Probing the microscopic structure and flexibility of oxidized DNA by molecular simulations

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ORIGINAL PAPER

Probing the microscopic structure and ﬂexibility of oxidized DNA

by molecular simulations

K B Chhetri

1,2

, S Naskar

and P K Maiti

Department of Physics, Center for Condensed Matter Theory, Indian Institute of Science, Bangalore 560012, India

Department of Physics, Prithvinarayan Campus, Tribhuvan University, Pokhara, Nepal

Received: 08 September 2021 / Accepted: 17 January 2022 / Published online: 17 March 2022

Abstract: The oxidative damage of DNA is a compelling issue in molecular biophysics as it plays a vital role in the epigenetic

control of gene expression and is believed to be associated with mutagenesis, carcinogenesis and aging. To understand the

microscopic structural changes in physical properties of DNA and the resulting inﬂuence on its function due to oxidative

damage of its nucleotide bases, we have conducted all-atom molecular dynamic simulations of double-stranded DNA

(dsDNA) with its guanine bases being oxidized. The guanine bases are more prone to oxidative damage due to the lowest value

of redox potential among all nucleobases. We have analyzed the local as well as global mechanical properties of native and

oxidized dsDNA and explained those results by microscopic structural parameters and thermodynamic calculations. Our

results show that the oxidative damage of dsDNA does not deform the Watson-Crick geometry; instead, the oxidized DNA

structures are found to be better stabilized through electrostatic interactions. Moreover, oxidative damage changes the

mechanical, helical and groove parameters of dsDNA. The persistence length, stretch modulus and torsional stiffness are

found to be 48.87 nm, 1239.26 pN and 477.30 pN.nm2, respectively, for native dsDNA and these values are 61.31 nm, 659.91

pN and 407.79 pN.nm2, respectively, when all the guanine bases of the dsDNA are oxidized. Compared to the

global mechanical properties, the changes in helical and groove properties are found to be more prominent, concentrated

locally at the oxidation sites and causing the transition of the backbone conformations from BI to BII at the regions of oxidative

damage.

Keywords: Oxidative damage; Stretch modulus; Persistence length; Torsional stiffness; Helicoidal parameters; Torsion

angles

1. Introduction

DNA oxidation, the oxidative damage of deoxyribonucleic

acid, is being the subject of interest due to the biological

repercussions like genome instability and mutation that it

brings to our body [1]. Among four DNA nucleobases,

guanine is more prone to oxidation since it has the lowest

redox potential [2,3]. During oxidative phosphorylation,

reactive oxygen species (ROS) are generated in the form of

super-oxides (O

2) and H2O2, which are responsible for

oxidation of biomolecules like protein, DNA, etc. [4]. ROS

can also be produced by ionizing or UV radiation. ROS

may interact with biological macromolecules such as DNA,

causing alteration and possibly severe repercussions to the

cell, regardless of their source (endogenous or exogenous)

[5]. The DNA alterations in mammalian chromatin caused

by free radicals are found to be associated with mutagen-

esis, carcinogenesis and aging [6–9].

Modiﬁcation of DNA bases is not only malignant but

also plays a vital role in the epigenetic control of gene

expression [10]. Practically, each of the DNA bases can be

modiﬁed; however, modiﬁcations of guanine and adenine

bases are most occurring due to their smaller redox

potentials [11]. The 7,8-dihydro-8-oxoguanine, also known

as 8-oxoguanine (8oxoG), is one of the most abundant

byproducts of oxidative DNA damage [12]. In addition to

being an output of DNA oxidative damage, 8oxoG has a

role in transcriptional regulation under oxidative stress

[13]. During hypoxia, intra cellular degrees of ROS are

raised, which facilitates the production of 8oxoG [14,15].

Excess oxidative DNA damage is linked to cancers and

*Corresponding author, E-mail: maiti@iisc.ac.in

Indian J Phys (July 2022) 96(9):2597–2611

https://doi.org/10.1007/s12648-022-02299-y

Ó2022 IACS

many other diseases, while a small amount of oxidized

nucleotides produced due to normal ROS levels is neces-

sary for memory and learning [16–18].

In 2016, while studying the effect of cytosine modiﬁ-

cations on DNA ﬂexibility, Ngo et al. [19] found that the

methylation of cytosine (5-methylcytosine) causes reduc-

tion of DNA ﬂexibility. During base analog substitution

(substituting either inosine for guanosine or 2,6-di-

aminopurine for adenine), Peters et al. [20] found minor

changes in global properties like persistence length, helical

repeat and torsional stiffness. The data obtained from cir-

cular dichroism spectroscopy in the same work of Peters

et al. showed some signiﬁcant changes in helical geometry

of the modiﬁed DNA compared to normal DNA. In another

work, Peters et al. [21] experimentally measured the

bending and twisting ﬂexibilities of DNA analog polymers

with one of the four regular bases of DNA substituted by

different cationic, anionic, or neutral analogs under low salt

buffers. They found only about 20%change in bending

rigidity but a large increase (about 5-fold) of twist ﬂexi-

bility on such modiﬁed DNA analogs in comparison to the

unmodiﬁed one. They suggested that such modiﬁcations of

regular bases make dsDNA to have transition to different

helical conformations other than canonical B-form and

effect is minimal as far as the mechanical properties are

concerned. It is reported that the methylation of the cyto-

sine base of DNA inﬂuence the DNA’s backbone structure

due to the steric hindrance between the methyl group and

ribose sugar that prevents the formation of hydrogen bonds

between the nucleobase and backbone and results in the

local increment of DNA ﬂexibility [22].

In another computational study by Miller et al. [23],

G19:C6 base pair of DNA oligonucleotide GGGAA-

CAACTAG:CTAGTTGTTCCC was replaced by 8oxoG.

They found that when 8oxoG replaced G19, the local

bending into the major groove is more probable than

changing the DNA’s global bending, which assists in the

formation of local kinks at the 8oxoG associated major

grooves. The oxidative damage of DNA can bring local

alterations on phosphate backbone and changes of sugar

puckers of the oxidized bases [24,25]. Cheng et al. [26]

conducted unrestrained molecular dynamics simulations

for several 13-mer DNA duplexes. In their results, the

B-form duplexes of oligomers with G:C and 8oxoG:C base

pairs are found to adopt proper Watson-Crick geometry

and the local and global ﬂexibilities of the duplexes are

increased. In the case of G:A mismatch, the Watson-Crick

geometry is found to be decreased with higher structural

ﬂuctuations. These simulations demonstrated that both

dynamic and equilibrium properties of DNA duplexes

change during their oxidative damage. All these works

inspired us to decipher how different amount of oxidation

in DNA can inﬂuence their microscopic structural and

mechanical properties.

In this work, we carried out all-atom MD simulations of

different oxidized double-stranded DNAs (dsDNAs) and

computed various mechanical properties such as stretch

modulus (cG), persistence length (lp), twist-stretch-cou-

pling (s) and torsional stiffness (C). As several earlier

works have shown more alterations of local parameters

than global ones, we have tried to explain the changes of

various microscopic structural parameters with the oxi-

dization of the DNA bases. We believe, it will help to

advance the understanding about the alterations of bio-

logical as well as physical properties brought due to

oxidative damage of DNA, speciﬁcally based on the

changes of mechanical properties and microscopic heli-

coidal parameters.

This article is organized as follows. We begin with the

methods describing the model building of oxidized DNA

and details of all-atom MD simulations. Then, we give

details of the theoretical models used to calculate the

elastic properties of nucleic acids. In the results and dis-

cussion section, we present the mechanical properties, such

as stretch modulus (cG), persistence length (lp), twist-

stretch-coupling (s), etc., of the dsDNAs. We also analyze

the various microscopic structural parameters. Finally, we

summarize all the results and provide a perspective and

utility of various results.

2. Materials and methods

2.1. Simulation setup

2.1.1. System build-up

The Dickerson-Drew dodecamer (d[CGCGAATTCGCG])

double-stranded DNA (dsDNA) was prepared using the

nucleic acid builder(NAB) [27] tool of Amber18 [28].

Using an in-house developed python script, the guanine

bases are oxidized as shown in Fig. 1(b). We prepared three

dsDNA molecules: one is native dsDNA, where no bases

are oxidized, another one is dsDNA(4oxG), whose four

guanine bases (two of each strand but in two opposite

halves) are oxidized and the third one is dsDNA(8oxG),

whose eight guanine bases (four of each strand or all

guanine bases) are oxidized.

The interactions of pure dsDNA are represented by

Amber ff10 force ﬁeld [29] while the interactions of oxi-

dized bases are taken from the work of Miller et al. [23].

The TIP3P water model [30,31] was used to solvate the

dsDNAs, resulting in a 15 A

˚TIP3P water buffer sur-

rounding the structure in each direction. Because the

2598 K B Chhetri et al.

phosphate backbones in dsDNA are negatively charged, the

systems needed to be neutralized, which was accomplished

by introducing the necessary amount of Naþions. The

Joung-Cheatham ion parameter set was used to character-

ize the interaction of ions with water and nucleic acids

[32]. In this way, three different solvated systems are

prepared, corresponding to dsDNA, dsDNA(4oxG) and

dsDNA(8oxG), respectively. Schematically these three

dsDNA duplexes are described by Fig. 1(a).

2.1.2. MD simulation methodology

To remove any bad contacts that arise during the system

preparation, we performed energy minimization of sys-

tems. We used the steepest descent algorithm (for 2500

steps), followed by the conjugate gradient algorithm (next

2500 steps). All of the solute atoms (nucleic acid atoms)

were kept ﬁxed, applying a harmonic potential of spring

constant 500 kcal.mol1A

˚2. The restraint applied to the

solute atoms was reduced to zero in ﬁve stages with 5000

steps of energy minimization in each stage. That means the

positional restraint applied to solute atoms was made zero

during the last 5000 steps of equilibration. The energy

minimized systems were then heated from 10 K to 300 K in

four steps: 10K to 50K, 50 K to 100 K, 100 K to 200 K and

200 K to 300 K. The dsDNA was position restrained using

a harmonic constant of 20 kcal.mol1A

˚2during the whole

heating process. We used Langevin thermostat [33,34]

with a coupling constant of 0.5 ps to control the tempera-

ture. To equilibrate, the systems were subjected to a 2 ns

NPT simulation following the heating. Berendsen weak

coupling method [35,36] with a coupling constant of 0.5 ps

was employed to maintain the pressure to 1 atm. At last,

500 ns long MD simulation was conducted in the NVT

ensemble with 2 fs integration time steps. During simula-

tion, the SHAKE algorithm [37] was adopted to constrain

the hydrogen bonds. To account for the electrostatic

interactions, we used the Particle Mesh Ewald (PME)

method [38]. We used an LJ potential cut-off of 10 A

˚.At

the cut-off, the van der Waals (vdW) and direct electro-

static interactions were terminated. Similar simulation

methodologies have been successfully implemented in

several of our previous studies involving DNA and DNA-

based nanostructures [39–44].

2.2. Theories on mechanical properties

2.2.1. Persistence length

In DNA, the persistence length is the elementary section of

its length over which the correlations in the tangent

directions are lost. The persistence length of DNA mea-

sures its bending rigidity. Molecules having larger persis-

tence lengths are considered to be stiffer to bend. There

exist many theoretical models to compute the persistence

length (lp) of dsDNA, described in the article of Garai et al.

[41]. One of the ways of calculating (lp) is through bending

angle distribution. If t

1be the unit vector to the ﬁrst base

pair and t

nbe the unit vector to the nth base pair then the

bending angle (h) of dsDNA is deﬁned as,

h¼cos1ðt

1:t

nÞ.

Figure 2describes end-to-end distance ðLeÞ, contour

length (L) and bending angle ðhÞof

dsDNA shown schematically. If libe the center to center

distance between two consecutive base pairs then the

average contour length is deﬁned as: L0¼\Pn

i¼1li[,

where \[ is the time average over all frames.

Fig. 1 (a) Schematics for the dsDNA duplexes with X as an oxidized

guanine. The dsDNA represents the DNA duplex with none of the

bases are oxidized, dsDNA(4oxG) represents the DNA duplex whose

four guanine bases (two of each strand but on opposite halves) are

oxidized and dsDNA(8oxG) represents the DNA duplex whose eight

guanine bases (four of each strand or all guanines) are oxidized.

(b) Conversion of guanine to oxoguanine (8oxoG) with its oxidative

damage. The larger dark silver spheres, smaller bright silver spheres,

blue spheres and red spheres represent carbon, hydrogen, nitrogen and

oxygen atoms, respectively

Probing the microscopic structure and ﬂexibility of oxidized DNA 2599

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ORIGINALPAPERProbingthemicroscopicstructureandﬂexibilityofoxidizedDNAbymolecularsimulationsKBChhetri1,2,SNaskar1andPKMaiti1*1DepartmentofPhysics,CenterforCondensedMatterTheory,IndianInstituteofScience,Bangalore560012,India2DepartmentofPhysics,PrithvinarayanCampus,TribhuvanUniversity,Pokhara,NepalRec...

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