J Fluoresc (2017) 27:2119–2130
DOI 10.1007/s10895-017-2151-x
ORIGINAL ARTICLE
Analytical Techniques Used to Detect DNA Binding Modes
of Ruthenium(II) Complexes with Extended Phenanthroline Ring
C. Shobha Devi1 · B. Thulasiram2 · S. Satyanarayana3 · Penumaka Nagababu2,4
Received: 11 April 2017 / Accepted: 31 July 2017 / Published online: 22 August 2017
© Springer Science+Business Media, LLC 2017
Abstract  This review describes the analytical techniques
used to detect DNA-probes such as Ru(II) complexes with
hetero cyclic imidazo phenanthroline (IP) ligands. Studies
on drug-DNA interactions are useful biochemical techniques
for visualization of DNA both in vitro and in vivo. The inter-
actions of small molecules that binds to DNA are mainly
classified into two major classes, one involving covalent
binding and another non-covalent binding. Covalent bind-
ing in DNA can be irreversible and may leads to inhibition
of all DNA processes which subsequently leads to cell death.
Usually, covalent interactions leads to permanent changes in
the structure of nucleic acids. The non-covalent interaction
of molecules with DNA can be due to electrostatic interac-
tion, intercalation and groove binding. These interactions of
DNA probes can be explored by various spectroscopic tech-
niques viz. UV–visible, emission, emission quenching spec-
troscopy, viscosity and thermal denaturation measurements.
Keywords  DNA-binding · Intercalation · Emission
studies · Ruthenium(II) complexes · Analytical techniques
C.S. Devi and Penumaka Nagababu contributed equally.
* Penumaka Nagababu
babupenumaka@gmail.com
1
Department of Chemistry, RGUKT, Basar, Telangana State,
India
2
Inorganic & Physical Chemistry Division, CSIR-Indian
Institute of Chemical Technology, Uppal Road, Tarnaka,
Hyderabad 500007, Telangana State, India
3
Department of Chemistry, Osmania University, Tarnaka,
Hyderabad, Telangana State, India
4
CSIR-NEERI Zonal Laboratory, I‑8, Sector C, East Kolkata,
Area Development Project, P.O. East Kolkata, Township,
Kolkata 700 107, India
 
Introduction
Heterocyclic systems are one of the most important bioactive
molecules found in nature. The compounds containing mainly
nitrogen, oxygen and sulphur atom constitute a large class of
compounds used in biological and medicinal applications [1].
An enormous number of heterocyclic systems that include
mainly 6- and 5- membered ring compounds represent a dif-
ferent group of molecular support. Several such heterocyclic
skeletons have been magnificently incorporated into many novel
drug leads and therapeutic agents [2–4]. Examples that illustrate
the biological importance and therapeutic utility of some het-
erocyclic compounds and its substitutions includes improved
1, 10, phenanathroline compounds like imidazo phenanthroline
(IP) ligands [5]. Bioinorganic chemistry (or) Medicinal inor-
ganic chemistry is rapidly increasing in significance in both
therapeutic and diagnostic medicine [6]. In the last few decades,
ruthenium(II) polypyridyl complexes with imidazo phenanathro-
line (IP) ligands have been developed as DNA structural probes
(Scheme 1) [7]. In this review we are describing octahedral metal
complexes containing an intercalating ligand (IP) which binds to
DNA three dimensionally. However, in-order to tune the DNA
binding ability ­(Kb) of the complexes, its ancillary ligands can
be modified or functionalized. A ligand with a small aromatic
or hetero aromatic ring can bind to the DNA in the double helix
through intercalating mode, thereby distorting the DNA back-
bone conformation and interfering the interactions of DNA–pro-
tein. Ruthenium(II) complexes are most effective sensitizers due
to their strong charge transfer absorption in the visible region.
Due to the interesting photo physical properties, chemical stabil-
ity and biological properties of ruthenium complexes, much of
research has been focused on these complexes. More recently,
attention has been paid to the use of these complexes as chemo-
therapeutic agents for cancer [8], DNA conformation probes and
DNA cleavage agents [9–12]. When the chemical compound
13
Vol.:(0123456789)

2120
Scheme 1  Structures of imidazo phenanthroline (IP) ligands as well as metal complexes
interacts with DNA, some modifications are experienced in mol-
ecule as well as in DNA. Detection and explanation of these
modifications make a great challenge for analytical techniques.
The aim of the modern research is to understand the specific
binding mode of drug-DNA complex and to find drug binding
sites, as well as conformational changes observed due to the
drug-DNA interaction. In this mini-review, we are mainly focus-
ing on the current advancements in the field of bio-analytical
methods using ruthenium polypyridine complexes with extended
phenantroline ring system. Our main focus is how ruthenium(II)
polypyridyl complexes are being used as DNA targeting agents
13
J Fluoresc (2017) 27:2119–2130
and the various analytical methods that can be used to determine
intercalative binding mode of ruthenium(II) complexes having
IP based ligands.
Deoxyribonucleic Acid (DNA)
DNA (deoxyribonucleic acid) is made up of molecules
called nucleotides and an important complex molecule
that carries the genetic code. Each nucleotide holds a
sugar group, a phosphate group, and a nitrogen base like;
adenine (A), thymine (T), guanine (G) and cytosine (C).
J Fluoresc (2017) 27:2119–2130 2121
The sides of the ladder as strands of alternating phosphate
and sugar groups-strands those run in conflicting direc-
tions. Each “String” of the ladder is made up of two base
pairs coupled together by hydrogen bonds due to its spe-
cific nature of chemical pairing, adenine always couple
with thymine, and cytosine always couple with guanine
(Fig. 1). Within this composition, one ladder mirrors to the
other as a result of the anti-aligned direction of the sugar-
phosphate backbones and also the complementary nature
of the adenine–thymine and cytosine-guanine base pair-
ing. The distances between the any two nitrogenous base
pairs are nearly equal, so that the distance across the DNA
double helix is similar. DNA double helix that can bind
with the small molecules having negatively charged phos-
phate backbone is called electrostatic binding, The sites
present in the major and minor grooves which are with
hydrogen donating and accepting groups is called hydro-
gen bonding, the aromatic hydrophobic components can
interact with the phosphate oxygen atoms is called van-
der Waals interactions. In generally the cations molecules
can interact with electron-rich sites on DNA double helix
such as N-donor ligands is called covalent binding and
guanine has the highest electron density of all other four
nitrogenous base pairs. It is accepted that this interaction
is answerable of cisplatin effect on cancer cells [9–12].
Mode of Drug‑DNA Interactions
Studies on Drug-DNA interactions are useful biochemical
tools for visualization and analysis of molecular networks
of DNA both inside and outside the cell [13]. Typically the
interactions of small molecules to DNA are characterized
into two major classes.
Fig. 1  Structure of double
stranded DNA exists of two
nucleotide chains whose
nitrogen base pairs are linked by
hydrogen bonds. (Copied from
http://www.SciLinks.org, web
code: cbn-4121)
Covalent Binding
Covalent Transition metal complexes can bind covalently to
DNA. They can occur within each linear strand and strongly
bond the sugars, bases, and phosphate groups [14]. It is
irreversibly bound DNA that leads to complete inhibition
of DNA processes and subsequent cell death. Chloraum-
bucil is a bi-functional alkylating agent of DNA [15] and
pyrrolo-(1,4)-benzodiazepines interacts with DNA via the
covalent bond formation [16]. One among the early exam-
ples of drugs that interacted covalently with DNA is the
well-known anticancer agent, Cisplatin [cis-diammine
dichloro platinum (II)]. The inhibition of cell replication is
brought out by the coordination of DNA on N7 and O6 sites
of guanine (Fig. 2) by means of the two labile chlorides of
the molecule. The distance between the two chloride atoms
(3.4 ­Ao) is the key factor in the coordination of this drug
with DNA.
Non‑covalent Interaction
The non-covalent mode of binding of small molecules-
DNA can be classified into three major categories
(Scheme 2):
a) Electrostatic interaction: Due to DNA double helix con-
figuration, negatively charged phosphate groups that are
found in the periphery, attract metal cation groups by
coulombic forces. These types of interactions are called
electrostatic interactions.
b) Intercalation: Intercalation occurs when a planar aro-
matic chromophore glide between two adjacent DNA
base pairs [17]. There are many proved metal polyp-
iridyl complexes are there to bind intercalative with
13

2122
Fig. 2  Structures of Guanine
(a), Cisplatin (b) and covalent
binding of Cisplatin (c)
DNA base pairs, which results in increasing in length
of DNA. DNA intercalators are widely used in anti-
neoplastic, antitumor, antibiotic, antimalarial and
antifungal agents, however, that does not mean that all
intercalators have genotoxicity (defined by the abil-
ity to alter the genetic material) in cells as a means of
inducing a toxic effect [17]. Intercalation changes the
base pair spacing from 3.4 to 6.8 Å and persuade local
structural changes to the DNA such as helix unwind-
ing and B-form to A-form transitions. These changes
can alter DNA-based processes. Intercalators such as
ethidium bromide and thiazole orange are known potent
mutagens. Similarly, due to their tight association with
DNA, many intercalators have clinical efficacy and
have been used as chemotherapeutic treatments for
DNA replication in growing cancer cells.
Several intercalators such as ethidium bromide and
thiazole orange (Fig. 3) are used as non-specific inter-
calating agents that display enhanced fluorescence upon
Scheme 2  Schematic rep-
resentation showing various
techniques to investigate DNA
binding of metal complexes
13
J Fluoresc (2017) 27:2119–2130
DNA binding. The intense fluorescence of these agents
upon DNA binding derives from electronic perturbations
in the ligand and result from the release of fluorescence
quenching water as well as the stabilization of overlap-
ping π-systems. The application of fluorescent intercala-
tors to examine the DNA binding characteristics of other
molecules is particularly useful [18]. In addition, ethidium
bromide is employed to visualize the presence or the loca-
tion of DNA fragments in agarose gel electrophoresis [19].
c) Groove binding: There are mainly two grooves in the
DNA double helix, which are called major groove and
minor groove. The minor groove occurs where they
are close together and the major groove occurs where
the backbones are far apart. Generally groove binding
drugs like distamycin, Hoescht 33258 are most often
favour to bind the minor groove of DNA double helix
[20]. As we know that all intercalative or groove bind-
ing drugs can be used for treatment of cancer, tumor
and infectious diseases induced by microbial organisms
J Fluoresc (2017) 27:2119–2130 2123
Fig. 3  Structures of ethidium bromide (1) and thiazole orange (2)
(both are proven Intercalators)
[21]. For instance, the anticancer drug mitomycin is
a typical groove binder and a class of clinically most
important compound anthracyclines with anti-bacterial
and anti-neoplastic properties, take advantage of both
modes of binding as a groove-binding as well as an
intercalative of DNA [22].
DNA Binding Studies of Metal Complexes
with Imidazo Phenanthroline (IP) Ligands
Over the past few years significant progress has been made
to evaluate transition metal complexes as structural probes
of DNA. The number of analytical techniques developed to
evidence metal complex-DNA interactions. Various physi-
cal, biochemical methodologies are employed to detect the
DNA interactions [23–26]. The classical UV–vis spectro-
photometry, fluorescence spectroscopy, viscosity and/or
thermal melting temperature studies have been extensively
reported for the detection of DNA interactions of different
metal complexes.
Absorption Spectroscopy
Absorption spectroscopy method is one of the much useful
for investigation on interplay of ruthenium (II) complexes
with CT-DNA. By utilizing absorption spectroscopy tech-
nique in DNA binding analysis with ruthenium (II) com-
plexes has been studied by various research groups [27, 28].
Generally transition metal ions are coloured in solutions due
d-orbitals of ions split in the higher and lower energy lev-
els, when d-orbitals of the ruthenium (II) complexes interact
with the negatively charged complexing agents (ligands) in
such a way they become non-degenerate. The colour of solu-
tions is affected by certain ligands which are coordinated
with coordination centre. For instance, imidazophenanthro-
line (IP) ligands, due to their extended conjugation increase
the absorption of metal complexes in visible region. In
Ru(II) polypyridyl complexes a metal and a ligand permit to
charge transfer band which is observed in the region between
420 and 490 nm [8–12, 29–33]. The π-π* transitions of the
IP ligands in metal complexes present in UV region i.e.
below 400 nm as shown in Fig. 4 (Table 1).
Bathochromism (red shift) as well as hypochromism were
observed when a polypyridyl complex with planar aromatic
ligands bound to DNA through intercalation mode and
strong π-π* stacking interaction between base pairs of the
DNA and the planar aromatic ligands occurred [34]. It is
generally agreed that the extent of the hypochromic shift in
the UV–Visible spectrum is consistent with the resistance of
intercalative interaction [35]. The mixture of ruthenium and
DNA solutions were allowed to stand for 2 to 3 min before
recording the spectra. Same titrations should be repeated
until there was no change is observed in the absorption
spectrum. In order to compare quantitatively the binding
strength ­(Kb) of the Ru-DNA were calculated by observ-
ing the changes in spectra at their respective metal–ligand
charge transfer (MLCT) with increasing DNA stock solu-
tion. Generally the following equation is used to calculate
the ­Kb [36].
/( ) /( ) /( ( ))
[DNA] εa − εf = [DNA] εb − εf + 1 Kb εb − εf ,
On plotting [DNA]/(Ɛa–Ɛf) vs [DNA], the binding con-
stant ­(Kb) is obtained by the ratio of the slope to the intercept
(Fig. 4). The concentration of DNA will be given as [DNA],
Ɛa, Ɛf and Ɛb corresponds to the apparent absorption coef-
ficient ­Aobsd/[Ru complex], the extinction coefficient for the
free complex, and the extinction coefficient for the complex
in the fully bound form respectively.
The Fig.  4 shows the absor ption spectra of
[Ru(phen)2ptip]2+ complex in the presence and absence of
DNA stock. Complex alone in aqueous solution showed
very low-energy MLCT absorption at wavelength 470 and
620 nm. On increasing the DNA, spectral changes showed
great hypochromism (Fig. 4) which suggested that the com-
plex was strongly bound to DNA, due to stacking interaction
between the aromatic intercalative ligand (IP ligand) and the
DNA base pairs.
Emission Spectroscopy
Fluorescence spectroscopy is most useful method to
study the ruthenium(II) complex-DNA binding interac-
tions. Complexes can strongly bind with DNA and be
guarded by DNA efficiently, since the hydrophobic envi-
ronment inside the DNA helix reduces the intelligibility
of solvent water molecules to the complex and their by
restricting the mobility of the complex at the binding site,
which results in increasing the intensity of fluorescence.
Emission quenching experiments of Ru(II) complexes are
usually performed using benchmark quencher like potas-
sium iodide and ferrocyanide to detect the binding of the
complexes with DNA. Further, the quenching constant
13

2124 J Fluoresc (2017) 27:2119–2130

Fig. 4  Absorption spectra of
complexes [Ru(phen)2ptip]2+ in
Tris–HCl buffer upon addition
of CT-DNA. Arrow shows the
hypsochromic and bathochro-
mic shift upon increase the
DNA concentration. Plots of
[DNA]/ (εa-εf) vs [DNA] for the
titration of DNA with Ru(II)
complexes

­(Ksv) can be calculated by using Stern–Volmer relation-
ship [37]. Ruthenium complexes with polypyridyl ligands
also possess a fascinating phenomenon called the light-
switch effect. When the polypyridyl ligand (IP) is inter-
calated to DNA, embedded in lipid membranes or in an
organic environment, the complex is strongly luminescent
but in aqueous solution the emission is totally quenched
[29–31].
This quality of the complexes makes them interest-
ing probes to study cells by using fluorescence micros-
copy. Emission spectral experiments were conducted at
ambient conditions to find the binding constant between
the ruthenium complexes and DNA. Emission titrations
were performed similar to the absorption titrations. To
the fixed concentrations of Ru(II) complex aliquots of
DNA were added until there is no change in the intensity
of spectrum. As shown in Fig. 5 with increase in concen-
tration of DNA, the intensities of [Ru(en) 2pyip]2+ com-
plex increased by around 2–10 times. The fluorescence
binding constants are calculated using the equation [38]
and substitution the data into the Scatchard plot of r vs r/
Cf. Where ‘r’ is the binding ratio C
­ b/[DNA] and C­ f is the
free complex. The affinity of binding for this complex
was reported as 5.02 × 10 4 ­M − 1. The emission binding
constants for various Ru(II) metal complexes is shown
in Table 1.
Fluorescence Quenching
Decrease in fluorescence intensity is called fluores-
cence quenching. It is very useful technique to evalu-
ate DNA binding affinity if a fluorophore (fluorescent
chemical compound that can re-emit light upon light
excitation) undergoes a change in an environment in the
binding process that is reflected by change intensity in
fluorescence. The molecular interactions between metal
complexes and DNA can lead to reduction of the fluo-
rescence intensity of fluorophore. This experiment is

Table 1  Results of absorption Complexes Absorbance λmax (nm) Kb ­(M− 1) from Kb ­(M− 1) from emission
and emission binding constants absorbance and references
­(Kb) for Ru(II) complexes
[Ru(phen)2(PtIP)]2+ 452,350, 275 7.01 × 105 6.10 × 105 [30]
[Ru(phen)2(IPPIP)]2+ 298, 366, 446 2.1 ± 0.2 × 105 [31]
[Ru(bpy)2(IPPIP)]2+ 256, 288, 446 8.5 ± 0.2 × 104 [31]
[Ru(bpy)2(PtIP)]2+ 451,342, 280 3.87 × 105 4.02 × 105
[30, 31]
[Ru(en)2(AIP)]2+ 437, 212 2.5 × 105 [32]
[Ru(en)2(PYIP)]2+ 452, 257 1.3 × 105 [32]
[Ru(en)2IP]2+ 277, 457 4.8 ± 0.2 × 104 [33]
[Ru(en)2PIP]2+ 257, 467 5.7 ± 0.1 × 104 [33]

13
J Fluoresc (2017) 27:2119–2130 2125

Fig. 5  Emission spectra of
[Ru(en)2pyip]2+ [32, 33] with
addition of DNA, well separated
band at 600 nm Scatchard plot
was drawn with r/Cf vs r, where
r is the ­Cb/[DNA] and C
­ f is the
concentration of free complex

usually performed using [Fe(CN)6]4− as quencher which DNA Photocleavage

further supports the above proposal in emission experi-
ment. Stern–Volmer plots of various complexes reported Electrophoresis through agarose is the standard method
indicates that ruthenium(II) complex alone (absence used to separate, identify or purify DNA fragments [26,
of DNA) efficiently quenched by [Fe(CN) 6] 4−. On the 42]. The technique is simple, rapid to perform and capable
other hand solution with complex + DNA (presence of resolving fragments of DNA that cannot be separated by
of DNA) quenching was less, because [Fe(CN) 6 ] 4− is other procedures. In general, electrophoretic separation is
highly negatively charged molecule it can repelled less carried out on agarose gels for DNA samples. Using this
negatively charged DNA phosphate backbone and would technique, bands containing as little 1–50 μg of DNA can
inhibit the quenching of the emission intensity of the also be detected by direct examination of the agarose gel
DNA bound complex. This may also explain by the fac-
tor that the bound metal cations of the ruthenium(II)
complexes protected from the anionic water-bound
[Fe(CN) 6 ] 4− (quencher) by DNA (negatively charged)
phosphate backbone hindering decreasing the emission
intensity of ruthenium(II) complexes [39, 40]. By using
Stern–Volmer equation the emission quenching constant
­K sv can be calculated [41]. The binding affinity can be
taken from measured slop.

I0 ∕I = 1 + Ksv [Q]

where I and I­ 0 are the fluorescence intensities in the

presence and absence of [Fe(CN)6]4− (quencher) respec-
tively, Q is the concentration of the [Fe(CN) 6] 4−, ­K sv is
a linear Stern–Volmer constant. In the quenching plot
of ­I 0/I versus [Q], K
­ sv is given by the slope. The ferro-
cyanide quenching plot for complex [Ru(phen) 2ptip] 2+
in the absence, presence and excess of DNA is shown
in Fig. 6 complex has shown linear Stern–Volmer plots. Fig. 6  Emission quenching of [Ru(phen)2ptip]2+ complex with
This confirms that the complex can bind to DNA more ­ 4[Fe(CN)6] in the absence (a) and presence (b) [Ru] = 20  mM and
K
strongly. excess of DNA (c)

13

2126
in the UV light [43, 44]. When an electric field is applied
across the gel, DNA, which is negatively charged at neu-
tral pH will migrates toward the anode. The interaction of
pBR322 DNA with ruthenium(II) polypyridyl complexes
was studied in order to determine the efficiencies of sensi-
tize DNA cleavage. This objective was achieved by moni-
toring the transition from the naturally occurring, cova-
lently closed circular form (Form I) to the nicked circular
relaxed form (Form II) by means of gel electrophoresis of
the plasmid. The cleavage of supercoiled DNA (Form I)
to the nicked circular DNA (Form II) was observed for all
the complexes regardless of different incubation periods
and different concentrations of the complexes. The results
of the experiments carried out in the concentration range
10–40 µM for all the three complexes after 1 and 2 h of
incubation. This study demonstrates that there is no sig-
nificant cleavage for controls. All complexes cleave DNA
and increase in concentration of the complex increases the
cleavage of the DNA (Fig. 7).
To Know Mode of Binding with DNA
Thermal Denaturation
The DNA mode of binding to metal complexes can moni-
tor with the thermal denaturation ­(Tm) method. When the
temperature of the (DNA + complex) solution increases
the double stranded DNA relatively separate in to single
strands, and intensity of absorption spectra is increases of
the DNA bases at around 260 nm [20]. As per previous lit-
erature reports [45, 46] the intercalation binding of a mol-
ecule (metal complexes) usually results in a considerable
increase of ­Tm. The duplex DNA structure can stabilize
when the metal complexes which can intercalate between the
base pairs of DNA. The hyperchromic effect in the absorp-
tion spectra of the DNA bases at 260 nm are recorded in
the temperature range 40 to 80 °C. The denaturation curves
are acquired by computing the absorbance at λmax 260 nm
Fig. 7  Photocleavage studies of
pBR322 DNA, in the absence
and presence of ruthenium(II)
complexes {[Ru(phen)2ptip]2+,
[Ru(bpy)2ptip]2+ and
[Ru(dmb)2ptip]2+} light after
30 min irradiation at 365 nm.
Lane 0 control untreated
plasmid DNA three sets of 4
lanes with 20, 40, 60 and 80 µM
respectively
13
J Fluoresc (2017) 27:2119–2130
for solutions of DNA with and without Ru(II) complex as a
function of temperature.
The thermal data are plotted as (A–A0)/(Af–A0) versus
thermal denaturation temperature, where A, A ­ 0, and ­Af are
the observed, initial, and the final absorbance at 260 nm,
respectively. This experiment can also be carried out with
DNA in the absence of metal complexes which reveals
that the Tm for the duplex is 60.6 ± 0.5 °C under same
experimental conditions. As shown in Fig. 8 the observed
change melting temperatures (∆T m) in the presence of
ruthenium(II) complexes including [Ru(phen) 2pyip] 2+
and [Ru(phen) 2aip] 2+ are ∼ 5–10  °C, respectively. The
large change in T ­ m suggests that the intercalative binding
affinities of ruthenium (II) complexes with DNA were very
strong [47].
Viscosity Measurements
Viscosity is a measure of a fluid’s resistance to flow. It
defines the internal friction of a moving liquid. A liq-
uid with little viscosity flows easily because its molecu-
lar makeup results in very slight friction when it is in
motion. A liquid with large viscosity resists motion since
its molecular makeup gives it a lot of internal friction.
In this study the viscosity measurements were carried
out for further clarification of the interaction between
the ruthenium complexes with DNA. As per Kelly et al.,
if increase of DNA length in between base pairs accom-
modate at intercalation locations, hence it will increase
the viscosity of DNA solution, which are properties of
intercalation binding mode of complexes. By contrast,
complexes that binds groove modes in the DNA by par-
tial/or non-classical intercalation typically cause less
or no change in DNA viscosity [48–50]. From the lit-
erature it is evident that with proven intercalative DNA
binder ethidium bromide under appropriate conditions
causes a significant increase in the DNA length. In con-
trast, a partial intercalation ligand could bend the DNA
helix, decreases its length and concurrently its viscosity.
J Fluoresc (2017) 27:2119–2130 2127

5 viscosity of DNA alone [5, 51]. A ligand that binds in

a the DNA groove causes a less pronounced change or no
b change in the viscosity of a DNA solution. The effect in
4 c presence of relative viscosity are calculated for a com-
plex to DNA ratio between 0.0 and 0.15 adopting the
d
method described by Cohen and Eisenberg [52]. Vis-
Relative OD

3 e cosity measurements can be carried out on calf thymus

DNA by varying the concentration of the added com-
plex. Effect of increasing amount of ethidium bromide
2
(A), Δ-[Ru(phen)3]2+ (E), Λ-[Ru(phen) 3]2+ (B), and rac-
[Ru(phen) 3] 2+ (D) and Hoechst 33,258 (C) on relative
viscosity of CT-DNA are shown in Fig. 9.
1
40 45 50 55 60 65 70 75 80 Ethidium Bromide Displacement Studies
Temperature 0C

Since 1950s the Ethidium bromide (EtBr) is used in medi-

Fig. 8  Plots of Relative O.D vs temperature of CT DNA (90  µm)
and complexes, DNA alone (a), DNA + complex [Ru(phen)2pyip]2+
(b), DNA + complex [Ru(phen)2aip]2+ (c), DNA + complex
[Ru(bpy)2pyip]2+ (d), and DNA + complex [Ru(bpy)2aip]2+ (e)
Viscosity experiments data will be presented as (η/ηo)1/3
vs the concentration of [Ru(II)]/[DNA] where η is the
viscosity of DNA in presence of complexes and ηo is the
cine to treat trypanosomiasis in veterinary for cattle a dis-
ease caused by trypanosomes [53]. It is very commonly used
as nucleic acid fluorescent tag in various laboratory tech-
niques. Due to its unique structure, it can easily intercalate
into DNA. The reason for its potent fluorescence after bind-
ing with DNA is the hydrophobic environment found in the
DNA base pairs [54]. Ethidium Bromide (EtBr) is a usually
used dye for DNA and RNA detection. It has characteristic

Fig.  9  a Effect of increasing amount of proven intercalator eth- (d). b Effect of increasing amount of complexes on the relative vis-
idium bromide (a), proven groove binder Hoechst 33,258 (c) and for cosities of CT-DNA increases at room temperature and models for
Δ-[Ru(phen)3]2+ (e), Λ-[Ru (phen)3]2+ (b), and rac-[Ru(phen)3]2+ intercalative binding of complexes with DNA at room temperature

13

2128
Fig. 10  Fluorescence intensity change on addition of ruthenium(II)
complexes (from Ref. [55]) in increasing amounts (0.8  µM in each
addition from 1 to 17  µM) to EtBr (9.1  µM)-CT-DNA (6.48  µM)
complex solution
UV absorbance maxima at 300 and 360 nm, in addition,
observation of the new excited peak at 260 nm radiation
when its reaction with nucleotides and re-emit at 590 nm
as yellow/orange light. Another useful strategy for compete
analyse was carried out for all complexes with constant of
DNA and EtBr concentrations with increasing ruthenium
complexes concentrations. As the complex concentration
increases the fluorescence of EtBr decreased (Fig. 10). This
may be ruthenium complexes compete with EtBr for the
DNA-binding sites thus displace the EtBr whose intensity
of fluorescence is increased upon DNA intercalative bind-
ing or it might be quenching on DNA. The results confirm
that all the ruthenium complexes bind robustly in intercala-
tive mode to DNA. The UV–vis spectral study on DNA-
intercalative binding of complexes to displacement of EtBr
from EtBr + DNA solution indicative of intercalative in
nature. However, the extensively studied DNA intercalative
agent, ethidium bromide has been used as a probe to identify
the unknown intercalative agent by displacement of bound
ethidium bromide. Results of ethidium bromide displace-
ment from DNA + ethidium bromide complex solution by
ruthenium(II) complexes illustrates the strong quenching
activity.
Conclusions
In this review we have discussed in detail about the tech-
niques which are useful to determine DNA-binding modes
of ruthenium (II) complexes with modified 1,10 phen-
anthroline lignads. We illustrated a few ruthenium (II)
13
J Fluoresc (2017) 27:2119–2130
polypyridyl complexes which were reported previously as
intercalators. Viscosity studies (Fig. 9) reveals that EtBr
as intercalator and Δ-[Ru(phen)3]2+, Λ-[Ru(phen)3]2+, and
rac-[Ru(phen)3]2+ are groove binders. However, when we
studied the extended ring system of phenanthroline such
as imidazo-phenanthroline(IP) ligands, they showed good
intercalation with DNA. This suggests that there is a bound-
ary between 1, 10-phenanthroline and imidazo-phenathro-
line (IP) ligands. Imidazo-phenathroline ligands having
moiety’s like phenyl called phenyl-imidazo-phenanthroline
(PIP) and substituted phenyls or extended PIP ligands (like:
AIP, PYIP and PTIP) are good intercalators as well as strong
DNA binders. It was further examined by DNA fragmenta-
tion assay to observe where DNA is cleaved into oligonu-
cleosomal fragments.
AIP, 2-(9-Anthryl)-1H-imidazo[4,5-f][1,10] phenanthro-
line; bpy, 2, 2′, bipyridine; en, ethylenediamine; CT DNA,
Calf thymus DNA; EtBr, Ethidium bromide; IP, Imidazo-
phenantrholine; phen, 1, 10, Phenanathroline; PIP, [2-phe-
nylimidazo [4,5-f][1,10] phenanthroline]; PPIP, [2-(4′-phe-
noxy-phenyl) imidazo[4,5-f][1,10] phenanthroline]; ptip,
(2-(5-phenylthiophen-2-yl)-1H-imidazo[4,5f][1,10] phen-
anthroline; PyIP, 2-(1-pyrenyl)-1H-imidazo[4,5-f][1,10]
phenanthroline
Acknowledgements  This work was suppor ted by CSIR-
NEERI/KZC and IICT Hyderabad, KRC No.: CSIR-NEERI/
KRC/2017/MAR/KZL/1.
References
1. Young DW (1975) Heterocyclic chemistry. Longman, London
2. Kumar A, Kumar R (2011) A review on synthesis of Schiff’s bases
of 2-amino 4-phenyl thiazole. Int Res J Pharm 2: 11–12
3. Patel NB, Shaikh FM (2010) New 4-thiazolidinones of nicotinic
acid with 2-amino-6-methylbenzothiazole and their biological
activity. Sci Pharm 78: 753–66
4. Bala S, Kamboj S, Kumar A (2010) Heterocyclic 1, 3, 4-oxadia-
zole compounds with diverse biological activities: a comprehen-
sive review. J Pharm Res 3:2993–2997
5. Nagababu P, Barui AK, Thulasiram B, Devi CS, Satyanarayana
S, Patra CR, Sreedhar B (2015) Antiangiogenic activity of mono-
nuclear copper (II) polypyridyl complexes for the treatment of
cancers. J Med Chem 58:5226–5241
6. Storr T, Thompson KH, Orvig C (2006) Design of target-
ing ligands in medicinal inorganic chemistry. Chem Soc Rev
35:534–544
7. Incesu Z, Bljnkli K, Akalin G, Gundogdukaraburu N (2013) The
effects of some phenanthroline ruthenium (Ii) complexes on A549
cell proliferation. Turk J Pharm Sci 10:193–203
8. Devi CS, Satyanarayana S (2012) Review: synthesis, characteri-
zation, and DNA-binding properties of Ru (II) molecular “light
switch” complexes. J Coord Chem 65:474–486
9. Devi CS, Nagababu P, Natarajan S, Deepika N, Reddy PV,
Veerababu N, Singh SS, Satyanarayana S (2014) Cellular uptake,
cytotoxicity, apoptosis and DNA-binding investigations of Ru (II)
complexes. Eur J Med Chem 72:160–169
J Fluoresc (2017) 27:2119–2130 2129
10. Berners-Price SJ, Appleton TG, Kelland LR (2000) The chemistry of
Cisplatin in aqueous solution. In: Kelland LR, Farrell NP (eds) Plati-
num-based drugs in cancer therapy. Humana Press, Totowa, pp 3–35
11. Chaires JB (1998) Drug-DNA interactions. Curr Opin Struct Biol
8:314–320
12. Jakupec MA, Galanski M, Keppler BK (2003) Tumour-inhibiting
platinum complexes-state of the art and future perspectives. Rev
Physiol Biochem Pharmacol 146:1–53
13. Schmidt CE, Möller J, Hesslau U, Bauer M, Gabbert T, Kremer B
(2005) Universitätskliniken im Spannungsfeld des Krankenhaus-
marktes. Anaesthesist 54:694–702
14. Thulasiram B, Kumar YP, Aerva RR, Satyanarayana S, Nagababu
P (2017) Correlation between molecular modelling and spectro-
scopic techniques in investigation with DNA binding interaction
of ruthenium (ii) complexes. J Fluoresc 27:587–594
15. Mattes WB, Hartley JA, Kohn KW (1986) DNA sequence selec-
tivity of guanine–N7 alkylation by nitrogen mustards. Nucleic
Acid Res 14:2971–2987
16. Hurley LH, Petrusek R (1979) Proposed structure of the anthramy-
cin–DNA adduct. Nature 282:529–531
17. Lerman LS (1961) Structural considerations in the interaction of
DNA and acridines. J Mol Biol 3:18IN13–30IN14
18. Boger DL, Winston CT (2001) Thiazole orange as the fluores-
cent intercalator in a high resolution fid assay for determining
DNA binding affinity and sequence selectivity of small molecules.
Bioorg Med Chem 9:2511–2518
19. Tse WC, Boger DL (2004) A fluorescent intercalator displacement
assay for establishing DNA binding selectivity and affinity. Acc
Chem Res 37:61–69
20. Waring MJ (1965) Complex formation between ethidium bromide
and nucleic acids. J Mol Biol 13:269–282
21. Crawford LV, Waring MJ (1967) Supercoiling of polyoma virus
DNA measured by its interaction with ethidium bromide. J Mol
Biol 25:23–30
22. Takenaka S, Takagi M (1999) Threading intercalators as a new
DNA structural probe. Bull Chem Soc Jpn 72:327–337
23. Wilson K, Walker J (2010) Principles and techniques of bio-
chemistry and molecular biology. Cambridge University Press,
Cambridge
24. Spiro TG (1980) Nucleic acid-metal ion interactions. Krieger Pub Co
25. Long EC, Barton JK (1990) On demonstrating DNA intercalation.
Acc Chem Res 23:271–273
26. Joseph S, David WR (2001) Molecular cloning: a laboratory
manual. Gold Spring Harbor, New York
27. Morgan RJ, Chatterjee S, Baker AD, Strekas TC (1991) Effects
of ligand planarity and peripheral charge on intercalative binding
of Ru (2, 2′-bipyridine) 2L2 + to calf thymus DNA. Inorg Chem
30:2687–2692
28. Tysoe SA, Morgan RJ, Baker AD, Strekas TC (1993) Spectro-
scopic investigation of differential binding modes of ∆-and
Λ-Ru (bpy) 2 (ppz) 2 + with calf thymus DNA. J Phy Chem
97:1707–1711
29. Devi CS, Nagababu P, Shilpa M, Kumar YP, Reddy MR, Gabra
NM, Satyanarayana S (2012) Synthesis, characterization and
DNA-binding characteristics of Ru (II) molecular light switch
complexes. J Iran Chem Soc 9:671–680
30. Srishailam A, Kumar YP, Reddy PV, Nambigari N, Vuruputuri U,
Singh SS, Satyanarayana S (2014) Cellular uptake, cytotoxicity,
apoptosis, DNA-binding, photocleavage and molecular docking
studies of ruthenium (II) polypyridyl complexes. J Photochem
Photobiol B Biol 132: 111–23
31. Nagababu P, Shilpa M, Latha JN, Bhatnagar I, Srinivas PN,
Kumar YP, Reddy KL, Satyanarayana S (2011) Synthesis, charac-
terization, DNA binding properties, fluorescence studies and toxic
activity of cobalt (III) and ruthenium (II) polypyridyl complexes.
J Fluoresc 21:563–572
32. Nagababu PE, Shilpa MY, Mustafa MD, Ramjee P, Satyanaray-
ana S (2008) DNA-binding and photocleavage studies of ethyl-
enediamine cobalt (III) and ruthenium (II) mixed ligand com-
plexes. Inorg React Mech 6: 301–11
33. Nagababu P, Latha J, Satyanarayana S (2006) DNA-binding
studies of mixed-ligand (Ethylenediamine) ruthenium (II) com-
plexes. Chem Biodivers 3:1219–1229
34. Tan LF, Chao H, Li H, Liu YJ, sun B, Wei W, Ji LN (2005)
Synthesis, characterization, DNA-binding and photocleavage
studies of [Ru (bpy) 2 (PPIP)] 2 + and [Ru (phen) 2 (PPIP)] 2+.
J Inorg Biochem 99:513–520
35. Pyle AM, Rehmann JP, Meshoyrer R, Kumar CV, Turro NJ,
Barton JK (1989) Trans-dichlorobis(N-p-tolylpyridin-2-amine)
palladium(II): synthesis, structure, fluorescence features and
DNA binding. J Am Chem Soc 111:3051–3058
36. Wolfe A, Shimer GH Jr, Meehan T (1987) Polycyclic aromatic
hydrocarbons physically intercalate into duplex regions of dena-
tured DNA. BioChemistry 26:6392–6396
37. Joseph R, Lakowicz GW (1973) Quenching of fluorescence by
oxygen. Probe for structural fluctuations in macromolecules.
BioChemistry 12:4161–4170
38. McGhee JD, von Hippel PH (1974) Theoretical aspects of DNA-
protein interactions: co-operative and non-co-operative binding
of large ligands to a one-dimensional homogeneous lattice. J
Mol Biol 86:469–489
39. Kumar CV, Barton JK, Turro NJ (1985) Photophysics of ruthe-
nium complexes bound to double helical DNA. J Am Chem Soc
107:5518–5523
40. Barton JK, Goldberg JM, Kumar CV, Turro NJ (1986) Tris
(phenanthroline) ruthenium (II) enantiomers with nucleic. J
Am Chem Soc 108:2081–2088
41. Ghosh BK, Chakravorty A (1989) Electrochemical studies of
ruthenium compounds part I. Ligand oxidation levels. Coord
Chem Rev 95:239–294
42. Fisher MP, Dingman CW (1971) Role of molecular conforma-
tion in determining the electrophoretic properties of polynu-
cleotides in agarose-acrylamide composite gels. BioChemistry
10:1895–1899
43. Aaij C, Borst P (1972) The gel electrophoresis of DNA. Biochim
Biophys Acta 269:192–200
44. Sharp PA, Sugden B, Sambrook J (1973) Detection of two restric-
tion endonuclease activities in Haemophilus parainfluenzae using
analytical agarose-ethidium bromide electrophoresis. BioChem-
istry 12:3055–3063
45. Neyhart GA, Grover N, Smith SR, Kalsbeck WA, Fairley TA, Cory
M, Thorp HH (1993) Binding and kinetics studies of oxidation of
DNA by oxoruthenium (IV). J Am Chem Soc 115:4423–4428
46. Liu YJ, Chao H, Tan LF, Yuan YX, Wei W, Ji LN (2005) Interac-
tion of polypyridyl ruthenium (II) complex containing asymmetric
ligand with DNA. J Inorg Biochem 99:530–537
47. McGhee JD (1976) Theoretical calculations of the helix–coil
transition of DNA in the presence of large, cooperatively binding
ligands. Biopolymers 15:1345–1375
48. Satyanarayana S, Dabrowiak JC, Chaires JB (1992) Neither delta-
nor lambda-tris (phenanthroline) ruthenium (II) binds to DNA by
classical intercalation. BioChemistry 31:9319–9324
49. Barton JK, Danishefsky A, Goldberg J (1984) Tris (phenanthro-
line) ruthenium (II): stereoselectivity in binding to DNA. J Am
Chem Soc 106:2172–2176
50. Satyanarayana S, Dabrowiak JC, Chaires JB (1993) Tris (phenan-
throline) ruthenium (II) enantiomer interactions with DNA: mode
and specificity of binding. BioChemistry 32:2573–2584
51. Chaires JB, Dattagupta N, Crothers DM (1982) Self-association
of daunomycin. BioChemistry 21:3927–3932
52. Cohen G, Eisenberg H (1969) Viscosity and sedimentation study
of sonicated DNA–proflavine complexes. Biopolymers 8:45–55
13

2130 J Fluoresc (2017) 27:2119–2130

53. Stevenson P, Sones KR, Gicheru MM, Mwangi EK (1995) Com- 55. Kundu S, Maity S, Bhadra R, Ghosh P (2011) Trans-
parison of isometamidium chloride and homidium bromide as dichlorobis(N-p-tolylpyridin-2-amine)palladium(II): synthesis,
prophylactic drugs for trypanosomiasis in cattle at Nguruman, structure, fluorescence features and DNA binding. Indian J Chem
Kenya. Acta Trop 59:77–84 50: 1443–9
54. Olmsted J III, Kearns DR (1977) Mechanism of ethidium bromide
fluorescence enhancement on binding to nucleic acids. BioChem-
istry 16:3647–3654

13