For Peer Review Only

Journal of Biomolecular Structure & Dynamics
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Dissociation of the Watson-Crick Base Pairs in Vacuum and

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in Aqueous Solution: a first-principles molecular dynamics
study
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Journal: Journal of Biomolecular Structure & Dynamics
Manuscript ID TBSD-2021-0894.R2
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Manuscript Type: Research Article
Date Submitted by the

23-Aug-2021
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Author:
Complete List of Authors: Ordóñez, Cristian; UNAH,

Martínez-Zapata, Daniel; UNAM, Theoretical Physics
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santamaria, ruben; UNAM, Theoretical Physics
DNA nucleic acid bases, hydrogen bridges, solvation effects, density

Keywords:
functional theory, steering molecular forces
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URL: http://mc.manuscriptcentral.com/jbsd Email: TBSD-peerreview@journals.tandf.com

Page 1 of 36 Journal of Biomolecular Structure & Dynamics
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14 Mexico City August 23, 2021
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16 Prof. Ramaswamy H. Sarma
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17 Editor in Chief
18 Journal of Biomolecular Structure and Dynamics
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Dear Editor
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23 We submit the revised manuscript with title “Dissociation of the Watson-Crick Base Pairs in Vacuum
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24 and in Aqueous Solution: a first-principles molecular dynamics study”, written by Cristian Ordóñez,
25 Daniel Martıı
nez-Zapata, and Ruben Santamaria, for publication as an article in the Journal of
26 Biomolecular Structure and Dynamics.
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We have taken all the recommendations of Reviewer-3 into consideration. The specific changes are
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30 explained in the following pages.

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32 We acknowledge your kind attention.
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34 Sincerely Yours
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37 Dr. Ruben Santamaria
Dept. of Theoretical Physics
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39 Institute of Physics, UNAM
40 rso@fisica.unam.mx
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Journal of Biomolecular Structure & Dynamics Page 2 of 36
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3 RESPONSE TO REVIEWER 3
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5 We have revised the manuscript according to the reviewer comments, and performed a thorough
6 revision of it. The reviewer can now find answers to its questions in this latest version of the
7 manuscript.
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10 REVIEWER 3
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12 The authors mention that STACKING forces between successive base pairs of DNA is important but
13 still does not attempt to calculate it. The flat faces of the bases are exposed to water, which is
14 unrealistic in double helical DNA.
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16 We recognize the importance of the stacking forces on the stability of DNA. Nonetheless, it is
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17 important to mention that the goal of the present paper is to exclusively evaluate the rupture
18 forces of the WC-base pairs in a solvated medium. Such forces can be used as reference forces to
19 eventually evaluate the rupture forces of the WC-base pairs in a stacking conformation. In other
words, the stacking effects on the rupture of forces may be evaluated after firstly dealing with the
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WC-base pairs alone and in a solvated medium (please, consider the following fact: in the paper
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the rupture forces in solvation were compared against the rupture forces in vacuum. In this way, it
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was possible to evaluate the solvation effects in the breaking of the H bonds). Thereby, the
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evaluation of the rupture forces of the WC-base pairs constitutes, as presented in the paper, a
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starting point for subsequent and more complex calculations.
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Placing these flat faces in contact with the flat faces of other stacked base pairs could have given
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more realistic simulation with not much extra cost (perhaps with same cost if periodic boundary
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condition is not invoked).
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The incorporation of more WC base pairs to have them in a stacking conformation also demands
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the incorporation of the sugar and phosphate molecules, since these compounds link the WC
32 bases. We estimate that it is necessary to increase the molecular system with 316 atoms to
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33 evaluate the breaking forces of the WC base pairs in a solvated medium. A description based on
34 ab-initio calculations demands a greater computing capacity and, as explained previously, such a
35 proposal represents a future research topic.
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It appears from Figure 2 that the authors selected C6 of Adenine and C4 of Thymine for application
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38 of steered force while used C5 of Guanine and C5 of Cytosine for same purpose to GC base pair. I
39 don't understand why the authors did not select similar atoms for application of stretch to AT and
40 GC base pairs.
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42 Paragraph 2 of Sect. 3.3 (Steering atomic forces) discusses the choice of atoms that determine the
43 lines of action of the AFM-like forces. The description of figure 2 was extended to include a bried
44 description on such forces.
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47 Unusual phrases used should be replaced by commonly accepted ones:
48 1. Hydrogen bridge --> Hydrogen bond
49 2. Double DNA helix --> DNA double helix
50 3. Van der Waals --> van der Waals
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52 1. A clarification of the term “hydrogen bridge” was inserted as a citation [2] in the first paragraph
53 of Sect. 1 (Introduction).
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55 2. The change: Double DNA helix --> DNA double helix was performed in the text.
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57 3. The change: Van der Waals --> van der Waals was performed in the text.
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6 NOTE: the colored paragraphs giving answers to the previous referees now appear without color,
7 since the refeeres are in agreement with our responses. The answers to the Referee-3’s
8 comments are shown in color in the text.
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3 Dissociation of the Watson-Crick Base Pairs
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5 in Vacuum and in Aqueous Solution:
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7 a first-principles molecular dynamics study
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11 Cristian Ordóñez, Daniel Martı́nez-Zapata, Ruben Santamaria
12 Dept. of Theoretical Physics
13 Institute of Physics, UNAM
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Abstract
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19 The damage of the DN A structure can affect the correct functioning of the cellular processes.
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20 This work investigates the required forces to dissociate the Watson-Crick (W C) base pairs AT
21 into A and T , and GC into G and C. The W C base pairs are immersed in water under realistic
22 conditions of temperature, volume, and density that reproduce the main characteristics of a
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biological system. The simulations are based on first-principles molecular dynamics combined
25 with steering atomic forces. In addition to the force intensities, the charge transfers between
26 the nucleic acid bases, energy variations, and temperature fluctuations in the cleavage moments
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27 are reported. With the purpose of evaluating the effects of the aqueous medium, simulations
28 of the W C base pairs in vacuum are included. The results considering the solvated medium
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are consistent with the experimental measurements, and show the importance of the aqueous
31 solution to regulate the structural modifications of the nucleic acid bases. The investigation
32 contributes with a novel molecular model in molecular simulations, and to better understand
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33 the biological processes where the DN A compounds play an active role in life forms.
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38 Keywords: DN A nucleic acid bases, hydrogen bridges, solvation effects, density functional
39 theory, steering molecular forces.
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1
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3 Contents
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1 Introduction 3
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8 2 Method 4
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10 3 Molecular Model 5
11 3.1 Confinement model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
12 3.2 NVT ensemble . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
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3.3 Steering atomic forces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
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16 4 Results 9
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17 4.1 AT and GC in vacuum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
18 4.2 AT and GC in aqueous solution . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
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20 5 Conclusions 17
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6 Acknowledgments 17
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25 Bibliografia 18
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3 1 Introduction
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The DN A molecule is a polymer that encodes the genetic information to produce the proteins
7 responsible for the optimal functioning of the cellular systems. Any damage on the constituent
8 elements of the DN A may alter the metabolic processes in humans [1]. The stability of the
9 DN A polymer depends of both, the hydrogen bridges that hold the Watson-Crick (W C) base
10 pairs together [2], and the stacking forces acting on the nucleic acid bases. The Watson-Crick
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base pairs possess a great degree of conformational flexibility due to their hydrogen bridges
13 with specific mechanical and elastic properties, making the DN A flexible, but also resistant to
14 fractures that may disrupt the genetic code [3, 4]. The hydrogen bridges allow the separation
15 of the DN A nucleotide chains in the replication and transcription mechanisms, resulting on
16 the formation of new DN A strands to preserve the genetic information [5, 6].
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The denaturation of the DN A molecule may be due to high temperature changes, the polarizing
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20 action of an acidic or alkaline solution, the destabilizing effects of external forces, etc. At the
21 cellular level, there are enzymes that participate in the metabolic processes and break the DN A
22 double helix into a pair of DN A strands [6]. Several experimental studies have investigated the
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separation of the chains of the double-stranded DN A with the application of external forces.
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Such experiments have used micro-needles [7], magnetic beads [8], optical tweezers [9], and the
26 atomic force microscope (AF M ) [10, 11, 12, 13]. For example, by using AF M techniques, the
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27 longitudinal and transverse stretchings of selected DN A fragments were performed to determine

28 the forces holding the complementary DN A chains together. It is common to average the
29 breaking forces according to the number of base pairs [14]. A different AF M experiment has
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31 considered the nucleic acid bases linked to a gold mesh immersed in aqueous medium. When
32 the AF M tip is linked to a complementary base, and moved closer and away from the DN A
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33 attached to the mesh, the attraction forces of the hydrogen bridges are measured and classified
34 in force intervals [15].
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36 There are theoretical calculations which have combined classical molecular dynamics with steer-
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ing atomic forces, with the goal of evaluating the DN A-polymer stretching and separation resis-
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39 tance of the complementary strands. Such studies simulate the AF M experiments [16, 17, 18].
40 The DN A molecule was immersed in an aqueous solution to establish the effects of the solvent.
41 The forces on the DN A molecule had harmonic character. The forces were applied making dif-
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42 ferent angles with the ends of the nucleotide chains. It was possible to record the tensile forces
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44 on the nucleotide chains, and the elongated effects on the DN A structure [19]. Simulations
45 with molecular mechanics that incorporate temperature effects through a Langevin thermo-
46 stat have been also performed with the purpose to analyze the breaking, self-assembly, and
47 behavior of the nucleic acid bases when these are deposited on different types of 2D materials
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[20, 21, 22, 23].
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51 The studies simulating the separation process of the Watson-Crick base pairs have been mainly
52 carried out with forcefields in classical simulations. Yet, those studies fail to include some subtle
53 effects, which are considered in quantum mechanical calculations [24]. For instance, by employ-
54 ing density functional theory (DFT), the interaction energies of the W C hydrogen bridges were
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3 found to be systematically underestimated in the classical approach, with average discrepan-
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cies of 1.9 and 3.3 kcal/mol for the adenine-thymine (AT ) and the guanine-cytosine (GC) base
6 pairs, respectively. The differences are attributed to the soft polarization and covalent effects of
7 the complementary nucleic acid bases [24]. The number of studies using first-principles molec-
8 ular dynamics, and simulating the AF M forces, are scarce due to the computational resources
9 required by this method. In spite of that, there are many studies that have characterized at
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the quantum mechanical level the Watson-Crick structures in their minimum energy states
12 [25, 26, 27, 28], and evaluated the hydrogen bridge strengths through binding energies [29].
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14 The main goal of this research is to estimate the binding forces of the hydrogen bridges that hold
15 the AT and GC Watson-Crick base pairs together. The simulations are performed for molecular
16 systems in aqueous solution, using first-principles molecular dynamics. In order to reproduce
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17 the essential conditions of a biological system, the thermodynamic variables of temperature T ,
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volume V , and density ρ are considered. The first-principles molecular dynamics method is
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20 combined with steering forces, derived from external potentials, in principle associated to the
21 action of the AF M microscope. With the purpose of establishing the aqueous medium effects,
22 the force values on the breaking process of the H bridges are evaluated not only in the aqueous
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solution but also in the vacuum. The present work considers no successive base pairs of the
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double helix, which stack on either side of the nucleic acid base pairs in a DN A. The stacking
26 interactions are important in the stability of the double-stranded DN A [30], nevertheless, we
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27 focus attention on the lateral base pairing interactions.

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29 The content of this work is as follows: the first-principles molecular dynamics approach is
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30 described in the following section. In the subsequent section, the details on the confinement
31 model, the way in which the thermodynamic variables are incorporated, and the steering forces
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are created, are provided. A following section discusses the magnitudes of the steering forces, the
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34 energy changes, temperature variations, charge transfers between the molecular compounds, and
35 the final molecular configurations of the nucleic acid bases. The last section briefly comments
36 on the main advances of this work.
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39 2 Method
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The DFT approach is a computational efficient and accurate method [31, 32, 33]. The DFT
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43 method has been widely used to investigate the energetic and structural properties of the DN A
44 molecule, and its basic components [34, 35, 36, 37, 38]. The DF T approach has been also used
45 in the study of the DN A nucleic acid bases in the presence of contaminating agents [39, 40].
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47 This work employs the B3 hybrid functional for the exchange energy, and the LY P functional
48 for the correlation energy [41, 42], which properly describe the properties of hydrogen bridges in
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the W C bases [43, 44]. The molecular orbitals are described with the atomic basis set 6 − 31G∗ .
51 The level of theory is denoted by B3LY P/6 − 31G∗ , which gives a precise description of the
52 biological molecules [45, 46], and the water molecules [47, 48].
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54 The molecular systems in this research are treated in the condensed state, and the van der
55 Waals (V DW ) forces require no consideration. However, the V DW dispersion effects under
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3 the DF T − D3 approach are included for the molecules in vacuum. The calculations are
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performed with the TeraChem software [49, 50] due to the use of GP U s (graphical process
6 units), also used to study the behavior of organic compounds in a solvent [51]. Large grid sets
7 are used in the numerical integrations of the energies, leading to a precision of 10−6 au. The
8 total charge of the system is zero, and the spin multiplicity is M = 1.
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10 In the Born-Oppenheimer approximation the atomic nuclei are immersed in the energy potential
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of the electrons, and the nuclei are evolved by means of Newtonian equations [52].
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Fi = −∇i V ({RA }) ; V ({RA }) = E DF T (2.1)
14 {RA }
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16 The electron energy E DF T plays the role of the potential energy, V , for the atomic nuclei. The
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17 forces are calculated every 1 f s. The positions and velocities of the nuclei are obtained by
18 applying the Verlet algorithm [53]. The Mulliken scheme is employed to determine the atomic
19 charges, and the charge exchange between compounds.
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23 3 Molecular Model
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25 The main DN A metabolic processes take place in the cell, where there is a specific physio-
26 logical environment. This section discusses the procedures to implement both, the elementary
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27 thermodynamic conditions of a biological system, and the representation of the steering forces.
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32 A 3D rectangular container is built from the potential Urect to keep the system with constant
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33 density. The potential along the x axis is defined by the expression:

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35 U (x) = (eax x + e−ax x ) / (eax bx + e−ax bx ) (3.1)
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The growth rate of the potentials is determined by the ax parameter: a large value of ax produces
an exponential wall with steep vertical growth, simulating a rigid wall potential, and a low value
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39 of ax produces an exponential wall with gradual increase, simulating a soft wall potential. By
40 suitably defining the value of ax , the potential decays rapidly towards the center of the container,
41 ensuring almost null interactions of the nucleic acid bases with the borders. At the boundaries
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x = bx and x = −bx , the value of the potential U (x) is 1 au. The 3D confining potential is the
44 sum of the individual components, Urect (R) = U (x) + U (y) + U (z). The limits (bx , by , bz ) give
45 the container dimensions in the x, y, z axes. The proposed confinement model avoids the use of
46 periodic-boundary-conditions, which presents complications in the conservation of the angular
47 momentum and the violation of the principle of energy equipartition [54, 55]. The effects of the
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potential are incorporated into the Newtonian equations of motion.
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3.2 NVT ensemble
53 The parameters taking part in Eq. 3.1 are ax = ay = az = 8 Å−1 , and bx = 15.1, by = bz = 7.6
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Å−1 . The rectangular container has volume V = 6977.41 Å3 , with space to carry the separation
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3 of the Watson-Crick pairs in the container. The aqueous solution consists of 218 water molecules
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surrounding the base pairs, which are positioned at the center of the container (Fig. 1). Two
6 different systems are created, AT @(H2 O)bulk and GC@(H2 O)bulk , with atom numbers N = 684
7 and N = 683. The density is 0.9968 g/cm3 and 0.9971 g/cm3 for the AT @(H2 O)bulk and
8 GC@(H2 O)bulk systems, respectively. These systems are immersed in a thermal bath, as the
9 atoms close to the container borders are in contact with the thermal bath. They are the α
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atoms, described by the Langevin equation of motion [56]:
12 d2 Rα (t)
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= −∇α E DF T (t) − ∇α Urect (t) − γMα Vα (t) + Gα (t) (3.2)
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dt
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34 aqueous solution (with 218 water molecules) confined in a rectangular box. The exponential functions employed
35 in the confinement of the molecular system are illustrated with the framework containing the water bulk.
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37 The atoms are considered Langevin atoms within a threshold distance of 3.5 Å from the
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39 container borders. The term −∇α E DF T is the systematic force due to the electronic cloud,
40 and −∇α Urect is the force exerted on the atoms by the confining potential. The contribution
41 −γMα Vα (t) is the viscous drag force of the thermal bath, where γ = 3.0 ps−1 is the friction
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42 coefficient. The contribution Gα is the stochastic force that simulates the collisions of the
43 thermal-bath particles with the Langevin atoms. The energy exchange with the thermal bath
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45 leads to the thermalization of the entire molecular system, and the Langevin atoms integrate
46 the Langevin thermostat [56].
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48 The atoms that are not part of the Langevin thermostat are recognized as the Newtonian atoms.
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50 d2 Ri (t)
Mi = −∇i E DF T (t) − ∇i Urect (t) − ∇i Usteer (t) (3.3)
51 dt 2
{WC bases}
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53 The steering force is −∇i Usteer , applied on selected atoms of the W C base pairs to induce the
54 breaking of the H bridges. The ordinary water atoms of the bulk satisfy the above equation of
55 motion, but omitting the steering force. The Langevin equations of motion are integrated using
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3 a Langevin integrator, and the Newtonian equations of motion are integrated using a Verlet
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integrator [57, 58, 59].
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7 The temperature of the molecular systems is 300 K. The temperature is imposed by a Marko-
8 vian distribution function that governs the stochastic forces, Gα [60]. Both, the friction coef-
9 ficient, γ, and the temperature, T , characterize the distribution function of the thermal-bath
10 fluid. The thermodynamic equilibrium is achieved when the statistical temperature is equal to
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the average mechanical temperature of the system, computed from the kinetic energies of the
13 Newtonian atoms (the mechanical temperature of the system plays the role of a mechanical
14 thermometer). The ergodicity of the simulations can be to some extent checked out. The sys-
15 tems in thermodynamic equilibrium of this type satisfy the first law of thermodynamics [56].
16 The N V T thermodynamic conditions of the system are imposed when the fixed number of
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atoms N = 684 for the AT @(H2 O)bulk system, N = 683 for the GC@(H2 O)bulk system, the
19 average volume V = 6977.41 Å3 , and the temperature T = 300 K are established.
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22 3.3 Steering atomic forces
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The AF M microscopy is a powerful technique for manipulating molecular structures under

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25 physiological conditions in real time [61, 62]. This technique is used in the study of the me-
26 chanical properties of biomolecules, in addition to characterizing the reactive features of the
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27 compounds at the nanometric scale [63, 64, 65]. The mechanical properties include, for in-
28 stance, the attraction and repulsion forces between molecular structures, the viscous effects of
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liquids, and the elasticity and hardness of materials [10, 66, 67]. The AF M microscope has
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31 been employed to evaluate the interactions that give shape to the DN A structure, like the
32 stacking forces and the H bridge forces of the nucleic acid bases [68, 69].
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46 Figure 2: The arrows in the image show the directions along which the AF M tips act on selected carbon
47 atoms of the base pairs. For AT the atoms C6 of adenine, and C4 of thymine were selected. For GC the atoms
48 C5 of guanine, and C5 of cytosine were selected. The lines of action minimize the torsional moments in the
49 separating process of the W C base pairs.
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51 In order to determine the forces responsible for the structural stability of the W C base pairs,
52 the basic operation principles of the AF M are enforced in this work [70, 71]. However, unlike
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the experiments that use a single AF M tip, the simulations consider two springs that simulate
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55 the actions of a pair of AF M tips. The pair of tips has no effect on the magnitudes of the
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3 rupture forces. Thus, the stretching of the H bridges is performed with two AF M tips acting
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on selected carbon atoms of the W C base pairs, in such a way to define a line of action centered
6 on the number of H bridges of each W C base pair (Fig. 2). In the case of AT , the line of action
7 is positioned at the center of the two H bridges. In the case of GC, with three hydrogen bridges,
8 the line of action is aligned with the central H bridge. The lines of action attempt to minimize
9 the torsional moments in the process of separating the W C base pairs. The stretching of the
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nucleic acid bases is carried out along the x longitudinal axis of the container by an external
12 potential of harmonic type, producing spring forces in opposite directions.
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Usteer = k[xi (t) − xoi (t)]2 /2 ; Fsteer = −k[xi (t) − xoi (t)] (3.4)
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15 The forces act on the i-th carbon atom, with instantaneous position xi . The term xoi is the origin
16 in the x axis of the spring acting on the i-th carbon. The spring origin moves away from the i-th
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18 atom in the form xoi (t) = ±xo ± vx t Å, with an initial spring elongation xo of 1.5 and 4.0 Å for
19 AT @(H2 O)bulk and GC@(H2 O)bulk [72]. The spring stiffness is k = 0.01 N/cm. The speed vx is
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20 fixed in the simulations with value vx = 0.0025 Å/f s. Such a value is adequate to display quasi-
21 static states, and observe the cleavage of the compounds in detail, establishing with relative
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precision the values of the energy, force, and net charge. The spring elongation increases the
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24 tensile stress on the selected carbon atoms. This method allows to introduce more spring forces
25 on the atoms of the molecule, and may be also used to determine the optimal position of the
26 AF M tip on the sample [16].
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3 4 Results
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The results to report are the energy, the charge, and the temperature changes of the molecular
7 systems, together with the forces involved in the separation of the nucleic acid bases.
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10 4.1 AT and GC in vacuum
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Force and Energy
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13 The AT and GC systems are studied in vacuum subjected to equal initial conditions of the
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steering forces (Fig. 2). The following results include the London-dispersion D3 expression in
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16 the DF T energy, nonetheless, some results without including such an energy contribution may
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17 be also given. The simulation times to observe the breaking of the H bridges are relatively
18 short, taking less than 7 ps in every case. The initial distance between the center of mass (CM )
19 of A and the CM of T is 6.0 Å, and between the CM of G and the CM of C is 5.6 Å (Fig.
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3). The large distance between A and T , also reported in [3], is defined by the positions of the
22 electrostatic potential wells. The initial distances between the hydrogen atoms and the acceptor
23 atoms forming the H bridges are on average 1.81 and 1.83 Å for AT and GC, respectively. As
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24 the steering forces pull the carbon atoms, the stress intensities on the H bridges increase. The
25 system A starts the separation from T at the instant of time 2900 f s, and G from C at 5560
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f s. At the instant of time 3675 the system A is completely separated from T . The respective
28 number for the systems G and C is 6394 f s. At such instants of time, the average distances
29 between the hydrogen atoms and the acceptor atoms forming the H bridges increased to 9.8
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30 and 10.8 Å for AT and GC, respectively (Fig. 4).

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51 Figure 3: Steering force vs distance between the CM of A and the CM of T , and the CM of G and the CM
52 of C in vacuum. The forces include dispersion effects.
53
54
55
56
57
58 9
59
1
2
3 The total energies with inclusion of dispersion effects of the AT and GC molecular systems in
4
5
vacuum are depicted in Fig. 4. The simulation is divided in three main stages there. In the
6 first stage, the nucleic acid bases remain linked by the H bridges. The region corresponding
7 to the first stage is shown at the left of the shadowed region. For AT this region covers the
8 time interval [1-2900] f s, and for GC we have [0-5560] f s. The average energies in these initial
9 regions are EiAT = −0.00513 and EiGC = −0.00320 au for AT and GC. Under conditions of
10
11
thermodynamic equilibrium, the inequality EiAT > EiGC is expected to be satisfied. Neverthe-
12 less, under the action of external steering forces, the molecular systems AT and GC are out
13 of thermodynamic equilibrium since the steering forces introduce energy into these molecular
14 systems. The dynamics of the nucleic acid bases change due to the pulling forces, making the
15 atoms vibrate beyond their equilibrium distances. In particular, the charges along the atomic
16
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17
bonds change dynamically, affecting the net charges of the individual AT and GC compounds.
18 In this context, the reversed inequality EiGC > EiAT is observed for the AT and GC systems
19 out of thermodynamic equilibrium.
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34 Figure 4: Molecular energies of the AT and GC systems in vacuum. The initial state, i, located on the left
35 side of the shadowed region, is characterized by nucleic acid bases forming W C base pairs. The final state,
36 f , located on the right side of the shadowed region, is characterized by dissociated nucleic acid bases. The
37 increasing energy in the shadowed region is associated to the i − f transition. The energies include dispersion
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38 effects.
39
40 The second stage is defined by the dissociation of AT into A and T , and of GC into G and
41
C. The region corresponding to the second stage is shown by the shadowed region. The
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42
43 molecular energy increases notably in that region (showing the shape of a deformed sigma
44 energy function). The cleavage of AT takes 775 f s and that of GC 834 f s with steering
45 forces of approximately 0.82 and 1.49 nN for AT and GC, respectively. The steering forces
46 without dispersion effects are 0.72 nN and 1.27 nN for AT and GC, respectively. In the
47
third stage, the nucleic acid bases have been separated. The region corresponding to the
48
49 third stage is shown at the right of the shadowed region. For AT this region covers the time
50 interval [3675-4550] f s, and for GC we have [6394-6950] f s. The average energies in these final
51 regions are EfAT = 0.02843 and EfGC = 0.05183 au. The energy change between the initial and
52 final stage of AT is |∆E AT | = 21.1 kcal/mol, and that for GC is |∆E GC | = 34.5 kcal/mol.
53
54
The respective energies without dispersion interactions are |∆E AT | = 16.54 and |∆E GC | =
55 29.8 kcal/mol. The experimental values for the energy change are 13 and 21 kcal/mol for
56
57
58 10
59
1
2
3 AT and GC, respectively [73]. According to the simulations in vacuum, we observe the scale
4
5
factor |∆E GC | = 1.64 |∆E AT | with dispersion effects, and |∆E GC | = 1.80 |∆E AT | with no
6 dispersion forces in going from AT with two H bridges to GC with three H bridges. Note
7 that the expression employed for estimating the dispersion energies has empiric character. It
8 depends, in fact, on the system under investigation, and may sometimes lead to energies in good
9 agreement with the experimental values and, in other situations, to overestimated dispersion
10
11
energies. In our situation, the dispersion corrections show no improvement over the energies
12 that include no dispersion energy correction [74, 75].
13
14 Charge
15
16 Fig. 5 shows four insets with the purpose to characterize the charge behavior of the W C nucleic
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17 acid bases in the dissociation process, with the D3 dispersion energy contribution included in
18
19 the total energy. The first two insets at the top of the figure exhibit the separation of the CM
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20 of A from the CM of T , and similarly for the G and C compounds. The separation of the CM s
21 is initially imperceptible, but the separation increases later due to the intense steering forces.
22 The plots at the bottom of the figure give the behavior of the electric charges of the individual
23
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nucleic acid bases. In the case of GC, there is an oscillatory response of the charges, which is
24
25 correlated with the oscillatory behavior of the GC energy, Fig. 4. In the case of AT , there is a
26 subtle polarization between A and T , which is inverted at the moments of the dissociation (refer
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27 to the plot in the colored region). A similar situation, but with different charge magnitudes is
28 observed in the case of the GC pair. After the dissociations occur, all the individual nucleic
29
acid bases remain stable and neutral, because the steering forces ceased their actions.
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32
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36
37
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58 11
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6
7
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9
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32 Figure 5: Insets at the top: relative distances between the CM of A and the CM of T , and similarly for G and
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33 C, in vacuum. Insets at the bottom: charge variations of the individual nucleic acid bases in the simulations.
34 The shadowed intervals correspond to the cleavage moments of the nucleic acid bases. The D3 dispersion
35 energy contribution is included in these simulations. The pulling forces induce the vibrations of the atoms
36 beyond their equilibrium distances, affecting the charges along the atomic bonds, together with the net charges
37 of the individual compounds.
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38
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40 4.2 AT and GC in aqueous solution
41
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42 Temperature
43
44 The Langevin thermostat imposes the 300 K equilibrium temperature of the molecular system.
45 The states of thermodynamic equilibrium are established with the physical observables: Teq =
46 300 K, N = 684 and N = 683 for AT @(H2 O)bulk and GC@(H2 O)bulk , respectively, and V =
47
48 6977.41 Å3 . After the thermodynamic equilibrium is achieved, three stages in the simulations
49 of AT @(H2 O)bulk are recognized:
50
51 • [0-350] f s: there are temperature fluctuations of ∼ 10% of Teq , and the nucleic acid bases
52 are free of the steering forces.
53
54 • [351-3850] f s: the steering forces are applied, and the maximum temperature is 344.6 K.
55
56
57
58 12
59
1
2
3 • [3851-6650] f s: the steering forces are switched off because the nucleic acid bases are
4
5
separated, and the temperature decreases down to ∼ 303.5 K.
6
7 In the case of GC@(H2 O)bulk , we similarly have:
8
9 • [0-250] f s: there is and average temperature of 303.2 K, and the nucleic acid bases are
10 free of the steering forces.
11
12 • [251-3200] f s: the steering forces are applied, and the maximum temperature is 331.6 K.
13
14 • [3201-7800] f s: the steering forces are switched off because the nucleic acid bases are
15
separated, and the temperature decreases down to ∼ 301.3 K.
16
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18 The different stages are divided with vertical bars in Fig. 6.
19
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Figure 6: Plots of temperature vs time of the molecular systems AT @(H2 O)bulk and GC@(H2 O)bulk . The
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33
regions in color indicate the time intervals in the separation process of the canonical bases. The temperature
34
increments are due to the steering forces acting on the AT and GC systems. When such external forces are
35
turned off, the systems return to the equilibrium temperatures of ∼ 300 K due to the effects of the Langevin
36
thermostat.
37
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40 Force and Energy
41
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42 The interest in this section is to analyze the breaking of the H bridges when the nucleic acid
43 bases are immersed in water. The simulation times to observe the breaking of the H bridges
44
45
take less than 7800 f s in every case (Fig. 7). In order to simplify the analysis, each simulation
46 is divided in three stages, as before. In the initial stage (i), the nucleic acid bases are free of
47 the steering forces, with average temperatures 297.8 K and 303.2 K for AT @(H2 O)bulk and
48 GC@(H2 O)bulk , respectively. The average energy of the wholes system in the time interval
49 [0-350] and [0-250] f s are EiAT = −0.5712 and EiGC = −0.2668 au, respectively (Fig. 8).
50
51
52
In the second stage, the systems AT and GC are subjected to initial forces of 1.00 and 1.08 nN
53 with the goal of increasing the kinetics of the systems. The temperature gradually increases,
54 while the Langevin thermostat works on stabilizing the temperature of the system to 300 K.
55 There are temperature peaks of 344.6 K and 331.6 K for AT @(H2 O)bulk and GC@(H2 O)bulk
56
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58 13
59
1
2
3 (Fig. 6). The dissociations of the nucleic acid bases are observed in the time intervals [351-
4
5
3850] and [251-3200] f s, with average steering forces 1.15 and 1.43 nN for AT @(H2 O)bulk and
6 GC@(H2 O)bulk , respectively (Fig. 7). The nucleic acid bases are completely separated, with a
7 maximum CM distance of 15.9 Å for A and T at the instant of time 3856 f s, and 14.6 Å for
8 G and C at the instant of time 3243 f s (Fig. 8). At such instants of time, the water molecules
9 already occupy the space between the individual nucleic acid bases. In the final stage (f ), the
10
11
steering forces are switched off, and the individual nucleic acid bases get slightly closer to each
12 other reaching a distance of 12.6 Å for A and T , and 11.9 Å for G and C. The nucleic acid bases
13 are not observed to reform the W C base pairs, yet, they are stabilized by forming H bridges
14 with the surrounding water molecules. A and T form 7 H bridges with the surrounding water
15 molecules. As a reference, the united nucleic acid bases show 9 and 6 H bridges, respectively.
16
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In the case of G and C separated from each other, 9 and 7 H bridges are formed with the
18 surrounding water molecules. When they are united, the number of H bridges with the water
19 molecules are 6 for each of them.
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37 Figure 7: Steering force vs distance between the CM of A and the CM of T , and the CM of G and the CM
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38 of C, when the nucleic acid bases are immersed in water.

39
40
41 The final energy of the whole molecular system is averaged in the time interval [5500-6650] and
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42 [6000-7800] f s for AT @(H2 O)bulk and GC@(H2 O)bulk . The energy values are EfAT = −0.6170
43 and EfGC = −0.3213 au. The respective energy changes are |∆E AT | = 28.8 and |∆E GC | =
44
34.2 kcal/mol. It is important to remark that such values include the reorganization of the water
45
46 molecules around the nucleic acid bases. We observe the scale factor |∆E GC | = 1.19 |∆E AT | in
47 going from AT with two H bridges to GC with three H bridges.
48
49 The dissociation of the W C base pairs in solution and in vacuum exhibit different features. In
50 solution, the nucleic acid bases show a soft and progressive separation because the surrounding
51
water molecules mitigate the separation process. This is, the water molecules avoid an abrupt
52
53 separation of the nucleic acid bases and, in consequence, it is difficult to establish specific
54 values of the steering forces that separate the nucleic acid bases (Fig. 7). On the other hand, in
55 vacuum, the nucleic acid bases are abruptly separated, and the steering forces exhibit specific
56
57
58 14
59
1
2
3 values (Fig. 3). A similar situation is observed in the case of the energies. Such energies change
4
5
in gradual manner when the nucleic acid bases are separated in aqueous solution (Fig. 8), but
6 show sudden and pronounced changes when the nucleic acid bases are in vacuum (Fig. 4). In
7 principle, the replication process of the DN A, which involves the cleavage of the W C base
8 pairs, appears to be less error-prone in water bulk than in vacuum. Table 1 summarizes the
9 steering forces and energy changes registered in the dissociation of the nucleic acid bases in
10
11
both vacuum and aqueous solution. Other values of the literature are also included.
12
13
14
15
16
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18
19
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21
22
23
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24 Figure 8: Molecular energies of AT @(H2 O)bulk and GC@(H2 O)bulk . The initial state, i, located on the left
25 side of the shadowed region is characterized by nucleic acid bases forming W C base pairs. The final state, f ,
26
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located on the right side of the shadowed region is characterized by dissociated nucleic acid bases. The energy
27 barrier in the gray region is associated to the i − f transition.
28
29
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30
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32
Table 1: Steering forces (nN ) and energy changes (kcal/mol) in the dissociation
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33 process of the W C base pairs.

34 W C Base Pair ∆E ∆Eexp Fsteer Fexp Ftheo
35 AT (in vacuum) a c
21.1 (13.4 , 15.0 )c
13.0 b
0.82 a
36
GC (in vacuum) 34.5a (25.4c , 27.5c ) 21.0b 1.49a
37
AT (in solution) 28.8a (12.8e , 13.6e ) 20.0d 1.15a 0.63d 0.55e
On
38
39 GC (in solution) 34.2a (28.0e , 25.5e ) 25.0d 1.43a 1.08d 0.86e
40 a Thiswork. b Mass spectrometry [73]. c Quantum mechanical calculations [76, 77].
41 d Atomic force microscopy [15]. e Forces with AMBER 4.1 [78], and energies with
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42
43 AMBER-4.1 and CHARMM-23 [79].
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 15
59
1
2
3 Charge
4
5 All the molecular systems simulated in this work are neutrally charged. Still, there are charge
6 fluctuations between the individual nucleic acid bases and the water bulk in the separation
7
8
process of the nucleic acid bases. The charge fluctuations are shown in Fig. 9. The separation
9 distances between the CM of A and the CM of T , and the CM of G and the CM of C are
10 also depicted in that figure to correlate the charge changes with the separation distances. The
11 shadowed regions covering the time intervals [351-3850] and [251-3200] f s correspond to the
12 dissociation of the nucleic acid bases AT @(H2 O)bulk and GC@(H2 O)bulk .
13
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15
16
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32
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33 Figure 9: Insets at the top: relative distances between the CM of A and the CM of T , and similarly for G
34 and C, in water solution. Insets at the bottom: charge variations of the individual nucleic acid bases in the
35 simulations. The shadowed intervals correspond to the cleavage moments of the nucleic acid bases. The pulling
36 forces induce the vibrations of the atoms beyond their equilibrium distances, affecting the charges along the
37 atomic bonds, together with the net charges of the individual compounds.
On
38
39
40 In the initial part of the AT @(H2 O)bulk simulation (time interval [0-350] f s), the total charges
41 of the nucleic acid bases are opposite to the total charge of the water bulk. The total charges of
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42 the systems fluctuate between [−0.25, 0.25]e. In the time interval [1000-3400] f s, the charges of
43 A and T show the largest difference with respect the charge of water bulk. When the systems
44 A and T exhibit the largest separation distance (time interval [3400-3800] f s), the net charges
45
46 of A, T , and water bulk are lower than |0.10 e|, indicating that some charge transfers occurred
47 between the nucleic acid bases and water bulk. Such systems tend to charge neutrality. The
48 A compound practically remains neutral after the dissociation (time interval [5000-6650] f s),
49 while the T molecule acquires a relatively small negative charge, always compensated by the
50
water bulk. The (almost) charge neutrality of the A and T compounds gives, to some extent,
51
52 evidence of the hydrophobic character of such nucleic acid bases. The nucleic acid bases are
53 surrounded by their own water molecules in the final part of the simulation.
54
55 The charge behaviors of G, C, and water bulk show less notorious changes in the GC@(H2 O)bulk
56
57
58 16
59
1
2
3 simulation. The C the compound essentially remains neutral when the nucleic acid bases are
4
5
separated.
6
7
The net charges of A, T , G and C show a symmetrical polarization in vacuum (Fig. 5). Such
8 a polarization fades out as the nucleic acid bases are separated from each other. On the other
9 hand, the nucleic acid bases show no polarization in aqueous solution, as they are able to share
10 charges with the surrounding water molecules (Fig. 9). In this connection, the water molecules
11 play the role of a regulatory factor in the cleavage of the nucleic acid bases, consequently
12
13
allowing subtle structural changes in those dissociation processes.
14
15
16 5 Conclusions
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17
18 This work investigated the dissociation of the nucleic acid bases by evaluating their energies,
19 forces, and electric charges. The first-principles molecular dynamics was employed in combi-
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20 nation with DF T , together with steering molecular forces. Such a level of theory is known
21
22 to provide good accuracy with a relatively low computational cost. The simulations were per-
23 formed for the AT and GC Watson-Crick base pairs in vacuum, with and without dispersion
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24 effects, and in aqueous solvent, where the ambient conditions of a biological system were re-
25 produced. The estimated dissociation energies of AT and GC in vacuum were 21.1 and 34.5
26
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kcal/mol, with average cleavage forces of 0.82 and 1.49 nN , respectively. Under conditions of
27
28 explicit solvent the estimated dissociation energies of AT and GC were, in that order, 28.8 and
29 34.2 kcal/mol, with forces of 1.15 and 1.43 nN . The estimated values are compatible with those
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30 obtained in experimental studies, particularly using the atomic force microscope, and with the
31 results from different theoretical approaches.
32
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33 This work also contributes with a novel theoretical scheme. The implementation of exponential
34
potentials to create the confinement of the molecular system, together with the Langevin ther-
35
36 mostat to simulate the contact with a thermal bath, which in turn imposes the thermodynamic
37 conditions of a N V T ensemble, and the harmonic potentials to simulate the action of an AF M
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38 tip, form all together a general and flexible approach that may be exploited to investigate, for
39 example, the stacking of the nucleic acid bases, the folding of proteins, the products of chemical
40
reactions, the thermodynamic potentials, etc.
41
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42
The present work has shown the importance of the aqueous solution in the simulations of DN A
43
44 fragments as, for example, the water molecules regulate the net charges of the A, T , C and G
45 compounds, generating less polarization effects, with soft energy changes, and subtle structural
46 modifications in the dissociation processes of the nucleic acid bases. The aqueous solution is
47 ignored in many simulations, yet, it appears to be a key factor in the preservation of the genetic
48
code by reducing drastic structural changes of the nucleic acid bases.
49
50
51
52 6 Acknowledgments
53
54 The authors acknowledge DGTIC for access to the Miztli computer, and express gratitude to
55 Carlos E. Lopez Nataren of IF-UNAM for valuable support.
56
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58 17
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1
2
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Journal of Biomolecular Structure & Dynamics

Dissociation of the Watson-Crick Base Pairs in Vacuum and

Journal: Journal of Biomolecular Structure & Dynamics

Manuscript Type: Research Article

Date Submitted by the

Complete List of Authors: Ordóñez, Cristian; UNAH,

santamaria, ruben; UNAM, Theoretical Physics

DNA nucleic acid bases, hydrogen bridges, solvation effects, density

URL: http://mc.manuscriptcentral.com/jbsd Email: TBSD-peerreview@journals.tandf.com

30 explained in the following pages.

27 longitudinal and transverse stretchings of selected DN A fragments were performed to determine

27 focus attention on the lateral base pairing interactions.

30 3.1 Confinement model

33 density. The potential along the x axis is defined by the expression:

The AF M microscopy is a powerful technique for manipulating molecular structures under

30 and 10.8 Å for AT and GC, respectively (Fig. 4).

38 of C, when the nucleic acid bases are immersed in water.

33 process of the W C base pairs.

33 (22), 11935–11940. doi:10.1073/pnas.94.22.11935

perties of DNA. Biophysical journal, 88 (3), 1684–1691. doi:10.1529/biophysj.104.046912

30 research, 34 (2), 564–574. doi:10.1093/nar/gkj454

27 J. Chem. Phys, 98 (7), 5648–6. doi:10.1063/1.464913

Quantum Mechanics to Fluid Dynamics. Cambridge: Cambridge University Press.

30 [54] Kuzkin, V. A. (2015). On angular momentum balance for particle systems

27 empirical potentials (AMBER, CFF95, CVFF, CHARMM, OPLS, POLTEV), semiem-

30 results. Journal of Computational Chemistry, 18 (9), 1136–1150. doi:10.1002/(SICI)1096-

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