22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Atomic-scale simulations of the oxidation of DNA by OH radicals using the
ReaxFF reactive force field
C.C.W. Verlackt, E.C. Neyts and A. Bogaerts
Research group Plasmant, Department of Chemistry, University of Antwerp, 2610 Antwerpen-Wilrijk, Belgium
Abstract: The use of cold atmospheric pressure plasmas for cancer treatment has provided
us with a successful alternative towards the often toxic conventional treatments. In this
work we investigate the interaction of ∙OH with DNA molecules as well as their influence
on the treated cells, by means of reactive molecular dynamic simulations using the ReaxFF
potential.
Keywords:
ReaxFF
molecular dynamics, plasma oncology, reactive oxygen species, DNA,
1. Introduction
Despite worldwide awareness, treating cancer remains a
tremendous challenge.
To date, a wide range of
treatments exist, aiming to kill cancerous cells by the use
of radiation or chemical therapy. However, each of the
conventional techniques faces obstacles which cannot be
overlooked. These treatments suffer from the lack of
selectivity in the sense that they prove to be harmful for
healthy cells also [1]. Moreover, many studies have
pointed to the growing resistance of the tumorous cells
towards these conventional therapies and to the body’s
own defense system [2-3]. But maybe the most important
drawback that scientists face to date is the inability of the
existing therapies to remove every malignant cell present
in the host body [1-2, 4]. Although most cancerous cells
can be destroyed, a small concentration may survive the
treatment, able to grow to a tumor which proves more
difficult to eradicate. For this purpose, new and more
selective treatments need to be investigated, which are
safer for the host body, treating the malignant cells
without damaging the normal, healthy cells.
Plasma medicine has become a fast growing discipline,
attracting the interest of many scientists worldwide.
Indeed, plasmas can be used in a wide range of
biomedical applications, such as wound and ulcer healing,
dental treatments, sterilization, blood coagulation but also
for cancer treatment [5-10]. Using cold atmospheric
pressure plasmas (CAPPs) a mixture of reactive species
(reactive oxygen and nitrogen species, i.e., RONS)
interacts with the surface of interest, resulting in
medically favorable results. Plasma generated reactive
species affect the delicate biochemical environment of the
treated cells by a series of chemical reactions (oxidations
and peroxidations) and by both inhibiting and activating
biochemical and cellular systems, enhancing specific
medical activities [5]. One example is the combination of
both wound healing and sterilization, thus stimulating
mammalian cells while damaging bacterial cells [6].
In light of oncology, the use of CAPPs has proven to
provide promising results for a vast range of different
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cancer lines, both in vitro and in vivo, able to attack the
tumorous cells while preserving their healthy counterparts
[11-12]. Among the investigated cancer lines are breast
cancer [13], cervical cancer [14], lung cancer [15], gastric
cancer [16], leukemia [17], pancreatic cancer [18], liver
cancer [19], ovarian cancer [20], melanoma [21],
neuroblastoma [22], glioblastoma and colorectal
carcinoma [23], etc. These studies conclude that the
plasma anti-tumor mechanisms, again, arise from the
synergetic activities of the reactive plasma species.
One of the well-known effects of plasma-cell treatments
is the increase of the cellular reactive oxygen species
(ROS) concentration, disturbing the oxidative balance
within the treated cells [14].
These species are
responsible for the oxidation of the cellular biomolecules,
e.g., proteins, DNA, lipids etc., but are countered by a
system of antioxidants, both chemical and enzymatic [24].
As cancerous cells show a higher steady-state
concentration of ROS, the increase in oxidants, as a result
of plasma treatment, may eventually overthrow the
antioxidant system of cancer cells, causing oxidative
damage within the treated malignant cells before their
healthy counterparts suffer any harm [25]. Indeed,
controlled plasma treatment is able to cause tumorselective damage by the oxidation of lipids, proteins and
DNA, able to trigger apoptosis or even necrosis in the
treated cancerous cells in a plasma-dose-dependent
manner.
Among the ROS, ∙OH is the most reactive towards
DNA causing a vast range of oxidation products, e.g.,
DNA strand breaks and H-abstraction reactions [26-27].
The oxidation of DNA and its consequences remain
subject of many studies and are not yet fully understood.
In this respect, computer simulations are ideally suited to
elucidate the dynamics of the above mentioned oxidation
reactions and their direct influence on the close
environment. Despite the advantages of these methods,
only a limited number of computational investigations
have been performed to study the oxidation of
biomedically relevant structures. An overview of the
1
most relevant works in this field is presented in the recent
review of Neyts et al. [28].
In this work we present the investigation of the
interaction of ∙OH with DNA by means of reactive
molecular dynamics using the ReaxFF potential. A range
of oxidation reactions will be presented along with their
combined influence on the cell in a dynamic manner.
2. Computational setup
We used the ReaxFF potential, as implemented in
LAMMPS. ReaxFF is a reactive force field. It allows one
to simulate reactive processes, i.e. bond formation and
dissociation reactions, providing fundamental atomicscale insight in the simulated systems. A modified
version of the force field published by Monti et al. [29] is
used, containing parameters for C/H/N/O/P-atoms which
were optimized for amino acids and later extended to
increase the stability of phosphate groups in the system in
solution [30].
Simulations were performed on a hexadecamer DNA
double helix in a ~33 Å x 33 Å x 64 Å periodic
rectangular cuboid.
The remaining space in the
simulation box was filled with water molecules
approaching a density of 1 mg/ml (Fig. 1). A series of
15 ∙OH were introduced and the interactions were
investigated over a course of 500 ps at 300 K in a
canonical ensemble (NVT) using the Nosé-Hoover
thermostat with a coupling constant of 0.25 fs. Prior to
the simulations, thermalization runs were performed to
both stabilize the system to the simulation parameters,
i.e., temperature, pressure and chemical environment, and
check the stability of the system using the mentioned
force field. Over the course of 300 ps, the total energy of
the system remained constant, with variations less than
0.1% of the total value.
3. Results
A total set of 15 independent simulations was
performed using the system as described above. After
500 ps, three sets of reactions were found: H-abstraction
reactions, OH-addition reactions on nucleotides and
OH-addition reaction on the phosphate-ribose backbone;
see Fig. 2. It is important to note that the reactions, as
discussed below, will be named after the reaction
products presented in Fig. 2.
The formation of both 1- and 2-N-centered radicals
(i.e., second and first product of Fig. 2) is a direct
consequence of an H-abstraction reaction caused by ∙OH,
and are observed in 4.5 and 10.5% of the simulated
reactions (65 in total over all 15 simulations),
respectively. The formation of the 2-N-centered radical
has been encountered for adenine (dAMP), cytosine
(dCMP) and guanine (dGMP) while the formation of the
1-N-centered radical has only been observed for dGMP.
It is important to state that the 1-N-centered radicals were
only seen at the nucleotides found on both ends of the
hexadecamer, thus being more solved in water compared
to the other nucleotides. This set of observations are in
2
Fig. 1. Snapshot of the 3D model of the simulated DNA
hexadecamer in solution (∙OH are not shown). A total of
5700 atoms is present in this simulation system.
Fig. 2. Overview of the oxidation reactions observed in
the MD simulations after 500 ps at 300 K in NVT. The
reactions and nomenclature are presented for purines for
the sake of consistency; please note that several reactions
have also been observed on pyrimidines. The reactions in
this work will be named after the reaction products as
presented in this figure.
line with multiple independent studies [27, 31].
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The OH-addition reaction on 8-C of purine (i.e., third
product in Fig. 2) has been observed for both dAMP and
dGMP in 32% and 47.5% of the total reactions simulated,
respectively. The addition of ∙OH results in a radical
inside the purine rings, able to react further with
molecules in close vicinity. 8-OH-adduct radicals form
the first step towards the formation of the oxidation
products 8-oxo-Gua/Ade and Fapy-Gua/Ade, widely
known as the markers for DNA oxidation [27]. The
introduction of these oxidation products has proven to be
responsible for DNA mutations and can result in the
activation of pro-apoptotic factors within the affected
cells [1, 27, 32]. The above mentioned oxidation products
have not been encountered in the present simulations due
to the absence of the necessary reactants and prohibitively
long simulation time, but can be found in literature [27].
The formation of the 5’-C-alcohol (i.e., fourth product
in Fig. 2) is a result of an addition-elimination reaction
where ∙OH binds with the 5’-C of a nucleotide, leading to
breaking of the C-OPO 3 bond, resulting in the desorption
of the phosphate group. This desorption leads to a break
in the affected DNA strand and is known as a single
strand break (SSB). SSBs are known as reversible
damage on DNA but are able to trigger double strand
breaks (DSBs), cleaving the DNA string as a whole,
which in turn activates pro-apoptotic factors within the
affected cells [7]. SSBs and DSBs are known as the main
anti-tumor responses of plasma treatment, leading to cell
death. The simulated reaction, however, is rather peculiar
as it is widely accepted that SSBs are a result of
H-abstraction reactions at the 5’-C or 4’-C, leading to the
desorption of a phosphate anion, thus breaking the DNA
strand [27].
The observed reactions are a direct
consequence of the limits of the used force field as it is
not specifically optimized for the precise simulation of the
thermodynamics of nucleotides.
The latter conclusion, combined with the absence of
specific, but well-known, oxidation products such as the
OH-addition reaction on sp² carbon atoms of all
nucleotides, with in particular cytosine and thymine [33],
raises the need for a more precise optimization of the used
force field, specifically for the thermo-dynamics of
nucleotides.
4. Conclusion
Reactive molecular dynamic simulations have been
performed using the ReaxFF code as implemented in
LAMMPS.
The interactions between ∙OH and a
hexadecamer DNA strand in solution have been
investigated, shedding light on the oxidation mechanisms
and their direct influences on the cell. Among the
oxidation products were the formation of 1- and
2-N-centered radicals and 8-OH-purine adduct radicals
which is in line with experimentally observed products.
However, the absence of certain reaction mechanisms
known from experimental work raises the need for further
optimization of the used computational method. Despite
this, it is clear that reactive molecular dynamics
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simulations, and the ReaxFF potential in particular,
provide us with indispensable atomic-scale insight in the
oxidation processes which occur within plasma treated
cancer cells and which are often very difficult to obtain
experimentally. This insight will prove to form a
contribution for a better understanding of cancer
treatments by means of cold atmospheric pressure
plasmas. However, more investigations are certainly
needed in the field of plasma assisted oncology to
elucidate the role of the plasma generated species in
activation of cancer-specific cell death.
5. Acknowledgements
Verlackt, Neyts and Bogaerts acknowledge financial
support from the Fund for Scientific Research – Flanders
(project number G012413N). The calculations were
performed using the Turing HPC infrastructure at the
CalcUA core facility of the Universiteit Antwerpen, a
division of the Flemish Supercomputer Center VSC,
funded by the Hercules Foundation, the Flemish
Government (department EWI) and the Universiteit
Antwerpen.
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