drug-dna complexes · 2019. 4. 16. · interaction that comprises stability of synthetic and...
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CONFORMATIONAL TRANSITIONS IN
DRUG-DNA COMPLEXES
By
Ghazala Yunus
1
CONTENTS
Abbreviations
List of tables
List of figures
CHAPTER 1: Introduction
CHAPTER 2: Theory
CHAPTER 3: Transition & Stability of DNA-Dipyrandium Complex
CHAPTER 4: Thionine Induced Thermal Stability of Natural DNAs
CHAPTER 5: Conclusion
2
ABBREVIATIONS
% Percentage
Conc. Concentration
DNA Deoxyribonucleic acid
DSC Differential scanning calorimetry
ed. Edited
edn. Edition
et al. et alia (Latin: and other)
etc. et cetera
FTIR Fourier transform IR
hr hours
i.e. That is
IR Infrared
ITC Isothermal titration calorimetry
K Kelvin
M Molar
MW Molecular weight
nm Nanometer
ºC Degree centigrade
pH Potential of hydrogen ion
RNA Ribonucleic acid
Temp. Temperature
Tm Melting temperature
3
UV Ultra violet
UV-VIS Ultra violet - visible
viz. vide licet
ΔCp Change in heat capacity
ΔG Changes in free energy
ΔH Changes in Enthalpy
ΔS Changes in Entropy
4
LIST OF TABLES
Table No. Title Page No.
1.1 Thermodynamic parameters for intercalators and
minor groove binders
00
3.1 Transition parameters for dipyrandium binding to
poly d(AT)
00
4.1 Transition parameters for thionine binding to
various natural DNAs
00
5
LIST OF FIGURES
Figure
No. Title Page No.
1.1 DNA structure 00
1.2 Major and minor groove of DNA 00
3.1 Molecular structure of dipyrandium 00
3.2 Heat capacity and transition profiles for unbounded
and bounded DNA with dipyrandium
00
3.3 Half width of the heat capacity curves 00
4.1 Chemical structure of thionine 00
4.2 Heat capacity and transition profiles for thionine
bounded and unbounded CP-DNA. (A) Unbounded
state, (B) Bounded state at saturation
00
4.3 Heat capacity and transition profiles for thionine
bounded and unbounded HT-DNA. (A) Unbounded
state, (B) Bounded state at saturation
00
4.4 Heat capacity and transition profiles (inset) for
thionine bounded and unbounded ML-DNA. (A)
Unbounded state, (B) Bounded state at saturation
00
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CHAPTER 1
INTRODUCTION
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1.1. Introduction
In the present thesis author included detailed study of Drug-DNA
interaction that comprises stability of synthetic and natural DNA on binding with
various drug. The present chapter provides basic information to the next four
chapters, which include the work on theoretical analysis of Dipyrandium and
Thionine binding with synthetic polynucleotide [poly d(AT)] and natural DNA
(CP-DNA, ML-DNA, HT-DNA). The concluding chapter of the thesis provides all
pros and cons of the present work and their significance in the present scenario.
1.2. Drug-DNA interaction: Significance and future
potential
The study of drug-DNA interactions begins around 1960s but the binding
of steroid-diamines and aromatic molecules to synthetic and natural nucleic
acids has received considerable attention over the past several years (Araya et
al., 2007; Bruylants et al., 2005; Gabbay and Glaser, 1971; Gonzalez-ruiz et al.,
2011; Islam et al., 2009; Kunwar et al., 2011; Lane, 2001; Mahler and Dutton,
1964; Mahler et al., 1966; 1968. Paul et al., 2010). Molecular interaction of the
drug with deoxyribonucleic acid (DNA) is a field of high topical interest and may
have a great importance to its biological activity. The molecules can interact
with DNA in a variety of ways such as surface binding to their minor or major
grooves, intercalation between adjacent base pairs, or electrostatic binding.
There are number and variety of techniques devoted to the study of drug-DNA
interactions which are increasing continuously.
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DNA is a molecule of great biological significance in which all the
information governing life processes of the organism are stored (Figure 1.1).
The total DNA content of a cell is termed as genome which is unique for each
and every organism. A segment of DNA that code for a polypeptide is known
as gene which contains the information necessary to produce a functional
product, usually a protein. DNA has two main functions in the cell viz.
transcription and translation. In transcription process, information is retrieved
from the DNA by RNA and utilized to synthesize proteins in the body. During
replication, DNA undergoes self-replication process and regenerates two
identical strains. DNA present in the form of a double helix, where each strand
is composed of a combination of four nucleotides, adenine (A), thymine (T),
guanine (G) and cytosine (C). Within a strand these nucleotides are linked via
phosphodiester linkages. The two strands are held together primarily via
Watson Crick hydrogen bonds where A forms two hydrogen bonds with T and
C forms three hydrogen bonds with G. Specific recognition of DNA sequences
by proteins/ small molecules is achieved via the combination of hydrogen bond
acceptor/donor sites available on the major groove or minor groove of DNA
(Figure 1.2).
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Figure 1.1. DNA structure
Figure 1.2. Major and minor groove of DNA
Transcription and replication plays a vital role in survival and proliferation
of the cell as well as for smooth working of all body processes. DNA starts
transcribing or replicating only when it receives a signal, which is often in the
form of a regulatory protein binding to a particular region of the DNA.
Therefore, if the binding specificity and strength of this regulatory protein can
be imitated by a small molecule, then DNA function can be artificially
modulated, inhibited or activated by binding this molecule instead of the
protein. Hence, this synthetic/natural small molecule can act as a drug when
activation or inhibition of DNA function is required to cure or control a disease.
DNA activation would produce more quantities of the required protein, or could
induce DNA replication; depending on which site the drug is targeted. DNA
inhibition would restrict protein synthesis, or replication, and could induce cell
death (Sheikh et al., 2004). Regions of DNA involved in vital processes such as
origin of replication, promotion of transcription, etc., are of particular interest
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as targets for a wide range of anticancer and antibiotic drugs (Chaires, 1997,
1998; Haq, 2002; Hurley, 2002.)
1.2.1. Mode of DNA binding with drugs/small molecules
Drugs and/or small molecules bind to DNA both covalently as well as
non-covalently. Covalent binding in DNA is irreversible and invariably leads to
complete inhibition of DNA processes and subsequent cell death. One of the
drug, anticancer antibiotic, covalently bind to DNA is cis-paltin. Non-covalently
bound drugs mostly classified into two classes viz. minor groove binders and
intercalators. Drugs with ability to minor groove binding (minor groove binders)
are usually crescent shaped, which complements the shape of the groove and
facilitates binding by promoting van der Waals interactions. Moreover, these
drugs can form hydrogen bonds to bases, typically to N3 of adenine and O2 of
thymine. Most of the minor groove binding drugs bind to A/T rich sequences.
This preference in addition to the designed propensity for the electronegative
pockets of AT sequences is probably due to better van der Waals contacts
between the ligand and groove walls in this region, since A/T regions are
narrower than G/C groove regions and also because of the steric hindrance in
the latter, presented by the C2 amino group of the guanine base (Sheikh et al.,
2004). Another group of Non-covalently bound drugs, intercalators, introduce
strong structural perturbations in DNA and contain planar heterocyclic groups
which stack between adjacent DNA base pairs. The complex is thought to be
stabilized by π-π stacking interactions between the drug and DNA bases. Non-
covalent binding is reversible and is typically preferred over covalent adduct
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formation keeping the drug metabolism and toxic side effects in mind. However,
the high binding strength of covalent binders is a major advantage. Some of
the minor groove binders and intercalators are netropsin, berenil, distamycin,
mithramycin, hoechst 33258, pentamidine, nogalamycin and menogaril. As the
number of DNA–drug complex structures that have been solved by X-ray
crystallography and NMR analysis is increasing, it is becoming possible to
identify structural features and their energetic consequences that guide
observed properties like affinity and sequence selectivity.
In drug-DNA interaction, energetics of DNA recognition is not fully
understood at a molecular level, due mainly to the strong electrostatic
interactions prevalent in the system and van der Waals interactions (Jayaram
and Beveridge, 1996; Jayaram et al., 2002). A detailed account and
quantification of these contributions can help in addressing issues of both
practical and fundamental interest (Lazaridis, 2002). The fundamental interest
lies in tackling challenges involved in predicting ligand binding free energies
qualitatively. Among the structural and energetic angles, binding of small
molecules to DNA and proteins differs significantly. Protein–drug binding has
been explained by various popular models such as lock and key model, induced
fit model etc., and it is also believed that hydrophobic effects play an important
role in the binding process. However, a straightforward extrapolation of these
interaction models to DNA–drug systems is not feasible since there is no formal
active site in DNA, unlike enzymes/proteins. Certain base sequence dependent
chemical, structural, and conformational characteristics of DNA double helix,
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nonetheless, carry sufficient information for recognition by regulatory proteins
as well as small molecules (Saenger, 1983; Sinden, 1994), which bind non-
covalently or introduce small covalent modifications. Fairly strong and specific
binding is observed between some ligands and DNA and this is attributed to
various factors like hydrogen bonding etc.
For understanding of DNA–drug interaction, experimental studies plays
basic role. The necessary information on binding constants and corresponding
free energy, entropy, enthalpy, and heat capacity changes on complex
formation can be obtained by thermodynamic studies (Chaires, 1997; Cooper,
1999). This information enormously used to authenticate investigation based
on theoretical predictions and structural analysis. Along with initial studies
focused on thermodynamics of drug-DNA complex formation, Breslauer and his
colleagues recommended significant observation of enthalpy–entropy
compensation in drug-DNA systems and moreover contributed considerably to
developing thermodynamic profiles of these complexes (Zaunczkowski et al.
1987; Chalikian and Breslauer, 1998; Pilch et al., 1999). Among the range of
previous studies in the field of DNA–drug complexes modeling, some of the
initial studies focused on netropsin (Zakrzewska et al., 1984; Caldwell and
Kollman, 1986). These studies proposed models to explain the accurate
functions played by formal charges and hydrogen bonds along with snug fit in
the netropsin–DNA complex. Basically molecular mechanical calculations based
on energy minimization protocols were employed to broaden the horizons and
study different classes of DNA-binding molecules, such as minor groove binding
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netropsin, bisintercalating drug, triostin (Singh et al., 1986), and the covalent
binder acridine (Rao and Singh, 1986). Along with this, the era of free energy
calculations and various components contributing to it became a key area of
interest (Tembe and McCammon, 1984; Jorgensen and Ravimohan, 1985;
Beveridge and DiCapua, 1989). In this regard, results of relative binding free
energies calculation for distamycin and its analog was in good agreement with
experimental one (Singh et al., 1994). However, computation of absolute
binding free energies is a quite complicated assignment and remains semi-
quantitative at present scenario. Out of these some successful attempts have
been reported in which the theoretically determined absolute binding free
energy is in correspondence with experiment (Singh and Kollman, 1999; Pineda
and Zacharias, 2002; Spackova et al., 2003). These theoretical studies on drug-
DNA interactions have resulted in important contributions to the understanding
of binding in a few DNA–drug complexes. Since these studies typically focused
on a single DNA–drug complex (Srivastava et al., 2004 Singh and Kollman,
1999; Pineda and Zacharias, 2002; Spackova et al., 2003), the need to examine
a series of complexes persists to facilitate origin of a set of general principles.
1.2.2. DNA-interactive agents
The polynucleotide, DNA, is also one of the receptors molecule with
which various drugs or small molecules can interact. These DNA-interactive
drugs/agents are generally very toxic to normal cells. Thus, these drugs are
reserved only for life-threatening diseases such as cancers. There are four
general types of compounds that interact with DNA:
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1. Intercalators
2. Groove binders
3. Alkylating agents
4. DNA cleaving agents
In general aromatic or hetero-aromatic molecules bind to DNA by
intercalating between the base pairs of the double helix. The principal driving
forces for intercalation are stacking and charge-transfer interactions, while
hydrogen bonding and electrostatic forces also play a role in stabilization
(Neidle and Abraham, 1984). Additional interactions may further stabilize the
complex but in many cases, no covalent bond is formed. The result of
intercalation is that the DNA double helix becomes geometrically distorted and
the translation of the genetic code may be unable to properly function.
Intercalation, first described in 1961 by Lerman, is a noncovalent interaction in
which the drug is held rigidly perpendicular to the helix axis (Lerman, 1961).
This causes the base pairs to separate vertically, thereby distorting the sugar-
phosphate backbone and decreasing the pitch of the helix. Intercalation is
energetically favored because the energy that holds the intercalated molecule
must be greater than normal base stacking. It is cleared that intercalation does
not disrupt normal DNA hydrogen bonding. However, it can destroy the regular
helical structure by unwinding the DNA at the binding site and importantly,
interferes with DNA-binding enzymes like polymerases. Some examples of the
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intercalators are echinomycin, triostin A, acridine, cis-Platin, daunomycin,
adriamycin, actinomycin D, ethidium and propidium.
Groove binders are highly sequence-specific that interact with DNA
groove, which are recognized by these molecules. The grooves of DNA (major
and minor) differ in hydrogen-bonding, electrostatic potential and degree of
hydration. Most of the large protein molecules exhibit high specific binding with
major groove region of the DNA and small molecules prefer the minor groove.
Minor-groove binding molecules have aromatic rings linked by bonds with
torsional freedom. Because groove-binding agents can extent many nucleic acid
base pairs, they can have highly sequence-specific recognition. Sequence-
specific DNA-binding proteins generally interact with the major groove of B-
DNA, because it exposes more functional groups that identify a base pair.
However, there are some known minor groove DNA-binding ligands such as
netropsin, distamycin, pentamidine etc. (Zimmer and Wahnert, 1986; Dervan,
1986)
Alkylation reaction mostly represents an irreversible binding to DNA
basically with guanine. Alkylating agents add methyl or other alkyl groups onto
molecules where they do not belong. This alkylation proceeds with different
mechanism. In the first mechanism an alkylating agent attaches alkyl groups
to DNA bases and results in DNA fragmented by repair enzymes in their
attempts to replace the alkylated bases. In second mechanism alkylating agents
caused DNA damage due to formation of cross-bridges and bonds between
atoms in the DNA. Cross-linking prevents DNA replication and finally
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transcription. In third type of mechanism, alkylating agents causes the
mispairing of the nucleotides leading to mutations. There are six groups of
alkylating agents viz. nitrogen mustards, ethylenimes, alkylsulfonates,
triazenes, piperazines, and nitrosureas. Cyclosporamide is a classic example
alkylating agent and is one or the most widely used agents.
DNA cleaving agents (strand breaker) initially intercalate into DNA, and
can react in such a way as to generate radicals depending on the local
environmental and cellular metabolism. These radicals typically extract
hydrogen atoms from the DNA sugar-phosphate backbone, leading to scission
of DNA strand. Hence, these DNA-interactive compounds are metabolically
activated radical generators. The anticancer drug bleomycin acts as a strand
breaker and other examples are anthracycline, tirapazamine and enediyne.
Bleomycin is actually a mixture of several glycopeptyde antibiotics isolated from
a strain of the fungus Streptomyces verticellus. Bleomycin cleaves double-
stranded DNA selectively at 5’-GC and 5’-GT sites in the minor groove by a
process that is both metal ion and oxygen dependent (Povirk, 1989; Steighner
and Povirk, 1990; Stubbe, 1996).
1.2.3. Thermodynamics of Drug-DNA Interactions
Nucleic acid-binding drugs are designed to transform gene activity
and/or inhibit protein translation. Therefore, these biomolecules represent a
major target in drug development strategies designed to manufacture next
generation therapeutics for diseases. In order to optimize the clinical efficacy
of drugs and also to discover or design new drugs, it is essential to understand
17
and characterize the molecular basis of drug-DNA interactions, including
structural details and thermodynamics of binding.
Thermodynamic study of drug-DNA interaction determines the forces
involve during the binding of drugs to its target DNA. For the calculation of
observed free energy change (ΔGobs) for a drug-DNA interaction, the following
relationship is used:
𝛥𝐺𝑜𝑏𝑠 = −𝑅𝑇𝑙𝑛𝐾𝐵
Where, R = universal gas constant
T = temperature
KB = equilibrium binding constant (The value of KB
is dependent on pH, salt concentration, and other
experimental parameters)
Isothermal titration calorimetry directly measures the enthalpy of
binding (ΔHB) and equilibrium binding constant (KB), and the entropy change
associated with binding (ΔSB) can also be calculated by using following
equation:
𝛥𝐺𝑜𝑏𝑠 = 𝛥𝐻𝐵 − 𝑇𝛥𝑆𝐵
To determine KB and ΔGobs experimentally, a number of methods are
available. These methods include isothermal titration calorimetry and
spectroscopic techniques. In addition to KB and ΔGobs, it is also important to
18
determine ΔHB and ΔSB for a binding interaction. Isothermal titration
calorimetry is the only experimental method available to directly measure ΔHB.
The driving forces involve in drug-DNA interaction depends on the nature of
reaction. When enthalpy is favorable, the driving forces are hydrogen bonding,
van der Waals interactions, and electrostatic interactions. When entropy is
favorable, binding is driven by hydrophobic interactions, whereas unfavorable
entropic changes are due to loss of conformational degrees of freedom.
Change in heat capacity (ΔCp) due to binding of drugs can also be
calculated by isothermal titration calorimetry, by determining the dependence
of ΔH on temperature. The heat capacity change can also be estimated from
change in accessible surface area (ΔASA), and one can correlate calculated heat
capacity values to experimentally-determined values. There are number of
reports on energetic contributions to the binding free energy for drug-DNA
interactions in which free energy was calculated by the data obtained from
isothermal titration calorimetry (Chaires, 1998a; 1998b; Haq and Ladbury,
2000; Haq et al., 2000; Haq, 2002). It is required to keep experimental
conditions in mind as the observed binding constant and ΔGobs is dependent
these conditions such as buffer, ionic strength, temperature and concentration.
For drug-DNA interactions following five free energy components have been
identified:
1. Unfavorable contribution from conformational changes in the drug or
DNA due to binding (ΔGconf).
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2. Unfavorable contribution due to loss of rotational and translational
degrees of freedom upon binding (ΔGr+t).
3. Contribution from hydrophobic transfer of the unbound drug to the
DNA binding site (ΔGhyd). This value can be determined from heat
capacity change.
4. Contribution from coupled polyelectrolyte effects due to binding of
cationic ligands (ΔGpe). Binding constant and observed ΔG are
dependent on salt concentration. ΔGpe can be determined
experimentally by measuring the binding constant at different salt
concentrations.
5. Contribution due to non-covalent interactions, such as hydrogen
bond formation and van der Waals interactions (ΔGmol).
These free energy terms can be experimentally determined by
isothermal titration calorimetry, in combination with recognized empirical
relationships from other experimental data. This information is used to modify
drug structure to improve binding affinity and specificity of the drug.
Differential scanning calorimetry is also used to study the binding of
various drugs to synthetic and natural DNA (Marky, et al., 1983a; Marky, et al.,
1983b; Leng, et al., 1998; 2003). UV melting profiles and DSC have been used
to measure the binding affinity of a new bisintercalating antibiotic, while other
methods were not successful. DSC was also used to characterize the binding of
echinomycin to DNA, a bisintercalator that is difficult to study due to its poor
solubility (Leng et al., 2003). Thermodynamic parameters for some of the
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intercalators and minor groove binders are given in table 1.1. On addition of
drug to DNA, the transition midpoint (Tm) increases, and when drug
concentration is high enough to saturate the binding sites on the DNA, McGhee
(McGhee and von Hippel, 1974) showed that:
1
𝑇𝑚0 −
1
𝑇𝑚=
𝑅
𝛥𝐻𝐷𝑁𝐴ln[(1 + 𝐾ℎ𝐿)
1/𝑛]
where 𝑇𝑚0 and 𝑇𝑚 are the melting temperature of drug-free and drug-bound
DNA, respectively,
1.2.4. Heat capacity changes associated with Nucleic acid folding
transitions
Besides the changes in free energy (ΔG), enthalpy (ΔH), and entropy
(ΔS) during the nucleic acid structures modification, heat capacity (CP) are also
changes significantly and affect the overall energetics of folding. Heat capacity
is defined as the amount of heat required to increase the temperature of a
substance by a unit degree and can be presented by:
𝐶𝑃 = [𝜕𝑞
𝜕𝑇]𝑃
Table 1.1. Thermodynamic parameters for intercalators and minor groove
binders
Drugs T ΔCp ΔGobs ΔHB TΔSB Reference
21
Intercalators:
Ethidium 25 -140 -6.7 -9.0 -2.3 Chaires, 1998b
Actinomycin 10 -364 -8.5 -2.7 +5.8 Chaires, 1998b
Doxorubicin 20 -150 -8.9 -7.4 +1.5 Chaires, 1998b
Daunorubicin 20 -160 -7.9 -9.0 +1.1 Chaires, 1998b
Propidium 25 -150 -7.5 -6.8 +0.7 Chaires, 1998b
Minor Groove Binders:
Netropsin 25 -213 -8.7 -5.8 +2.9 Haq, 2002
Distamycin 20 NA -10.1 -12.3 -2.2 Chaires, 1998b
Berenil 25 -146 -8.0 +0.6 +8.6 Haq, 2002
Hoechst 33258 25 -330 -7.7 +4.4 +12.1 Haq et al., 1997
NA=Not available
Heat capacity is related to other fundamental thermodynamic
parameters also. Accordingly, different expressions relating CP to these other
parameters may be selected as a function of experimental ease or to highlight
some physical aspect of the system. For example, the most commonly
employed relationships of CP are
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𝐶𝑃 = [𝜕𝐻
𝜕𝑇]𝑃= [
𝜕𝑆
𝜕𝑇]𝑃
Hence, CP can be calculated as the temperature dependence of the
entropy or enthalpy. Changes in heat capacity (ΔCP), associated with the folding
or unfolding of a biopolymer can be expressed as:
𝛥𝐶𝑃 = [𝜕𝛥𝐻
𝜕𝑇]𝑃= [
𝜕𝛥𝑆
𝜕𝑇]𝑃
The above equation demonstrates the most common experimental
method for determining ΔCP, by measuring ΔH as a function of temperature.
The same equation also implies that ΔH and ΔS for a given transition change
together and in the same direction as a function of the ΔCP. Additionally, to
describe ΔCP with respect to ΔH and ΔS, we can also calculate change in heat
capacity with respect to ΔG that may be calculated as:
𝛥𝐶𝑃 = [−𝑇𝜕2𝛥𝐺
𝜕𝑇2]𝑃
Change in heat capacity is the second derivative of ΔG with respect to
temperature. Therefore, folding transitions associated with large ΔCP values
should exhibit significant curve in a plot of ΔG versus temperature. In terms of
physical origins, the heat capacity changes of a nucleic acid in solution derives
from a combination of solvent interactions (both solute–solvent and solvent–
solvent) and internal solute effects, such as conformational entropy,
electrostatics, vibrational modes, and others (sturtevant, 1977).
23
To find the impact of heat capacity changes on nucleic acid folding we
must go through the basics of heat capacity and its relation with the change in
temperature. In the absence of a ΔCP, the free energy associated with nucleic
acid unfolding is linear with temperature, as expressed by the Gibbs equation:
𝛥𝐺 = 𝛥𝐻 − 𝑇𝛥𝑆
However, introduction of a ΔCP introduces curvature in the temperature-
dependent stability profile, as reflected by the fact that CP is the second
derivative of G with respect to temperature. In the case of nucleic acid folding
transitions, it is highly likely that ΔCP itself significantly depends on temperature.
To accommodate a temperature-dependent ΔCP, the Gibbs law can be
modified:
∆𝐺 = (𝑇𝑟𝑒𝑓 − 𝑇
𝑇𝑟𝑒𝑓)∆𝐻𝑟𝑒𝑓 +∫ ∆𝐶𝑃𝑑𝑇
𝑇
𝑇𝑟𝑒𝑓−∫ ∆𝐶𝑃𝑑(𝑙𝑛𝑇)
𝑇
𝑇𝑟𝑒𝑓
where ΔHref is the change in enthalpy upon unfolding at an arbitrary reference
temperature (Tref) which is often set at the high-temperature melting midpoint
(TM) for convenience. Two related consequences of the temperature-dependent
curvature in ΔG imparted by ΔCP are i) that there exists a temperature of
maximum stability, Tmax, and ii) that unfolding can in principle be induced by
sufficiently deviating in temperature either above or below Tmax. The
phenomenon of cold unfolding or cold denaturation has been sufficiently
demonstrated for proteins and linked to the presence of a large, positive ΔCP
for unfolding (Privalov, 1990).
24
Since temperature-dependent changes in ΔH and ΔS largely offset one
another in ΔG, the zero ΔCP approximation is usually adequate for routine
stability estimations of short stretches of duplex at 37°C. The same may not be
true for applications that require high-precision estimates of ΔG for
hybridization of short duplexes. The temperature dependence of ΔCP conferred
by linked single strand equilibria further complicates the prediction of folding
energetic at temperatures distant from those at which parameter data are
collected. Predicted values of ΔH and ΔS are less accurate than those for ΔG
and vary especially between oligomeric and polymeric duplexes; inclusion of
ΔCP within stability calculations can reconcile these differences. A more recent
study found that explicit consideration of ΔCP was necessary to calculate
adequate differential thermodynamic parameters between various duplexes in
an effort to understand the physical basis of their different stabilities
(Tikhomirova et al., 2004; Mikulecky and Feig, 2006). Change in heat capacity
for nucleic acid structural changes have been obtained by different methods.
One of the methods is purely computational method with hydration of solvent
accessible surfaces. Some of the major experimental approaches used to
determine ΔCP are van’t Hoff method, differential scanning calorimetry (DSC)
method and isothermal titration calorimetry (ITC) method.
1.2.5. Thermodynamics in Drug Design
Drugs that interact with DNA have clinical significance especially for the
treatment of solid tumors, lymphoma, and leukemia. The interruption of DNA
replication is effective against rapidly reproducing cancer cells. More efficient
25
and targeted DNA-binding drugs can be achieved by designing new compounds
with multiple binding moieties that bind to specific DNA sequences. Application
of calorimetric methods to study the thermodynamics of nucleic acid-folding
transitions has gradually been improved in recent years. These improvements
escort to the production of high-precision microcalorimeters. Therefore, the
amount of high-resolution structural results about nucleic acids has developed
and clarified the physico–chemical interactions, which force the formation of
nucleic acid structures (Mikulecky and Feig, 2006; Gill et al., 2010). DSC
monitors the excess Cp of a nucleic acid in solution, and the temperature is
increased or decreased with a constant rate. Measuring the differential heat
flow between the sample and a reference accomplishes the excess Cp. When
sample and reference are scanned simultaneously, folding and unfolding
reactions occur in the nucleic acid as a consequence of absorbed or released
heat. This differential heat is gained with subtraction from the reference
thermal profile. Peaks obtained from types of DSC thermograms represent
folding transitions, and melting points of the transition occur near the jump
point of the curve. The melting point corresponds to a single molecular
transition, where ΔCp is equal to zero (Mikulecky and Feig, 2006). Peak results
are fitted in such ways that accommodate Limited ΔCp or ignore them. The later
leads to ΔHVH. The ratio of ΔHcal to ΔHVH could be used for study of intermediate
folding states, which occur near the melting point (Pilch, 2000; Breslauer et al.,
1992). Therefore, the DSC calorimeter is ideal for determining ΔCp and
measuring Cp directly (Mikulecky and Feig, 2006).
26
Latest drug discovery efforts have been dominated by structure-based
design concepts in which lead compounds are sought by attempting to match
their shapes with the complementary shapes of active sites of receptors using
known structures obtained by X-ray crystallography. However, it was formerly
noted that thermodynamic studies are an essential and necessary complement
to structural studies in drug design (Henry, 2001). Structural data alone, even
when coupled with the most sophisticated current computational methods,
cannot fully define the driving forces for binding interactions. Thermodynamics
provides quantitative data of use in elucidating these driving forces and for
evaluating and understanding at a deeper level the effects of substituent
changes on binding affinity (Weber and Salemme, 2003; Chaires, 2008).
1.3. Previous research work done
Ligands that can bind to nucleic acids have been the subject of much
research for the past five decade because DNA/RNA has been presumed to be
the intracellular targets for various small ligands and the ligand-nucleic acid
interaction creates the potential for regulating gene expression resulting in
therapeutic treatment for tumors and other bacterial and viral diseases.
Single strand nucleic acids do not form unique structure but rather
partially stacked helixes or random coils (Fresco and Klempere,r 1959; Saenger,
1984), with the degree of stacking being strongly temperature, ion-strength or
27
pH dependent (Fresco and Klempere,r 1959; Saenger, 1984). Among those
single nucleic acids, poly(A) and poly(U) have attracted much attention due to
their ability to form higher order or other secondary nucleic acid structures, and
the importance of their biological functions in cell growth. It is known that
poly(A) structure play an important role in gene expression in eukaryotic cells.
All mRNAs in eukaryotic cells contain a poly(A) tail at the 3’ end that is important
for the maturation and stability of mRNA and for the initiation of translation
(Zarudnaya and Hovorun, 1999; Munroe and Jacobson, 1990). Post-
translational polyadenylation of mRNA is catalyzed by the enzyme poly(A)
polymerase. Recent studies have found that neoPAP, a human polyadenylic
polymerase, is significantly over-expressed in human cancer cells (Topalian et
al., 2001; 2002). Thus, the new cancer therapeutic agents can be developed,
ones that bind to the poly(A) tail of mRNA and thereafter interfering with mRNA
processing by polyadenylic polymerase. However, very few molecules are
known to bind to a poly(A) single structure or to stabilize the poly(A)•poly(A)
duplex at neutral pH. Of this small number of ligands, berberine (Yadav et al.,
2005), coralyne (Xing et al., 2005, and palmatine (Giri et al., 2006) have been
studied.
The various conformations that DNA can adopt exhibit different binding
properties upon interacting with small ligands. As discussed previously, the
small ligands bind to duplex DNA/RNA mainly via intercalation and groove
binding. Since the major groove is larger than the minor groove, the majority
of groove binders selectively bind to the minor groove because their charges
28
and shapes are complementary. In addition to the groove size, the binding of
small ligands to the duplex is also sequence dependent. For example, Hoechst
33258 prefers AT-rich duplexes (Harshman and Dervan, 1985), with imidazole-
pyrrole-pyrrole (Bremer et al., 2000) recognizes the GC base pair. For most
drug-DNA interactions, these small molecules essentially act as a rigid body,
with little conformational change upon binding. However, the DNA/RNA duplex
conformation may be changed upon binding. In describing the B-form DNA
minor groove recognition, the lexitropson family of compounds, typically
netropsin and distamycin, have been studied extensively due to their inherent
high affinity for DNA duplex. Because of the changes in the minor groove width
and the unusually flexible TpA base step, the particularly binding preference for
these compounds is 5’-AATT and 5’-ATAT rather than 5’-TTAA and 5’-TATA
(Abu-Daya et al., 1995; Lane et al., 1993). Structural analyses obtained through
NMR and crystallographic studies have indicated that the recognition of a ligand
by DNA arises from H-bonded contacts with A/T, electrostatic interaction with
the backbone of the DNA, and the van der Waals interaction with the floor and
the walls of the groove. It has also been observed that the stacking interaction
between the O4’ atom of sugar and the pyrrole ring of the ligand contributes
to the stability of the complex formation (Pelton and Wemmer, 1988). In
addition to the structural findings, binding of lexitropsin with the DAN minor
groove is also accompanied by high binding affinity, favorable enthalpy changes
and binding entropy (Marky and Breslauer 1987).
29
The thermodynamics of drug-DNA interaction has been the focus of
much past research; however, the recent introduction of the high sensitive
micro-calorimetric technique has made this study in this yield more
straightforward. Consistent with other minor groove binding ligands, the
interaction between lexitropson and DNA are characterized by enthalpy-entropy
compensation mechanisms. Studies have shown that netropsin has a binding
affinity of 4.3×107 M-1 at 1:1 binding to the A3T2T3 oligomer, with the entropy
change contributing more to the binding free energy than enthalpy (Rentzeperis
et al., 1995). However, significant differential effects have been observed in
the binding of netropsin to poly(dA)•poly(dT) and poly(dA-dT)2 duplex systems,
suggesting that conformational change plays an important role in the binding
(poly(dA).poly(dT) experiences a conformational change) (Marky and Kupke,
1989). Additional classic B form DNA minor groove binders such as berenil,
Hoechst 33258, and propamidine have also been studied, results indicating
binding affinities in the range of 105 – 107M-1 and the heat capacity changes
(ΔCp) in the range of -150 ~ -400 cal/mol.K. The ΔCp value for minor groove
binders is typically higher than for intercalators, for which this value is usually
less than -200 cal/mol.K (Lane and Jenkins, 2000). Recently, the study
developed in Arya’s laboratories has found B-form DNA recognized by
aminoglycosides, which were previously thought to have no effect on B-form
DNA. This recognition, specifically dual recognition, was achieved by
synthesizing the neomycin-Hoechst 33258 conjugate (Willis and Arya, 2006) or
the neomycin-neomycin dimer (Arya et al., 2004) and then binding them to the
B-form duplex, in particular the AT-rich sequences. It was found that this
30
binding affinity is significantly higher than the classic B-form DNA binders
mentioned previously. These findings open the door for the development of
compounds that target the B-form DNA duplex. Recognition of A-form nucleic
acid, in particular the RNA duplex, by small ligands, however, exhibits a higher
binding affinity than the B-form DNA systems. A good example is the 16S A site
rRNA interaction with aminoglycosides (Kaul and Pilch, 2002). As discussed
above, this system was found to achieve a high binding affinity in the
nanomolar range, but different from the B-form DNA-minor groove binder
system, this binding is predominately enthalpy driven and the binding site is
within the major groove of the RNA. Very few small molecules, however, have
been found to be able to bind to the DNA•RNA hybrid duplex. Recent report
has shown that ethidium bromide is the preferred catalyst for this hybrid
recognition (Ren and Chaires, 2001). The binding affinity is in low micromolar
range. Aminoglycosides, were also found by Arya to bind to this hybrid in the
100 nM range. The methidium-Neomycin conjugate was subsequently
synthesized to achieve a high binding affinity and specificity of drug binding to
DNA•RNA hybrid. Initial results have shown the powerfulness of this conjugate,
which binds to the DNA•RNA hybrid specifically at an affinity of approximately
0.1 nm.
To date, the only full and detailed conformational information about the
DNA triplex comes from NMR studies. In addition to the conformational change,
the stability of the triplex has found to be much lower than its corresponding
duplex because of the charge neutralization resulting from the high
31
electronegative potential formed when three backbones are associated with
one another. Bringing in a positive charge on the third strand (Plum and
Breslauer, 1995), replacing the phosphodiester with a peptide at the backbone
(Cherny et al., 1993), introducing the ionic strength of the solution (Cheng and
Pettitt, 1992), and applying polycations (aminoglycosides) (Arya et al., 2001),
all have been shown to increase the stability of the triplex effectively. Even
though the formation of triplex is slow and less stable than the formation of
duplex, this structure has received much attention as an antigene strategy.
Triplex forming oligonucleotide has served as a potential genetic drug
regulating the gene expression after binding. Triplex formation also performs
numerous functions in biological systems. The replication of polynucleotide is
inhibited by the third-strand binding. In addition, HIV DNA has been reported
to be inhibited from integrating into host genomes (Bouziane et al., 1996).
Cooney and his coworkers have demonstrated the specific inhibition of
transcription by means of triplex formation at the poly(purine).poly(pyrimidine)
sites in the promoter regions (Cooney et al., 1988). Because of the numerous
activities the triplex may exhibit in the biological systems, increasing its stability
has been the focus of much research, with many endeavors being conducted
using small ligands. Intercalators such as acridines, ethidium, proflavine, and
ruthenium complexes (Wilson et al.,1994; Cassidy et al., 1994; Choi et al.,
1997; Mergny, et al., 1991), as well as DNA minor groove-binding ligands such
as berenil, netropsin, and Hoechst 33258 (Durand et al., 1994a; Park and
Breslauer, 1992; Durand et al, 1994b), have been shown to bind to DNA triplex.
However, those ligands do not bind to the triplex selectively, and some of them
32
even destabilize it. Benzo(e)pyridoindole, the first triplex-specific ligand
described (Mergny et al., 1992), binds to the triplex through an intercalation
mechanism, increasing the melting temperature of triplex over the duplex
significantly. Aminoglycosides have been shown in Arya’s laboratories to
stabilize triplex nucleic acids both selectively and significantly. Among these
aminoglycosides, neomycin exhibits the most potent effect, while, neomycin is
the first groove-binding ligand to result in triplex stabilization effect. Molecular
modeling has suggested that neomycin binds within the Watson-Hoogsteen
groove (Arya et al., 2003) formed between the third strand and the pyrimidine
strand.
The formation of a tetraplex or a quadruplex DNA structure has been
suggested as a potential chemotherapeutic target of a new antitumor
compound. This structure is formed from the tandemly repeating guanine
clusters, present primarily in the single stranded segment at the 3’ ends of
chromosomal DNA and characterized by a stacked arrangement of several G4
planar tetrads in which each G interacts with the adjacent one via forces of the
Hoogsteen bonds. These G-rich sequences occur frequently in human genomes
and in many other organisms (Huppert and Balasubramanian, 2005; Todd et
al., 2005). The formation of G-tetraplex has been reported to exhibit many
biological functions. For example, G-tetraplex has been found to exist at the
promoter region of oncogenes such as c-myc (Simonsson et al., 1998). Of the
many G-rich sequences in the organisms, telomere has been the primary focus
over the past few years due to its important biological role and its potential as
33
an antitumor target. Targeting the G-tetraplex with small ligands to improve its
stability has been the subject of much recent research. One of the first studies
involved the binding of ethidium bromide to the G-tetraplex d(T4G4)4 (Guo et
al., 1992). Arya’s laboratory has studied the binding of aminoglycosides,
specifically neomycin, to the G-tetraplex d(T2G20T2)4 using sodium and a pH of
6.8.
A huge number of studies have been reported on vibrational dynamics
of biopolymer system having different confirmations (Prasad et al., 1996; Gupta
et al., 2008; Srivastav et al., 1997; Krishnan and Gupta, 1970a, 1970b; Singh
and Gupta, 1971; Dwivedi and Gupta, 1972; Gupta et al., 1973; Burman et al.,
1995a, 1995b; Gupta et al., 1995). The analysis in most of these cases is based
on separation of the vibrational spectrum into group and skeletal vibrations.
The former are taken from computationally fitted IR and Raman data and the
latter by using the two-parameter Tarasov model (Wunderlich and Bu, 1987)
and fitting to low temperature heat capacities. The vibrational dynamics of
poly(L-glutamine) was interpreted by LaVerne and coworkers from the
dispersion and dispersion profile of the normal modes of poly(L-glutamine) as
obtained by Higg’s method for infinite systems (LaVerne et al., 2009).
Additionally, the dispersion curves give information of the degree of coupling
and the dependence of the frequency of a given mode on the sequence length
of ordered conformation. These curves also facilitate a correlation of the
microscopic properties, such as specific heat, free energy and enthalpy.
1.4. Methods used in previous studies and in present work
34
Advancement in the innovation of nucleic acid binding drugs obviously
relies on the availability of analytical methods or procedures that judge the
efficiency and nature of interactions between nucleic acids and their putative
ligands. A range of novel methods for these studies have emerged in current
years, and methods for studying DNA/Drug interactions highlights new and
non-conventional methods for exploring nucleic acid/ligand interactions. The
structure of these systems can be studied experimentally as well as
theoretically. From the conventional UV-VIS spectrophotometry to the
improved gel mobility electrophoresis assay or the powerful HPLC-MS, the
number of novel specific assays and methodologies is overwhelming (Tian et
al., 2005). Her, I will describe some of the most useful techniques used for
drug-DNA interaction studies. Vibrational spectroscopy such as Infrared
spectroscopy (IR) has been widely used for the structural analysis of DNA
because it can distinguish among A-, B-, and Z-forms of DNA, triple stranded
helices, and other structural motifs. It has also been a useful tool to study
interactions of nucleic acids with drugs and the effects of such interactions in
the structure of DNA, providing some insights about the mechanism of drug
action. A major advantage is that samples can be analyzed in different
aggregation states as solids or crystals, and also in solution. In addition, small
quantities of sample are needed and collection of spectra is very fast. The
region of interest in IR studies dealing with DNA in aqueous solutions is
between 1800 and 800 cm-1. Due to interfering absorption bands of water at
1650 cm-1 and below 950 cm-1, spectra are generally recorded in D2O, where
these bands move to 1200 cm-1, and below 750 cm-1. Fourier transform IR
35
(FTIR) has been used alone or supporting other techniques to determine drug
binding sites and sequence preference, as well as conformational changes due
to drug-DNA interaction (Jangir et al., 2010; Mandeville et al., 2010; Neault and
Tajmir-Riahi, 1996). Raman spectroscopy also depends on the vibrational
frequencies of characteristic groups and has been used sometimes in
conjunction with infrared spectra to study DNA-drug interactions.
Nuclear Magnetic Resonance (NMR) spectroscopy is based on the fact
that atomic nuclei endowed with a property called nuclear spin will align with
an applied magnetic field. The degree of this alignment depends not only on
the strength of the magnetic field, but also on the type of nucleus and its
chemical environment. Every nucleus with spin gives rise to a signal or peak
which represents a transition between a ground and an excited state. Each
magnetically active nucleus is characterized by different parameters such as
chemical shift, multiplicity, J-couplings and relaxation data that can be used to
obtain detailed structural information about the molecule under study. Among
the atomic nuclei available for the study of DNA (1H, 13C, 15N and 31P), 1H is
the most common, but 31P NMR is especially useful for studying the effects of
ligand binding on the phosphate groups of DNA. NMR experiments are very
versatile and the information can be obtained at different temperatures,
solvents, pH values, ionic strengths and dielectric constants. DNA-binding drugs
specific for the minor groove of DNA generally prefer AT-rich, rather than GC-
rich regions of DNA. These drugs are usually planar with crescent shapes. The
DNA minor groove possesses an electrostatic potential minimum attractive to
36
many such ligands. Many anti-tumour drugs bind to the major groove, and they
usually do it covalently through N-7 of guanine but their modes of interaction
have been studied with techniques different from NMR (González-Ruiz et al.,
2011).
The interaction of drugs with DNA can be also detected by UV-VIS
absorption spectroscopy on the basis of absorption properties of the drug or
the DNA molecules. The UV-VIS absorption spectrum of DNA exhibits a broad
band (200-350 nm) in the UV region with a maximum absorbance at 260 nm.
This maximum absorbance is a result of the chromophoric groups present in
purine and pyrimidine moieties responsible for the electronic transitions. The
application of this versatile and simple technique allows estimating the molar
concentration of DNA on the basis of the measurement of the absorbance value
at 260 nm. In practice, the molar concentration of DNA is evaluated in terms
of the concentration of base pairs. The absorbance ratios (A260/A280) can also
characterize the DNA molecules (Paul et al., 2010). Slight changes in the
absorption maximum as well as the molar absorptivity can be appreciated with
the variations in pH or ionic strength of the media. The ε values (λmax= 260
nm) of free oligonucleotides are higher than the ones corresponding to the
same oligonucleotides in single strand DNA (ss-DNA) and double strand DNA
(ds-DNA) because base-base stacking results in a hypochromic effect. This
behavior can be exploited to verify denaturalization of DNA by measuring its
absorbance values before and after denaturing treatment. The hypochromic
effect can also be employed to verify the existence of drug-DNA interactions,
37
due to the fact that the monitoring of the absorbance values allows studying
the melting behaviour of DNA. Melting temperature (Tm) is the temperature
value corresponding to the conversion of 50 % of the double strands into single
strands. An increase in the absorbance value with the increase of temperature
is observed because the ε (260 nm) of ss-DNA is higher than the ε (260 nm) of
ds-DNA. When a drug–DNA interaction exists, Tm is shifted to values different
from native ds-DNA. The magnitude of the shift depends on the type of
interaction. Thus, for intercalating agents the increase observed in the Tm value
is higher than in the case of agents interacting through the DNA minor or major
grooves (González-Ruiz et al., 2011). The changes in the Tm value can be
followed by other techniques such as fluorescence, circular dichroism, NMR or
calorimetry, but UV-Vis absorption spectrometry is the most frequently
employed method due to its good sensitivity, versatility and simplicity. Drug-
DNA interactions can be resolved by comparison of UV-VIS absorption spectra
of the free drug and drug-DNA complexes, which are usually different.
Circular dichroism (CD) spectroscopy is valuable techniques to explore
non-covalent drug-DNA interactions, which affect the electronic structure of the
molecules and also modify their electronic spectroscopic behavior. Circular
dichroism spectroscopy allows to rapidly characterize drug-DNA complexes
using a small amount of sample. Circular dichroism is defined as the difference
in absorption of left and right circularly polarised light. Circular dichroism
provides additional structural details of the complex. When electromagnetic
radiation reaches DNA, the macromolecules present a certain degree of
38
alineation in the direction of the electric field vector, and this molecular
alignment is measured by the light polarised absorbance. When a drug binds
to DNA, its spectrum will be modified if this binding causes changes in DNA
conformation. When a drug binds to DNA, an induced CD spectrum is observed
because of the interaction with DNA. This may result from either a geometric
change in the drug or from coupling between its electronic transitions and those
of the DNA. Particularly the electronic CD has become very significant for
qualitative characterization of conducting biopolymers.
Differential scanning calorimetry (DSC) is a technique capable to study
thermally induced transitions and particularly, the conformational transitions of
biological macromolecules. There are number of literature support applications
of this technique (Robertson and Murphy, 1997; Privalov and Potekhin, 1986;
Biltonen and Freire, 1978; Freire, 1995; Lopez and Makhatadze, 2002;
Jelesarov and Bosshard, 1999; Cooper and Johnson, 1994; Hinz and Schwarz,
2001). DSC is used to understand the molecular mechanism and energetics of
drug-DNA interactions. Statistical mechanical theories were developed by
Crothers and McGhee allowing interpretation and calculation of DNA melting
curves in the presence of ligands (Spink and Wellman, 2001; Crothers, 1971;
McGhee, 1976). The combination of DSC and temperature dependent UV
melting methods allowed the first direct determination of the binding enthalpy
for echinomycin (Leng et al., 2003). DSC was also applied to understand
interaction of distamycin with ColE1 DNA, a well characterized plasmid (Ueta et
al., 2001). DSC measurements were conducted to characterize the thermally
39
induced denaturation of the 20-bp duplex with the specific goal of elucidating
the thermal and thermodynamic consequences of modifying and constraining
DNA via a single, site-specific interstrand cross-link of antitumor cis-platin or its
clinically ineffective trans isomer (Hofr and Brabec, 2001). The interaction of
other drugs, like chlorobenzylidin (Zhong et al., 2003), phenoxazone (Veselkov
et al., 2003) or CP-31398 (Rippin et al., 2002), an anticancer drug, with DNA
has been also investigated by differential scanning calorimetry.
In the present work, we have attempted to understand the effect of
drugs/small molecules binding on synthetic and native deoxyribonucleic acids
through the theoretical analysis. We used amended Zimm and Bragg theory,
initially considered for helix coil transitions in polypeptides, to explain lambda
point anomalies in heat capacities and order-disorder transition in drug
bounded and unbounded DNAs (Zimm and Bragg, 1959). The effect of drugs
or small molecules binding is reflected in the change in nucleation parameter,
which is an inverse measure of binding strength. Our study is aimed at
providing such a theoretical protocol for complementing experimental
techniques and facilitating a minute study of the structure-energy relationships
in DNA-drug complexes.
1.5. Chapter wise presentation of the work done
There are total five chapters in the present thesis which compiled all the
methodology and results of the present work including conclusion. Chapter 1
40
consists of detailed introduction of the thesis and chapter 2 comprises complete
experimental and theoretical approaches used in the next three chapters.
The third chapter of this thesis deals with theoretical analysis of steroid
diamine (Dipyrandium), binding with DNA duplex by using an amended Zimm
and Bragg theory, to explain the melting behavior and heat capacity of DNA
with and without dipyrandium binding. The experimental models of Marky and
coworkers (1983) have been used for the study. Chapter 4 of the thesis deals
with the similar work done on theoretical analysis of tricyclic heteroaromatic
molecule (Thionine) binding with natural DNAs of varying base composition by
using an amended Zimm and Bragg theory, to explain the melting behaviour
and heat capacity of DNAs with and without thionine binding. We used
experimental models of Paul et al. for implementing this study (Paul et al.,
2010). Chapter five presented the conclusion of the entire work. This is the last
chapter which includes all the pros and cons of the present work.
41
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53
CHAPTER 2
THEORY
54
Molecular interaction of the drugs with DNA and peptides is a field of
high topical interest and may have a great importance to its biological activity.
In the present chapter the theoretical methods for phase transition studies are
discussed. In this section the theory for order–disorder phase transition is
given. It includes the theoretical formalism of phase transition in telomeres as
a function of temperature and concomitant changes in the heat capacity at the
transition point which shows lambda anomaly. The study includes methods
involve in theoretical analysis of Dipyrandium and Thionine binding with
different natural and synthetic DNA. We used amended Zimm and Bragg theory
(Zimm and Bragg, 1959), to elucidate the order-disorder phase transition in
drug bounded and unbounded DNA duplexes, which is considered initially for
the helix coil transitions in polypeptides. The effect of drug binding is reflected
in the change in nucleation parameter, which is an inverse measure of binding
strength.
Phase Transition Theory
The models of drug-DNA complex determined by the various techniques
suggest that the Watson-Crick hydrogen bonds are intact in the neighbor-
exclusion complex and that every other set of base pairs partially unstacks and
the drugs are partially inserts at this site. However, the system remains a highly
co-operative one therefore the co-operative transition theory could be applied
to explain the melting profile and temperature dependence of thermodynamical
parameter, such as heat capacity. Briefly, Zimm and Bragg theory consists of
55
writing an Ising matrix for a two-phase system, the bounded state and
unbounded state. The Ising matrix M can be written as:
𝑓𝑟 𝑓𝑘 𝑓ℎ
M =
𝑓𝑟𝑓𝑘𝑓ℎ
|
𝑓𝑟1/2𝑓𝑟
1/2 𝑓𝑟1/2𝑓𝑘
1/2 0
𝑓𝑟1/2𝑓𝑘
1/2 0 𝑓𝑘1/2𝑓ℎ
1/2
𝑓ℎ1/2𝑓𝑟
1/2 0 𝑓ℎ1/2𝑓ℎ
1/2
| (1)
where fr, fh and fk are corresponding base pair partition functions’ contributions
in the three states i.e. ordered, disordered and boundary or nucleation. The
eigen values of M are given by:
𝜆1 = [(𝑓𝑟+𝑓ℎ) + {(𝑓𝑟−𝑓ℎ)2 + 4𝑓𝑟𝑓𝑘}
1/2]/2
𝜆2 = [(𝑓𝑟+𝑓ℎ) − {(𝑓𝑟−𝑓ℎ)2 + 4𝑓𝑟𝑓𝑘}
1/2]/2𝜆3 = 0
(2)
Since we are dealing with a finite system hence the effect of initial and
final states becomes important. The contribution of the first segment to the
partition function is given by:
𝑈 = (𝑓𝑟1/2, 0,0) (3)
where the column vector V gives the state of the last segment,
V = |
𝑓𝑟1/2
𝑓𝑘1/2
𝑓ℎ1/2
| (4)
The partition function for an N-segment chain is given by;
56
𝑍 = 𝑈𝑀𝑁−1𝑉 (5)
The matrix T which diagnolizes M consists of the column vectors given by:
𝑀𝑋 = 𝜆𝑋 (6)
Where 𝑋 = |𝑋1𝑋2𝑋3
|
By substituting the values of M from Eq. (6), we get:
𝑇 = ||
1 1 1(𝜆1−𝑓𝑟)
(𝑓𝑟1/2
𝑓𝑘1/2
)
(𝜆1−𝑓𝑟)
(𝑓𝑟1/2
𝑓𝑘1/2
)−(𝑓𝑟
1/2𝑓𝑘1/2
)
(𝑓ℎ1/2
𝑓𝑟1/2
)
(𝜆1−𝑓ℎ)
(𝑓ℎ1/2
𝑓𝑟1/2
)
(𝜆1−𝑓ℎ)−(𝑓ℎ
1/2𝑓𝑟1/2
)
|| (7)
Similarly, we get T-1 from the matrix equation
𝑌𝑀 = 𝜆𝑌 (8)
where, 𝑌 = [𝑌1,𝑌2,𝑌3]
Again by substituting the values of M from Eq. (1) in Eq. (8), we get:
𝑇1 =|
|𝐶1
𝐶1(𝑓𝑟1/2
𝑓𝑘1/2
)
𝜆1
𝐶1(𝑓𝑘𝑓𝑟1/2
𝑓ℎ1/2
)
𝜆1(𝜆1−𝑓ℎ)
𝐶2𝐶2(𝑓𝑟
1/2𝑓𝑘1/2
)
𝜆2
𝐶2(𝑓𝑘𝑓𝑟1/2
𝑓ℎ1/2
)
𝜆2(𝜆2−𝑓ℎ)
𝐶3𝐶3(𝑓𝑟
1/2𝑓𝑘1/2
)
𝜆3
𝐶3(𝑓𝑘𝑓𝑟1/2
𝑓ℎ1/2
)
𝜆3(𝜆3−𝑓ℎ)
|
| (9)
The normalization constants are:
57
𝐶1 = (𝜆1 − 𝑓ℎ)/(𝜆1 − 𝜆2), 𝐶2 = (𝜆2 − 𝑓ℎ)/(𝜆2 − 𝜆1), 𝐶3 = 0 (10)
If we let Λ = 𝑇−1𝑀𝑇 be the diagonalized form of M, the partition
function can be written as:
𝑍 = 𝑈𝑇𝛬𝑁−1𝑇−1𝑉 (11)
On substituting the values from Eqs (1), (3), (4), (7), (9) and (10) in
Eq. (11), the partition function becomes:
𝑍 = 𝐶1𝜆1𝑁 + 𝐶2𝜆1
𝑁 (12)
The fraction of the segments in the disordered form is given by
𝑄𝑟 = [𝛿𝑙𝑛𝑍/𝛿𝑙𝑛𝑓𝑟]/𝑁
Solving the above equation, we get:
𝑄𝑟 = 1/2 + (1 − 𝑠)(2𝐴 − 1)/2𝑃 + (1 + 𝑠){(2𝐴 − 1)𝑃 − 1 + 𝑠}/2𝑃2𝑁
……………….(13)
where 𝑃 = (𝜆1 − 𝜆2)/𝑓𝑟 , 𝑠 = 𝑓ℎ/𝑓𝑟 , 𝜎 = 𝑓𝑘/𝑓𝑟 , 𝐴 = [(𝑓𝑟 − 𝑓ℎ)2 +
4𝑓𝑘𝑓𝑟]−2
Here s is propagation parameter, which for simplicity is assumed to be
1. In fact, in most of the systems, it is found to be close to unity. If Ar and Ah
are the absorbance in disordered and ordered states, respectively, the total
absorption can be written as:
58
𝐴 = 𝑄𝑟𝐴𝑟 + (1 − 𝑄𝑟)𝐴ℎ (14)
The extension of this formalism to specific heat (Cv) is straight forward.
The specific heat is related to the molar enthalpy and entropy changes in the
transition from state I to II. From the well-known thermodynamic relations,
free energy and internal energy are 𝐹 = −𝐾𝑇𝑙𝑛𝑍 and𝑈 = −𝑇2(𝛿/𝛿𝑇)(𝐹/
𝑇), respectively. Differentiating internal energy with respect to temperature
we get the specific heat:
𝐶𝑣 = 𝛿𝑈/𝛿𝑇 = 𝑁𝑘(Δ𝐻/𝑅𝑇𝑚)2(𝑆𝛿𝑄𝑟/𝛿𝑆) (15)
Where ∆H is the molar change in enthalpy about the transition point, S is
entropy which is equal toS = exp[(∆H/R){(1/T) − (1/Tm)}], Tm is the
transition temperature, and
𝛿𝑄𝑟/𝛿𝑆 = (1/2𝑃2)[2𝑃(1 − 𝑆)𝛿𝐴/𝛿𝑆 − 𝑃(2𝐴 − 1)− (1 − 𝑆)(2𝐴
− 1)𝛿𝑃/𝛿𝑆] + (1/2𝑃3𝑁)[𝑃{(𝑆 + 1){(2𝐴 − 1)𝛿𝑃/𝛿𝑆
+ 2𝑃𝛿𝐴/𝛿𝑆 + 1} + {(2𝐴 − 1)𝑃 − 1 + 𝑆}} − {(2𝐴 − 1)𝑃
− 1 + 𝑆}2(𝑆 + 1)]
with 𝛿𝐴/𝛿𝑆 = {(𝑆 − 𝜎)𝑁/(𝑍/𝑓𝑟𝑁)2} × (𝜎/𝑃3) × [−2 + {𝑁(𝑆 − 2𝜎 −
1)/𝑆 − 𝜎}]
𝛿𝑃/𝛿𝑆 = (𝑆 − 1)/𝑃 and 𝜎 = 𝑓𝑘/𝑓𝑟; σ is the nucleation parameter
and is a measure of the energy expanded/released in the formation (uncoiling)
of first turn of the ordered/disordered state. It is related to the uninterrupted
59
sequence lengths (Zimm and Bragg, 1959). The volume heat capacity Cv has
been converted into constant pressure heat capacity Cp by using the Nernst-
Lindemann approximation (Roles and Wunderlich, 1991) :
𝐶𝑝 − 𝐶𝑣 = 3𝑅𝐴0(𝐶𝑝2𝑇/𝐶𝑣𝑇𝑚) (16)
where A0 is a constant often of a universal value [3.9×10-9 (Kmol)/J-1] and Tm
is the melting temperature.
References:
Pan R., Verma M.N. and Wunderlich B. (1989). J. Therm. Anal., 35: 955.
Roles K.A. and Wunderlich B. (1991). Biopoly., 31: 477-487.
Tandon P., Gupta V.D., Prasad O., Rastogi S. and Gupta V.P. (1997). J. Polym.
Sc. Part B Polym. Phys. 35: 2281.
Zimm B.H. and Bragg J.K. (1959). J. Chem. Phys., 31: 526-535.
60
CHAPTER 3
TRANSITION AND
STABILITY OF DNA–
DIPYRANDIUM COMPLEX
61
3.1. Introduction
Molecular interaction of the drug with DNA is a field of high topical
interest and may have a great importance to its biological activity. The
molecules can interact with DNA in a variety of ways such as surface binding
to their minor or major grooves, intercalation between adjacent base pairs,
covalent attachments to the double helix, or electrostatic binding. The study of
drug-DNA interactions began since 1960s but the binding of steroid-diamines
to synthetic and natural nucleic acids has received considerable attention over
the past ten years (Mahler and Dutton, 1964; Mahler et al., 1966, 1968; Waring,
1970; Gabbay and Glaser, 1971; Waring and Chisholm, 1972; Lane, 2001;
Bruylants et al., 2005; Araya et al., 2007; Islam et al., 2009; Paul et al., 2010;
Gonzalez-Ruiz, 2011; Kunwar et al, 2011). Thus, some more theoretical and
experimental studies are conducted based on the assumption that the steroid
diamine binding may ‘kink’ DNA as suggested by Sobell et al. (Waring and
Henley, 1975; Sobell, 1977; Saucier, 1977; Dattagupta, 1978; Manning, 1979;
Patel and Canuel, 1979; Patel, 1979; Lane, 2001; Bruylants et al., 2005; Araya
et al., 2007; Islam et al., 2009; Paul, 2010; Gonzalez-Ruiz, 2011; Kunwar et al,
2011). The naturally occurring steroidal diamine, dipyrandium, is amphiphilic in
nature, and its biophysical features include increasing the duplex denaturation
temperature and altering of the UV and CD spectra of DNA.
Dipyrandium (Fig. 3.1) is an aminosteroid neuromuscular blocking agent
that revolutionized the performance of anesthesia. The relaxant action of
dipyrandium chloride in man was studied by (Mushin and Mapleson, 1964). The
62
binding of dipyrandium to DNA is proposed to occur in the minor groove in
conjunction with 5'-d(TA) kinks (Patel and Canuel, 1979). In view of the
potential importance of such a molecular distortion to drug binding, interest in
and investigations of steroid diamine-DNA complexation has greatly increased.
The aminosteroid-DNA complex model suggested that the Watson-Crick
hydrogen bonds are intact in the neighbor-exclusion complex and that every
other set of base pairs partially unstacks and the steroid diamines partially
inserts at this site (Sobell et al., 1976). Hui and his co-workers studied binding
modes analysis of dipyrandium to the double-stranded hexanucleotides
d(TATATA)2, d(ATATAT)2, and d(CGCGCG)2 by the energy minimization
procedure JUMNA. The binding of dipyrandium to d(CGCGCG)2 is found to be
considerably less favourable than for either of the two (AT) sequences (Hui et
al., 1989). The interaction of dipyrandium with DNA and its dependence on the
base sequence was also studied by Vorlícková et al. using circular dichroism
(Vorlícková et al., 1985). They suggested that calf thymus DNA and
polynucleotide duplexes underwent similar alterations in the chiroptical
properties upon binding of dipyrandium. These results recommend that these
DNAs have similar B-type structures which may kink at the dipyrandium binding
sites. Alternatively, poly(dA-dT).poly(dA-dT) and especially poly(dA-
dU).poly(dA-dU) exhibit some features of A-type structure. Poly(dA-
dT).poly(dA-dT) changes its chiroptical properties little when complexed with
dipyrandium, as if it contained some type of kinks as equilibrium structural
elements (Vorlickova et al., 1985).
63
The calorimetric analysis of aminosteroid binding with DNA duplex was
reported for the first time by Marky et al. (1983). They recommended that
increasing the concentration of bound steroid increases the thermal stability of
the duplex and at saturation; the duplex melts with a Tm some 20°C above that
of the free duplex (Marky et al., 1983). They also concluded that dipyrandium
binding to poly d(AT) is an endothermic process and this binding increases the
melting temperature of the duplex. In the present investigation, we have
attempted to understand the effect of steroid diamine binding on a DNA duplex
by using the experimental model of Marky and his coworkers (Marky et al.,
1983) who studied the thermal and thermodynamic behaviour of dipyrandium
binding to poly d(AT). We used amended Zimm and Bragg theory, to elucidate
the order-disorder transition in dipyrandium bounded and unbounded DNA
duplexes, which is considered initially for the helix coil transitions in
polypeptides (Zimm and Bragg, 1959; Srivastava et al., 1999, 2001). We also
explained lambda point anomalies in heat capacities by using the same theory.
The effect of dipyrandium binding is reflected in the change in nucleation
parameter, which is an inverse measure of binding strength.
Figure 3.1. Molecular structure of dipyrandium
64
3.2. Theory
The model proposed by Sobell for the aminosteroid-DNA complex on the
basis of NMR study, as mentioned above, suggested that the Watson-Crick
hydrogen bonds are intact in the neighbor-exclusion complex and that every
other set of base pairs partially unstacks, and the steroid diamines partially
inserts at this site (Sobell et al., 1977). However, the system remains a highly
co-operative one therefore the co-operative transition theory could be applied
to explain the melting profile and temperature dependence of thermodynamical
parameter, such as heat capacity. Therefore, we used amended Zimm and
Bragg theory (1959) which is presented in chapter 2.
3.3. Experimental model
In this study, we used experimental model of Marky et al. (1983) to
understand the effect of dipyrandium binding to poly d(AT).
3.4. Results and discussion
As stated above the DNA structure still remains a very much co-
operative once binds to dipyrandium and consequently two-state theory of
order-disorder transition is applicable. The hypothesis given by Zimm and
Bragg (1959) is amended so as to consider ordered (bounded/unbounded) and
disordered states as the two states which co-exist at the transition position.
The transition is characterized mainly by the nucleation parameter and overall
change of enthalpy/entropy, which are also the main thermodynamic forces
65
driving the transition. The change in enthalpy obtained from differential
scanning calorimetric (DSC) measurements and changes in other transition
parameters, such as nucleation parameter (σ) and melting point (Tm), takes
all this into account. This may be further elaborated that, the enthalpy change
(∆H) on binding of dipyrandium is maximum (7.6 Kcal/M bp) in case of
nucleotide to drug ratio infinity and minimum (5.09 Kcal/M bp) at the ratio of
5:1, as shown in table 3.1. It may also be concluded that by increasing the
drug concentration the change of enthalpy is decreases gradually (Table 3.1).
The stability of duplex may be determined by its melting temperature (Tm) and
it is increases with increasing concentration of bounded dipyrandium. The
melting temperature for free duplex was 46.5°C and when the duplex was
saturated with dipyrandium, it melts with a Tm some 28°C above that of the
unbounded duplex (Table 3.1). The drug-induced increase in the thermal
stability of the duplex is accompanied by a decrease in the overall transition
enthalpy of the duplex. The sharpness of the transition can be looked at in
terms of half width and a sensitivity parameter defined as (∆H/σ). The variation
of these parameters systematically reflects that the transition is sharpest in
case of unbounded state and goes in order to nucleotide to dipyrandium ratio
of 17:1, 10:1 and 5:1. In case of λ-transition, the same trend in the sharpness
of transition is seen between the dipyrandium bounded as well as unbounded
curves. As expected, the sharpness is better in unbounded, as compared to
bounded state. The various parameters, which give transition profiles in best
agreement with the experimental measurements for binding of dipyrandium,
are presented in table 3.1.
66
The heat capacity and transition profiles for unbounded nucleotide, poly
d(AT), and with different concentration of aminosteroid, dipyrandium, are
shown in figure 3.2. In case of heat capacity curve some insignificant
divergence are reported at the tail ends may be due to presence of different
disordered states, as these states cannot be distinctively defined. Additionally,
these variations could also be arises due to the presence of short helical
segments found in the random coil states. The experimental and calculated
transition curves for the duplex to single strand transition of poly d(AT) in the
absence of dipyrandium are shown by the graph ‘A’ in figure 3.2. Similarly,
graph ‘B’ and ‘C’ shows the transition in the presence of dipyrandium at
nucleotide to drug ratios of 17:1 and 10:1, respectively.
67
Table 3.1. Transition parameters for dipyrandium binding to poly d(AT)
Parameters
Phosphate/Drug (P/D) ratio
∞* 17/1 10/1 5/1
Melting Temperature (K)
319.5 323.2 331.57 347.5
∆H Kcal/M bp 7.6 6.61 6.43 5.09
σ 4x10-4 1x10-3 3x10-3 3.5x10-3
N 106 106 106 50
Ah 0 0 0 0
Ar 1 1 1 1
Sensitivity
parameter (∆H/σ)
19000 6610 2143.33 1454.28
*Unbounded
68
Fig. 3.2. Heat capacity and transition profiles (inset) for unbounded and
bounded DNA with dipyrandium. (A) Unbounded state, (B) Bounded state with
P/D ratio 17/1, (C) Bounded state with P/D ratio 10/1, (D) Bounded state with
P/D ratio 5/1. [(—) calculated and (••••) experimental values].
69
In figure 3.2, graph ‘D’ shows the poly d(AT) transition when the duplex
is saturated with dipyrandium (nucleotide to drug ratio of 5:1). There is a
cooperative transition profile in between calculated and experimental data that
was anticipated from the results. The results presumed from the theoretical
data is reliable with the experimental data of Marky et al. (1983) that was
directly measured binding enthalpy through differential scanning calorimetric
method. The presented data of theoretical analysis also demonstrated that
dipyrandium binding to DNA duplex is an endothermic process and this binding
increases the melting temperature of the duplex as supported by differential
scanning calorimetric measurement. Marky and coworkers have used DSC to
investigate the binding of dipyrandium to the poly d(AT) duplex (Marky et al.,
1983). However, Patel and Canuel used NMR for the same study (Patel and
Canuel, 1979).
In view of that, we can interpret our theoretical analysis results in the
perspective of the particular structural features of the drug-DNA complex as
deduced from their experimental data obtained through DSC/NMR study. It is
evident from figure 3.2 (graph A and D) that the transition of the dipyrandium
saturated DNA duplex is significantly broader than the transition of the
dipyrandium free DNA duplex. Therefore, it is clear that besides affecting the
enthalpy and melting temperature, binding of dipyrandium also alters the
nature of the transition as reflected by the increase in transition width in
experimental as well as calculated data (Figure 3.2). Heat capacity is a second
derivative of the free energy and has been calculated by using equation 15
70
(described in chapter 2). By using heat capacity, the dynamical and
conformational states of a macromolecular system are characterized. The heat
capacity curves, obtained from theoretical and experimental data, along with
their transition profile are shown in figure 3.2. From these curves it may be
concluded that the theoretically obtained heat capacity profiles agreed with
the experimentally reported ones and could be brought almost into coincidence
with the use of scaling factors, which is very close to one in transition profiles
and slightly higher for the heat capacity curves. In addition, the sharpness of
the transition can also be characterized by the half width of the heat capacity
curves. The half width of these heat capacity curves are also calculated which
is in good agreement in both theoretical and experimental graphs (Figure 3.3).
Fig. 3.3. Half width of the heat capacity curves
2
3
6.5
8.5
2
3
6.5
8.5
0
1
2
3
4
5
6
7
8
9
10
∞ 17/1 10/1 5/1
Half w
idth
valu
e
P/D ratio
Half width (Exp.)
Half width (Theo.)
71
3.5. Conclusion
In recent years, the study of nucleic acid interaction with molecules is of
high interest (Araya et al., 2007; Gonzalez-Ruiz et al., 2011; Islam et al., 2009;
Kunwar et al., 2011; Paul et al., 2010). The present study concluded that the
DNA molecule is an extremely co-operative structure and when dipyrandium
binds to it the co-operativity of DNA is not so much disturbed. Therefore, the
amended Zimm and Bragg theory can be effectively applied to it. It generates
the experimental transition profile and λ-point heat capacity anomaly
successfully. The results obtained will allow us to evaluate the thermodynamic
profile of dipyrandium interaction with DNA (dipyrandium-DNA complex). This
theoretical analysis of steroid diamine binding with nucleotide is being extended
to other synthetic and natural DNA polymers. This theoretical analysis can also
be useful in order to understand bimolecular interaction and thus can be applied
in the process of drug design and development.
72
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75
CHAPTER 4
THIONINE INDUCED
THERMAL STABILITY OF
NATURAL DNAS
76
4.1. Introduction
In recent years the study of small molecules interaction with
deoxyribonucleic acids is a research area of prominent interest. There are
number and variety of techniques devoted to evidence drug-DNA interactions
which are increasing continuously (Perez-Montoto et al., 2009; Gonzalez-Diaz
et al., 2009; Panigrahi and Desiraju, 2007; Rohs et al., 2005). To understand
the interactions between small molecules and biomacromolecules, Tian et al.
summarize some general methodology used in these studies (Tian et al., 2005).
Over the past several years, the interaction of aromatic heterocyclic molecules
with deoxyribonucleic acids has received considerable attention due to their
relevance in a range of biological applications including cancer chemotherapy
(Hurley, 2001, 2002; Martinez and Chacon-Garcia, 2005; Palchaudhuri and
Hergenrother, 2007; Maiti and Kumar, 2007; Paul, et al., 2010; Bhadra and
Kumar, 2011). Accordingly, a number of experimental studies are conducted to
understand the nature and thermodynamics of heterocyclic aromatic molecules
and nucleic acid interaction (Ihaya and Nakamura, 1971; Kumar and Asuncion,
1993; Tuite and Kelly, 1993, 1995; Jockusch, et al., 1996; Dohno, et al., 2003;
Du et al., 2005; Xu, 2006).
Thionine (3,7-Diamino-5- phenothiazinium), a tricyclic heteroaromatic
molecule (Fig. 4.1), has been studied for its intercalative binding with DNA
duplex (Tuite and Kelly, 1995) and its photo-induced mutagenic actions on
binding to DNA (Dohno et al., 2003). According to Chang et al. (2005) thionine
binds specifically with guanine–cytosine (GC) content of DNA duplex as
77
suggested by satellite hole spectroscopy study. However, Tuite and Kelly (1995)
recommended that (GC) specificity of thionine binding was not very marked.
The other study suggested that thionine exists in two different tautomeric forms
viz. amino form and imino form and only amino form is intercalated while the
imino form is not (Hecht et al., 2004, 2005). Another study carried out through
pressure tuning hole burning spectroscopy has conclusively shown an external
stacking mode of thionine binding to quadruplex structures (Hecht et al., 2004).
All these studies provide somewhat contradicting data on the GC specificity of
thionine binding to linear double stranded DNA. Although some structural data
is available, the same is not conclusive and the thermodynamics of thionine
binding to DNA was not elucidated. Recently, the calorimetric analysis of
thionine binding to natural DNAs of varying base composition along with the
structural and thermodynamic aspects of the interaction has been reported by
Paul et al. (2010). These authors recommended strong binding of thionine with
DNAs which increases the thermal stability of the duplex. They also concluded
that binding of thionine to DNA of varying base composition is an exothermic
process and there is involvement of multiplicity of non-covalent interactions in
the binding process. In the present study, we have attempted to understand
the effect of thionine binding on native deoxyribonucleic acids through the
theoretical analysis using the experimental model of Paul et al. who studied the
thermal and thermodynamic behaviour of thionine binding to three natural
DNAs of varying base composition (Paul et al., 2010). We used amended Zimm
and Bragg theory, initially considered for helix coil transitions in polypeptides,
to explain lambda point anomalies in heat capacities and order-disorder
78
transition in thionine bounded and unbounded DNAs (Zimm and Bragg, 1959;
Srivastava et al., 1999, 2001). The effect of thionine binding is reflected in the
change in nucleation parameter, which is an inverse measure of binding
strength.
Fig. 4.1. Chemical structure of thionine
4.2. Theory
The spectroscopic and calorimetric study, carried out by Paul et al.
(2010) for the complex formation between thionine and three natural DNAs of
varying base composition, concluded that thionine binds strongly with DNAs of
different GC content (Clostridium perfringenes DNA, herring testes DNA and
Micrococcus lysodeikticus DNA) which resulted in thermal stabilization of the
complex. The binding of thionine to the DNAs was intercalative and was non-
cooperative that resulted in significant perturbation of the conformation of the
DNA as well as thermal stabilization (Paul et al., 2010). However, the system
remains a highly co-operative one; therefore the co-operative transition theory
could be applied to elucidate the melting profile and temperature dependence
79
of thermodynamic parameters, such as heat capacity. Therefore, amended
Zimm and Bragg theory (1959) may be used theoretical approach which is
described in chapter 2.
4.3. Experiment model
In this study, we used experimental model of Paul et al. (2010) to
understand the effect of thionine binding to natural DNA of various base
specificity.
4.4. Results and discussion
The spectroscopic and fluorescence studies of binding affinity of
thionine with Clostridium perfringenes DNA (Type XII, 27 mol% GC), herring
testes DNA (Type XIV, 41 mol% GC) and Micrococcus lysodeikticus DNA (Type
XI, 72 mol% GC) showed strong intercalative binding and enabled the
assumption of two state systems consisting of bound and free thionine (Paul
et al., 2010). When thionine, a phenothiazinium dye, binds with natural DNAs
of varying base composition as mentioned above, the structure of DNAs duplex
still remains a highly co-operative and thus two-state theory of order-disorder
transition is applicable. The Zimm and Bragg theory (1959) is amended so as
to consider ordered (bounded/unbounded) and disordered states as the two
states which co-exist at the transition point. The transition is characterized
mainly by the nucleation parameter and overall change of enthalpy/entropy,
which are also the main thermodynamic forces driving the transition. The
change in enthalpy obtained from differential scanning calorimetric (DSC)
80
measurements takes all this into account. This is evident from the change in
enthalpy and other transition parameters, such as nucleation parameter (σ)
and melting point as shown in table 4.1. The results obtained from theoretical
analysis recommended that the binding of thionine increases melting
temperature of DNAs at saturation point.
As shown in table 4.1, the melting point of Clostridium perfringenes DNA
(CP DNA), herring testes DNA (HT DNA) and Micrococcus lysodeikticus DNA
(ML DNA) was increased with 6.5, 9 and 9.7 K, respectively in comparison to
free duplexes. The sharpness of the transition was defined in terms of half
width and a sensitivity parameter (∆H/σ). The variations in half width and
sensitivity parameter scientifically reflect that the transition is sharp in case of
unbounded state of CP DNA, HT DNA and ML DNA; and goes blunt when these
DNAs are saturated with thionine. In case of λ-transition, the similar trends in
the sharpness of transition are also seen between the thionine bounded as
well as unbounded curves. As anticipated, the sharpness of transition is better
in unbounded CP DNA, HT DNA and ML DNA, as compared to bounded state
of these DNAs with thionine. The various parameters, which give transition
profiles in best agreement with the experimental measurements for binding of
thionine to DNAs of various base compositions, are given in Table 4.1.
81
Table 4.1. Transition parameters for thionine binding to various natural DNAs
Parameters CP DNA HT DNA ML DNA
Unbounded Saturated* Unbounded Saturated* Unbounded Saturated*
Tm (K) 345 351.5 353 362 365.5 375.2
∆H Kcal/M bp 3.06x103 3.24x10
3 6.41x10
3 9.72x10
3 4.84x10
3 5.81x10
3
Σ 3x10-4 5x10
-4 9x10
-3 2x10
-2 6x10
-4 7.5x10
-4
N 98 90 28 16 82 66
Ah 0 0 0 0 0 0
Ar 1 1 1 1 1 1
Half width (Exp.) 5 6.5 11 12 4.5 5
Half width (Theo.) 5 6.5 11 12 4.5 5
Sensitivity parameter (∆H/σ) 102x105 648x104 712x103 486x103 807x104 775x104
*Saturated with thionine
82
The transition profiles and heat capacity for unbounded CP DNA, HT DNA
and ML DNA; along with bounded with thionine are shown in figure 4.2, 4.3
and 4.4, respectively. As per transition profiles shown in figure 4.2, 4.3 and 4.4;
there are slight insignificant deviation at the tail ends of profiles that may be
primarily due to the presence of various disordered states and short helical
segments found in the random coil states. The curve ‘A’ in figure 4.2, 4.3 and
4.4, shows experimental and calculated transition curves for the CP DNA, HT
DNA and ML DNA, respectively in the absence of thionine. However, curve ‘B’
in figure 4.2, 4.3 and 4.4, shows the transition when CP DNA, HT DNA and ML
DNA was saturated with thionine in the ratio of thionine/CPDNA=0.4,
thionine/HTDNA=0.5 and thionine/MLDNA=0.5, respectively. As it was
expected, a cooperative transition profiles is obtained with calculated data
obtained through theoretical analysis for all the DNAs of varying base
composition. The results obtained from the theoretical analysis are in good
agreement with the binding enthalpy determined through DSC by Paul et al.
(2010).
83
Fig. 4.2. Heat capacity and transition profiles (inset) for thionine bounded and unbounded CP-DNA. (A) Unbounded state, (B)
Bounded state at saturation. [(—) calculated and (••••) experimental values]
84
Fig. 4.3. Heat capacity and transition profiles (inset) for thionine bounded and unbounded HT-DNA. (A) Unbounded state, (B)
Bounded state at saturation. [(—) calculated and (••••) experimental values]
85
Fig. 4.4. Heat capacity and transition profiles (inset) for thionine bounded and unbounded ML-DNA. (A) Unbounded state, (B)
Bounded state at saturation. [(—) calculated and (••••) experimental values]
86
The conformational and dynamical states of thionine binding to CP DNA,
HT DNA and ML DNA are characterized by heat capacity which is second derivative
of the free energy and has been calculated by using equation 15 (described in
chapter 2). These heat capacity curves along with λ-point anomaly and their
transition profiles for CP DNA, HT DNA and ML DNA are shown in figure 4.2, 4.3
and 4.4, respectively which recommended that the theoretically calculated heat
capacity profile agreed with the experimentally reported ones and could be
brought almost into agreement with the use of scaling factors, which is very close
to one in transition profiles and slightly higher for the heat capacity curves. The
sharpness of the transition can be characterized by the half width of the heat
capacity curves. As shown in table 4.1 and figure 4.2 half width of transition for
unbounded and saturated CP DNA are in good agreement in both experimental
and theoretical graphs. Similar results are also obtained in case of HT DNA and
ML DNA in which half width are in good agreement (table 4.1), as shown in figure
4.3 and 4.4, respectively.
This analysis recommended that natural DNAs viz. CP-DNA, HT-DNA and
ML-DNA are very much co-operative structure and their co-operativity is not
greatly disturbed when thionine bind to these DNAs. Accordingly, the modified
Zimm and Bragg hypothesis can be applied to it which generates the experimental
transition profile and λ-point heat capacity anomaly effectively. The data obtained
by theoretical analysis of thionine binding with CP DNA, HT DNA and ML DNA also
demonstrated that the binding of thionine to DNAs are an endothermic process
and this binding increases the melting temperature of the DNAs as suggested by
Paul et al. (2010) through calorimetric study. The specific binding and intercalation
87
of thionine with DNA have been studied by using spectroscopic methods (Tuite and
Kelly, 1995; Chang et al., 2005). However, Paul et al. used optical melting and DSC
techniques to understand the interaction of thionine with three natural
deoxyribonucleic acid viz. CP DNA, HT DNA and ML DNA containing different
composition of GC contents (Paul et al., 2010). In accordance to the finding of Paul
and coworkers, the present theoretical analysis also suggested that the binding of
thionine stabilized DNAs and the melting temperature (ΔTm) of CP DNA, HT DNA
and ML DNA was increases about 6.5, 9 and 9.7 K, respectively under saturating
condition. Consequently, we can interpret results of our theoretical analysis in the
context of the specific structural features of thionine-DNAs complex as supposed
from the spectroscopic/DSC data of Paul et al. (2010). Assessment of curve ‘A’ and
‘B’ from figures 4.2, 4.3 and 4.4, reveals that the transition of the thionine-
saturated DNAs duplex are significantly broader than the transition of the thionine-
free DNAs. Thus, besides affecting the transition enthalpy and melting
temperature, binding of thionine also alters the nature of the transition as revealed
by the increase in transition width in experimental and calculated (theoretical) both
data. The transition width was increases from 5.0 to 6.5, from 11 to 12 and from
4.5 to 5.0 for CP DNA, HT DNA and ML DNA, respectively in theoretical and
experimental both data.
In recent years, an augmented understanding of the role played by nucleic
acids in biological systems made DNA as an alternative candidate for the
development and formulation of new drugs. The successful applications of
molecular modeling in virtual ligand screening and structure-based design of
organic and inorganic molecules that target specific DNA are highlighted by Ma et
88
al. (2011). The interactions of drugs and calf thymus DNA were also investigated
by using non-linear fit analysis (Yuan et al., 2011). Further recent literatures also
reviewed that the study of DNA interaction with a small molecule is of high interest
(Paul et al., 2010; Araya et al., 2007; Islam et al., 2009; Gonzalez-Ruiz et al., 2011;
Kunwar et al., 2011). Therefore, the theoretical studies of small molecules binding
presented in this study may also be used to other synthetic and natural DNAs and
can be applied to understand bimolecular interactions.
4.5. Conclusion
The present theoretical analysis concluded that these natural DNA
molecules are a highly co-operative structure and when thionine binds to these
DNAs the structure of DNA remains a highly co-operative one. Consequently, the
amended Zimm and Bragg (phase transitions) theory can be successfully applied to
it. These results will allow us to calculate the thermodynamic profile of the binding
process. Thus, the present theoretical studies of thionine (small molecules) binding
are being extended to other synthetic and natural DNAs and can be applied to
understand bimolecular interactions. This study may also be used in the
investigation process of drug development and in biomedical industries.
89
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CHAPTER 5
CONCLUSION
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In the previous chapters (Chapter 3 and 4) author included theoretical study
of Drug-DNA interaction that comprises stability of synthetic and natural DNA on
binding with drugs/small molecules. Molecular interaction of the drug with
deoxyribonucleic acid is a field of high topical interest. Therefore, a number of
techniques committed to drug-DNA interactions are evolved. In fact, the
transcription or translation of DNA starts only when it receives a signal, which is
often in the form of a regulatory protein binds to a particular region of the DNA.
Thus, if the binding specificity and strength of this regulatory protein can be
emulated by a small molecule, then DNA function can be artificially modulated,
inhibited or activated. Therefore, this synthetic/natural small molecule can act as
a drug when activation or inhibition of DNA function is required to cure or control
a disease. The specific sites of DNA molecule involved in vital processes, such as
origin of replication etc., are of particular interest as targets for a wide range of
antibiotic and anticancer drugs (Haq, 2002; Hurley, 2002.) Drugs bind to DNA both
covalently as well as non-covalently. Covalent binding of drugs with DNA is
irreversible and inhibit DNA processes that leads to cell death. Non-covalent
binding is reversible and is typically preferred over covalent adduct formation
keeping the drug metabolism and toxic side effects in mind.
In drug-DNA interaction, energetics of DNA recognition is not fully
understood at a molecular level, due mainly to the strong electrostatic interactions
prevalent in the system and van der Waals interactions. Protein–drug binding has
been explained by various popular models but a straightforward extrapolation of
those interaction models to DNA–drug systems is not feasible since there is no
formal active site in DNA, unlike enzymes/proteins. However, for understanding of
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DNA–drug interaction, experimental studies play major role and the essential
information on binding constants and corresponding free energy, entropy,
enthalpy, and heat capacity changes on complex formation can be obtained by
thermodynamic studies. This information extremely used to authenticate
investigation based on theoretical predictions and structural analysis. These
theoretical studies on drug-DNA interactions have resulted in important
contributions to understand binding of DNA–drug complexes.
In the present investigation as described in above chapters (chapter 3 and
4), theoretical analysis of steroid diamine (Dipyrandium) and tricyclic hetero-
aromatic molecule (Thionine) binding with DNA duplex by using an amended Zimm
and Bragg theory are reported. These studies explain the melting behavior and
heat capacity of DNA with and without drugs binding. The results of both the
studies (with Dipyrandium and Thionine) suggested that the DNA molecule is an
extremely co-operative structure and when drugs bind to it the co-operativity is
not so much disturbed. Thus, the amended Zimm and Bragg theory can be
effectively applied to it. It generates the experimental transition profile and λ-point
heat capacity anomaly successfully. The results obtained will allow us to evaluate
the thermodynamic profile of the binding process. This theoretical analysis of small
molecules binding with nucleotide is being extended to other synthetic and natural
DNA and can also be useful in order to understand bimolecular interaction and in
the process of drug development.
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References
Haq I. (2002). Arch. Biochem. Biophys., 403: 1-15.
Hurley L.H. (2002). Nat. Rev. Cancer, 2: 188–200.