**10. Mechanisms of anticancer drug-DNA interaction**

The addition of anticancer drugs to a DNA molecule creates a new bond. Some examples for these mechanisms include intercalating agents, intercalating reagents (II), and bleomycins.

### **10.1 Intercalating agent**

This agent contains planar aromatic or heteroaromatic ring systems (dactinomycin as an example), binding to sugar phosphate backbone by cyclic peptide or by NH3. The planar systems slip between the layers of nucleic acid pairs and disrupt the shape of the helix. The preference is often shown for the minor or major groove. The intercalation prevents replication and transcription. In addition, the intercalation inhibits

#### **Figure 10.**

*Diagrammatic model illustrating intercalation of the flat part of the molecule of adriamycin (in black) into DNA, presenting the local unwinding of the helical structure.*

**85**

**Figure 12.**

**Figure 11.**

*Stabilizations of DNA-topo (II) complex.*

*The intercalating region (in blue color) of bleomycin A2 via the bithiazole moiety to DNA.*

*Anticancer Drugs' Deoxyribonucleic Acid (DNA) Interactions*

topoisomerase II (an enzyme that relieves the strain in the DNA helix by temporarily cleaving the DNA chain and crossing an intact strand through the broken strand). Another example is the intercalation of the flat part of the molecule of Adriamycin into DNA, presenting the local unwinding of the helical structure (**Figure 10**) [23].

During replication, supercoiled DNA is unwound by the helicase. The thereby created tension is removed by the topoisomerase II (topo II) that cuts and rejoins the DNA strands. When doxorubicin is bound to the DNA it stabilizes the DNA-topo (II) complex at the point where the enzyme is covalently bound (**Figure 11**) [1, 24].

*DOI: http://dx.doi.org/10.5772/intechopen.85794*

**10.2 Intercalating reagents (II)**

*Anticancer Drugs' Deoxyribonucleic Acid (DNA) Interactions DOI: http://dx.doi.org/10.5772/intechopen.85794*

topoisomerase II (an enzyme that relieves the strain in the DNA helix by temporarily cleaving the DNA chain and crossing an intact strand through the broken strand). Another example is the intercalation of the flat part of the molecule of Adriamycin into DNA, presenting the local unwinding of the helical structure (**Figure 10**) [23].

## **10.2 Intercalating reagents (II)**

*Biophysical Chemistry - Advance Applications*

**10. Mechanisms of anticancer drug-DNA interaction**

*Schematic interaction between DNA and echinomycin.*

(II), and bleomycins.

**Figure 9.**

**10.1 Intercalating agent**

The addition of anticancer drugs to a DNA molecule creates a new bond. Some examples for these mechanisms include intercalating agents, intercalating reagents

This agent contains planar aromatic or heteroaromatic ring systems (dactinomycin as an example), binding to sugar phosphate backbone by cyclic peptide or by NH3. The planar systems slip between the layers of nucleic acid pairs and disrupt the shape of the helix. The preference is often shown for the minor or major groove. The intercalation prevents replication and transcription. In addition, the intercalation inhibits

*Diagrammatic model illustrating intercalation of the flat part of the molecule of adriamycin (in black) into* 

*DNA, presenting the local unwinding of the helical structure.*

**84**

**Figure 10.**

During replication, supercoiled DNA is unwound by the helicase. The thereby created tension is removed by the topoisomerase II (topo II) that cuts and rejoins the DNA strands. When doxorubicin is bound to the DNA it stabilizes the DNA-topo (II) complex at the point where the enzyme is covalently bound (**Figure 11**) [1, 24].

**Figure 11.** *Stabilizations of DNA-topo (II) complex.*

## **10.3 Bleomycin A2**

The bleomycin A2 intercalate via the bithiazole moiety (DNA-binding domain) (**Figure 12**). The bithiazole moiety intercalates into the double helix and the attached side chain containing a sulfonium ion is attracted to the phosphodiester backbone. In addition, the N-atoms of the primary amines, pyrimidine ring and imidazole ring chelate Fe, which is involved in the formation of superoxide radicals, which subsequently act to cut DNA between purine and pyrimidine nucleotides [25].

## **11. Techniques for studying drug-DNA interactions**

Various analytical techniques have been used for studying drug-DNA interactions (interaction between DNA and small ligand molecules that are potentially of pharmaceutical importance). Several instrumental techniques (emission and absorption spectroscopic) such as infrared (IR), UV-visible, nuclear magnetic resonance (NMR) spectroscopies, circular dichroism, atomic force microscopy (AFM), electrophoresis, mass spectrometry, viscosity measurements (viscometry), UV thermal denaturation studies, and cyclic, square wave and differential pulse voltammetry, etc., were used to study such interactions. These techniques have been used as a major tool to characterize the nature of drug-DNA complexation and the effects of such interaction on the structure of DNA. In addition, these techniques are regularly applied to monitor interactions of drugs with DNA because these optical properties are easily measured and tend to be quite sensitive to the environment. Moreover, these techniques provide various types of information (qualitative or quantitative) and at the same time complement each other to provide full picture of drug-DNA interaction and aid in the development of new drugs. In addition, the information gained from this part might be useful for the development of potential survey for DNA structure and new therapeutic reagents for tumors and other diseases. In this part of the chapter, we will focus on FT-IR, UV-Visible, NMR, AFM and viscosity measurements [5].

#### **11.1 Fourier transform infrared spectroscopy**

Fourier transform infrared (FT-IR) spectroscopy is a widely used technique to study interactions of nucleic acids (DNA and RNA) and proteins with anticancer drugs and other cytotoxic agents in solutions [26, 27]. In addition, it can generate structural information of the whole molecule in a single spectrum as a photograph of all conformations present in the sample that can distinguish among A-, B- and Z-forms of DNA, triple stranded helices, and other structural patterns. In addition, it is a powerful tool to study interactions of DNA with drugs and the effects of such interactions in the structure of DNA, and providing some insights about the mechanism of drug action. The technique is ideal for systematic studies of nucleic acids (e.g., sequence variations, covalent modifications), since it is fast, nondestructive, and requires only small amount of sample [28].

IR spectrum can be divided into four characteristic spectral ranges. The region between 1800 and 1550 cm<sup>−</sup><sup>1</sup> corresponds to the in-plane double bond vibrations of the nucleic bases (C〓O, C〓N, C〓C and N▬H bending vibrations of bases). These bands are sensitive to changes in the base stacking and base pairing interactions. Bands occurring in the interval 1500–1250 cm<sup>−</sup><sup>1</sup> assigned to vibrations of the bases and base-sugar connections are strongly related to the conformational changes of the backbone chain and glycosidic bond rotation. The range 1250–1000 cm<sup>−</sup><sup>1</sup> involves sugar phosphate vibrations, such as, PO2 symmetric and asymmetric stretching

**87**

**Figure 13.**

*Anticancer Drugs' Deoxyribonucleic Acid (DNA) Interactions*

vibrations and C▬O stretching vibrations. These vibrations show high sensitivity to

, spectra are generally recorded also in D2O, where these bands move to

for bands associated with vibrations of sugars which correlate with the various

obtaining a complete spectrum. The use of D2O also causes shifts in nucleic acid absorptions, resulting from deuterium exchange of labile NH protons, and these can be used to monitor H–D exchange processes. A method to remove water signals in the spectra is water subtraction, using a sodium chloride (NaCl) solution as reference. D2O is used to allow shifts in the absorption of nucleic acid in order to monitor H–D exchange processes. Four regions, each having marker bands showing either nucleic acid interactions or conformations, are presented

The ring vibrations of nitrogenous bases (C〓O, C〓N stretching), PO2 stretching vibrations (symmetric and asymmetric) and deoxyribose stretching of DNA backbone are confined in the spectral region between 1800 and 700 cm<sup>−</sup><sup>1</sup>

denote phosphate asymmetric and symmetric

(G), thymine (T), adenine (A) and cytosine (C) nitrogenous bases, respectively.

vibrations, respectively. These are the prominent bands of pure DNA, which are monitored during carboplatin-DNA interaction at different ratios. Changes in these bands are shown in **Figure 14** [33]. After carboplatin addition to DNA solution, G-band at 1710 shifts to 1702–3, T-band at 1662 shifts to 1655 and A-band at 1613

to direct platin binding to G (N7), T (O2), and A (N7) of DNA bases. No major shifting is observed for phosphate asymmetric and symmetric vibrations indicating no external binding. The plots of the relative intensity (*R i*) of several peaks of DNA

against the compound concentrations can be obtained after peak normalization

*l <sup>968</sup>*

where *Ri* is the relative intensity, *Ii* is the intensity of absorption peak for pure DNA and DNA in the complex with *i* concentration of compound, and *l968* is the

peak (internal reference) [35].

*The characteristics IR bands of DNA and aqueous solvents. (a) 1800–1500 cm-1 region is sensitive to effects of base pairing and base stacking; (b) 1500–1250 cm-1 region is sensitive to glycosidic bond rotation, backbone conformation, and sugar pucker; (c) 1250–1000 cm-1 region is sensitive to backbone conformation; and* 

is characteristic

. The

are assigned to guanine

<sup>−</sup> stretching vibra-

<sup>−</sup> groups),

(1)

(PO2

. These shifting can be attributed

and below

. Combination of results from both spectra allows

conformational changes in the backbone. The range 1000–800 cm<sup>−</sup><sup>1</sup>

vibrational bands of DNA at 1710, 1662, 1613 and 1492 cm<sup>−</sup><sup>1</sup>

in-plane vibrations related to A–T, G–C base pairs and the PO2

tions such as 1717 (G), 1663 (T), 1609 (A), 1492 (C), and 1222 cm<sup>−</sup><sup>1</sup>

shifts towards lower wave number 1609–10 cm<sup>−</sup><sup>1</sup>

*Ri* = \_\_\_\_ *Ii*

*(d) 1000–800 cm-1 region is sensitive to sugar conformation.*

nucleic acid sugar puckering modes (C2'-endo and C3'-endo) [29, 30]. Due to interfering absorption bands of water at 1650 cm<sup>−</sup><sup>1</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.85794*

, and below 750 cm<sup>−</sup><sup>1</sup>

950 cm<sup>−</sup><sup>1</sup>

1200 cm<sup>−</sup><sup>1</sup>

in **Figure 13** [31, 32].

Bands at 1228 and 1087 cm<sup>−</sup><sup>1</sup>

using formula (1) [5, 34]:

intensity of the 968 cm<sup>−</sup><sup>1</sup>

*Biophysical Chemistry - Advance Applications*

and viscosity measurements [5].

**11.1 Fourier transform infrared spectroscopy**

and requires only small amount of sample [28].

Bands occurring in the interval 1500–1250 cm<sup>−</sup><sup>1</sup>

between 1800 and 1550 cm<sup>−</sup><sup>1</sup>

The bleomycin A2 intercalate via the bithiazole moiety (DNA-binding domain) (**Figure 12**). The bithiazole moiety intercalates into the double helix and the attached side chain containing a sulfonium ion is attracted to the phosphodiester backbone. In addition, the N-atoms of the primary amines, pyrimidine ring and imidazole ring chelate Fe, which is involved in the formation of superoxide radicals, which subse-

Various analytical techniques have been used for studying drug-DNA interactions (interaction between DNA and small ligand molecules that are potentially of pharmaceutical importance). Several instrumental techniques (emission and absorption spectroscopic) such as infrared (IR), UV-visible, nuclear magnetic resonance (NMR) spectroscopies, circular dichroism, atomic force microscopy (AFM), electrophoresis, mass spectrometry, viscosity measurements (viscometry), UV thermal denaturation studies, and cyclic, square wave and differential pulse voltammetry, etc., were used to study such interactions. These techniques have been used as a major tool to characterize the nature of drug-DNA complexation and the effects of such interaction on the structure of DNA. In addition, these techniques are regularly applied to monitor interactions of drugs with DNA because these optical properties are easily measured and tend to be quite sensitive to the environment. Moreover, these techniques provide various types of information (qualitative or quantitative) and at the same time complement each other to provide full picture of drug-DNA interaction and aid in the development of new drugs. In addition, the information gained from this part might be useful for the development of potential survey for DNA structure and new therapeutic reagents for tumors and other diseases. In this part of the chapter, we will focus on FT-IR, UV-Visible, NMR, AFM

Fourier transform infrared (FT-IR) spectroscopy is a widely used technique to study interactions of nucleic acids (DNA and RNA) and proteins with anticancer drugs and other cytotoxic agents in solutions [26, 27]. In addition, it can generate structural information of the whole molecule in a single spectrum as a photograph of all conformations present in the sample that can distinguish among A-, B- and Z-forms of DNA, triple stranded helices, and other structural patterns. In addition, it is a powerful tool to study interactions of DNA with drugs and the effects of such interactions in the structure of DNA, and providing some insights about the mechanism of drug action. The technique is ideal for systematic studies of nucleic acids (e.g., sequence variations, covalent modifications), since it is fast, nondestructive,

IR spectrum can be divided into four characteristic spectral ranges. The region

the nucleic bases (C〓O, C〓N, C〓C and N▬H bending vibrations of bases). These bands are sensitive to changes in the base stacking and base pairing interactions.

and base-sugar connections are strongly related to the conformational changes of the

sugar phosphate vibrations, such as, PO2 symmetric and asymmetric stretching

backbone chain and glycosidic bond rotation. The range 1250–1000 cm<sup>−</sup><sup>1</sup>

corresponds to the in-plane double bond vibrations of

assigned to vibrations of the bases

involves

quently act to cut DNA between purine and pyrimidine nucleotides [25].

**11. Techniques for studying drug-DNA interactions**

**10.3 Bleomycin A2**

**86**

vibrations and C▬O stretching vibrations. These vibrations show high sensitivity to conformational changes in the backbone. The range 1000–800 cm<sup>−</sup><sup>1</sup> is characteristic for bands associated with vibrations of sugars which correlate with the various nucleic acid sugar puckering modes (C2'-endo and C3'-endo) [29, 30].

Due to interfering absorption bands of water at 1650 cm<sup>−</sup><sup>1</sup> and below 950 cm<sup>−</sup><sup>1</sup> , spectra are generally recorded also in D2O, where these bands move to 1200 cm<sup>−</sup><sup>1</sup> , and below 750 cm<sup>−</sup><sup>1</sup> . Combination of results from both spectra allows obtaining a complete spectrum. The use of D2O also causes shifts in nucleic acid absorptions, resulting from deuterium exchange of labile NH protons, and these can be used to monitor H–D exchange processes. A method to remove water signals in the spectra is water subtraction, using a sodium chloride (NaCl) solution as reference. D2O is used to allow shifts in the absorption of nucleic acid in order to monitor H–D exchange processes. Four regions, each having marker bands showing either nucleic acid interactions or conformations, are presented in **Figure 13** [31, 32].

The ring vibrations of nitrogenous bases (C〓O, C〓N stretching), PO2 stretching vibrations (symmetric and asymmetric) and deoxyribose stretching of DNA backbone are confined in the spectral region between 1800 and 700 cm<sup>−</sup><sup>1</sup> . The vibrational bands of DNA at 1710, 1662, 1613 and 1492 cm<sup>−</sup><sup>1</sup> are assigned to guanine (G), thymine (T), adenine (A) and cytosine (C) nitrogenous bases, respectively. Bands at 1228 and 1087 cm<sup>−</sup><sup>1</sup> denote phosphate asymmetric and symmetric vibrations, respectively. These are the prominent bands of pure DNA, which are monitored during carboplatin-DNA interaction at different ratios. Changes in these bands are shown in **Figure 14** [33]. After carboplatin addition to DNA solution, G-band at 1710 shifts to 1702–3, T-band at 1662 shifts to 1655 and A-band at 1613 shifts towards lower wave number 1609–10 cm<sup>−</sup><sup>1</sup> . These shifting can be attributed to direct platin binding to G (N7), T (O2), and A (N7) of DNA bases. No major shifting is observed for phosphate asymmetric and symmetric vibrations indicating no external binding. The plots of the relative intensity (*R i*) of several peaks of DNA in-plane vibrations related to A–T, G–C base pairs and the PO2 <sup>−</sup> stretching vibrations such as 1717 (G), 1663 (T), 1609 (A), 1492 (C), and 1222 cm<sup>−</sup><sup>1</sup> (PO2 <sup>−</sup> groups), against the compound concentrations can be obtained after peak normalization using formula (1) [5, 34]:

$$R\_i = \frac{I\_i}{I\_{\text{ges}}} \tag{1}$$

where *Ri* is the relative intensity, *Ii* is the intensity of absorption peak for pure DNA and DNA in the complex with *i* concentration of compound, and *l968* is the intensity of the 968 cm<sup>−</sup><sup>1</sup> peak (internal reference) [35].

#### **Figure 13.**

*The characteristics IR bands of DNA and aqueous solvents. (a) 1800–1500 cm-1 region is sensitive to effects of base pairing and base stacking; (b) 1500–1250 cm-1 region is sensitive to glycosidic bond rotation, backbone conformation, and sugar pucker; (c) 1250–1000 cm-1 region is sensitive to backbone conformation; and (d) 1000–800 cm-1 region is sensitive to sugar conformation.*

**Figure 14.** *Intensity ratio variations for DNA as a function of different carboplatin/DNA molar ratios.*

Similarly, Raman spectroscopy, which also depends on characteristic group vibrational frequencies, can be used together with infrared spectra to study vibrations in DNA. It is useful because Raman and IR spectroscopy provide complementary information.

#### **11.2 UV-visible spectroscopy**

UV-visible absorption spectroscopy can be utilized to detect the DNA-ligand interaction by measuring the changes in the absorption properties of the DNA molecules or the ligand. The UV-vis absorption spectrum of DNA displays a broad band in the range of 200–350 nm in the UV region, with a maximum situated at 260 nm. The maximum is due to the chromophoric groups in pyrimidine and purine moieties responsible for the electronic transitions. The utilization of this simple and versatile technique enables an accurate estimation of the DNA molar concentration based on absorbance measurement at 260 nm. To measure the interaction between ligands and DNA, a hypochromic shift is utilized because the monitoring of the values of absorbance enables studying of the melting action of DNA. Apart from versatility, other major advantages of UV-vis absorption spectroscopy include simplicity, reproducibility, and good sensitivity [36, 37].

#### **11.3 Nuclear magnetic resonance spectroscopy**

Binding between ligands and the molecules of DNA causes a significant change in the chemical shift of the values presented in **Table 2** [32]. For example, applying thermal denaturing in order to un-stack the base-pair double-helical DNA to form two ss-DNAs is often accompanied by the 1 H resonances' downfield shift for nonexchangeable protons.

The broadening of <sup>1</sup> H NMR resonances of DNA upon addition of an appropriate minor groove binding compound is one type of evidence of complex formation in DNA 31P-NMR spectroscopy has also been used to provide important information concerning the binding of intercalators to DNA. The 31P chemical shifts are sensitive DNA conformational changes, and hence intercalating drugs cause downfield shift, while divalent cations causes up field shifts in the 31P signal [38].

**89**

*Anticancer Drugs' Deoxyribonucleic Acid (DNA) Interactions*

**shifta**

 **(ppm)**

3.70

 *Chemical shifts relative to internal TSP (3-(trimethylsilyl)propionic acid).* 

T5 (CH3) 1.00–2.00 A 2 (CH); A 8 (CH); G 8 (CH) T 6

Mass spectrometry (MS) has become one of the most common techniques adopted to study interactions between DNA and small ligand molecules. The ability of mass spectrometry to investigate drug-DNA interactions have been reviewed recently. The binding stoichiometry, the relative binding affinities and the binding constants for DNA double helices of various sequences may be determined. Electrospray ionization (ESI) is the most common ionization method used in the study of biomolecules due to its soft ionization. Using ESI techniques, biomolecules can be transferred from the solution to the mass spectrometer with minimal fragmentation and, so, both the mass of the DNA and the mass of the DNA-ligand complex can be determined, as the noncovalent interactions that formed the complex are not altered during the electrospray process [39–41]. Focusing on the use of ESI-MS to study complexes, MS gives a signal for each species with a different mass and so it is very straightforward to establish the stoichiometry of the complexes. ESI-MS signals enable several calculations to be performed. The number of DNA strands involved, the number of bound cations (if present) and the number of bound ligands, among others. Taking into account the structure of the nucleic acids, ESI-MS studies are performed using negative polarity. It is well known that the phosphodiester backbone of DNA is fully deprotonated under usual working conditions. In general, in order to preserve their structure, nucleic acid solutions are prepared with monovalent ions. Perylene derivatives, such as, N,Nbis-(2-(dimethylamino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide, favor π-π interactions with the G-tetrad surface. Moreover, 5,10,15,20-tetrakis-(1-methyl-4-pyridyl)-21H,23H-porphine is an effective telomerase inhibitor, also binds to the

*H NMR spectra of nucleic acids.*

Sugar 3′ (CH) 4.50–5.20 C 4 (NH2) (H-2)b 8.30–8.50 ppm Sugar 1′ (CH) 5.30–6.20 G 1 (NH) 12.50–13.00 ppm C 5 (CH) 5.30–6.20 T 3 (NH) 13.50–14.00 ppm

**Proton type Expected chemical** 

(CH) C 6 (CH)

4.00–4.50 C 4 (NH2) (H-1)b 6.40–6.80

**shifta**

 **(ppm)**

6.50–8.20

Atomic force microscopy (AFM) can be used to distinguish proteins bound to nucleic acid templates. One of the great advantages of the atomic force microscope, particularly with respect to the imaging of biological specimens, is that it can work in fluid, so that experiments can be performed under near physiological conditions and allowing the imaging of interactions and transactions between molecules in real time [43]. AFM techniques will play a larger role in studying interactions between biological specimens, such as ligand-receptor and protein-DNA systems,

*DOI: http://dx.doi.org/10.5772/intechopen.85794*

**Proton type Expected chemical** 

Sugar 2′ (CH2) 2.00–3.00

Sugar 5′ terminal

Sugar 5′ (CH2); 4'(CH)

(CH2)

*a*

*b*

**Table 2.**

**11.4 Mass spectrometry**

 *For Watson-Crick base pairs (CG).*

*Typical ranges of chemical shifts for <sup>1</sup>*

G-quadruplex in the c-myc promoter [42].

**11.5 Atomic force microscopy**



*b For Watson-Crick base pairs (CG).*

#### **Table 2.**

*Biophysical Chemistry - Advance Applications*

tary information.

**Figure 14.**

**11.2 UV-visible spectroscopy**

Similarly, Raman spectroscopy, which also depends on characteristic group vibrational frequencies, can be used together with infrared spectra to study vibrations in DNA. It is useful because Raman and IR spectroscopy provide complemen-

*Intensity ratio variations for DNA as a function of different carboplatin/DNA molar ratios.*

UV-visible absorption spectroscopy can be utilized to detect the DNA-ligand interaction by measuring the changes in the absorption properties of the DNA molecules or the ligand. The UV-vis absorption spectrum of DNA displays a broad band in the range of 200–350 nm in the UV region, with a maximum situated at 260 nm. The maximum is due to the chromophoric groups in pyrimidine and purine moieties responsible for the electronic transitions. The utilization of this simple and versatile technique enables an accurate estimation of the DNA molar concentration based on absorbance measurement at 260 nm. To measure the interaction between ligands and DNA, a hypochromic shift is utilized because the monitoring of the values of absorbance enables studying of the melting action of DNA. Apart from versatility, other major advantages of UV-vis absorption spectroscopy include

Binding between ligands and the molecules of DNA causes a significant change in the chemical shift of the values presented in **Table 2** [32]. For example, applying thermal denaturing in order to un-stack the base-pair double-helical DNA to form

minor groove binding compound is one type of evidence of complex formation in DNA 31P-NMR spectroscopy has also been used to provide important information concerning the binding of intercalators to DNA. The 31P chemical shifts are sensitive DNA conformational changes, and hence intercalating drugs cause downfield

shift, while divalent cations causes up field shifts in the 31P signal [38].

H resonances' downfield shift for non-

H NMR resonances of DNA upon addition of an appropriate

simplicity, reproducibility, and good sensitivity [36, 37].

**11.3 Nuclear magnetic resonance spectroscopy**

two ss-DNAs is often accompanied by the 1

exchangeable protons. The broadening of <sup>1</sup>

**88**

*Typical ranges of chemical shifts for 1 H NMR spectra of nucleic acids.*

### **11.4 Mass spectrometry**

Mass spectrometry (MS) has become one of the most common techniques adopted to study interactions between DNA and small ligand molecules. The ability of mass spectrometry to investigate drug-DNA interactions have been reviewed recently. The binding stoichiometry, the relative binding affinities and the binding constants for DNA double helices of various sequences may be determined. Electrospray ionization (ESI) is the most common ionization method used in the study of biomolecules due to its soft ionization. Using ESI techniques, biomolecules can be transferred from the solution to the mass spectrometer with minimal fragmentation and, so, both the mass of the DNA and the mass of the DNA-ligand complex can be determined, as the noncovalent interactions that formed the complex are not altered during the electrospray process [39–41]. Focusing on the use of ESI-MS to study complexes, MS gives a signal for each species with a different mass and so it is very straightforward to establish the stoichiometry of the complexes. ESI-MS signals enable several calculations to be performed. The number of DNA strands involved, the number of bound cations (if present) and the number of bound ligands, among others. Taking into account the structure of the nucleic acids, ESI-MS studies are performed using negative polarity. It is well known that the phosphodiester backbone of DNA is fully deprotonated under usual working conditions. In general, in order to preserve their structure, nucleic acid solutions are prepared with monovalent ions. Perylene derivatives, such as, N,Nbis-(2-(dimethylamino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide, favor π-π interactions with the G-tetrad surface. Moreover, 5,10,15,20-tetrakis-(1-methyl-4-pyridyl)-21H,23H-porphine is an effective telomerase inhibitor, also binds to the G-quadruplex in the c-myc promoter [42].

#### **11.5 Atomic force microscopy**

Atomic force microscopy (AFM) can be used to distinguish proteins bound to nucleic acid templates. One of the great advantages of the atomic force microscope, particularly with respect to the imaging of biological specimens, is that it can work in fluid, so that experiments can be performed under near physiological conditions and allowing the imaging of interactions and transactions between molecules in real time [43]. AFM techniques will play a larger role in studying interactions between biological specimens, such as ligand-receptor and protein-DNA systems,

and can be applied to the study of drug interactions with a variety of biological specimens [5].

Drug-DNA complexes have been studied with AFM to determine the binding force between them. This is of considerable interest since nucleic acid ligands are commonly used as anticancer drugs and in the treatment of genetic diseases. However, determining whether they bind to DNA by intercalation within major and/or minor grooves, by normal modes, or by a combination of these modes can often be difficult. AFM was used to study drug binding mode, affinity, and exclusion number by comparing the length of DNA fragments that have and have not been exposed to the drug. It is well known that if intercalative binding is occurring, the DNA strand increases in length. Moreover, the degree of lengthening is informative in determining the binding affinity and the site-exclusion number. AFM was shown to be an effective means of seeing and measuring any changes in the DNA strand. For example, when it exposed to ethidium, the DNA strand was shown through AFM to have increased in length from 3300 to 5250 nm, this indicating the intercalative mode of binding. Similarly, AFM intercalative binding studies showed the increase in the DNA strand, from 3300 to 4670 nm, upon exposure to daunomycin. This technique has also successfully been applied to new drugs in which the mode of binding was unclear. For example, exposure of 2,5-bis(4-amidinophenyl) (APF), did not produce lengthening of the DNA strands, indicating that the drug binds by non-intercalative modes. The different structural changes and binding processes of the DNA occur because of interactions with these two components [5].

#### **11.6 Viscosity measurements**

DNA viscosity is sensitive to DNA length change, for this reason, its measurement upon the addition of a compound is often concerned as the least ambiguous and most critical method to clarify the interaction mode of a compound with DNA and this will provide reliable evidence for the intercalative binding mode. Relative viscosity measurements have proved to be a reliable method for the assignment of the mode of binding compounds to DNA. In the case of classical intercalation, DNA base pairs are separated in order to host the bound compound resulting in the lengthening of the DNA helix and subsequently increased DNA viscosity. On the other side, the binding of a compound exclusively in DNA grooves by means of partial and/or non-classic intercalation, under same conditions, causes a bend or kink in the DNA helix and reducing its effective length and, as a result, DNA solution viscosity is decreased, or it remains unchanged.

**Figure 15** show the interaction of three Schiff base compounds of N′-substituted benzohydrazide and sulfonohydrazide derivatives: (1) N′-(2 hydroxy-3-methoxybenzylidene)-4-tert-butylbenzohydrazide, (2) N′-(5-bromo-2 hydroxy-benzylidene)-4-tert-butylbenzohydrazide and (3) N′-(2-hydroxy-3 methoxy-benzylide-ne)-4-methylbenzenesulfonohydrazide with SS-DNA [44]. This can be explained by the insertion of the compounds in between the DNA base pairs, leading to an increase in the separation of base pairs at intercalation sites and, thus, an increase in DNA length [45].

The viscosity data show that there are at least two phases of binding between the complex and CT-DNA. At lower concentration of the complex, the viscosity first decreases and then increases at higher concentration of complex. This slow increase in viscosity is an indication of groove binding [11].

**Figure 16** indicate that with increasing amount of (3-(3,5 dimethyl-phenylimino) methyl)benzene-1,2-diol (HL), the relative viscosity of DNA first remains constant and then increases [46]. This observation supports that HL bind through intercalation mode but with different affinity, i.e., also show some affinity for

**91**

**Figure 17.**

*Anticancer Drugs' Deoxyribonucleic Acid (DNA) Interactions*

binding with grooves of DNA through hydrogen bonding, typically to N3 of adenine and O2 of thymine. However, strong binding is presumably due to interca-

*Effects of increasing amount of compounds (1–3) on relative viscosity of SS-DNA at 25 ± 0.1°C.* 

*Effects of increasing amount of HL on relative viscosity of CT-DNA at 25 ± 0.1°C. [DNA] = 2.37 × 10<sup>−</sup><sup>5</sup>*

*(1) Effect of increasing amount of the complexes [Ni(hhmh)2], (2) [Ni(bhmh)2], (3) [Ni(ihmh)2], (4) [Ni(PPh3) (hpeh)], (5) [Ni(PPh3)(bpeh)] and (6) [Ni(PPh3)(ipeh)] on the relative viscosity of HS-DNA at 16(±0.L)°C.*

 *M.*

*DOI: http://dx.doi.org/10.5772/intechopen.85794*

lation with DNA [11].

**Figure 15.**

**Figure 16.**

*[DNA] = 7.2 μM, r = 0, 6.9, 13.9, 20.8, 27.8.*

binding with grooves of DNA through hydrogen bonding, typically to N3 of adenine and O2 of thymine. However, strong binding is presumably due to intercalation with DNA [11].

**Figure 15.**

*Biophysical Chemistry - Advance Applications*

**11.6 Viscosity measurements**

thus, an increase in DNA length [45].

in viscosity is an indication of groove binding [11].

specimens [5].

and can be applied to the study of drug interactions with a variety of biological

Drug-DNA complexes have been studied with AFM to determine the binding force between them. This is of considerable interest since nucleic acid ligands are commonly used as anticancer drugs and in the treatment of genetic diseases. However, determining whether they bind to DNA by intercalation within major and/or minor grooves, by normal modes, or by a combination of these modes can often be difficult. AFM was used to study drug binding mode, affinity, and exclusion number by comparing the length of DNA fragments that have and have not been exposed to the drug. It is well known that if intercalative binding is occurring, the DNA strand increases in length. Moreover, the degree of lengthening is informative in determining the binding affinity and the site-exclusion number. AFM was shown to be an effective means of seeing and measuring any changes in the DNA strand. For example, when it exposed to ethidium, the DNA strand was shown through AFM to have increased in length from 3300 to 5250 nm, this indicating the intercalative mode of binding. Similarly, AFM intercalative binding studies showed the increase in the DNA strand, from 3300 to 4670 nm, upon exposure to daunomycin. This technique has also successfully been applied to new drugs in which the mode of binding was unclear. For example, exposure of 2,5-bis(4-amidinophenyl) (APF), did not produce lengthening of the DNA strands, indicating that the drug binds by non-intercalative modes. The different structural changes and binding processes of the DNA occur because of interactions with these two components [5].

DNA viscosity is sensitive to DNA length change, for this reason, its measurement upon the addition of a compound is often concerned as the least ambiguous and most critical method to clarify the interaction mode of a compound with DNA and this will provide reliable evidence for the intercalative binding mode. Relative viscosity measurements have proved to be a reliable method for the assignment of the mode of binding compounds to DNA. In the case of classical intercalation, DNA base pairs are separated in order to host the bound compound resulting in the lengthening of the DNA helix and subsequently increased DNA viscosity. On the other side, the binding of a compound exclusively in DNA grooves by means of partial and/or non-classic intercalation, under same conditions, causes a bend or kink in the DNA helix and reducing its effective length and, as a result, DNA solution viscosity is decreased, or it remains unchanged. **Figure 15** show the interaction of three Schiff base compounds of N′-substituted benzohydrazide and sulfonohydrazide derivatives: (1) N′-(2 hydroxy-3-methoxybenzylidene)-4-tert-butylbenzohydrazide, (2) N′-(5-bromo-2 hydroxy-benzylidene)-4-tert-butylbenzohydrazide and (3) N′-(2-hydroxy-3 methoxy-benzylide-ne)-4-methylbenzenesulfonohydrazide with SS-DNA [44]. This can be explained by the insertion of the compounds in between the DNA base pairs, leading to an increase in the separation of base pairs at intercalation sites and,

The viscosity data show that there are at least two phases of binding between the complex and CT-DNA. At lower concentration of the complex, the viscosity first decreases and then increases at higher concentration of complex. This slow increase

**Figure 16** indicate that with increasing amount of (3-(3,5 dimethyl-phenylimino) methyl)benzene-1,2-diol (HL), the relative viscosity of DNA first remains constant and then increases [46]. This observation supports that HL bind through intercalation mode but with different affinity, i.e., also show some affinity for

**90**

*Effects of increasing amount of compounds (1–3) on relative viscosity of SS-DNA at 25 ± 0.1°C. [DNA] = 7.2 μM, r = 0, 6.9, 13.9, 20.8, 27.8.*

**Figure 16.** *Effects of increasing amount of HL on relative viscosity of CT-DNA at 25 ± 0.1°C. [DNA] = 2.37 × 10<sup>−</sup><sup>5</sup> M.*

**Figure 17.**

*(1) Effect of increasing amount of the complexes [Ni(hhmh)2], (2) [Ni(bhmh)2], (3) [Ni(ihmh)2], (4) [Ni(PPh3) (hpeh)], (5) [Ni(PPh3)(bpeh)] and (6) [Ni(PPh3)(ipeh)] on the relative viscosity of HS-DNA at 16(±0.L)°C.*

#### **Figure 18.**

*(1) Effects of increasing amount of tri-n-butyltin (IV) 3-[(3′,5'dimethylphenylamino)] propanoate and (2) triphenyltin(IV) 3-[(3′,5'dimethylphenylamino)]propanoate on relative viscosity of SS-DNA at 25 ± 0.1°C, [DNA] = 1.86 × 10<sup>−</sup><sup>4</sup> M.*

**Figure 17** [47] and **Figure 18** [48] shows the electrostatic binding mode of nickel and organotin(IV) complexes with DNA, respectively. The viscosity of DNA remains essentially unchanged on the addition of the nickel complexes while it decreases in case of organotin(IV) complexes [11].

#### **12. Conclusions**

This chapter has focused on drug-DNA interactions and their study by various analytical techniques such as IR spectroscopy, viscosity measurements, MS and AFM. These techniques are used to evaluate the binding mode as well as binding strength of the complex formed between drug and DNA. The study should be useful for the development of potential survey for DNA structure and new therapeutic reagents for tumors and other diseases. Fundamentally, drugs interact with DNA through two different ways, covalent and/or non-covalent modes. Covalent binders act as alkylating agents as they alkylate the nucleotides of DNA, while, the non-covalent binders interact by three different ways: (i) intercalation, (ii) groove binding, and (iii) external binding (on the outside of the helix). Different spectroscopic techniques are generally, powerful tools to study interactions of DNA with drugs and the effects of such interactions in the structure of DNA, providing some insights about the mechanism of drug action. The binding stoichiometry, the relative binding affinities and the binding constants for DNA double helices of various sequences.

**93**

**Author details**

Saudi Arabia

Saad Hmoud Alotaibi1

University of Bahri, Sudan

and Awad Abdalla Momen1,2\*

1 Department of Chemistry, Turabah University College, Taif University,

2 Department of Chemistry, College of Industrial and Applied Science,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: aamomena@yahoo.com

provided the original work is properly cited.

*Anticancer Drugs' Deoxyribonucleic Acid (DNA) Interactions*

*DOI: http://dx.doi.org/10.5772/intechopen.85794*

*Anticancer Drugs' Deoxyribonucleic Acid (DNA) Interactions DOI: http://dx.doi.org/10.5772/intechopen.85794*

*Biophysical Chemistry - Advance Applications*

**Figure 17** [47] and **Figure 18** [48] shows the electrostatic binding mode of nickel and organotin(IV) complexes with DNA, respectively. The viscosity of DNA remains essentially unchanged on the addition of the nickel complexes while it

*(1) Effects of increasing amount of tri-n-butyltin (IV) 3-[(3′,5'dimethylphenylamino)] propanoate and (2) triphenyltin(IV) 3-[(3′,5'dimethylphenylamino)]propanoate on relative viscosity of SS-DNA at 25 ± 0.1°C,* 

This chapter has focused on drug-DNA interactions and their study by various analytical techniques such as IR spectroscopy, viscosity measurements, MS and AFM. These techniques are used to evaluate the binding mode as well as binding strength of the complex formed between drug and DNA. The study should be useful for the development of potential survey for DNA structure and new therapeutic reagents for tumors and other diseases. Fundamentally, drugs interact with DNA through two different ways, covalent and/or non-covalent modes. Covalent binders act as alkylating agents as they alkylate the nucleotides of DNA, while, the non-covalent binders interact by three different ways: (i) intercalation, (ii) groove binding, and (iii) external binding (on the outside of the helix). Different spectroscopic techniques are generally, powerful tools to study interactions of DNA with drugs and the effects of such interactions in the structure of DNA, providing some insights about the mechanism of drug action. The binding stoichiometry, the relative binding affinities and the binding constants for DNA double helices of various

decreases in case of organotin(IV) complexes [11].

**12. Conclusions**

**Figure 18.**

*[DNA] = 1.86 × 10<sup>−</sup><sup>4</sup>*

 *M.*

**92**

sequences.
