Chemical Physics Letters 495 (2010) 117–120

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Chemical Physics Letters

journal homepage: www.elsevier .com/ locate /cplet t

An in silico study of the protonated DNA triplex: In vivo stability of C+GCin a DNA triple helix

Jiwon Jung, Sang Hak Lee *

Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea

a r t i c l e i n f o

Article history:Received 12 May 2010In final form 17 June 2010Available online 22 June 2010

0009-2614/$ - see front matter Crown Copyright � 2doi:10.1016/j.cplett.2010.06.045

* Corresponding author. Fax: +82 2 889 5719.E-mail address: shlee@skkim.snu.ac.kr (S.H. Lee).

a b s t r a c t

Protonated DNA base pairs, particularly the Cytosine–Guanine–Cytosine (CGC) triple pairs, were investi-gated its relative energetics by theoretical calculations. The energetic stabilities of C+GC and CG+C arealmost the same in the gas phase, although the C+G pair is more stable than CG+ by ca. 1150 cm�1

(3.34 kcal/mol). It is to be noted, however, that only C+GC was found in vivo, which seems to indicatedifferent chemical environments for the triple pairs in vivo.

Crown Copyright � 2010 Published by Elsevier B.V. All rights reserved.

1. Introduction

The main function of DNA is the long-term storage of geneticinformation; thus its structural stability, including a chemical as-pect, plays a vital role in preserving the genetic informationthrough specific base pairing [1]. However, DNA can be modifiedchemically or structurally by external stimuli, or it can react toenvironmental toxins [2,3], which can lead to its damage. Usually,most DNA damage can be fixed by repair enzymes in an healthyorganism [4,5]. But if much unrepaired damage accumulates, theresult might be mutations, which are a causative factor of an inher-ited disease [6].

Actually, Friedreich’s ataxia is a genetic disease caused by DNAstructural mutation, especially of the DNA triple helix [7]. In fact,the triple helix structure is generated by a simple chemical changeon DNA, such as protonation and deprotonation. H-DNA and stickyDNA among the triplex helices are from the protonation of DNAbases, especially cytosine [7,8]. Protonation and deprotonationare simple chemical reactions; thus they often occur in a chemicalenvironment. However, though the triple helix is caused by pro-tonation, its molecular properties are rarely considered.

A DNA triple helix is made by triple base pairing, which is calleda triplex. Triplexes can be classified into two types; a (Y)-pyrimi-dine–(R)-purine–(Y)-pyrimidine pairing and a Y–R–R pairing [8].In the case of Y–R–R, divalent metal cation plays a vital role inenergetically stabilizing the triplex [9]. Otherwise, a key factor tostabilize the Y–R–Y type triplex is not a divalent metal ion but achange in intrinsic molecular properties. The previous studies, ithas been discovered that there were two requirements to stabilizethe Y–R–Y type triplex; the first is cytosine protonation [10,11] and

010 Published by Elsevier B.V. All r

the other is the Hoogsteen base pairing of the third strand [12].Actually, in an in vivo study [8] it was found that the Y–R–Y typetriplex always had a protonated cytosine, which is a prerequisiteto the Hoogsteen base pairing.

However, it is not easy to explain why only the protonated cyto-sine, though the guanine in the triplex also is able to be protonatedas the previous result [13], is essential to stabilize the Y–R–Y typetriplex, because there are many considerable factors, such as thepH condition, electrostatic interaction with the phosphate back-bone, and the solvation effect. Actually, only by using experimentalmethods it is difficult to select which one is the key factor to sta-bilize the Y–R–Y triplex. However, an in silico study can providethe answer to the question of how to stabilize the triplex. Thuswe employed the in silico method to figure out the molecular prop-erties, such as the energetics, structure, and vibration, of a DNA tri-ple helix.

2. Computational methods

In this study we carried out theoretical calculation for the pro-tonated DNA triplex at the levels of HF(6-31+G*), B3LYP(6-31+G(d)and 6-311+G(2d,p)) using the GAUSSIAN program suite [14]. Firstly,we obtained the optimized geometry of the gas phase throughthe full optimization. And from this geometry solvation energieswere obtained by re-optimization using solvation models, suchas the Onsager model, Polarizable Continuum model (PCM), Con-ductor-like PCM (CPCM) and Integral-Equation-Formalism PCM(IEFPCM) implemented in the GAUSSIAN program suite. At all levelswe specified water (dielectric constant, e = 78.39) as the solvent.Solvation energies for various solvents were calculated at the levelof B3LYP/6-311+G(2d,p) using IEFPCM. Also, the vibration frequen-cies in water were obtained by using CPCM with B3LYP/6-311+G(2d,p).

ights reserved.

PCM

IEFP

CM

Gas

phas

e

+G

CC

G+C

C+G

CC

G+C

C+G

CC

G+C

1325

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75.

38

118 J. Jung, S.H. Lee / Chemical Physics Letters 495 (2010) 117–120

3. Results and discussion

Among the several effects that may perturb the triplex, such asstructure and stability, we paid attention to the solvation effect asmany studies have carefully considered the solvation effect to fig-ure out the energetics and the structure for DNA [15], amino acid[16] and protein folding [17]. In fact, all biomolecules were instate of solution, and their function was determined by theirstructure in the solution.

In this Letter, we explain why only cytosine is protonated inthe Y–R–Y triplex of in vivo through an in silico study using thesolvation model. Firstly, according to the previous computationalstudy [13], the energy difference between two protonated dimers(C+G and CG+) was 1154 cm�1 (3.35 kcal/mol), a value obtained atthe level of B3LYP/6-311+G(2d,p), in the gas phase. Otherwise, atthe same level, C+GC(CH+� � �GC) and CG+C(C� � �H+GC) had little en-ergy difference in the gas phase. The energy difference was lessthan 0.01 kcal/mol after basis set superposition error (BSSE) cor-rection. However, there was no drastic structural difference be-tween the two isomers, as shown in Fig. 1a. This point is inaccord with the vibration analysis, in which the frequency ofthe proton stretching vibration was quite similar to each other,2374 cm�1 (C+GC) and 2392 cm�1 (CG+C), respectively.

Interestingly, though there is no energy difference betweenC+GC and CG+C in the gas phase, only the C+GC form is foundin vivo. As previously mentioned, we believe that this result isdue to the solvation energy difference. Thus we employed severalsolvation models such as the Onsager model, PCM, CPCM andIEFPCM, which are implemented in the GAUSSIAN 03 program suite.

C+G

CG+

CG+C

C+GC

C+GC

CG+C

gas phase solution

E

C

G

C

H+

CH+...GC (C

(a)

(b)

+GC)

C HG+C (CG+C)

H+

C

G

C

1154 cm-1862 cm-1

Fig. 1. (a) Optimized geometries of CH+� � �GC(C+GC) and CGH+� � �C(CG+C). (b)Schematic energy diagram for comparing stability in the gas phase and in thesolution. Ta

ble

1Ca

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ated

ener

gies

for

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Can

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+ Ctr

iple

x.

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(cm�

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ues

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ent

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der

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itof

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0 10 20 30 40 50 60 70 80100

200

300

400

500

600

700

800

900

En

erg

y d

iffe

ren

ce (

cm-1

)

Dielectric constant

Ar

Cyc

lohe

xane

Isoq

uino

line Et

hano

l

Met

hano

l

Wat

er

Fig. 2. Plot of energy differences between C+GC and CG+C obtained at varioussolvents. From the ethanol (e = 24.55) energy difference was saturated.

J. Jung, S.H. Lee / Chemical Physics Letters 495 (2010) 117–120 119

We carried out the theoretical calculation at three different lev-els (HF/6-31+G*, B3LYP/6-31+G* and B3LYP/6-311+G(2d,p)) usingfour kinds of solvation models. In this calculation, we obtainedmeaningful results for the energy difference between C+GC andCG+C. As mentioned above, in the gas phase, there is no energy dif-ference. For the Onsager model, energy differences were observedat approximately 523 cm�1 (1.52 kcal/mol), 525 cm�1 (1.52 kcal/mol) and 450 cm�1 (1.31 kcal/mol) in a water solvent. These valuesare meaningful if we consider the room temperature energy,227 cm�1 (0.65 kcal/mol). Additionally, as summarized in Table 1,the values of the energy difference obtained using other solvationmodels are at least twofold larger than those obtained using the

Rel

ativ

e In

tens

ity (a

. u.)

CG+C

C+GC

Rel

ativ

e In

tens

ity (a

. u.)

0 500 1000 1500 2000 2500 3000 3500 4000

0 500 1000 1500 2000 2500 3000 3500 4000

Wavenumber (cm-1)

Gas Phase

νproton

νproton

Fig. 3. Predicted IR spectra in the gas phase and in the condensed phase. These spectra wthe proton stretching mode in the triplex.

Onsager model. In other models, energy differences were predictedfrom 1482 cm�1 (4.30 kcal/mol) to 855 cm�1 (2.48 kcal/mol). Thesevalues are more reliable than those of Onsager, because the Onsag-er model treats the solute molecule as only one sphere. Thus theobtained energy difference from the Onsager model might under-estimate the solvation effect. Nevertheless, the entire energetictendency, in which C+GC is more stable than CG+C in a solution,was coincident at all solvation models and calculation levels asshown in Fig. 1b.

Furthermore, we carefully investigated the dipole moment oftwo isomers. The dipole moment can be a direct clue to explainthe energetic difference in a solution. In fact a dipole moment doesnot perturb the intrinsic energetics of a molecule, but in a solutionthe dipole moment is a crucial factor to stabilize the molecule be-cause solvation energy is a result of the interaction between themolecular charge distribution and the reaction field of the solvent.Thus the isomer of a larger dipole moment is in a more stable formin a solution, especially in a polar solvent. In this calculation, thedipole moment of C+GC was predicted to be twice as large as thatof CG+C. A difference of the dipole moment also strongly supportsthe idea that the C+GC form was more stable in vivo.

We carried out the calculation for obtaining the energy differ-ence in the various solvent to more clearly investigate thesolvation effect for C+GC and CG+C using IEFPCM. From this calcu-lation, we found that the energy difference was increased as thefunction of the dielectric constant of the solvent increased asshown in Fig. 2. And from e = 24.55 (ethanol), the energy differenceis saturated, which is the typical phenomenon for solvation. Thuswe believe that the solvation effect plays a key role in observingonly the C+GC form in Y–R–Y triplex in vivo unlike in the gas phase.

Fig. 3 shows the simulated IR spectra for two species in the gasphase as well as in water solution. In the gas phase, the energeti-cally similar, vibrational frequencies of the proton stretching mode

0 500 1000 1500 2000 2500 3000 3500 4000

Wavenumber (cm-1)

0 500 1000 1500 2000 2500 3000 3500 4000

Rel

ativ

e In

tens

ity (a

. u.)

CG+C

C+GC

Rel

ativ

e In

tens

ity (a

. u.)

Condensed Phase

νproton

νproton

ere convoluted by the Lorentzian function with 10 cm�1 bandwidth. mproton indicates

120 J. Jung, S.H. Lee / Chemical Physics Letters 495 (2010) 117–120

of C+GC and CG+C are remarkably similar 2370 cm�1 and2390 cm�1, respectively. However, the proton stretching frequen-cies are blue shift, 2774 cm�1 (C+GC) and 2590 cm�1 (CG+C) in awater solution, which means that both triplex forms exist in a sta-ble state in a solution. In these spectra, we found that the fre-quency shift of the proton stretching mode of C+GC, 409 cm�1,was twice as large as that of CG+C, 200 cm�1. It also directly sup-ports the idea that the C+GC form is energetically more stable insolution.

4. Conclusions

In summary, we carried out theoretical calculation using solva-tion model to elucidate that the solvation effect plays a major rolein stabilizing the protonated DNA triplex. In this calculation, wefound that, in the protonated CGC triplex, C+GC form is energeti-cally more stable than CG+C in the solution. This observationstrongly supports why only the C+GC form was found in vivo. Thus,we concluded that the solvation factor should be mainly consid-ered to figure out the molecular property of the DNA triplex ofin vivo. Additionally, if the solvation effect is studied, includingthe electrostatic interaction between the triplex and the phos-phate-sugar backbone, more light would be shed on the physicaland chemical properties of DNA triple helix in vivo.

Acknowledgements

S.H. Lee thanks Prof. Seong Keun Kim for stimulating discus-sions. This work was supported by Grant No. KSC-2008-S04-0002from Korea Institute of Science and Technology Information.

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