Ro ¼ 8:8 1025 k /D N4 J where k2 denotes orientation factor, /D is the fluorescence quantum yield of the donor in the absence of FRET, N is the refractive index of the medium and J represents the spectral overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor which was calculated using equation given below [90]:

R JðkÞ ¼

FðkÞeðkÞk4 dk R FðkÞdk

where F(k) is the fluorescence intensity of the donor in the wavelength range of k to k+ dk and is dimensionless and 2(k) is the extinction coefficient (in M1 cm1) of the acceptor at k. E can also be estimated using the fluorescence emission intensity both in the absence and presence of acceptor by using the equation:


10


Fig. 7. Studying drug–DNA interaction with the help of cyclic voltammetry and 1H NMR. (A) Cyclic voltammogram of a small molecule in absence and presence of increasing concentration of DNA. With increasing concentration of DNA, there is a decrease in the peak current with an initial cathodic shift followed by anodic shift. The initial cathodic shift is related to electrostatic interaction while the anodic shift is observed due to groove binding and partial intercalation. Figure is reproduced with permission from the manuscript of Shah et al. [78]. (B) 1H NMR spectra used to study the binding mode. The 1H NMR spectra of chlorobenzylidine in absence (above) and presence (below) of DNA is shown here. There is an extensive broadening of different peaks in the presence of DNA due to intercalation of chlorobenzylidine in the DNA helix. NMR technique is regularly used in different studies [83,84]. Figure is reproduced with permission from the manuscript of Zhong et al. [85].

EFRET ¼ 1 ðF=F 0 Þ Spectral overlap, the Forster distance, distance between the energy donor and acceptor and FRET efficiency was calculated from these equations and it was found to be 3.2 1015, 1.92 nm, 2.11 nm and 35%, respectively [5]. Since the distance between the donor and acceptor (RDA) is close to the critical distance at which the energy transfer is 50% (Ro), the energy transfer from naproxen–DNA to EB could occur with high probability. This also

Fig. 8. Studying the interaction of drug with DNA using FRET. (A) Spectral overlap between the energy donor and energy acceptor. The figure shows the overlap of normalised absorption spectrum of EB and the emission spectrum of naproxen in presence of DNA. (B) Interaction of naproxen with EB. Emission spectra of naproxen in the presence of increasing concentration of EB show no changes. This suggests absence of any direct interaction between naproxen and EB. (C) Changes in the emission spectrum of naproxen in presence of DNA with addition of EB. Figure is reproduced with permission from the manuscript of Husain et al. [5].

provides an indirect evidence for intercalation mode of binding for naproxen with DNA. Also, DNA bending or looping caused by interacting proteins can be easily studied using FRET [91,92].



11

Fig. 9. Molecular docking study to decipher drug–DNA interactions. Representative figure of a molecule interacting with DNA via groove binding mode. Figure is adapted from Sarwar et al. [25] and reproduced by permission of The Royal Society of Chemistry.

Molecular docking

Conflict of interest

Molecular docking plays an important role in establishing the interaction between different molecules and helps in structure based drug discovery. Docking is usually performed between small molecules and a target macromolecule like protein and DNA. The use of automated molecular docking software helps in predicting the binding mode, formation of energetically favourable complex and predicting binding affinity. Molecular docking provides information about the mechanism of binding of various drugs and hence contributes significantly in designing new drugs [5,51,61]. Use of molecular modelling techniques prior to experimental screening of various compounds reduces labour and is cost effective. Molecular docking also corroborates experimental results and helps in understanding the mechanisms of interaction. Molecular modelling programs are commonly employed in determining the binding mode of various drugs where intercalators and groove binders are easily distinguished (Fig. 9) [5,25,26,41,42,51]. The beginning step of docking process involves retrieving the structure of the receptor or macromolecule (DNA) from Protein Data Bank (PDB) available at http://www.rcsb.org/ pdb. Ligands are then made flexible to attain different conformations in order to predict the best fit orientation for binding to DNA helix and best energy docked structures are further analysed. Several runs of search are carried out to determine the best ligand-receptor orientation [93]. There are various modelling theories and algorithms that have been evolved in the last decades. Discussing these in details is beyond the scope of this review article.

The authors declare that there is no conflict of interest in this work.

Concluding remarks With the introduction of various biophysical techniques to study drug–DNA interactions, there has been tremendous increase in number of published articles focussing on these interactions. These studies involve a number of techniques employed to confirm the interaction and binding mode of novel and existing drugs with DNA and provide an insight into the functioning of these drugs and therefore help in novel drug designing. The current review article provides an overview of these commonly used techniques and their applications. Author contributions Conceived, designed and wrote the manuscript: SUR TS MAH HMI MT.

Acknowledgements Authors are thankful to CSIR, New Delhi, for the award of CSIR-SRF fellowship to SUR (File no - 09/112(0470)/2011-EMR1) and UGC for the award of MANF to TS and MAH. Department of Biochemistry, Faculty of Life Sciences, AMU, Aligarh is also acknowledged for providing the necessary facilities. We are also thankful to DBT, New Delhi for providing generous funding to MT (Project number: BT/PR8032/BID/7/443/2013). References [1] S. Sobha, R. Mahalakshmi, N. Raman, Spectrochim. Acta A Mol. Biomol. Spectrosc. 92 (2012) 175–183. [2] X.L. Li, Y.J. Hu, H. Wang, B.Q. Yu, H.L. Yue, Biomacromolecules 13 (2012) 873– 880. [3] W.D. Sasikala, A. Mukherjee, J. Phys. Chem. B 116 (2012) 12208–12212. [4] A. Rescifina, C. Zagni, M.G. Varrica, V. Pistarà, A. Corsaro, Eur. J. Med. Chem. 74 (2014) 95–115. [5] M.A. Husain, Z. Yaseen, S.U. Rehman, T. Sarwar, M. Tabish, FEBS J. 280 (2013) 6569–6580. [6] W. Hu, S. Deng, J. Huang, Y. Lu, X. Le, W. Zheng, J. Inorg. Biochem. 127 (2013) 90–108. [7] McKnight RE, in: Amal Ali Elkordy (Ed.), Isothermal Titration Calorimetry and Microcalorimetry, ISBN: 978-953-51-0947-1, InTech, doi: 10.5772/54061, 2013. [8] M. Ganeshpandian, S. Ramakrishnan, M. Palaniandavar, E. Suresh, A. Riyasdeen, M.A. Akbarsha, J. Inorg. Biochem. 140 (2014) 202–212. [9] G. Zhang, P. Fu, L. Wang, M. Hu, J. Agric. Food Chem. 59 (2011) 8944–8952. [10] M. Khorasani-Motlagh, M. Noroozifar, S. Mirkazehi-Rigi, Spectrochim. Acta A Mol. Biomol. Spectrosc. 75 (2010) 598–603. [11] N. Shahabadi, S. Hadidi, Spectrochim. Acta A Mol. Biomol. Spectrosc. 96 (2012) 278–283. [12] C. Wei, J. Wang, M. Zhang, Biophys. Chem. 148 (2010) 51–55. [13] K. Bhadra, G.S. Kumar, Biochim. Biophys. Acta 1810 (2011) 485–496. [14] K. Akdi, R.A. Vilaplana, S. Kamah, F. Gonzalez-Vilchez, J. Inorg. Biochem. 99 (2005) 1360–1368. [15] Y. Song, D. Zhong, J. Luo, H. Tan, S. Chen, P. Li, L. Wang, T. Wang, Luminescence 29 (2014) 1141–1147. [16] F. Arjmand, S. Parveen, M. Afzal, L. Toupet, T. Ben Hadda, Eur. J. Med. Chem. 49 (2012) 141–150. [17] L. Wang, Y. Wu, T. Chen, C. Wei, Int. J. Biol. Macromol. 52 (2013) 1–8. [18] B. Jana, S. Senapati, D. Ghosh, D. Bose, N. Chattopadhyay, J. Phys. Chem. B 116 (2012) 639–645. [19] C.V. Kumar, R.S. Turner, E.H. Asuncion, J. Photochem. Photobiol. A 74 (1993) 231–238. [20] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed., Springer, Berlin, 2006. 278–282. [21] X. Ling, W. Zhong, Q. Huang, K. Ni, J. Photochem. Photobiol. B 93 (2008) 172– 176.


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