Study of Interactions Between 3-benzoyl-4-hydroxy-2-methyl-2H-1, 2-benzothiazine and Human DNA by Theoretical, Spectroscopic and Viscometric measurements

From the last few years mode of interactions between drugs and DNA is an attractive research area as it bridges chemistry, molecular biology and medicinal science. Interactions between small heterocyclic molecules and human DNA is a noteworthy feature in pharmacology for investigation of drugs mechanism and designing of more effective and target specific drugs with fewer side effects. The present research work focuses on the theoretical investigations of 3-benzoyl-4-hydroxy-2-methyl-2H-1, 2-benzothiazine (SASA) by using Gaussian (16 W) software to predict optimized geometry, HOMO–LUMO gap, bond length, bond angle, dihedral angle, electronic and vibrational spectra. Possible reaction site observed in SASA was C7, C9 and C18 as these atoms show maximum charge density. Later the interactions of SASA with human DNA was explored spectroscopic investigations and viscometric investigations at physiological buffers of pH of 4.7 (stomach pH) and 7.4 (blood pH) respectively. Maximum absorbance between SASA-DNA complex was observed in buffer solution of pH 3.4 at wavelength of 370 nm, whereas at 7.4 has maximim absorbance between. Spectroscopic results reflects the bathochromic and hyperchromic shift succeeding the addition of human DNA. During viscosity measurement, intercalation and electrostatic mode of interaction were detected at low and high concentration of drug in solution respectively. Increase in the value of rate constant was observed with the increase in concentration of drug. Larger values of rate constant were observed at pH 7.4 in comparison to pH 3.5. Rate constant, thermodynamic parameters and viscometric analysis prefers the intake of SASA via blood.


Introduction
Study of chemical compounds with the help of computer programs for the estimation of their chemical properties is called the computational chemistry or the computational study of that particular chemical compound. Numbers of software have been developed for the evaluation of chemical properties by researchers [1].
Mode of binding of cisplatin with DNA is different from other drugs and the way of intake of cisplatin is by mouth. Prediction of binding of cisplatin with DNA by Raman spectroscopy removed all confusions about the Gaussian Raman spectra. The main purpose of this work was the investigation 1 3 of validity of mPW1PW basis set for spectral calculations [2].
Moleculer orbitals, minimum energy structure, stable conformation, strength of hydrogen bond and many other properties of the Ketoprofen was investigated by DFT calculations. GUASSIAN 98 W was used for that work.
The E HOMO -E LUMO was smallest for 2-SHBI and other properties like dipole moment and electronegativity calculated by DFT method gave prediction that the deterioration inhibition efficiency was decreased as following 2mercaptobenzimidazole > 2-methylbenzimidazole > benzimidazole.
Calculations were done on Gaussian software and P 1 derivative have high deterioration inhibition efficiency. In P 1 the CH 3 group was replaced by Cl and bond length, bond distance and dihedral angle were changed. Electron availability due to Sulfur atom was increased which provide evidence of high inhibition efficiency of P 1. P 1 structure was less distorted than P 2 . That is why P 1 have high inhibition efficiency [3].
DFT technique was used on Gaussian to predict that amino acid was the most excellent solvent for CO 2 capturing. As CO 2 was important greenhouse gas so it was essential to imprison this gas form different man mad resources. Computational studies predict that amino acids were more established resource and amino acids have high efficiency for Post-combustion capture because carbamates of amino acids were more stable [4].
DFT methods were used to found the regioselectivity of drugs and molecules processed by aldehyde oxidase which is a molybdenum hydroxylase that catalyzes the oxidation of aldehydes and nitrogen containing heterocycles. Using density functional theory methods, accurate geometry of tetrahedral intermediates of drugs and drug like compounds were investigated to anticipate the expected metabolites of aldehyde oxidase. All geometries and energies found in that study were computed using the B3LYP density functional theory method implemented in the Gaussian03 program package. Reliable geometry was obtained using the 6-31G(d,p) basis set and single-point calculations on the accurate geometries were performed with the bigger basis set 6-311 + G(2d,2p) to obtain more perfect energy values [5].
Computational study of diketopyrrolopyrrole-based organic dyes for dye sensitized solar cell applications was completed. Four diketopyrrolopyrrole (DPP)-based organic dyes were observed by density functional theory (DFT) and time-dependent DFT (TDDFT) approaches. Four dyes were designed by different donor groups, i.e. indoline, carbazole, triphenylamine, and coumarin. They determined the effects of the DPP unit and different contributors on the spectra and electrochemical properties of the dyes, individually. The DPP dyes exhibited curiously improved spectral responses in the visible region of the solar spectrum in association with the model dye having minimum LUMO levels and therefore band gap was reduced. They explored some significant properties including absorption spectra, light-harvesting efficiency, molecular orbital distributions, and injection time of electrons from the excited state of dye. The dye DPP-I with indoline moiety as the electron donor specified wanted energetic, electronic, and spectroscopic parameters for dye sensitized solar cells (DSSCs) applications.
By applying density functional theory (DFT) calculations, the structural, vibrational and electronic properties of two familiar model compounds of polyaniline pernigraniline have been inspected. Polypernigraniline was individual polyaniline to inhabit a doubly degenerate ground state. A comprehensive vibrational analysis of phenyl-end-capped dimer (B2Q1) and phenyl-end-capped tetramer (B3Q2) was presented and complete assignments were described. DFT calculations with 6-31G** basis set provided accurate results of IR and Raman spectra. TD-DFT calculations for HOMO-LUMO analysis give awareness to the electronic structure of these model compounds.
Investigations on the interactions between small heterocyclic molecules acting as drugs and human DNA has gathered attention of many scientists as it helps in understanding the mode of interactions and discovery of new drugs [6][7][8][9].
Examination of structure of DNA shows the presence of large numbers of positive and negative ions which can react with the drug molecules. As a result of interactions between drug and DNA complex structural changes are observed in both drug and DNA molecules [10,11]. Association between targeted drug and DNA takes place at definite discrete sites, known as binding sites and resultantly the solvent molecules are dislocated. Both drug and DNA possess opposite charges and due to the reorientation of charges between drug-DNA complex and solvent molecules, solvation of complex is observed, which leads to extra stability [12]. The heterocyclic compounds containing nitrogen and sulphur atoms play a very vital role in the field of medicinal chemistry. Derivatives of Benzthiazine are target of interest due to their incredible biological and pharmacological properties [13][14][15][16][17][18][19]. A number of these derivatives show remarkable properties as blocker of various receptors of nicotinic, muscarinic, histamine H and serotonine 5HT3 receptors. 1,2 -benzothiazine nucleus possess exceptional anti-inflammatory [20], antimalarial [21], antipro-liferative [22] and analgesic activities [23]. Various derivatives of 1,2-benzothiazine are excellent drugs for treatment of vascoocclusive disorders [24]. 3-benzoyl-4-hydroxy-2-methyl-2H-1,2 benzothiazine (SASA) was synthesized by multistep reactions [25]. SASA shows remarkable potential as antimicrobial [26], antioxidant [27] and anti-HIV [28]. Most of the derivatives of thiazine are insoluble in water so the co-solvent immiscible with water i.e. benzene, toluene and DMSO [29] was used. Since the derivatives of 1,2-benzothiazine possesses outstanding pharmaceutical properties so the investigation of mode of interaction with DNA will be subject of interest.
Theoretical predictions help to design new compounds. Computational chemistry is rapidly developing subfield of theoretical chemistry, where the main attention is on solving chemical problems by calculations. Computational study is mainly the study of predictions given by the theoretical overview. Hyperchem and Gaussian software were used to examine optimize molecular geometry, bond length, bond angle, torsion angle, energy calculation, kinetics of the reactions, IR, UV-Visible, and NMR spectra of the chemical compound under investigation.
In DFT calculations entire energy is expressed in terms of the total electron concentration, instead of the wave function. Computational chemistry is used to describe the precise structure and strength of chemical system, to investigate energy variances between different shapes, and to elucidate reaction pathways and mechanism at atomic level. It is also used to examine the properties of solids in materials science. Semiconductors, superconductors, plastics, ceramics; all these have been examined with the aid of computational chemistry.
In pharmacological field computational chemistry has vital role for studying the interaction of drugs with biomolecules, structure of active site of proteins for synthesizing their compatible substrate to bind on these active site to cure or to control the disease. New drugs could be synthesized by the information of theoretical study. The drug control or inhibition of disease is done by making interaction with DNA. Binding of small molecules of drug with DNA by covalent and non-covalent bonds depend upon the interaction between them [30].
The drug-DNA interaction is a significant feature in pharmacological field and three types of mode of interaction were investigated by non-covalent interaction.
• Electrostatic interaction: It is the binding of small molecules with negatively charged sugar-phosphate back bone of DNA. • Hydrogen bonding and electrostatic interaction: It is caused by binding of small molecules on minor and major grooves. • Intercalation: It is the binding of small planer organic molecules with DNA base pairs [31].
DNA interacts with the drug by the charged sugar-phosphate groups, through its base pairs that was named intercalation and through minor and major groove binding [32]. Intercalation is the strong binding mode between different biomolecules and DNA [16,33]. Intercalation mode of interaction was first time proposed by Lerman and he also predicted that planer molecules show intercalation mode of interaction with DNA [34]. More efficient anticancer and antitumor drugs could be designed by the Study of binding of small molecules with DNA [35]. Experimentally a number of spectroscopic techniques like UV-Visible, fluorescence, Raman, Nuclear magnetic resonance and atomic force microscopy could be used to investigate the drug-DNA interactions [6,[36][37][38]. Viscosity measurement, thermal denaturation and DNA-foot printing could be utilized for the same purpose [39][40][41] Predictions during UV-Visible study for interactions between Drug-DNA complex could be obtained by observing the variations in the absorption spectra of the drug, the DNA molecules and Drug-DNA complex [42]. Hypochromism and red shift could be the sign of intercalation while hyperchromism might be the sign of electrostatic interactions. Extension, unwinding and rigidity of DNA resulted due to the intercalative binding of drug with DNA. The UV-Visible absorption spectrum of DNA shows a broad band (200-350 nm) in the UV region with a maximum absorption at 260 nm [43]. Strength of binding interaction could be investigated by calculating binding constant (K f ) [44]. Way of collaboration of small molecules with DNA could also be visualized by calculating viscosity of the drug and drug-DNA complexes [45].
The viscosity measurement is based on the flow rate of a DNA solution through a capillary viscometer [46] Intercalation way of interaction is predicted by the increase in viscosity due to the increase in the length of DNAdouble helix . Evidence about the electrostatic mode of interaction is given by the decrease in viscosity value if the concentration of drug in solution is decreased. Strong argument for mode of interaction could be obtained by Viscometric study [47]
SASA was synthesized by Dr. Sana Aslam during her PhD in university of Punjab, Lahore, Pakistan and it was provided in pure form after complete purification followed by characterization [25]. This heterocyclic compound under investigation contains nitrogen and sulphur atoms. This compound is of significant importance in the field of medicinal chemistry. Benzthiazine is a diverse compound having biological and pharmacological importance. The
Analytical grade Dimethylsulfoxide was purchased from Sigma Aldrich Chemical Co.USA. Human DNA was purchased from molecular lab, Agriculture University Faisalabad, Pakistan. Stock solution of selected SASA drug was prepared by adding 0.004 g selected drug in 100 ml DMSO. All investigations were carried out in buffers having pH 3.5 (stomach pH) and pH 7.4 (blood pH).

Spectrophotometric Study
UV/Vis absorbance spectra were obtained by UV752PC UV-VIS spectrophotometer with a quartz micro-colorimetric vessel of 1-cm path length. Absorption of solution was recorded at constant concentration of DNA and varying the concentration of SASA. Observed Spectral shifts were used to predict the mode of interaction of drug with DNA. Rate constant was calculated with the help of Benesi-Hidebrand equation to observe the strength of binding with the increase in concentration of drug [49]. Rate constant as a function of temperature was calculated to observe the effect of temperature on binding affinity of drug with DNA [50].

Viscometric Measurements
Viscosity measurements were carried out by using Fungi lab S.A ADVR 212,008 viscometer. All the calculations were carried out at pH 3.4 and pH 7.4. Viscosity measurements provide further clues about the drug-DNA interaction. Plot of viscosity versus DNA concentration represents the mode of interaction either by intercalation, groove or electrostatic interaction [51]

Computational Studies
Theoretical study of SASA was carried out to get complete information about the structure, energetics, molecular orbitals (HOMO-LUMO gaps), UV-visible spectra, IR spectra, reactive sites and thermodynamic parameters [52]. Theoretical studies were carried out by using Gaussian 09 program package. All geometric parameters (bond length, bond angle, and dihedral angles) were computed with the help of DFT and HF functional by using 3-21G, 6-311G, and 6-311 + G basic sets.

Optimized Geometric Parameters of SASA
The optimized geometry of SASA is represented in Fig. 2. Selected optimized parameters including bond lengths, bond angles and dihedral angles were calculated. Bond length, the average distance between two atoms is a transferable property of a bond between atoms of fixed types, relatively independent of the rest of the molecule. Figure 3a,b shows the bond lengths of SASA calcualted by DFT and HF methods by using a 3-21G, 6-31G and 6-311G basic sets. Calculated bond angles are represented in Fig. 4a, b. the optimized ring angles proves benzene ring as a planar. The computational analysis was executed in gas phase, whereas the SASA exists in the solid-state. Small differences between computed and experimental results might be due to the small Vander Waals interactions missing in the gaseous phase [53,54]. Optimized structure and geometric parameters suggest that SASA has a symmetrical structure in terms of substitutions. Possible reaction site in SASA was predicted at C 7, C 9 and C 18 atoms due to possession of maximum charge density.

HOMO-LUMO Orbitals
HOMO-LUMO orbitals, the main chemical participants of a reaction represents the HOMO as electron donor while LUMO as electron acceptor species [55] (Fig. 5). Hyperpolarizability studies provide significant information regarding the unsaturation in organic compounds. Frontier molecular orbital, expression of several energies along with various electronic transitions is sketched in Fig. 6. The energy gap between HOMO and LUMO reflects the chemical reactivity and bioactivity of the molecule [56,57]. It plays a vital role in examination of the molecular electrical transport properties [58]. Smaller is the band gap between HOMO-LUMO, lesser will be the stability of molecule and greater will be the stabilization of LUMO as a result of its strong capability to accept electrons. Therefore it plays a vital role in deciding the most probable reaction site in a conjugated system.
The  [59]. Charge density on HOMO also affected the effectiveness of amides and sulfur presented more charge density on HOMO for electrophilic attack.
The electronic absorption corresponds to transitions from ground to excited states and is mainly described by one electron jump from HOMO to LUMO.

ESP Diagram of SASA
Distribution of electron density surfaces depends on the type of substitutions and also depends on negative and positive charges (Fig. 7). Electrostatic potential, represented by different colours in the MEP expresses the size, shape, positive and negative potential. Eelectrophile attack is expected near nitrogen and nucleophile attack near carbon atoms. The upper scale represents the value varying from red to blue region [60] According to this picture, the density distribution on SASA molecule is homogenous Green region is representative of negative charges with high electron density indicate the repulsion while blue is electropositive indicate the attraction with low electron density.

IR Spectra of SASA
Results of calculated IR spectrum of SASA providing complete information about functional group in-plane and out of plane bending of N-H, C-H groups, stretching of C-O, C-C bonds is represented in Table 1. The obtained results were compared with literature values and close resemblance was observed between computed and experimental results.

UV-Visible Spectra of SASA
Spectroscopy, a reliable technique to investigate the interaction of different pharmaceutical compounds with DNA clearly predicts the binding interaction between drugs and DNA in the form of spectral shifts [67]. Figure 8 represents the UV visible spectrum of SASA calculated by HF method with 3-21G, 6-31G, 6-31G / and 6-311G basic sets.
A number of basic sets for HF model involving various variables were used for calculations of visible spectra. 3-21 G is very economical and based on split valence basic set using one basic function per valence electron. 6-31 G is based on Cartezian Gaussian polarization function. 6-31G + is based on valence double-zeta polarized basic set and 6-311 G incorporates d and p orbitals as well. Thus greater is the basic set greater is incorporation of interactions between molecule and photons and it is represented by the spectral shifts in figure number 8.
Prominent peaks with strong oscillation strength, wavelength, excitation energies and absorption were observed in computed peaks. The HF calculations predicted that maximum absorption peak occurred near at 275 nm of set 6-31G and 6-31G / with strong oscillator strength. Other two peaks of singlet states occur at 260 nm and 264 nm of set 3-21G and 6-311G respectively. While DFT calculations predicted that maximum 4 absorption peaks occurred at 573 nm, 392 nm, 392 nm and 392 nm with strong oscillator strength of 6-311G set with value of 573.

UV-Vis Spectra of SASA-DNA Complexes at 3.4pH and 7.4pH
In experimental spectra the SASA shows maximum absorbance at 370 nm at 3.5 pH and at 380 nm at pH 7.4. The difference between the experimental and theoretical

C7-C8
C13-C15 C26-C28 C10-O4 N6-C12 S1-O2  results was due to the presence of weak Van der Waals interactions between solvent and SASA in solution, which are missing while theoretical calculations in gas phase [54]. Later the absorption spectra of SASA with DNA was also calculated with varying concentration of SASA. Increase was noticed in absorption with increase in concentration of SASA at fixed DNA concentration (Fig. 9). The observed spectral shifts predicts binding affinity of SASA with DNA to form SASA-DNA complex [68].
Bathochromic shift and hyperchromic effect was observed at both pH. Bathochromic effect predicts the intercalation mode of interaction between SASA and DNA. This effect is also related with the reduction in the energy gap between the highest (HUMO) and the lowest molecular orbitals (LUMO) after the interaction of SASA to DNA [52]. It is based on the principles that nucleic acids absorb ultraviolet (UV) light at a specific wavelength. For pure

SASA-DNA Binding Constant
The interactions between SASA and DNA results is spectral shifts in absorption spectra and binding constant K f was calculated by using the Benesi-Hildebrand equation [69].
Initially increase was observed in the value of binding constant with the increase in the concentration of SASA but at much higher concentration decrease was observed in the value of binding constant. It is clearly seen that K f values at 7.4 pH is large than at 3.5 pH. (Table 2). Significant value of binding constant reflects the interactions between SASA and DNA. These interactions might be hydrogen bonding, electrostatic forces, van der waals Variation in the value of binding constant with change in concentration of SASA reflects the alteration in the nature of interactions between SASA-DNA complex [70]. Later the effect of temperature was investigated on the rate of binding constant and it was observed to be decreasing showing the weakening of SASA-DNA complex (Table 3). This behaviour might also be due to the deformation in the DNA conformation in turn weakening the SASA-DNA complex and thus affecting the binding ability of drug with the base pairs of DNA.

Thermodynamic Parameters
Negative value of Gibbs free energy was noticed for SASA-DNA complex (Table 4) at both pH representing that the complexation reaction under investigation is spontaneous and thermodynamically favourable. Set of five temperatures (303 K till 323 K), based on the limitation of apparatus were used for the calculation of Gibbs energy and the resulting value of Gibbs energy are presented in Table 4. Thermodynamic results clearly reflect the spontaneous complexation between SASA and DNA at both pH. pH 7.4 was found to be more favourable in comparison to pH 3.5. Main binding forces between SASA and DNA in complex, predicted from negative value of enthalpy and positive value of entropy (Table 5) was vander waals force of attraction [71].
The interaction of drugs with DNA is a significant feature in pharmacology and plays a vital role in the determination of the mechanisms of drug action and designing of more efficient and specifically targeted drugs with lesser side effects. Several instrumental techniques are used to study such interactions like UV-Visible spectroscopy and fluorescence spectroscopy etc. The interaction between DNA and drugs can cause chemical and conformational modifications and, thus, variation of the electrochemical properties of nucleobases, due to vander waals force of attraction.

Viscometric Measurements
An effective way to predict the mode of interaction between SASA molecule and DNA is viscosity measurement. In present case initially increase was observed in the viscosity with increase in concentration of drug representing the intercalation mode of interaction and, at further higher concentration of drug decrease was observed in the viscosity behaviour representing the electrostatic mode of interaction. The mode of interaction was found to be dependent upon the concentration of SASA. Consequence of intercalation might be the conformational changes in DNA and result of electrostatic interaction might be damage of DNA structure.
Viscosity plots in Fig. 10 initially predicts the intercalation mode of interaction but at higher concentration of SASA decrease in viscosity, not equal to zero, reflects the change in conformation and development of electrostatic interaction. On the other hand, at pH 7.4, after initial decrease in viscosity, approaching zero, upon further increase in concentration of SASA reflects the damage in DNA structure. Maximum suitable interaction between SASA and DNA was observed at pH 7.4 thus preferring the intake of SASA via blood (Arshada et al. 2014).

Cooperativity
The sequence-dependent interaction of DNA with molecules that bind in the minor groove involves a delicate interplay between enthalpic and entropic components of the recognition process. It has been shown through calorimetric studies that in some cases the process is entropy-driven, being related to the solvation term. In general though, examples of cooperativity in DNA recognition appear to owe this characteristic to enthalpic factors, which are generally fairly evident, at least in qualitative form, from structure determinations. These typically reveal close physical contact between the two ligands, and a major structural deformation of the DNA that requires both ligands to stabilize it. In this case, NMR structure determination has shown that neither of these factors is operating. Binding of the first ligand restricts the flexibility of the DNA well beyond the actual binding site. Binding of the second ligand has little further effect. Both sites are already structurally fairly well predisposed toward ligand binding and the small adjustments required bear a modest enthalpic penalty and, though anticooperative, are outweighed by the entropic term. The results of this investigation provide an example of the general hypothesis of allosteric communication without conformational change. Cooperativity could be the consequence of ligand-induced changes in the dynamic behavior of a receptor. Statistical thermodynamic arguments were used to calculate the possible magnitude of the cooperative effect that could be produced by purely dynamic changes. The approach was based on estimates of the increase in the frequency at each binding step, rather than measurements from computer simulation. The analysis showed that considerable differences in binding free energy could be obtained from changes in conformational flexibility alone, without the need for changes in the time averaged structure of the macromolecule; others have presented similar arguments. The molecular origins of cooperativity in the absence of conformational change cannot be understood unless the dynamic properties of the system are taken into account. The results presented in this study illustrate the power of molecular simulation methods to investigate such phenomena, and highlight the general importance of flexibility in determining the properties of biomolecules such as DNA. Yet an element of rigidity, as well, is the key to the ability of this dodecamer to transmit "information" between the two drug binding sites.

Chirality
A compound that cannot be superposed on its mirror image by any combination of rotations, translations, and some conformational changes. This geometric property is called chirality.
Chirality plays a fundamental role in the binding affinity and interactions between the drug and its target, thus shaping the drug's pharmacology. For this reason, in 1992 the Food & Drug Administration (FDA) outlined a series of guidelines for the pharmaceutical development of single enantiomers and racemates. Since then, most drugs in the market are chiral, and the number of single-enantiomer and single-diastereomer drugs has consistently increased. Chirality is an innate property of some molecules derived from an absence of an internal plane of symmetry. Many naturally occurring molecules (amino acids, sugars) and biologically relevant molecules (DNA, RNA, proteins) are chiral. Drugs interact in a chiral environment, and biological targets recognize their ligands in a three-dimensional (3D) fashion. It is logical that molecules with a higher degree of 3D shape will interact with their targets with an increased affinity and higher levels of specificity, thus making more efficacious and safer drugs.
From biology to nanotechnology, chirality is an intrinsic property of any DNA system. In biological systems, this chirality plays a major role in prescribing the preferred structure and dynamics to biological assemblies containing nucleic acids, the proteins they interact with, and the organization of genetic material within the cell. The most pertinent unanswered questions in this area relate to the very origins of life. Is it really a work of chance that life on Earth exclusively utilizes righthanded nucleic acids or was that itself a product of natural selection? Future quests for traces of biological life on Mars and beyond may provide important clues to that. Another unchartered area is the structural organization of genomes in eukaryotic organisms, where we are only now starting to supplement biochemical and bioinformatic data with structural physical models. Finally, the practical utility of chiral structures in the macroscopic world is often to couple the motion along a linear path to a rotation about the path's axis. Interestingly, such coupling is rarely observed in biological systems, where molecular motors are found to take a linear path on a seemingly chiral track, such as in the motion of RNAP along a DNA duplex or the hand-over-hand motion of a kinesin on a microtubule. The chiral LC phases formed by dense DNA mixtures provide a stringent test of our understanding of ordering in soft matter systems. Despite significant effort, the path by which the broken mirror symmetry of a molecule propagates from atoms to macroscopic material has only been partially charted. Strongly chiral, well-studied, and programmable DNA systems provide the perfect testbed for studying the fundamental statistical mechanics of chiral LC phases. The ongoing theoretical, methodological, and experimental advancements in this area can be expected to result in a continuing stream of insights and, likely, new technologies that exploit the chiral phase behavior of DNA.
With regard to using DNA as a building material, the chirality of DNA is a mixed blessing. Because the building material is intrinsically chiral, the nanostructures based on DNA are constructed from chiral junctions arranged in chiral patterns, even when the goal is to develop a symmetric structure. The chirality of DNA and of the junctions have been exploited in many distinct ways to achieve diverse results, ranging from actuation to selection of a knotted stereoisomer. In larger DNA assemblies, sometimes the chirality of DNA manifests as an unwanted twist through a nanostructure. Other times, a custom chirality is intentionally introduced into the assembly. Whether desired or not, chirality is a fundamental attribute of DNA nanostructures that needs to be taken into account to achieve optimal functionality of a self-assembled DNA system.
Harnessing chirality of DNA nanostructures for practical applications has been so far marked with some technological achievements, with the most promising applications being in the area of configurable plasmonic nanostructures and nanosensors. Although the chirality of DNA provides a natural mechanism for interacting with circularly polarized light, the majority of such applications use DNA either as a building material or as a sensitive transducer of molecular binding events into structural motion. In the field of spintronics, both the chirality of the DNA structure and the ability of DNA to self-assembly into orderly larger-scale structures could have a competitive advantage over solid-state systems, which are difficult to assemble with the same precision and density at the nanoscale [72].

Conclusion
The drug under investigation shows band in absorption spectra at both pH (3.5 and 7.4) representing π → π * transitions. Predominant hypochromic and very small red shifts were observed in absorption spectra on addition of DNA. Strong binding was observed between SASA and DNA and this was reflected from the values of binding constant. Association decreases between SASA and DNA with the increase in temperature from 293 till 323 K. Complex formations between SASA and DNA was a spontaneous process and this was reflected from the negative value of free energy. Strong and stable complex was formed at pH 7.4 as evident from the shifts in absorption peaks, binding constant and thermodynamic parameters. Thus the intake of medicine via blood at pH 7.4 is preferred over the oral intake i.e. at pH 4.7. The interpretation of physical mode to intake the newly synthesized SASA drug into human body through blood rather than oral intake is the novelty of this research work.
Author's Contribution Concept and design of this article is collective contribution of all authors. They all read and approve the final manuscript of this research article. Sana Fatima long with Sadia Asim plays a vital role in material preparation, data collection, and analysis. All theoretical calculations were performed by Asim Mansha. The first draf of manuscript was written by Sana Fatima which was later refined by Sadia Asim and Asim Mansha.

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Conflict of Interest
The review article entitled "Study of Interactions Between 3-benzoyl-4-hydroxy-2-methyl-2H-1, 2-benzothiazine and Human DNA by Theoretical, Spectroscopic and Viscometric Measurements." is carried out with the financial help from Higher Education Commission, Pakistan (Project number: 9922). All the authors involved in the write up of this article do not have any conflict of Interest.