Indian Journal of Chemistry
Vol. 49A, August 2010, pp. 1003-1015
Advances in Contemporary Research
Interaction of DNA with small molecules: Role of copper histidyl peptide
complexes in DNA binding and hydrolytic cleavage
Pulimamidi Rabindra Reddy* & Addla Shilpa
Department of Chemistry, Osmania University, Hyderabad 500 007, India
Email: [email protected]
Received 19 March 2010; revised and accepted 17 July 2010
Deoxyribonucleic acid is the site of storage and retrieval of genetic information through interaction with proteins and
other small molecules. Hydrolysis of the phosphodiester bond of DNA is of critical importance at several stages in a cell
cycle. Thus, the development of metal complexes that cleave nucleic acids hydrolytically at physiological conditions is of
great interest in the field of artificial metallonucleases. Among transition metals, Cu(II) complexes have been extensively
studied in promoting hydrolysis of DNA. Histidine is a biologically important ligand for Cu(II) binding in many biological
systems. Since imidazole is an efficient catalyst for ester hydrolysis at neutral pH and histidine residues are often involved in
neutral hydrolytic metalloenzyme active centres and have the potential to bind to DNA, an attempt is made in this review to
highlight the importance of DNA binding and cleavage and the role of a few copper histidyl peptide complexes in DNA
binding and hydrolytic cleavage.
Keywords: Bioinorganic chemistry, DNA cleavage, Copper, Hydrolysis, Metalloenzymes
IPC Code: Int. Cl.9 CO7F1/08
Prof. P. Rabindra Reddy is
Emeritus
Fellow
in
the
Department
of
Chemistry,
Osmania University, Hyderabad,
India. His research interest
includes interaction of metal ions
with biomolecules including
nucleotides,
peptides
etc.
assigning functional roles for
DNA and RNA. Presently his
research group is involved
in the development of new metallonucleases for efficient
cleavage of DNA. Prof. Reddy is the recipient of several
awards including British Council Award (1976), UGC Young
Scientist (1977), Indian Chemical Society (1984) and
Department of Biotechnology Overseas Award (1985). He
was a Visiting Scholar at Harvard University (1986-88) and
Visiting Professor at University of Utah, USA in 1992.
Ms. Addla Shilpa is a senior
research scholar at Department of
Chemistry, Osmania University,
Hyderabad, India. She is
pursuing her Ph.D degree under
the guidance of Prof. P. Rabindra
Reddy. Her research topic
includes design and synthesis
of
metallonucleases
and
investigating their DNA binding
and cleavage abilities.
The deoxyribonucleic acid (DNA) plays the key
informational role in the cell. The DNA in the nucleus
bearing the genetic blue print is frequently copied
and transcribed by DNA and RNA polymerases.
Yet DNA is wrapped up in chromatin structure and
must be manipulated by topoisomerases and helices
to allow its access and usage. Like proteins DNA has
primary, secondary and tertiary structures. Since
primary and secondary structures are well known
a brief mention of tertiary structure is made here.
The tertiary structure of DNA is important in view
of its interaction with several anticancer agents.
DNA is an extremely long molecule, so long in fact
that it would not fit into the nucleus of the cell if it
existed as a linear molecule. It has to be coiled into a
more compact three dimensional shape which can fit
into the nucleus – a process known as supercoiling.
This process requires the action of a family of
enzymes called topoisomerases. Supercoiling allows
the efficient storage of DNA, but the DNA has to be
uncoiled again if replication and transcription are to
take place. The same topoisomerase enzymes are
responsible for catalyzing this process, so inhibition
of these enzymes can efficiently block transcription
and replication1. Therefore, cleavage of supercoiled
(SC) DNA into nicked circular (NC) and linear forms
INDIAN J CHEM, SEC A, AUGUST 2010
1004
are necessary biological process. Mimicking such a
process in the laboratory is not only important but
also challenging to the scientists. Another important
property of DNA is its ability to interact reversibly
with a broad range of chemical species that include
water, metal ions and their complexes, small organic
molecules and proteins. All the intricate DNA
conformations which exist are stabilized and are only
possible because of these reversible interactions.
Because of their relative simplicity, the interactions
of small molecules with nucleic acids have
provided accurate information about nucleic acid
binding specificity. Incidentally, this (specificity)
differentiates between the two types of DNA cleavage
i.e. oxidative and hydrolytic. Hence it is important
to know the types of reversible interactions.
Molecules and ions interact with DNA in three
primary ways which are significantly different2
(Scheme 1).
(a) binding along the exterior of the helix through
interactions which are generally non-specific
and are primarily electrostatic in origin,
(b) groove binding interactions which involve
direct interactions of the bound molecule with
edges of base pairs in either of the major (G-C)
or minor (A-T) grooves of nucleic acids, and,
(c) intercalation of planar or approximately planar
aromatic ring system between base pairs.
The cleavage of nucleic acids may be considered as
an enzymatic reaction which comprises various
biological processes as well as the biotechnological
manipulation of genetic material. One of the most
important approaches to drug development and
Three primary binding modes with a double helical DNA
Scheme 1
current chemotherapy against some cancers and viral
and parasitic diseases involve drugs which interact
reversibly with DNA. Therefore, design of new metal
complexes which can bind with specificity to DNA
and bring about its (DNA) cleavage are of importance
in the development of new antitumor agents3,4.
In particular the development of reagents which
cleave nucleic acids hydrolytically under mild
conditions is attracting great interest in the field of
artificial metallonucleases5,6. Possessing higher
reactivity in the cleavage of DNA than other
transition metal complexes, Cu(II) complexes have
attracted the most interest. Many Cu(II) complexes
have been synthesized as artificial nucleases7-10.
A thorough literature survey reveals that much of
the attention has been focused on complexes of
copper and histidine/histidyl peptides11-13 due to their
coordinating abilities and their biological importance.
In view of the above an attempt has been made
to review the importance of DNA binding and
cleavage and compare the DNA binding and
hydrolytic cleavage activities of some ternary
copper complexes of histidyl peptides and phen/Hist/
En (where phen=phenanthroline, Hist=histamine,
En=ethylenediamine).
While several inorganic and organic systems that
mediate the hydrolysis of RNA or simple phosphate
monoesters have been developed, the hydrolysis of
the DNA backbone has been much more difficult to
achieve. This is due to the absence of 2'-OH group,
which when deprotonated, can assist in the cleavage
of phosphodiester bonds between ribonucleotides.
The half-life of the phosphodiester linkage in DNA at
pH 7.0 and 30 °C is estimated to be 200 million
years14. The exceptional stability of phosphodiester
backbone of DNA has been suggested as one reason
why nucleic acids have evolved as genetic material15.
However DNA cleavage is an essential phenomenon
in an organism.
Importance of DNA cleavage
Hydrolysis of phosphodiester bond of DNA is
of critical importance at several stages in a cell
cycle (including DNA repair excision, integration
and signal transduction). Hence, DNA strand
scission reactions are of considerable interest both
in understanding the ubiquitous phosphate ester
hydrolysis reactions carried out in nature and also
in designing new artificial restriction enzymes16.
Double strand breaks in duplex DNA are thought
REDDY & SHILPA : INTERACTION OF DNA WITH SMALL MOLECULES
to be more significant sources of cell lethality
than single strand breaks, as they appear to be less
readily repaired by DNA repair mechanisms17. Metal
ion mediated hydrolysis of phosphate esters by
metallonuclease enzymes is a common catalytic
pathway in nucleic acid biochemistry, and such
enzymes are ubiquitous and essential for living
organisms. Hence, there is a considerable interest
in the design, synthesis and characterization of
molecules that target RNA or DNA motifs, either
to inhibit the binding of cognate proteins or enzymes,
or to mediate strand scission18. DNA cleavage
agents find many potential applications in molecular
biology, biotechnology and medicine19-21, DNA
footprinting22, for locating base mismatches and loop
regions23, conformational variations in DNA24 and
as chemotherapeutic agents25.
The interaction of transition metal complexes
with nucleic acids is a major area of research due
to the utility of these complexes in the design and
development of synthetic restriction enzymes,
spectroscopic probes, site specific cleavers and
molecular photoswitches26. The reason for using
transition metal complexes as artificial nucleases is
because of their diverse structural features, and the
possibility to tune their redox potential through the
choice of proper ligands27,28. Inorganic constructs such
as cis platin and bimetallic rhodium acetate, etc., exert
antitumor activity by inner sphere coordination29.
There are some distinct advantages of chemical
nucleases over conventional enzymatic nucleases.
The former are smaller in size rendering quick
stretching in the hindered sites of a macromolecule30
and have a quick reach to sterically hindered
regions of DNA31. Hence, considerable attention has
been focused towards the development of DNA
cleavage agents7-10,32. Ce4+ complexes have been
reported to hydrolyze single and double strand
DNA33, several dinuclear complexes have been
reported to be very efficient in hydrolytic cleavage
of plasmid DNA34-36, while Cu(II) complexes of linear
or macrocyclic polyamines, aminoglycosides and
histidine show good nuclease activity. It has also been
demonstrated that many compounds of transition
metals are capable of promoting efficient hydrolytic
cleavage of DNA37-40.
The DNA cleavage can occur by two major
pathways, i.e., hydrolytic and oxidative:
(a) Hydrolytic DNA cleavage involves cleavage of
phosphodiester bond to generate fragments
1005
which can be subsequently religated.
Hydrolytic cleavage active species mimic
restriction enzymes.
(b) Oxidative DNA cleavage involves either
oxidation of the deoxyribose moiety by
abstraction of sugar hydrogen or oxidation of
nucleobases. The purine base guanine is most
susceptible for oxidation among the four
nucleobases41. Oxidative cleavage of DNA
occurs in the presence of additives or
photoinduced DNA cleavage agents42,43.
Photocleavers require the presence of a
photosensitizer that can be activated on
irradiation with UV or visible light. The redox
active ‘chemical nucleases’ are effective
cleavers of DNA in the presence of a reducing
agent or H2O2 as an additive44,45.
Oxidative cleavage agents require the addition
of an external agent (e.g. light or H2O2) to initiate
cleavage and are thus limited to in vitro applications.
Since these processes are radical based22,46 and deliver
products lacking 3' or 5' phosphate groups47 that
are not amenable to further enzymatic manipulation,
the use of these reagents has been limited in the
field of molecular biology and their full therapeutic
potential has not been realized. Hydrolytic cleavage
agents do not suffer from these drawbacks. They
do not require co-reactants and, therefore, may be
more useful in drug design. Also, they produce
fragments that may be religated enzymatically.
The metal complexes that catalyze DNA hydrolytic
cleavage may be useful not only in gene manipulation
but also in mimicking and elucidating the important
roles of metal ions in metalloenzyme catalysis48.
Metal ions in biology
Hydrolytic degradation of nucleic acids by
nuclease enzymes is a critical biological reaction
and metal ions play a central role in mediating
such cleavage pathways. Many hydrolytic enzymes
contain metal ions in their active sites42,49,50, which
have an important roles in enzymes and all ribosome
mediated scission reactions42,51,52. Small metal
complexes that promote the hydrolytic cleavage
of DNA, therefore, are useful not only in molecular
biology and drug design but also in elucidating
the precise role of metal ions in enzyme catalysis.
Hydrolysis of DNA by small molecules is
primarily hindered by the repulsive interaction of
negatively charged phosphate group towards an
incoming nucleophile. However, various transition
1006
INDIAN J CHEM, SEC A, AUGUST 2010
metal and lanthanide cations have been shown to
alleviate this repulsive interaction by direct inner
sphere binding of the phosphate ester. The redox
properties in metal centers are essential to generate
reactive oxygen species for DNA cleavage19,44. By
changing the metal ions, the geometry of the complex
(square planar, tetrahedral, octahedral, etc) and
consequently its photophysical properties, may be
modified and may affect its interaction with nucleic
acids53,54.
The key role of metal ions in promoting
hydrolysis of phosphate esters appears to be an
intramolecular delivery of nucleophile by reducing
the pKa of coordinated water molecules, resulting
in good nucleophiles at neutral pH55. Therefore,
the design of metal complexes as potential synthetic
hydrolytic agents requires:
(a) two cis oriented labile coordination sites in
order to bind both the phosphodiester
substrate and a water molecule in an
orientation appropriate for intramolecular
attack, and
(b) a strong lewis acid metal in order to facilitate
deprotonation of the coordinated water
molecule in order to generate the hydroxide
nucleophile43,55,56.
Role of copper in DNA cleavage
Copper, a natural constituent of the cell nuclei, has
been suggested to play a key role in structural
organization57 and function58 of chromosomes. It is
one of the most abundant transition metals found
in living systems, and is an integral component
of many enzymes, e.g., superoxide dismutase,
tyrosinase, ceruloplasminn, etc. A driving force in the
activity of these enzymes is the folding of a peptide
which consists of amino acid side chains around
the Cu(II) cation. It is involved in mixed ligand
complex formation in a number of biological
processes59.
The biological role of Cu(II) and its synergetic
activity with drugs have been the focus of a large
number of research studies60. The antifungal and
antibacterial properties of a range of Cu(II) complexes
have been evaluated against several pathogenic fungi
and bacteria61,62. Coordination compounds of copper,
in both its oxidation states (+1 and +2), have been
extensively used in metal mediated DNA cleavage
through the generation of hydrogen abstracting
activated oxygen species63,64. Cu(II) complexes under
certain ligand environments have also been shown
to bring about photocleavage of DNA as well as
RNA and DNA scission via phosphodiester
transesterification65,66. In recent years there has been
substantial interest in the design and study of lewis
acidic, redox potential and spectroscopically active
Cu(II)/Cu(I) complexes of synthetic and naturally
occurring ligands as nuclease mimics66,67.
Cu(II) complexes have been extensively studied in
the past decade in promoting hydrolysis of DNA or
model substrates68 because of its similar coordination
chemistry, superior lewis acidity to that of Zn(II)
which has been observed as cofactor in many
nucleases69, biologically accessible redox potential
and relatively high affinity for nucleobases70,71.
Sigman and co-workers72,73 have shown that the
cationic complex [CuI(phen)2]+ in the presence of
molecular oxygen and a reducing agent, acts as an
efficient nuclease by oxidative cleavage mechanism
with high preference for the double stranded DNA.
There are also reports of copper complexes cleaving
DNA hydrolytically74 and several other copper based
synthetic nucleases have also been reported75-77.
A wide range of ligand environments for Cu(II)
has been found to be effective in DNA and
phosphodiester hydrolysis. Some examples include
simple macrocycles78, amino acid residues68 and
complex polysachcharides79, etc. Many Cu(II)
complexes including mononuclear80,81, dinuclear82,83,
trinuclear84,85 and macromolecular catalytic systems86,87
have been synthesized as artificial nucleases.
Role of 1, 10 phenanthroline and histidine in
DNA cleavage
1,10 Phenanthroline (phen) is the parent molecule
of an important class of chelating agents. The
consequence of the planar nature of phen is its ability
to participate as either an intercalating or a groove
binding species with DNA and RNA. There has been
substantial interest in understanding the DNA binding
properties of transition metal polypyridyl complexes
in the hope of developing novel probes of nucleic
acid structures, DNA cleavage agents and antitumor
drugs88. Transition metal complexes of bipy, phen
or their modified variants are widely employed in
studies of DNA in view of their applications
in several research areas including bioinorganic and
biomedicinal chemistries89,90.
Copper complexes containing phen bases have
received considerable current interest in the nucleic
acids chemistry due to their various applications
REDDY & SHILPA : INTERACTION OF DNA WITH SMALL MOLECULES
following the discovery of the “chemical nuclease”
activity of bis-phen complex of Cu(I) in presence of
H2O2 and a reducing agent by Sigman and
coworkers72,73,91. Subsequent studies have shown that
molar ratio of 1:2 for Cu and phen is necessary to
observe efficient DNA cleavage activity92. Yang and
coworkers81 have reported that the mono-(phen)Cu
complex in the presence of H2O2, exhibits higher
nuclease activity than bis-(phen)Cu species. However
mono-phen Cu(II) complexes cleave SC DNA less
effectively in presence of a reducing agent.
Copper complexes containing polypyridine
ligands and their derivatives exhibit numerous
biological activities such as antitumor93, anticandida94,
antimicrobacterial95 and antimicrobial96 activities, etc.
Cu(II) complexes of phen have been used to inhibit
DNA and RNA polymerase activities, to induce
strand scission of DNA in the presence of H2O2
or thiol97 and also in DNA footprinting experiments,
which are important for the detailed study of
DNA-protein interactions44. The substitution of phen
in complexes has been shown to influence their
reactivity with DNA98.
Among the amino acids, L-histidine (His) is one of
the strongest metal coordinating ligands and plays an
important role in metal-protein binding99. L-His has
three potential metal-binding sites, namely, the
carboxylate oxygen (Ocarboxyl), the imidazole imido
nitrogen (Nim) and the amino nitrogen (Nam). The pKa
values are 1.8, 6.0, and 9.18 respectively100. L-His can
bind as a mono-, bi-, and tridentate ligand and its
mode of coordination implicitly depends on the pH of
the solution.
A driving force in the activity of galactose oxidase
and superoxide dismutase is the folding of peptide
which consists of amino acid side chains around the
Cu(II) cation. His moieties are known to be of major
importance in the process because this type of
N-coordinating ligand forms stable complexes with
the copper cation. To unravel the formation processes
of these types of enzymes and to find the exact
coordination geometry of the Cu/His moieties,
extensive research has been carried out in this
area much of which has been summarized by
Sarkar et al11,12. The coordination ability of His
containing peptides towards Cu(II) ion has been
discussed by Kozlowski et al13.
His residues are important binding sites for Ni(II).
In human serum albumin, the binding site for nickel
is the N-terminal Asp-Ala-His. Serum albumin is
1007
believed to be the primary nickel transport protein in
human blood. Additionally, Ni(II) complexes with
bound histidine nitrogens are present in, at least, one
nickel containing enzyme. Enzyme superoxide
dismutase contains histidyl nitrogen coordinated to
the axial position of nickel Ion101.
Due to the presence of an imidazole ring, histidine
exhibits metal binding and DNA binding activities
different from those of other amino acids102. It has
been reported that Cu(II)-L-His complex aggregates
in a groove or along a phosphodiester chain of the
double helical DNA102 and shows hydrolytic cleavage
activity of plasmid DNA and a dinucleotide at
physiological pH and temperature68. His also shows
specific interaction with DNA as a protein residue103.
Peptides containing histidine residues at the third
position from the N-terminus are well characterized
specific sequences for metal binding in several
proteins104. Ni(II) complexes of Xaa-Xaa-His have
shown sequence selective DNA cleaving activities105
and have been employed as a specific metal binding
and DNA cleaving motif in several DNA binding
molecules106,107.
Ni(II) peptide complexes containing His as the
third amino acid residue has been used in site specific
DNA cleavage108, RNA cleavage109, protein-protein
cross linking110 and protein affinity labelling111.
DNA cleavage activity of histidine68,112, histidine
derivative113, Cu(II)-Histidine complex114,115, diironhistidine complex116, seryl-histidine 117,118, serylhistidine derivative119,120, etc., has been reported.
Cu(II)-L-His systems
Cu(II)–L-His systems which are more stable in
comparison with the other metal(II)-L-His complexes
(metal=Co, Ni, Zn, Cd) are of particular interest due
to the biochemical and pharmacological properties, as
well as the different possible coordination modes12.
Since the discovery in 1966 of the Cu(II)–L-His
species in human blood, extensive research has been
carried out to determine its role in copper transport12.
Copper transport in the tissues is facilitated by
L-His121 while albumin inhibits the copper uptake into
the cells122,123. The exchange of Cu(II) between L-His
and albumin modulates the availability of copper to
the cell. Evidence has been presented for the existence
of a ternary coordination complex between albumin,
Cu(II) and L-His under physiological conditions124.
The physiological importance of Cu(II)–L-His and its
therapeutic applications are well known11,125. Studies
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INDIAN J CHEM, SEC A, AUGUST 2010
on the transport properties of Cu(II)–L-His has led to
its application in the treatment of Menkes disease, a
fatal genetic neurodegenerative disease125. Recently,
the use of Cu(II)–L-His in the treatment of infantile
hypertrophic cardioencephalomyopathy has also
been reported126. Experiments with hepatic tissues
demonstrated that L-His enhances the uptake of
copper in hepatic cells127. Similar results have been
obtained in placental cells128. In vitro studies have
demonstrated that L-His can facilitate copper uptake
in mammalian brain129.
Considering the fact that L-His has significant
importance in biology, we have reported studies on
copper complexes of histidyl peptides, viz., histidyl
serine (HisSer), histidyl phenylalanine (HisPhe) and
histidyl leucine (HisLeu) (Fig. 1) and reported their
DNA binding and cleavage activity.
DNA binding and cleavage activity of ternary
Cu(II) complexes of histidyl peptides
The DNA binding and cleavage activity of
[Cu(II)(HisSer)(phen)]+(1)130,[Cu(II)(HisLeu)(phen)]+
(2)130, [Cu(II)(Hist)(HisPhe)]+ (3)131, [Cu(II)(Hist)(HisSer)]+(4)131, [Cu(II)(Hist)(HisLeu)]+(5)132, [Cu(II)(En)(HisLeu)]+(6)132 have been reported from our
laboratory. Since DNA binding by nuclease is a
prerequisite for its DNA cleavage activity, an
attempt is made here to summarize some of the
important and commonly used DNA binding
techniques along with the trends observed for the
complexes (1-6).
DNA binding
The mode and extent of binding of complexes
to DNA can be determined by various techniques i.e.,
absorption spectroscopy, thermal denaturation and
fluorescence spectroscopy133-142. Natural biomolecules
often contain chromophoric functional groups that
have characteristic UV spectra. For example, DNA
has significant absorption in the UV range because
of the presence of aromatic bases like adenine,
guanine, cytosine and thymine. Thus, by monitoring
the absorbance at a fixed wavelength with changes
in the concentration of reactants, the biomolecular
interactions have been assessed. Hyperchromism
and hypochromism are both the spectral features of
DNA concerning its double helix structure. The UV
absorption spectra of the complexes in the absence
and presence of calf-thymus DNA (CT-DNA) were
monitored. Figure 2. shows absorption spectra of (1)
in the absence and presence of increasing amounts of
CT-DNA (peak at 394 nm was monitored). In the
presence of CT-DNA, a decrease in absorption
intensities (hypochromism) was observed for (1), (2),
(5) and (6) while hyperchromism was observed
Fig. 1 – Geometry of Cu(II) complexes of histidyl pepetides. [1, [Cu(II)(HisSer)(Phen)]+; 2, [Cu(II)(HisLeu)(Phen)]+;
3, [Cu(II)(Hist)(HisPhe]+; 4, [Cu(II)(Hist)(HisSer]+; 5, [Cu(II)(Hist)(HisLeu]+; 6, [Cu(II)(En)(HisLeu]+]. [Reproduced from Refs 130-132].
REDDY & SHILPA : INTERACTION OF DNA WITH SMALL MOLECULES
for (3) and (4). Hypochromism was suggested to
arise due to the interaction between the electronic
state of the intercalating chromophore and that of
the DNA bases. These spectral changes are consistent
with the intercalation of (1), (2), (5) and (6) into
the DNA base stack. Hyperchromism for (3) and
(4) indicates base binding. The intrinsic binding
constants (Kb) of the complexes (Table 1) with
CT-DNA were determined using following function
equation143.
[DNA]/ (εa-εf) = [DNA]/ (εb-εf ) + 1/ Kb(εb-εf ) ...(1)
The ‘apparent’ extinction coefficient (εa) was
obtained by calculating Aobsd / [Cu]. The terms εf
and εb correspond to the extinction coefficients of
free (unbound) and the fully bound complexes,
respectively. A plot of [DNA]/ (εa-εf) versus [DNA]
will give a slope 1/(εb-εf) and an intercept 1/Kb (εb-εf ).
Kb is the ratio of the slope and the intercept. The
Kb values of complexes (1-6) are presented in Table 1.
Fig. 2 – Absorption spectra of [Cu(II)(HisSer)(Phen)]+ in the
absence (…….) and presence (  ) of increasing amounts of
CT-DNA. [Conditions: [Cu] = 10 µM, with increasing CT-DNA
concentration. Inset: linear plot for the calculation of the intrinsic
DNA binding constant, Kb. Arrow (↓) shows the absorbance
changes upon increasing concentration of CT-DNA. [Figure taken
from Ref. 130].
1009
The melting of CT-DNA can be used to distinguish
between those molecules which bind via intercalation
and those binding externally. The interaction of small
molecules with double helical DNA may increase or
decrease Tm, the temperature at which double helix is
broken up into single stranded DNA. An increase in
Tm value is indicative of an intercalative or phosphate
binding, while a decrease is attributed to base binding.
The thermal denaturation profiles of DNA in the
absence and presence of complexes were monitored
(Fig. 3. for 1). An increase of 5 – 7 °C was observed
in the Tm profile of (1), (2), (5) and (6) whereas a
decrease of 4 – 6 °C was observed for (3) and (4) as
compared to that of free DNA. These results provide
an evidence for intercalative and/or phosphate binding
for (1), (2), (5) and (6) and base binding for (3) and
(4) with DNA.
Fluorescence spectroscopy is an emission
phenomenon, which depends upon radiation
excitation rather than thermal excitation, of an atomic
vapor. Fluorescence quenching experiments were
Fig. 3 – Thermal-denaturation profile of CT-DNA (75 µM) in
aqueous buffer (pH 7.5) before (curve a) and after (curve b)
addition of [Cu(II)(HisSer)(Phen)]+ (75 µM), D is the difference
in absorbance. Inset: differential melting curves. [Figure taken
from Ref. 130].
Table 1 – Binding and cleavage of copper complexes of histidyl peptides
Complexes
[Cu(II) (HisSer)(Phen)]+(1)
[Cu(II)(HisLeu)(Phen)]+(2)
[Cu(II)(Hist)(HisPhe]+(3)
[Cu(II)(Hist)(HisSer]+(4)
[Cu(II)(Hist)(HisLeu]+(5)
[Cu(II)(En)(HisLeu]+(6)
Intrinsic binding
constant Kb (M-1)
4.9×103
4.2×103
2.9×103
2.2×103
7.8×102
5.9×102
[Complex]
(µM)
125, 187, 378, 437, 500
125, 187, 250, 312, 378,437, 500
13.3, 21.4, 26.7, 31.2, 62.2, 93.7
13.3, 21.4, 26.7, 31.2, 62.2, 93.7
125, 250, 375
125, 250, 375, 500
NC (Linear) DNA
(%)
45, 54, 69,95,97
50, 53, 55, 60, 65, 85,90
38, 56, 69, 97, 98, 100
35, 44, 64, 95, 97, 100
43(15), 34 (27), 27(43)
58, 62, 64, 70
Rate constant
(h-1)
1.40
1.32
1.29
1.24
0.97
0.81
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INDIAN J CHEM, SEC A, AUGUST 2010
performed with ethidium bromide (EB) bound DNA
with increasing concentrations of complexes to
determine the extent of binding between the second
molecule and DNA. EB, a typical indicator of
intercalation strongly fluoresces in the presence of
DNA due to complete intercalation between the
adjacent DNA base pairs, a process that can be
reversed by addition of a competing molecule
(fluorescence quenching)144. Two mechanisms have
been proposed in this context; the replacement of
molecular fluorophores, and/or electron transfer. The
quenching extent of fluorescence EB bound to DNA
can be used to determine the DNA-binding strength of
a given molecule. The fluorescence quenching curves
of EB bound to DNA in the absence and the presence
of complexes were monitored (Fig. 4 for 1). The
addition of complex to DNA pretreated with EB
caused appreciable reduction in emission intensity,
indicating that the complex binds to DNA at the sites
occupied by EB. The quenching plots (Fig. 4 inset
for 1) indicate that the quenching of EB bound to
DNA by the complexes is in good agreement with
the linear Stern-Volmer equation, which indicates
that the complexes bind to DNA. In the plot of Io/I
versus [complex]/[DNA], Ksq is given by the ratio
of slope to intercept.
Fluorescence Scatchard plots for the binding of
EB to CT-DNA in the presence of complexes
were obtained and rEB values were determined
(Fig. 5 for 1). The term rEB = [EB]b/DNA is
the concentration ratio of bound EB to total DNA
and [EB]f is the concentration ratio of free EB
(i.e. [EB] = [EB]b + [EB]f). The binding mode
of complexes with the DNA can be inferred from
the trends of Scatchard plots.
Fig. 4 – Fluorescence-quenching of EB bound to CT-DNA
in the absence (…….) and presence (___) of complex
[Cu(II)(HisSer)(Phen)]+. ([Cu]/DNA) = 0.24, 0.48, 0.72, 0.94,
1.18. λex = 540 nm. Inset: Stern-Volmer quenching curves.
[Figure taken from Ref. 130].
Fig. 5 – Fluorescence Scatchard plots for the EB to CT-DNA
in the absence (curve a) and presence (curve b) of
[Cu(II)(HisSer)(Phen)]+. The term rEB is the concentration ratio of
bound EB to total DNA, and Cf is the concentration of free EB.
[Figure taken from Ref. 130].
A decrease of slope and intercept upon the addition
of complexes indicates an intercalative and covalent
binding of the complexes with DNA, while a decrease
of slope with no change in intercept as compared
to complex free plot confirms an intercalative binding
of complexes with DNA.
DNA cleavage
Even though atomic force microscopy145-147 is
being used nowadays for monitoring DNA cleavage,
gel electrophoresis technique148-156 is still widely used
as it provides information regarding the percentage
of cleavage as a function of concentration of nuclease.
Hydrolytic cleavage which started in a modest way
of converting SC DNA to the NC form,is now being
used for identifying the % of cleavage as a function
of concentration of nuclease. Recent advances in
this area have also provided information regarding
the site specific cleavage and religation of the cleaved
products. Kinetic experiments also showed rate
enhancements of DNA cleavage in comparision
to non-catalyzed DNA cleavage. Despite the fact that
rate enhancements produced by natural enzymes
are much higher, the enhancement of rate constant for
DNA cleavage by metal complexes in the range
REDDY & SHILPA : INTERACTION OF DNA WITH SMALL MOLECULES
0.09–0.25 h-1 was considered impressive157,158. Several
complexes have been reported which show higher rate
enhancement than the 0.09-0.25 h-1 range.
The cleavage reaction on SC pUC19 DNA by
Cu(II) complexes has been monitored by agarose gel
electrophoresis. In general, when DNA is subjected to
electrophoresis, a relatively fast migration will be
observed for the intact SC DNA. If scission occurs on
one strand (nicking), the SC DNA will relax to
generate a slower moving NC form. If both strands
are cleaved, a linear form that migrates between SC
form and NC form will be generated159,160. When
DNA was incubated with increasing concentration
of the complexes, SC DNA was degraded to the
NC form. The cleavage efficiency was found to
increase with increase in the concentration of
Cu(II) complexes. The agarose gel electrophoresis
patterns for the cleavage of pUC19 DNA by (1)
is shown in Fig. 6.
After confirming the cleavage, attention was
focused towards the kinetic aspects of the reactions.
The observed distribution of SC and NC DNA forms
in an agarose gel provides a measure of the extent of
hydrolysis of phosphodiester bond in each plasmid
DNA. The time course plot for the decrease of form I
(SC DNA) and formation of form II (NC DNA)
during the reaction under physiological conditions
by (1) is shown in Fig. 7. The decrease of form I
and the increase of form II DNA fits well to a
single exponential curve. From these curve fits,
the hydrolysis rate constants at 37 °C and at a fixed
concentration of the Cu(II) complexes (1-6) have been
determined (Table 1).
The experimental results suggested that the
above complexes effectively bind and cleave
DNA under mild conditions. The order of DNA
binding ability and the rate constants were found
to be 1 > 2 > 3 > 4 > 5 > 6. The order of DNA binding
ability is also reflected in their cleavage activity.
Histidyl peptide in complexes (1-6) plays an
Fig. 6 – Agarose gel electrophoresis patterns for the cleavage
of pUC19 DNA by [Cu(II)(Phen)(HisSer)] +(1). [Lane 1, DNA
control; Lane 2, (125 µM); Lane 3, (187 µM); Lane 4, (378 µM);
Lane 5, (437 µM); Lane 6, (500 µM)]. [Figure taken from
Ref. 130].
1011
important role in DNA binding and cleavage due
to the presence of planar imidazole group which
actively participates in DNA binding through
intercalative mode and also provides an additional
donor site in metal chelation through N-3 atom
of imidazole ring.
When the binding (Kb) and DNA cleavage
constants of phen (1 and 2), Hist (3, 4 and 5), En (6)
systems are compared, it can be seen that the binding
ability (Kb) and DNA cleavage rate constants follow
the order: phen>Hist>En. Phen systems (1 and 2)
shows higher efficiency due to the presence of planar
aromatic system with 3 rings which can show
effective stacking interaction with the DNA bases.
However in Hist systems (3, 4 and 5) stacking
interactions are weak due to the presence of only one
ring in histamine (imidazole ring). The En system
(6) shows lower efficiency since it is an aliphatic
system and cannot participate in stacking interactions
with DNA bases.
Within the phen systems (1 and 2), (1) show a
slightly higher Kb and cleavage ability due to the
OH side chain of HisSer dipeptides, which can
involve in cleavage thereby enhancing the rate.
To explain this, the following tentative mechanism
for the hydrolytic pathway has been proposed:
The α amino group of His forms H-bonds with
the phosphate and the hydroxyl group of serine
moiety forms one H-bond with oxygen atom of
another phosphate. There are two other H-bonds
between the amide or the imidazole group of
Fig. 7 – Disappearance of supercoiled form I plasmid DNA (■)
and formation of nicked circular form II DNA (▲) in the presence
of [Cu(II)(Phen)(HisSer)] +. [Cond.: [Cu] = 378 µM; in aq. buffer
(pH 8.0) at 37 °C]. [Figure taken from Ref. 130].
1012
INDIAN J CHEM, SEC A, AUGUST 2010
DNA cleavage by [Cu(II)(HisSer)(Phen)]+(1). [(----) represent H-bonds].
Scheme 2
His moiety and phosphate group of DNA. These
interactions may play an essential role in the
binding and facilitate nucleophilic attack by bringing
the DNA closer to the complex. This is schematically
depicted in Scheme 2. Similar mechanism for
DNA cleavage has been reported for SerHis
peptide117.
Within the Hist systems (3, 4 and 5), (5) shows
weak binding which is also reflected in its cleavage
ability whereas (3) and (4) have comparable Kb which
is again reflected in their cleavage activity. However,
the slightly higher binding and cleavage ability of
(3) may be attributed to the presence of an aromatic
phenyl ring, which is known to involve in stacking
interaction with DNA bases. In (4), OH side chain of
HisSer dipeptide is involved in phosphodiester bond
hydrolysis thereby showing higher DNA cleavage
rate constant than (5). In (5), the leucine part of
HisLeu peptide has a simple carbon chain which
cannot involve in stacking interaction with DNA,
thereby showing less efficiency than (3) and (4).
Conclusions
In recent years copper containing complexes have
received considerable interest in nucleic acid
chemistry, following the discovery of the chemical
nuclease activity of [Cu(phen)2] in the presence
of H2O2 and a reducing agent. In particular, the
investigations on Cu(II) peptide complexes are of
scientific and technological importance, since
such systems may be regarded as models for both
protein-DNA and antitumor agent-DNA interactions.
Therefore, it is important to rationalize how such
complexes with peptides affect DNA binding and
cleavage.
Future prospects in this area lie in the development
of new chemical nucleases which are capable
of achieving 100 % DNA cleavage with minimum
concentration of nuclease. Further they should be able
to enhance the rate of hydrolysis as close to that of
natural enzymes but in the absence of external agents
and under physiological conditions. This is important
since these conditions are close to in vivo systems.
Moreover, higher concentration of complexes and
addition of external agents will have adverse effects
on the target molecule. This will pave way for new
drug designing which covers a wide spectrum of other
disciplines and technologies such as chemistry,
biochemistry, genetics, etc. leading to new products
and technologies in the future.
Acknowledgement
PRR thanks the Council of Scientific and Industrial
Research (CSIR), New Delhi, for financial assistance.
He also thanks UGC, New Delhi, for the award of
Emeritus Fellowship.
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