Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19

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Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier.com/locate/jphotobiol

Short Review

Drug–DNA interactions and their study by UV–Visible, fluorescence

spectroscopies and cyclic voltametry
Muhammad Sirajuddin 1, Saqib Ali ⇑, Amin Badshah 2
Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: The present paper review the drug–DNA interactions, their types and applications of experimental tech-
Received 7 February 2013 niques used to study interactions between DNA and small ligand molecules that are potentially of phar-
Received in revised form 26 March 2013 maceutical interest. DNA has been known to be the cellular target for many cytotoxic anticancer agents
Accepted 28 March 2013
for several decades. Understanding how drug molecules interact with DNA has become an active research
Available online 6 April 2013
area at the interface between chemistry, molecular biology and medicine. In this review article, we
attempt to bring together topics that cover the breadth of this large area of research. The interaction
Keywords:
of drugs with DNA is a significant feature in pharmacology and plays a vital role in the determination
Drug–DNA interaction
UV–Visible spectroscopy
of the mechanisms of drug action and designing of more efficient and specifically targeted drugs with les-
Fluorescence spectroscopy ser side effects. Several instrumental techniques are used to study such interactions. In the present
Cyclic voltammetry review, we will discuss UV–Visible spectroscopy, fluorescence spectroscopy and cyclic voltammetry.
The applications of spectroscopic techniques are reviewed and we have discussed the type of information
(qualitative or quantitative) that can be obtained from the use of each technique. Not only have novel
techniques been applied to study drug–DNA interactions but such interactions may also be the basis
for the development of new assays. The interaction between DNA and drugs can cause chemical and con-
formational modifications and, thus, variation of the electrochemical properties of nucleobases.
Ó 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Drug–DNA interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. Types of Drug–DNA interaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
4. Modes of Drug–DNA binding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
4.1. Covalent mode of binding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
4.1.1. Alkylating agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
4.2. Non-covalent mode of binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
4.2.1. Intercalation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
4.2.2. Groove binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4.2.3. External binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5. Techniques used to study Drug–DNA interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5.1. UV–Visible absorption spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5.2. Fluorescence emission spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.3. Cyclic voltammetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
6. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
7. Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

⇑ Corresponding author. Tel.: +92 51 90642130; fax: +92 51 90642241.

E-mail addresses: m.siraj09@yahoo.com (M. Sirajuddin), drsa54@yahoo.com (S. Ali), aminbadshah@yahoo.com (A. Badshah).
1
Tel.: +92 51 90642208; fax: +92 51 90642241.
2
Tel.: +92 51 90642131; fax: +92 51 90642241.

1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotobiol.2013.03.013
2 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19
1. Introduction
DNA is present in body in the form of a double helix (Fig. 1A),
where each strand is composed of a combination of four nucleo-
tides (a nucleotide is a nucleoside with one or more phosphate
groups covalently attached to the 30 - and/or 50 -hydroxyl group(s)),
adenine (A), thymine (T), guanine (G) and cytosine (C) (Fig. 1B).
Within a strand these nucleotides are connected through phospho-
diester (deoxyribose sugars joined at both the 30 -hydroxyl and
50 -hydroxyl groups to phosphate groups in ester links is called
phosphodiester linkage) linkages (Fig. 1C). The two strands are
held together primarily through Watson Crick hydrogen bonds
where A forms two hydrogen bonds with T and C forms three
hydrogen bonds with G (Fig. 1D) [1–3]. The region where the
two strands are close to each other (deep–narrow) is called minor
grove while the region where they are away from each other
(shallow–wide) is called major groove.
2. Drug–DNA interactions
DNA is the pharmacological target of many of the drugs that are
currently in clinical use or in advanced clinical trials. Targeting
DNA to regulate cell functions by modulating transcription (gene
expression and protein synthesis) or by interfering with replication
(a major step in cell growth and division) seems logical, intuitively
appealing and conceptually straightforward. Small ligand mole-
cules bind to DNA and artificially alter and/or inhibit the function-
ing of DNA. These small ligand molecules act as drug when
alteration or inhibition of DNA function is required to cure or con-
trol a disease [4].
The study of interaction of drug with DNA is very exciting and
significant not only in understanding the mechanism of interac-
tion, but also for the design of new drugs. However, mechanism
of interactions between drug molecules and DNA is still relatively
little known. It is necessary to introduce more simple methods for
investigating the mechanism of interaction. By understanding the
mechanism of interaction, designing of new DNA-targeted drugs
and the screening of these in vitro will be possible [5].
3. Types of Drug–DNA interaction
There are primarily three different ways by which anticancer
drugs interact with DNA (Scheme 1), (i) through control of tran-
scription factors and polymerases. Here, the drugs interact with
proteins that bind to DNA, (ii) through RNA binding to DNA double
helix to form nucleic acid triple helix structures or RNA hybridiza-
tion (sequence specific binding) to exposed DNA single strand
forming DNA–RNA hybrids that may interfere with transcriptional
activity and (iii) through binding of small aromatic ligand mole-
cules to DNA double helical structures. The binding of small mole-
cules to DNA involves electrostatic interaction, intercalation
between base pairs and minor and major DNA grooves binding
interaction [6].
4. Modes of Drug–DNA binding
There are two modes of drug–DNA binding, covalent and non-
covalent. Some examples of DNA-interacting agents are given in
Table 1[7].
4.1. Covalent mode of binding
Many anticancer drugs in clinical use function by interacting
with DNA, not only those that bind to DNA through non-covalent
interactions but also those that form covalent adducts such as
via alkylation or inter- and intrastrand crosslinking [8]. The cova-
lent mode of binding of drug–DNA is irreversible and invariably
causes the complete inhibition of DNA processes and subsequent
cell death (if the reaction is not directly chemically reversible). A
major advantage of covalent binders is the high binding strength.
Moreover, covalent bulky adducts can cause DNA backbone distor-
tion, which in turn can affect both transcription and replication,
such as by disrupting protein complex recruitment [9].
Cis-platin [cis-dichlorodiammineplatinum(II)] is a famous cova-
lent binder used as an anticancer drug, and makes an intra/inter-
strand cross-link through the chloro groups with the nitrogens
on the DNA bases (Scheme 2).
The covalent attachment of organic intercalators to transition
metal complexes, producing metallointercalators, can lead to novel
DNA interaction that affect biological activity. Metal complexes hav-
ing r-bonded aromatic side arms can acts as dual-function com-
plexes: they bind to DNA both by metal coordination and through
intercalation of the attached aromatic ligand. These aromatic side
arms introduce new mode of DNA binding, involving mutual inter-
actions of functional groups held in close proximity [10].
The covalent binders are also called alkylating agents due to ad-
duct formation because they are used in cancer treatment to attach
an alkyl group (CnH2n+1) to DNA [11].
4.1.1. Alkylating agents
DNA alkylating agents have been used as anticancer agents by pro-
ducing significant DNA damage to kill cancer cell [12]. Alkylating
agents involve reactions with guanine in DNA. However, some alkylat-
ing drugs exert their strongest effects through alkylation of other posi-
tions and other types of cross-links. These drugs add methyl or other
alkyl groups onto molecules where they do not belong. This in turn
inhibits their correct utilization by base pairing and causes a miscod-
ing of DNA. Alkylating agents can interact via three mechanisms.
In the first mechanism, an alkylating agent attaches alkyl groups
to DNA bases. This alteration results in the DNA being fragmented by
repair enzymes in their attempts to replace the alkylated bases.
A second mechanism by which alkylating agents cause DNA
damage is the formation of cross-links, bonds between atoms in
the DNA. In this process, two bases are linked together by an alkyl-
ating agent that has two DNA binding sites. The cross-linking of the
two strands of DNA produced by the bifunctional alkylating agents
prevents the use of that DNA as a template for further DNA and
RNA synthesis leading to inhibition of replication and transcription
and, then, to cell death. A large number of chemical compounds are
alkylating agents under physiological conditions, and a variety of
such compounds have exhibited antitumor activity.
The third mechanism of action of alkylating agents causes the
mispairing of the nucleotides leading to mutations. The alkylating
agents, by chemical interactions, form covalent links with DNA.
This causes ‘‘mistakes’’ in the DNA that may result in mispairing,
substitutions, or excision. The cellular response of these mistakes
may inhibit DNA synthesis and proliferation or they may result
in apoptosis (process of programmed cell death that may occur
in multinuclear organisms) [11].
Alkylating drugs are the oldest class of anticancer drugs still
commonly used; they play an important role in the treatment of
several types of cancers. Most alkylating drugs are monofunctional
methylating agents (e.g., temozolomide [TMZ], N-methyl-N0 -nitro-
N-nitrosoguanidine [MNNG], and dacarbazine), bifunctional alkyl-
ating agents such as nitrogen mustards (e.g., chlorambucil and
cyclophosphamide), or chloroethylating agents (e.g., nimustine
[ACNU], carmustine [BCNU], lomustine [CCNU], and fotemustine)
[13]. The alkylation of N3 of guanine by (+)-CC1065 (antitumor
antibiotic) is shown in Scheme 3 [14]. (+)-CC1065 induces both
DNA bending toward the minor grooves and widing equivalent to
about one base pair per covalent alkylation event. (Scheme 4)
 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19 3

Fig. 1. (A) Structure of DNA molecule, (B) bases (adenine, guanine, thymine and cytosine) of DNA. (C) Watson–Crick pairing between purine and pyrimidine bases in
complementary DNA strands. (D) Watson Crick base pairing, A–T and G–C base pairing. Arrows indicates potential nitrogen and oxygen coordination sites.

4.2. Non-covalent mode of binding
Non-covalent DNA interacting agents, DNA-groove binders and
DNA intercalators, are generally considered less cytotoxic than
agents producing covalent DNA adducts and other DNA damage.
Although the impact of non-covalent compound–DNA interactions
on convoluted molecular and biochemical pathways is not well
characterized, the most important effects include DNA conforma-
tional and related structural perturbations, interference with nor-
mal DNA protein interactions, such as topoisomerases, as well as
effects on mitochondrial DNA and function. The non-covalent bind-
ing mode is reversible and is typically preferred over covalent
4 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19
Scheme 1. Summary of mechanism of action of anticancer drugs.
adduct formation keeping the drug metabolism and toxic side effects in
mind. Non-covalent DNA interacting agents can change DNA confor-
mation, change DNA torsional tension, interrupt protein–DNA interac-
tion, and potentially lead to DNA strand breaks. All of these events can
have substantial effects on gene expression [8].
The non-covalent mode of drug–DNA binding is further classi-
fied into three types, intercalation, groove binding and external
binding (on the outside of the helix) [15].
4.2.1. Intercalation
Intercalation of planar organic compounds to DNA was first pro-
posed by Lerman to explain the strong affinity of certain heterocy-
clic aromatic dyes such as acridines for DNA [16]. Planar
heterocyclic compounds act as intercalators which stack between
adjacent DNA base pairs leading to significant p-electron overlap.
Intercalators are molecules that stack perpendicular to the DNA
backbone without forming covalent bonds and without breaking
up the hydrogen bonds between the DNA bases. The only known
forces that sustain the stability of the DNA–intercalator complex,
even more than DNA alone, are van der Waals, hydrogen bonding,
hydrophobic, and/or charge transfer forces [17–21]. DNA intercala-
tors are used in chemotherapeutic treatment to inhibit DNA repli-
cation in rapidly growing cancer cells. DNA-intercalator complex is
stabilized by p–p stacking interaction and is thus less sensitive to
ionic strength relative to the two other binding modes (groove
binding and external binding). Structural changes are induced in
DNA by intercalators. Intercalation stabilizes, lengthens, stiffens,
and unwinds the DNA double helix [22]. In order for an intercalator
to fit between base pairs, the DNA must dynamically open a space
between its base pairs by unwinding. The degree of unwinding var-
ies depending on the intercalators. The ethidium cation unwinds
DNA by about 26° and proflavine by about 17°. These structural
modifications can lead to functional changes, often leading to inhi-
bition of transcription and replication and DNA repair processes,
which make intercalators potent mutagens [23,24].
Intercalation is usually independent of the DNA sequence con-
text (a slight GC specificity has been observed). This mode of bind-
ing is usually favored by the presence of an extended fused
aromatic ligand as PHEHAT [25] or DPPZ [26]. With less extended
aromatic systems, the intercalation is usually prevented through
clashing of the ancillary ligands with the phosphodiester backbone,
so that only partial intercalation can occur as it is the case for
 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19 5

Table 1
Examples of DNA-interacting agents.

Non-covalent binding agents Covalent DNA-adducts and direct DNA damage

Groove binding DNA intercalators
agents
Berenil Aminoacridines Busulfan
Bisbenzimadoles Arylaminoalcohols Camptothecin
Bleomycin Coumarins Chlorambucil
Chloroquine Cystodytin J Cis-platinum
Chromomycin A3 Diplamine Clomesone
Diamidine-2- YO-1 and YOYO-1 Cyclodisone
phenylindole
Distamycin A Daunomycin Ionizing radiation
Guanyl Quinolines and Nitrogen mustard
bisfuramidine quinoxalines
Mithramycin Ethidium bromide Nitrosoureas: 1,3-bis (2-chloroethyl)-1-nitrosourea; 1-(2-chloroethyl)-3-cyclohexyl-nitrosourea; 1-(2-chloroethyl)3-
Netamycin Proflavine (2,6-dioxo-3-piperidyl)-1-nitrosourea
Netropsin Echinomycin
Pentamidine Chlorpheniramine
Pilcamycin Methapyrilene
SN6999 Tamoxifen
SN7167 Bis-naphthalimide Oxidative-stress agents
Hoechst 33258 Doxorubicin UV radiation
M-AMSA Busulfan
Indoles Camptothecin
Cis-platinum

Scheme 2. (A) Cisplatin covalently bonded to DNA. B. (a) Modes of binding of cisplatin to guanine (G) and adenine (A); (b) 1,2-intrastrand GpG (structure a), 1,2-intrastrand
ApG (structure b), 1,3-intrastrand GpNpG (structure c), and 1,2-interstrand GpG (structure d).

[Ru(phen)3]2+ [25,26] (Fig. 2A and B) whereas Fig. 2C shows the
intercalation of Co and Cu complexes into the DNA base pairs
[27]. Intercalation of a planar ligand of the complex in the DNA
base pairs stack is shown in Fig. 2A [28,29].
4.2.1.1. Modes of intercalation. There are two major modes of inter-
calation: classical intercalation and threading intercalation [9,30].
4.2.1.1.1. Classical intercalation. Classical intercalators, such as ben-
zo[a]pyrene (BP) (Fig. 3A) [31], bind to DNA duplexes with essen-
tially all of their aromatic system inserted between GpG base
pairs that form the top and bottom of the intercalation site.
4.2.1.1.2. Threading intercalation. Threading intercalators usually
have two side chains on opposite sides of a planar aromatic ring
system, the process of complex formation with DNA is more com-
plicated. In such cases, one of the side-chains must slide through
the intercalation cavity in order to form the complex. Favorable
interactions of the side-chains with both the major and minor
grooves contribute to the complex stability of the threading inter-
calators [32]. A threading intercalator occupies and interacts
strongly with both the minor and major grooves of DNA simulta-
neously. For example, the threading intercalation of acridine-4-
carboxamides into the duplex 50 -d(CG(5-BrU)ACG)2-30 (Fig. 3B)
[33]. Acridine compounds are able to inhibit topoisomerase I and
II enzymes (topoisomerases are essential DNA-targeting enzymes
that initially induce a DNA strand cleavage), render DNA damage,
disrupt DNA repair and replication, and induce cell death [34].
4.2.2. Groove binding
Some small compounds bind to the minor groove of DNA by van
der Walls interaction and hydrogen bonding. Minor groove binding
drugs typically have several aromatic rings, such as pyrrole, furan
or benzene connected by bonds possessing torsional freedom. Min-
or groove binding drugs are usually narrow curved shaped, which
is isohelical to the curve of the minor groove, and facilitates
6 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19

Scheme 3. Proposed mechanisms for the alkylation of N3 guanine by (+)-CC1065: (a) tautomerization of the G–C base pair to give the enol-G–imino-C tautomer; (b) a
concerted mechanism.

Scheme 4. Structures of the seven small molecules studied (P1–P7).

binding by promoting van der Waals interactions. Additionally,
these drugs can form hydrogen bonds to bases, typically to N3 of
adenine and O2 of thymine. The groove-binding molecules are
commonly specific to adenine–thymine (AT) rich sequences. This
preference in addition to the designed propensity for the electro-
negative pockets of AT sequences is probably due to better van
der Waals contacts between the ligand and groove walls in this re-
gion, since AT regions are narrower than GC groove regions and
also because of the steric hindrance in the latter, presented by
the C2 amino group of the guanine base [35]. However, a few
 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19 7

Fig. 2. (A) Intercalation of a planar ligand of the complex in the DNA base pairs stack. (B) Intercalated ruthenium complex, [Ru(L)2(L0 )]2+ (L = bipyridine, 1,10-phenanthroline
(phen) and 1,4,5,8-tetraazaphenanthrene (tap) and L0 = dipyridophenazine (dppz) in double-stranded DNA fragment (CG–GC). (C) Intercalation of Co(II) and Cu(II) metal
complexes of N1,N5-bis[pyridine-2-methylene]-thiocarbohydrazone.

synthetic polyamides like lexitropsins and imidazole-pyrrole

polyamides have been designed which have specificity for GC
and CG regions in the grooves. Hydrophobic and/or hydrogen-
bonding are usually important components of this binding process,
and provide stabilization. The antibiotic netropsin is a model
groove-binder (Fig. 4A). Fig. 4B shows the generally proposed bind-
ing of acridine bis-imidazolidinones (R = ethyl) into adjacent Cyto-
sine: Guanine base pairs from minor groove side [36]. Geometric
and steric factors also play a role as shown with [Ru(TMP)3]2+,
where the methyl groups prevent intercalation [37] (Fig. 4C). Un-
like to intercalators, groove-binding drugs induce little or no struc-
tural rearrangement of the DNA helix. Fig. 4D shows the Groove
binding of Ni(II) and Zn(II) metal complexes of N1,N5-bis[pyri-
dine-2-methylene]-thiocarbohydrazone [28].

4.2.3. External binding

This type of binding is electrostatic in nature. Some ligands are
also capable of forming non-specific, outside edge stacking interac-
tions with the DNA phosphate backbone. This mode usually occurs
where the ligand self-associates to form higher-order aggregates,
which may stack on the anionic DNA backbone in order to reduce
charge–charge repulsion between ligand molecules. Some metal
complexes also interact with DNA through external binding, e.g.,
binding of Ru(II) complexes which are 2+ positive charge with Fig. 3. (A) NMR solution structure of the d(C4A5C6)-d(G13G14G15) segment of the
the DNA phosphate sugar backbone which is negatively charged. 10S-[BP]dAdG 9-mer duplex (50 -G1G2T3C4-[BP]A5C6G7A8G9-30 )/(50 -C10T11C12G13G14-
This association mode was proposed for [Ru(bpy)3]2+ as the lumi- G15A16C17C18-30 ), where the BP group attached to the A5 base intercalates
nescence enhancement of this complex upon binding to DNA is classically between the G13G14base step of the opposite strand. (B). X-ray structure
showing threading intercalation of 9-amino-6-bromo-DACA (space-filling model)
strongly dependent on the ionic strength. Cations like Mg2+, usu- into the DNA duplex 50 -d(C|G(5-BrU)AC|G)2-30 (wireframe), where | indicates
ally also interacts in this way (Fig. 5) [38]. intercalation sites.

5. Techniques used to study Drug–DNA interactions
This section consists of a list of the different instrumental tech-
niques that have been used to study interactions between DNA and
small ligand molecules. For each technique, we have provided a
brief theoretical introduction to how it is applied to the study of
the interactions, followed by a discussion of some examples that
are characteristic of the technique or that are of specific interest.
Various techniques that are used to study the binding of drug
molecules with DNA includes Infrared (IR), Raman, Circular
8 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19

Fig. 4. (A) DNA complexed with netropsin, a minor groove binder. (B) Putative binding of acridine bis-imidazolidinones (R = ethyl) into adjacent cytosine:guanine base pairs
from minor groove side. (C) Adsorption of the [Ru(TMP)3]2+ in the DNA grooves. (D) Groove binding of Ni(II) and Zn(II) metal complexes of N1,N5-bis[pyridine-2-methylene]-
thiocarbohydrazone.

Fig. 5. External association of the complex in the atmosphere of ions of the DNA polyelectrolyte.

dichroism, UV–Visible, Nuclear magnetic resonance (NMR) spec-
troscopies, Atomic force microscopy (AFM), Electrophoresis, Mass
spectrometry, Viscosity measurements, Thermal denaturation
studies, Cyclic, Square wave and Differential pulse voltammetry,
etc. These techniques have been used as a major tool to character-
ize the nature of drug–DNA complexation and the effects of such
interaction on the structure of DNA. In this review article we will
focus on UV–Visible, fluorescence spectroscopies and cyclic vol-
tammetry as these are of common use.
5.1. UV–Visible absorption spectroscopy
UV–Visible absorption spectroscopy is perhaps the simplest and
most commonly employed instrumental technique for studying
both the stability of DNA and their interactions with small ligand
molecules. The study of drug–DNA interactions could be carried
out by UV–Visible absorption spectroscopy by monitoring the
changes in the absorption properties of the drug or the DNA mol-
ecules. Usually, molecules used as ligands show an absorption
band that can be clearly distinguished in the visible region. An easy
way to determine whether there is any interaction between the
DNA and the drug is therefore to examine the shifting of the posi-
tion of the maximum of this band from when the ligand is free in
solution to when the ligand is bound with the DNA. It has been as-
sumed that the magnitude of this shifting could be interpreted as
an indication of the strength of the interaction between the DNA
structure and the ligand considered [39–42].
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. This maximum is a consequence of the chromophoric
groups in purine (adenine and guanine) and pyrimidine (cytosine
and thymine) moieties responsible for the electronic transitions.
The probability of these transitions is high and thus the molar
absorptivity (e) is of order of 104 M1 cm1 [3]. This technique al-
lows determining the molar concentration of DNA on the basis of
the measurement of the absorbance value at 260 nm. The absor-
bance ratios (A260/A280 and A260/A230) can also describe the purity
of DNA. This ratio should be in the range of 1.8–1.9 to ensure that
DNA is sufficiently free of protein [43]. Slight changes in the
absorption maximum as well as the molar absorptivity can occur
with the variations in pH or ionic strength of the media. Drug–
DNA interactions can be studied by comparison of UV–Visible
absorption spectra of the free drug and drug–DNA complexes,
which are usually different. Compounds binding with DNA through
intercalation usually results in hypochromism and bathochromism
(red shift). Because of the intercalative mode involving a stacking
interaction between an aromatic chromophore and the base pair
of DNA, the extent of the hypochromism is usually consistent with
 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19 9

the strength of intercalative interaction [44]. The strength of this
electronic interaction is expected to decrease as the cube of the
distance between the chromophore and the DNA bases decreases.
By decreasing the distance between intercalated compound (drug)
and DNA bases, hypochromism take place apparently. Thus, this is
consistent with the combination of compound p electrons and p
electrons of DNA bases. Consequently, the energy level of the p–
p electron transition decreases, which causes a red shift. This con-
tributes to the hypochromic effect discussed above [45,46]. In case
of electrostatic attraction between the compound and DNA, hyper-
chromic effect is observed that reflects the corresponding changes
of DNA in its conformation and structure after the complex–DNA
interaction has occurred. The hyperchromic effect is the outstand-
ing increase in absorbance of DNA upon denaturation. The two
strands of DNA are held together mainly by the stacking interac-
tions, hydrogen bonds and hydrophobic effect between the com-
plementary bases. The hydrogen bond limits the resonance of the
aromatic ring so the absorbance of the sample is limited as well.
When the DNA double helix is treated with denaturing agents,
the interaction force holding the double helical structure is dis-
rupted. The double helix then separates into two single strands
which are in the random coiled conformation. At this time, the
base–base interaction will be reduced, increasing the UV absor-
bance of DNA solution because many bases are in free form and
do not form hydrogen bonds with complementary bases. As a re-
sult, the absorbance for single-stranded DNA will be 40% higher
than that for double stranded DNA at the same concentration. Fur-
thermore, the hyperchromic effect arises mainly due to the pres-
ence of charged cations which bind to DNA via electrostatic
attraction to the phosphate group of DNA backbone and thereby
causing a contraction and overall damage to the secondary struc-
ture of DNA [47]. The hyperchromic effect may also be attributed
to external contact (electrostatic binding [48]) or to partial uncoil-
ing of the helix structure of DNA, exposing more bases of the DNA
[49]. If there is a weaker interaction then only hypochromic or
hyperchromic effects are observed without significant changes of
shifts in the spectral profiles [3,50].
Based upon the variation in absorbance, the intrinsic binding
constant/association constant (K) of the drug with DNA can be
determined according to Benesi–Hildebrand equation [43]:
A0 eG eG 1
¼ þ 
A  A0 eHG  eG eHG  eG K½DNA
where K is the association/binding constant, A0 and A are the absor-
bances of the drug and its complex with DNA, respectively, and eG
and eH–G are the absorption coefficients of the drug and the

Fig. 6. (A) Absorption spectra of [{SnPh3(O2CCH2SXyl)}1, where Xyl = 3,5-Me2C6H3] in the presence of increasing amounts of DNA. Arrow indicates that absorbance changes
upon increasing DNA concentrations. Inset: plot of [DNA]/eo  ef = [DNA]/eo  ef + 1/Kb(/eo  ef). (B) Electronic spectral of [Cu(L)(H2O)2](NO3)2 (L = 2,6-bis(benzimidazol-
yl)pyridine) through titration with CT-DNA in tris–HCl buffer; C. Plot of [DNA]/(eo  ef) vs. [DNA] for the titration of CT-DNA with [Cu(L)(H2O)2](NO3)2 for the determination of
Kb (0.8  105 M1).
10 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19

Fig. 7. UV–Visible absorption spectra of complexes C10H22N2O5SnCl2 (A) and C10H22N2O5ZrCl2 (B) in Tris–HCl buffer upon addition of CT-DNA. Plots of [DNA] vs. [DNA]/ea  ef
for the titration of CT-DNA with complexes C10H22N2O5SnCl2 (C) and C10H22N2O5ZrCl2 (D).

drug–DNA complex, respectively. The association constant can be ob-

tained from the intercept-to-slope ratios of A0/(A  A0) vs. 1/[DNA]
plots. The K can also be determined from the intercept-to-slope ratios
of the plot of [DNA] vs. [DNA]/ea  ef, where ea (or eG) and ef (or eH–G)
are the absorption coefficients of the drug and the drug–DNA com-
plex, respectively.
Fig. 6A, describes the interaction of triphenyltin(IV) carboxylate
complex [{SnPh3(O2CCH2SXyl)}1, where (Xyl = 3,5-Me2C6H3)] with
DNA. The inset graph represents the plot of [DNA]/eo  ef = [DNA]/
eo  ef + 1/Kb(/eo  ef), used for the determination of the intrinsic
binding constant, Kb (Kb = 1.68  105 M1) [51]. As it is shown in
Fig. 6A, with increasing concentrations of DNA, the absorption
bands of the complex were affected, resulting in the tendency of
hyperchromism and a very slight blue shift. The complex [{SnPh3(-
O2CCH2SXyl)}1] may be charged (R3Sn+), The R3Sn+ moieties are
assumed to directly affect DNA [52] as well as binding to mem-
brane proteins or glycoproteins, or to cellular proteins, and there
could be classical electrostatic interactions with the negatively
charged oxygen of the phosphate group of DNA, which may be
responsible for the spectral changes observed. However, other
electrostatic effects such as hydrogen bonding between the com-
plexes and the base pairs in DNA may also be present [44,53,54].
Fig. 6B represent the interaction of copper(II) complex with Fig. 8. Absorption spectra of Zn(Pyimpy)2](ClO4)2C6H5CH30.5H2O in 0.1 M phos-
DNA. With increase in DNA concentration (indicated by an arrow) phate buffer (pH 7.2) containing 5% DMF in the presence of increasing amounts of
DNA. Arrows show the absorbance changes upon increasing DNA concentration.
 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19 11

Fig. 9. Absorption spectra of 0.2 mM Ph2SnL (A), Me2SnL (B) and Bu2SnL (C) in the absence and presence of 5–25 mM DNA. The arrow direction indicates increasing
concentrations of DNA. (D). Plots of A0/(A – A0) vs. 1/[DNA] for the determination of binding constants of Complex-DNA adducts. Absorption spectra of 3 mM Bu3SnL (E),
Cy3SnL (F) and Ph3SnL (G) in the absence and presence of 20 lM, (b) 30 lM, (c) 40 lM, (d) 50 lM, (e), 60 lM (f), 70 lM (g) and 80 lM DNA (h) in 10% aqueous DMSO at 25 °C.
The arrow direction indicates increasing concentrations of DNA.

hyperchromic effect is observed. A significant hyperchromism ef-
fect centered at the 330 nm absorption maximum, suggest a strong
interaction between the copper(II) complex and DNA. This spectral
change might be indicative of groove binding. Groove binding mol-
ecules contain unfused aromatic ring systems connected by bonds
with torsional freedom in order to adopt appropriate conformation
that closely matches the helical turn of DNA grooves, while fused
polyaromatic systems containing protonated nitrogen atoms or
having protonated side chains attached to the ring system are typ-
ically intercalators. Here, the organic ligand L, having coplanar
atoms upon copper coordination, likely facilitates the formation
of Van der Waals contacts or hydrogen bonds during interaction
with DNA grooves [55].
The complexes C10H22N2O5SnCl2 (1) and C10H22N2O5ZrCl2 (2)
exhibit intraligand absorption bands in the UV region at 248 and
253 nm, respectively, as shown in Fig. 7A and B. By the addition
of increasing amounts of CT-DNA ((0.066–0.333)  104 M) to
complexes C10H22N2O5SnCl2 and C10H22N2O5ZrCl2, a sharp hyper-
chromic effect in the absorption bands with a moderate red shift
of 5 and 3 nm, respectively is observed. Hyperchromic effect re-
flects the corresponding changes of DNA in its conformation and
structure after the complex–DNA interaction has occurred. Fur-
thermore, the hyperchromic effect arises mainly due to the pres-
ence of charged cations Sn(IV)/Zr(IV) which bind to DNA via
electrostatic attraction to the phosphate group of DNA backbone
and thereby causing a contraction and overall damage to the sec-
ondary structure of DNA [47].
Fig. 8 describes the interaction of Zn complex with CT-DNA. A
blue shift of 4 nm and hyperchromic shift of the absorption band
were observed in case of complex Zn(Pyimpy)2](ClO4)2C6H5CH3-
0.5H2O. Isosbestic point near 350 nm was observed for the com-
plex. Such type of spectral changes describes the covalent
binding of zinc complex with DNA. Zinc complexes in the presence
of DNA get attached to the nucleic acid base(s) and a new species
generates during DNA interaction. The intrinsic binding constant K
for the complex was found to be 2.86  103 M–1. The observed
binding constant is smaller than the classical intercalators and
metallointercalators where binding constant was reported to be
in the order of 107 M–1[56,57].
Fig. 9 describes the effect of varying concentration of DNA (5–
25 mM) on the electronic absorption spectra of 0.2 mM of Me2SnL
(A), Bu2SnL (B) and Ph2SnL (C) (L = N0 -(2-hydroxybenzylidene)
12 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19
Fig. 10. UV–Visible absorption spectra of 2.5  105 M GMB in presence of different
concentrations of DNA 0 (1), 7.5 (2), 15 (3), 22.5 (4), 30 (5), 37.5 (6), 45 (7), 52.5 and
(8) 60 lM.
formohydrazide. The strong absorption of these compounds in the
near UV region (292–330 nm) is due to the long-living triplet ex-
cited state of the aromatic system. The broad absorption bands in
the region (350–470 nm) is due the intraligand transition between
p and p energy levels of the tridentate ligand N0 -(2-hydroxyben-
zylidene)formohydrazide. The absorption spectra of Me2SnL, Bu2-
SnL and Ph2SnL recorded a 68.26%, 62.83% and 65.91% decrease
in peak intensities accompanied with 2, 5 and 3 nm red shift
respectively, by the addition of 25 mM DNA. The peculiar hypo-
chromic effects can be associated with the interaction of electronic
states of the intercalating chromophore and those of the stacked
base pairs of DNA. The slight bathochromic shifts can best be de-
scribed by the lowering in p and p transition energy of the ligands
in diorganotin(IV) complexes due to their ordered stacking be-
tween the DNA base pairs after intercalation. After binding to the
DNA, the p orbital of the binding ligand could couple with p orbi-
tal of base pairs in the DNA. The coupling p orbital was partially
filled by electrons, thus decreasing the transition probabilities,
and hence resulting in the hypochromicity. The binding constants,
with values of 1.54  104, 8.19  103 and 2.59  104 M1 for the
interaction of Me2SnL, Bu2SnL and Ph2SnL with DNA were obtained
from the slope to intercept ratio of the plots (D) between
A0/(A  A0) vs. 1/[DNA] [58,59].
Fig. 11. UV–Visible absorption spectra of 30 lM NFC in the absence of DNA (a) and
presence of 10–60 lM DNA (b–g) in 10% aqueous ethanol buffered at pH 6.
A similar intercalation mode of interaction of Bu3SnL (E), Cy3SnL
(F) and Ph3SnL (G) (L = 4-(4-nitrophenyl)piperazinium 4-(4-nitro-
phenyl)piperazine-1-carbodithioate) with DNA is shown in
Fig. 9E–G. The binding of complexes Bu3SnL, Cy3SnL and Ph3SnL
to DNA caused a progressive blue shift of 10 (401–391), 8 (400–
392) and 4 nm (398–394), respectively. Such spectral characteris-
tics are indicative of their binding to DNA. The peculiar hypochro-
mism observed here is attributed to the intercalation of these
drugs into the DNA base pairs. The binding constant value deter-
mined for these compounds are 6.05  103, 2.30  103 and
3.25  103 M1, respectively. The greater value of K for the Bu3SnL
is due to additional hydrophobic nature of butyl group with the
bases of DNA [60].
Absorption spectrum of GMB exhibited bands at 207, 232, 248,
283 and 331 nm (Fig. 10). With the addition of increasing amounts
of DNA the absorbance of GMB increased at 207, 232, 248 and 283
(with red shift at 207 and 248 nm), while those at 331 nm de-
creased slightly. These results revealed that there is strong interac-
tion between drug and DNA. The appreciable shift in wavelength
(red shift P 15 nm) indicates the intercalative binding mode of
molecules to DNA helix due to the interaction of a DNA p stack
with ligand, while outside binders (groove binders) shows a smal-
ler red shift (Dk 6 8 nm). The red shift for absorption peaks at 207
and 248 is about 1 and 5 nm, respectively, so groove binding mode
between GMB and DNA is also there. The binding constant, K, cal-
culated is 1.97  106 M1[61].
Fig. 11 shows the effect of different concentration of DNA (10–
60 lM) on the electronic absorption spectrum of 30 lM NFC. The
rationale behind the band in the UV region (328 nm) is the proba-
ble charge transfer between the non-bonding or antibonding orbi-
tal of the cyclopentadienyl ring and the iron atom of NFC. The
maximum absorption of the drug at this wavelength exhibited
slight bathochromic and pronounce hypochromic shifts by the
incremental addition of DNA. The bathochromic effect is associated
with the decrease in the energy gap between the highest (HUMO)
and the lowest molecular orbitals (LUMO) after the interaction of
NFC to DNA. The compactness in the structure of either the drug
alone and/or DNA after the formation of drug–DNA complex may
result in hypochromism [62].
5.2. Fluorescence emission spectroscopy
Fluorescence spectroscopy is probably one of the techniques
most commonly used to study interactions between small ligand
molecules and DNA. The advantages of molecular fluorescence
over other techniques are its high sensitivity, large linear concen-
tration range and selectivity. The most intense and the most useful
fluorescence is found in compounds containing aromatic func-
tional groups with low-energy p ? p transition levels. Com-
pounds containing aliphatic and alicyclic carbonyl structures or
highly conjugated double-bond structures may also show fluores-
cence, but the number of these transitions is small compared with
those in aromatic systems [40,63].
The mode of binding of drugs to DNA can be determined by
fluorescence spectroscopy and the various analytical tools based
on fluorescence emission can also provide particularly useful infor-
mation. The orientation of fluorophoric ligands and their closeness
to the DNA pairs of bases can be studied by fluorescence anisotropy
or fluorescence resonance energy transfer.
Fluorescence quenching experiments give additional informa-
tion concerning the localization of the drugs and their mode of
interaction with DNA [3].
Fluorescence emission is very sensitive to the environment, and
hence the fluorophore transfer from high to low polarity environ-
ments usually causes spectral shifts (10–20 nm) in the excitation
and emission spectra of drugs [64]. Moreover, the effective
 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19 13

Fig. 12. Emission spectra of EB bound to CT-DNA in the presence of the Mn complex [C42 H40 Mn N14 O16] (a) and Zn complex [C78 H62 N26 O28 Zn2] (c). The arrows show the
intensity changes upon increasing concentrations of the complexes. Fluorescence quenching curves of EB bound to CT-DNA by the Mn complex (b) and Zn complex (d). Plots
of I0/I vs. [Complex].

interaction with DNA usually causes a significant enhancement
of the fluorescence intensity as a consequence of different fac-
tors. Thus, in the case of intercalating drugs, the molecules are
inserted into the base stack of the helix. The rotation of the free
molecules favors the radiationless deactivation of the excited
states, but if the drugs are bound to DNA the deactivation
through fluorescence emission is favored, and a significant in-
crease in the fluorescence emission is normally observed. In case
of groove binding agents, electrostatic, hydrogen bonding or
hydrophobic interactions are involved and the molecules are
close to the sugar-phosphate backbone, being possible to observe
a decrease in the fluorescence intensity in the presence of DNA
[65]. The use of well-established quenchers, i.e., halide ions, pro-
vides further information about the binding of drugs to DNA. The
groove binders are more sensitive to the quenching effect by ha-
lides than the intercalating agents, because the pairs of bases
hinder the accessibility of the drug by the quenchers. Besides,
the electrostatic repulsive forces among phosphate groups on
DNA and anionic quenchers collaborate to protect the drug from
the quencher effects. Thus, in the case of intercalating agents
reduction in the KSV {Stern–Volmer equation, Fo/F (or I0/
I) = 1 + KSV [Q], where Fo or I0 is the fluorescence intensity in
the absence of quencher while F or I is the fluorescence intensity
in the presence of quencher, KSV is the Stern–Volmer quenching
constant, [Q] is the concentration of the quencher} values is ob-
served in the presence of quencher. KSV can be obtained from the
slope of the plot of Fo/Fvs. [DNA] [3,66].
Ethidium bromide (EB) is a common fluorophore that bind to
DNA. The fluorescene of EB increases in the presence of CT-DNA,
due to its strong intercalation between the adjacent CT-DNA base
pairs. It was previously reported that the enhanced fluorescence
can be quenched by the addition of a second molecule. Thus if
the second molecule intercalates into DNA, it leads to a decrease
in the fluorescence intensity of the EB-DNA because it will compete
with EB in binding with DNA. The extent of fluorescence quenching
of EB bound to CT-DNA can be used to determine the extent of
binding between the second molecule and CT-DNA [66–68]. The
emission spectra of EB bound to DNA in the absence and presence
of complex are shown in Fig. 12a–d, where the fluorescence inten-
sity of EB-DNA decreases in the presence of a second molecule that
intercalates itself into the base pair of the DNA. The fluorescence
quenching of EB bound to CT-DNA by Mn complexe [C42 H40 Mn
N14 O16] and Zn complex [C78 H62 N26 O28 Zn2] is shown in
Fig. 12a and c. The quenching of EB bound to CT-DNA by both com-
plexes is in good agreement with the linear Stern–Volmer equa-
tion, which provides further evidence that the complexes bind to
DNA. The KSV values for Mn and Zn complexes and are
2.9  103 M1 and 1.9  103 M1, respectively. The data suggest
that the interaction of Mn complex with CT-DNA is stronger than
that of Zn complex [67].
14 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19
Fig. 13. Emission spectra of EB bound to CT-DNA in the presence of free HL [N-(2-hydroxylacetophenone)-3-oxapentane-1,5-diamine] (A) and Ni complex [Ni2(L)2(NO3)2] (C),
kex = 520 nm. The arrows show the intensity changes upon increasing concentrations of the complexes. Fluorescence quenching curves of EB bound to CT-DNA by the free HL
(B) and Ni complex (D). (Plots of I0/I vs. [Compound]).
Similarly Fig. 13 describes the interaction of N-(2-hydroxylace-
tophenone)-3-oxapentane-1,5-diamine (HL) and its Ni complex
with EB-DNA. The KSV values of compound are (1.7 ± 0.105)
9  103 M1 and (7.0 ± 0.097)  103 M1 for HL and the complex,
respectively. These values show that both the free ligand and com-
plex can compete for DNA binding sites and so displace EB from the
DNA, which is usually characteristic of the intercalative interaction
of compounds with DNA. Fig. 14 describe the interaction of DNA
with copper complexes [55,68,69].
5.3. Cyclic voltammetry
The application of electrochemical methods to the study of met-
allointercalation and coordination of metal ions and chelates to
DNA provides a useful complement to the previously used meth-
ods of investigation, such as UV–Visible spectroscopy. Small mole-
cules which are not amenable to such methods, either because of
weak absorption bands or because of overlap of electronic transi-
tions with those of the DNA molecule, can potentially be studied
via voltammetric techniques. Multiple oxidation states of the same
species as well as mixtures of several interacting species can be ob-
served simultaneously. Equilibrium constants (K) for the interac-
tion of the metal complexes with DNA can be obtained from
shifts in peak potentials, and the number of base pair sites involved
in binding(s) through intercalative, electrostatic, or hydrophobic
interactions by the reliance of the current passed during oxidation
or reduction of the bound species on the amount of added DNA. In
some cases it should also be possible to obtain kinetic data from
current and potential measurements, since voltammetric methods
are sensitive to chemical reactions (e.g., ligand dissociation) cou-
pled to the electron-transfer steps [70].
Cyclic voltammetry (CV) is widely used for the evaluation of mode
of action and binding strength of drug–DNA interaction. This technique
is predominantly useful for metal based compounds due to their acces-
sible redox states. As in CV the scan is revered so the fate of the species
in the backward scan can also be studied. The peak potential and peak
current of the compound changes in the presence of DNA if the com-
pound interacts with it. The variation in peak potential and peak cur-
rent can be exploited for the determination of binding parameters.
The decay in peak current (Ip) of the drug by the addition of increasing
amount of DNA can be used for the determination of binding constant
and binding site size, whereas the shift in peak potential can be used to
ascertain the mode of interaction. The binding constant is quantified
by the following equation [71]:
logð1=½DNAÞ ¼ log K þ logðI=ðI0  IÞÞ
where K is the binding constant, I0 and I are the peak currents of the
drug in the absence and presence of DNA, respectively. The binding
constant, K is obtained from the intercept of the plot of log (1/
[DNA]) vs. log (I/(I0  I)).
 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19 15

Fig. 14. (A) Emission spectrum of EtBr bound to DNA in the presence of [Cu2L(phen)](ClO4)2 where L = 6,60 -piprazine-1,4-diyldimethylene bis (4-methyl phenol). The arrow
shows the intensity change upon increasing Cu(II) complex (3) concentrations. Inset shows the plots of emission intensity I0/I vs. [DNA]/[complex for determining KSV. (B).
Emission spectra of the CT-DNA–EB system in tris–HCl buffer in the presence of [Cu(L)(H2O)2](NO3)2, where L = 2,6-bis(benzimidazolyl)pyridine; kex = 522 nm. The arrow
indicates the increase of the complex concentration. (C). Plot of I0/I vs. [complex] for the titration of CT-DNA–EB system with [Cu(L)(H2O)2](NO3)2 for determining KSV
(10.0  104). (D) Plot of Io/I vs. [complex] for the titration of CT-DNA–EB system with 1 using spectrofluorimeter; linear Stern–Volmer quenching constant (Ksv) for 1 =
10.0104; (R = 0.99596 for five points).

Another equation which can be used for the determination of

binding constant is given as [72]:
1 Kð1  AÞ
¼ K
½DNA 1  I=I0
where A is proportionality constant. The binding constant is calcu-
lated from the intercept of the plot of 1/[DNA] vs. 1/(1  I/I0).
A simple site binding model used to fit the cyclic voltammetric
data acquired from the interaction between the drug and DNA is
given as [73]:
C b =C f ¼ Kf½free base pairs=sg
where s is the binding site size in terms of base pairs, Cf is the con-
centration of the free species, Cb denotes the concentration of DNA-
bound species and K represents binding constant.
Measuring the concentration of DNA in terms of nucleotide
phosphate, the concentration of the base pairs can be expressed
as [DNA]/2 and the above equation can be written as
C b =C f ¼ Kf½DNA=2sg
The Cb/Cf was determined by the equation given below [74]:
Fig. 15. Square wave voltammograms for 0.09 mM quinacrine in the ‘‘a’’ absence
C b =C f ¼ ðI0  IÞ=I and ‘‘b’’ presence of 0.5 mM DNA in 100 mM Tris–HCl buffer at pH 7.0. Equilibrium
time: 10 s, frequency: 10 Hz, step potential: 5 mV, amplitude: 25 mV.
The right hand side of the above equation can be obtained di-
rectly from the experimental peak currents. The experimental data
can therefore be expressed as plots of Cb/Cf against the total con- It has been reported in the literature that the binding constants
centration of DNA keeping concentration of the drug constant. for redox active species can be obtained from straightforward
16 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19

Fig. 16. CV behavior of 3 mM dibutyltin(IV) bis(4-nitrophenylethanoate) at GC
electrode in the absence (a) and presence of 10 (b), 20 (c), 30 (d), 40 (e), 50 (f) and
60 lMDNA (g) in 10% aqueous DMSO with 0.1 M TBAFB as supporting electrolyte at
0.1 V/s scan rate.
voltammetric experiments in which the DNA is titrated against the
redox active molecule. The measurement of diffusion currents in
the presence of excess nucleic acid has shown that the diffusion
coefficient of DNA-bound species is more than one order of magni-
tude lower than that of the free species [74–76].
Carter and Bard have reported three kinds of modes for small
molecules binding to DNA, if E1/2 shifted to more negative value
when small molecules interacted with DNA; the interaction mode
was electrostatic binding. On the other hand, if E1/2 shifted to more
positive value, the interaction mode was intercalative binding [70].
Fig. 15 shows the effect of the addition of DNA to a solution of
0.09 mM quinacrine in 100 mM Tris–HCl buffer, pH 7, on the
square wave voltammetry of quinacrine. The current drops on
the addition of DNA owing to the binding of quinacrine. Further-
more, the peak potential shifted to a more positive value in the
presence of DNA. The shift in peak potential is typical of the inter-
calation of species into DNA [21,30]. In the presence of nucleic
acids, the current is mainly due to free species, as the diffusion rate
of bound species is small [26]. The cause for the decrease in the
peak current was that the obvious diffusion coefficient and the
obvious concentration of electroactive species decreased [75,77].
Fig. 16 describes the cyclic voltammetric behavior of dibutyl-
tin(IV) bis(4-nitrophenylethanoate) on glassy carbon electrode in
the absence and presence of DNA, in 10% aqueous DMSO at
25 °C. Due to the instability of Sn3+, the voltammetric response is
attributed to the 2e redox process of Sn2+/Sn4+ couple. In the ab-
sence of DNA, dibutyltin(IV) bis(4-nitrophenylethanoate) regis-
tered a cathodic peak at 1.212 V and anodic peak at 1.014 V.
The large peak to peak potential difference (DEp) of 198 mV is sug-
gestive of electrochemical reaction coupled with a chemical reac-
tion. With the increase in concentration of DNA in a constant
amount of dibutyltin(IV) bis(4-nitrophenylethanoate), the voltam-
metric response of the compound changed as is evidenced by the
sequential drop in peak current and gradual peak potential shift
in positive direction. The shift of peak potential to less negative
values is suggestive of intercalation of dibutyltin(IV) bis(4-nitroph-
enylethanoate) into DNA. The observed decrease in peak current

Fig. 17. Cyclic voltammograms of 3 mM: (A) Ph2SnL (1), (B) Me2SnL (2) and (C) Bu2SnL (3) in 10% aqueous DMSO with 0.1 M TBAP as supporting electrolyte in the absence (a)
and presence of 50 mM DNA (b) at 25 °C. (D) Plots of log (IH–G/(IG  IH–G)) vs. log (1/[DNA]) used to calculate the binding constants of 1-DNA, 2-DNA and 3-DNA adducts. (E)
Plots of I vs. m1/2, for the determination of diffusion coefficients of the free drugs (3 mM 1–3). Scan rates 0.1–0.6 V/s with a difference of 0.1 V/s. (F) Plots of I vs. m1/2, for the
determination of the diffusion coefficients of the DNA bound drugs by taking 3 mM 1–3 and 60 mM DNA. Scan rate 0.1–0.6 V/s with a difference of 0.1 V/s.
 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19 17

Fig. 18. (A) Cyclic voltammograms of the copper(II) complex in the absence (a) and presence (b) of HS-DNA. (B) The plot of 1/[DNA] vs. 1/(1  i/i0).

indicates the formation of large and slowly diffusing dibutyltin(IV) In UV–Visible spectroscopy, hypochromism and red shift are the
bis(4-nitrophenylethanoate)-DNA adduct due to which the free signs of intercalation while hyperchromism is the sign of electro-
drug concentration (which is mainly responsible for the conduc- static mode of interaction. Similarly in cyclic voltammetry shift
tion of the current) is lowered [78]. of E1/2 towards more positive value is the indication of intercala-
Similarly Fig. 17 describes the intercalative mode of interaction tion mode of interaction while that towards more negative value
of organotin(IV) complexes with DNA. The shift in peak potential is the indication of electrostatic mode of interaction. In the case
toward positive side could be attributed to the intercalation of of fluorescence, a significant increase in the fluorescence emission
compounds A–C into the stacked base pair pockets of DNA [58]. is normally observed for intercalative mode of interaction while a
Fig. 18 describes the interaction of [Cu2(dmaeoxd)(ox)]nnH2O decrease in the fluorescence intensity is observed for groove bind-
with HS-DNA via groove binding. It can be seen from Fig. 18, in ing agents, electrostatic, hydrogen binding or hydrophobic
the absence of DNA (curve a), the copper(II) polymer has been interactions.
found to show a quasi-reversible redox process corresponding to
Cu(II)/Cu(I) with the cathodic (Epc) and anodic peak potential 7. Abbreviations
(Epa) being 0.181 and 0.097 V (DEp = 0.084 V), respectively.
The formal potential (Eo0 ) was found to be 0.139 V. In the pres-
ence of DNA (curve b) with R = 10 (R = [DNA]/[complex]) the vol- A adenine
tammetric peak currents decreased, suggesting that there exists T thymine
an interaction between the copper(II) polymer and HS-DNA. The G guanine
peak-to-peak separation becomes larger with DEp = 0.087 V, indi- C cytosine
cated that the electron-transfer process seems to become less FEBS Federation of European Biochemical Societies
reversible in the presence of the DNA. The ratio of the constants TMP 3,4,7,8-tetramethyl phenanthroline
for binding the Cu(I) and Cu(II) ions to HS-DNA is estimated to byp bipyridine
be 0.9–1, indicating that both Cu(I) and Cu(II) interact with HS- DPPZ dipyridophenazine
DNA almost to the same extent. Since the complex has no charged PHEHAT 1,10-phenanthrolino[5,6-b]-1,4,5,8,9,
groups and aromatic rings, therefore, it may bind to HS-DNA in a 12-hexaazatriphenylene
groove-binding mode [72]. EB ethidium bromide
CT-DNA calf thymus DNA
6. Conclusions HS-DNA herring sperm DNA
Tris tris(hydroxymethyl)aminomethane
The review article has focused on drug–DNA interactions and MLCT metal-to-ligand charge transfer
their study by various techniques like UV–Visible, fluorescence GMB gemcitabine hydrochloride
spectroscopies and cyclic voltammetry. These techniques are used Pyimpy (2-((2-phenyl-2-(pyridin-2-
to evaluate the binding mode as well as binding strength of the l)hydazono)methyl)pyridine)
complex formed between Drug and DNA. Basically small ligand Hnap naproxen
molecules (drug) interact with DNA via two different ways: cova- Nadicl sodium diclofenac
lent and non-covalent modes. Covalent binders act as alkylating NFC 4-nitrophenylferrocene
agents as they alkylate the nucleotides of DNA. The non-covalent dmaeoxd N,N0 -bis[2-(dimethylamino)ethyl]oxamide
binders interact with via three different ways: intercalation, ox oxalate
groove binding and external binding (on the outside of the helix).
18 M. Sirajuddin et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 1–19

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Acknowledgment polypyridyl DNA intercalators: structure and electronic absorption
spectroscopy of [Ru(phen)2(dppz)]2+ and [Ru(tap)2(dppz)]2+ complexes
The author is acknowledged the HEC Islamabad, Pakistan for intercalated in guanine–cytosine base pairs, J. Inorg. Biochem. 104 (2010)
893–901.
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