Properties of the Thiobase Pairs Hydrogen Bridges: A Theoretical

Jan 23, 2009 - Properties of the Thiobase Pairs Hydrogen Bridges: A Theoretical Study. Giovanni Villani†. Istituto per i Processi Chimico-Fisici, IP...
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J. Phys. Chem. B 2009, 113, 2128–2134

Properties of the Thiobase Pairs Hydrogen Bridges: A Theoretical Study Giovanni Villani† Istituto per i Processi Chimico-Fisici, IPCF-CNR, Via G. Moruzzi, 1, I-56124 Pisa, Italy ReceiVed: August 28, 2008; ReVised Manuscript ReceiVed: NoVember 24, 2008

The characteristics of the hydrogen bridges in the base pairs with S f O substitution have been studied and the comparisons with the unmodified systems considered. The modifications induced by the sulfur atom in the structures, energies, and atomic charges have been evidenced and the energetic effects revaluated for the first time. Their effects on the hydrogen transfer from a base to the other and the relevance of these static and dynamical features for biological properties have been suggested. 1. Introduction Nowadays, there has been continuous interest in the structural and functional properties of modified nucleic acid bases since most of them are widely implicated for a variety of biological activities. In particular, chemically modified bases are frequently studied for their numerous pharmacological, biochemical, and biological capabilities. For example, 6-thioguanine is one of the most important therapeutic agents used in the clinical treatment of acute childhood lymphoblastic leukemia, AIDS, and some other pathologies,1-6 or 2-thiouracil and 4-thiouracil can be used as mutagenic, anticancer, and antithyroid drugs.7-11 Thiobases influence the structure of DNA, though the exact picture of such changes is not known at the molecular level. Also, the changes of the interactions of metal cations with thiobases are well documented.12 For example, the presence of a sulfur atom increases the ability of thiogaunines to interact with soft metal cations, such as mercury and cadmium, compared to that of guanine.13 It is well-known that the G-C thiobase pairs are likely the most biologically significant with respect to the therapeutic effects of thiopurines. In fact, several DNA-protein interactions and enzyme activities involved in DNA replication and repair have been shown to be altered in the presence of G-C thiobase pairs. Since in the pair G-C there are two oxygen atoms, one on guanine and the other on cytosine, the G-C thiobase pairs can be obtained both from the thioG opposite cytosine and thioC opposite guanine. Here, we have studied both cases. In the literature, the activities of RNase H,2,14 DNA ligase,15 and topoisomerase II (Topo II),1 key enzymes involved in DNA repair and replication, have been shown to be significantly altered in the presence of nucleic acid substrates containing single thioG modifications opposite cytosine. These findings show that thioG in the context of a G-C thiobase pairs modifies the functions of specific DNA-processing and recognition proteins. In the literature, there are no systematic experimental studies planning the improvement of these base pair stabilities because it is difficult and time-consuming to prepare a wide variety of nucleic acid base analogues and to experimentally measure their base pair formation ability. Thus, to improve the base pair stability, theoretical calculations of nucleic acid base analogues are highly demanded. These studies are also helpful for understanding the nature and the dynamics of the hydrogen † E-mail: [email protected].

bonds of these modified base pairs and to suggest a mechanism for biological properties. In particular, the thiobases have the same distribution of hydrogen donors and acceptors as the standard bases; however, the sulfur atom may induce changes in the properties of bases and their interactions, and although the tautomeric equilibria are of interest, the modified biochemical activity of thiobases is mainly due to their altered molecular interactions. For example, the therapeutic effects of the thioguanines are believed to be related to their incorporation into the DNA,16-20 but not many details regarding structural and functional consequences of such incorporation are known. In this paper, we have systematically studied the effect of the replacement of one or two oxygen atoms with sulfur in G-C and A-T base pairs as a function of energies and atomic charges. A complete study of thiobase pairs is not present in the literature, and the biological importance of these systems suggests the necessity of this study. Moreover, it is well-known17 that the substitution of oxygen atom with sulfur in the thiopurines has a local effect on the chemical environment that can be extended from one to two base pairs beyond this modification, but the most significant change in the chemical environment occurs at the base pair containing the sulfur atom. Also, the effects of sulfur atom substitution on base pair dynamics is highly localized, and the base pair lifetimes involving adjacent residues are similar.17 For this reason, a theoretical study of one modified base pair can give interesting indications in the nature of different chemical and biological properties of this type of system. The gas-phase properties and molecular interactions of 6-thioguanine have been already studied by means of ab initio methods21 some years ago. It was found that 6-thioguanine has tautomeric properties very similar to those of guanine and that its electrostatic distribution is also similar, as demonstrated by the dipole moment oriented in the same direction, and only slightly larger for 6-thioguanine than for guanine (7.8 D vs 6.6 D). Kawahara et al.22,23 have studied the base pairs with a similar base set. Here, we have studied these base pair systems with a more accurate basis set, and this paper can also be used to verify these previous results. 2. Results, Discussion, and Biological Considerations First of all, we would like to clarify the notation that will be used in this paper. The base pairs are named A-T and G-C in this order in every case, and hence, the thiobase pairs are A-thioT, A-(thio)2T, thioG-C, G-thioC, and thioG-thioC. The first system (A-thioT) has been obtained by the S f O

10.1021/jp807670f CCC: $40.75  2009 American Chemical Society Published on Web 01/23/2009

Thiobase Pairs Hydrogen Bridges

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TABLE 1: Atomic Distances (in Å) in the Hydrogen Bridges of the Thiobase Pairs and in the Corresponding Structures of Unmodified Base Pairs NsH-N

N-HsN

A-T 1.782 1.055 A-thioT 1.861 1.053 A-(thio)2T 1.889 1.054 G-C 1.039 1.877 thioG-C 1.033 2.092 G-thioC 1.032 2.087 thioG-thioC 1.031 2.171

N-HsO(S)

(S)OsH-N

1.027 1.024 1.024 1.027 1.032 1.027 1.031

1.723 2.223 1.667 2.208

1.897 2.406 2.392 1.890 1.776 2.335 2.291

1.045 1.041 1.047 1.043

TABLE 2: Angles (in deg) in the Hydrogen Bridges of the Thiobase Pairs and in the Corresponding Structures of Unmodified Base Pairsa

A-T A-thioT A-(thio)2T G-C thioG-C G-thioC thioG-thioC

N-H-N

N-HO(S)

179.6 170.9 175.7 177.0 174.2 172.6 173.7

174.2 175.6 168.7 178.3 174.1 176.3 174.1

(S)OH-N

179.2 174.4 176.1 171.6

(S)O(NH-N)

(N-H-N) O(S)

176.0 170.9 159.4 122.1

179.5 179.4 140.7 177.3 176.3 160.3 88.4

a The first three columns are bond angles and the last two dihedral angles.

substitution of the oxygen atom of the hydrogen bridge in the thymine. There is also another A-thioT base pair generated by the S f O substitution of the oxygen atom not directly involved in the hydrogen bridge in the thymine, but this case is considered only in Table 2 and explicitly named A-(thio)iT. The (A-(thio)2T) base pair is obviously obtained by substituting both the oxygen atoms of the thymine with sulfur atoms. The meaning of the other thiobase pairs is evident since both G and C have only one oxygen atom that can be substituted with S atom. The hydrogen bridges that bonds the two bases are N(A)sH-N(T) and N(A)-HsO(T) in the A-T structure and N(G)-HsO(C), N(G)-HsN(C), and O(G)sH-N(C) in the G-C one, where the long bond is the hydrogen one, of course. This notation can be easily simplified (without loss of precision) if we assume that the first heavy atom of the bridge is in A and G base and the second in the T and C one in every case (for example, N(A)sH-N(T) becomes NsH-N and N(G)-HsO(C) becomes N-HsO). Our theoretical investigation of these systems is based (in Gassian 2003 package24) on the density functional theoretical (DFT) approach (b3lyp) in cc-pVDZ basis set since the DFT is an efficient alternative to conventional ab initio theory for accurately describing the hydrogen bonds involved in DNA base pairs, and the basis set used is reliable for these systems.25-28 As demonstrated by Floria´n et al.29 in the studies of these systems, the use of the adiabatic potential that allows the geometry relaxation of the system during the hydrogen transfer process is necessary; hence, we have performed the calculation of the PES in this approach. With this approach, we have computed the full optimized structure of these systems. Tables 1 (distances) and 2 (angles) collect the main geometric parameters of the two or three hydrogen bridges of the thiobase pairs in the equilibrium structure, and the data of the corresponding unmodified base pairs are shown therein for comparison. By the analyses of these tables, we can emphasize the following conclusions: 1. In both A-T and G-C base pairs, the substitution of the oxygen atom with the sulfur generates a HsS hydrogen bond ∼0.5 Å larger than the OsH one.

Figure 1. Potential energy curves of A-T base pairs with oxygen or sulfur atoms. The distance in the horizontal axis is the variation of the hydrogen position in the bridge from the initial heavy atom to the other one. The labels “1” and “a” are related to one S f O or all oxygen atoms substitution, respectively.

2. In both base pairs the hydrogen bond between the two nitrogen atoms (N-H-N), indirectly involved in the substitution, is lengthened (∼0.1 Å in the A-T base pair and ∼0.2 Å in the G-C one). 3. The OsH-N hydrogen bond of the G-C base pair, indirectly involved in the substitution, is shortened by ∼0.1 Å. 4. When a second S f O substitution is added, and all oxygen atoms of the base pairs have been substituted with sulfur, the changes are very important in the G-C base pair (both sulfur atoms are involved in the hydrogen bonds) and little in the A-T base pair. In particular, in the case of G-C base pair, the N-H-N hydrogen bridge, not involved in the substitution, is lengthened by ∼0.3 Å. 5. After the S f O substitution, the planarity of the base pair is distorted (from a few degrees to 20 deg) in every case. In the cases with two sulfur atoms (A-(thio)2T and thioG-thioC), the base pair is a largely nonplanar system. Several modifications can also be found in the potential energy curves of the hydrogen bonds and in the atomic charges. In the following figures we have shown these modifications as a function of the hydrogen atom position in the bridge. In particular, in Figure 1 we have shown the potential energy curves of A-T and in Figure 2 of G-C modified/unmodified base pairs (in Figure 2a those of the hydrogen bridges, N-HsO and O-HsN, directly involved in the S f O substitution, and in Figure 2b that of the N-H-N hydrogen bridge, only indirectly involved in this substitution). In these figures the zero point energy of all systems has been assumed equal in order to evidence the differences among the curves. Figures 1 and 2 highlight the following considerations: 1. In the A-T system, the S f O substitution reduces the energy of the NsH-N bridge (indirectly involved) and increases that of the directly involved N-HsO one. The addition of the other S f O substitution, that of the oxygen atom not involved in the hydrogen bridges, gives an ulterior reduction of the energy of the NsH-N bridge, but it is irrelevant for the N-HsO one. In this system, the energy difference between the soft and hard29-32 hydrogen bridge is increased. 2. In the G-C system, the S f O substitution on the guanine base increases the energy of all (directly and indirectly involved) hydrogen bridges. The addition of the other S f O substitution, that of the oxygen atom of the cytosine, gives a reduction of

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Villani

Figure 4. Charges of the atoms involved in the NsH-N hydrogen bridge of A-T or modified base pairs.

Figure 2. (a) As in Figure 1, but for the N-HsO(S) and (S)OsH-N hydrogen bridges of the G-C base pairs and thiobase pairs. (b) As in Figure 1 but for the N-HsN hydrogen bridge of the G-C systems.

Figure 3. Charges of the atoms involved in the N-HsO(S) hydrogen bridge of A-T or modified base pairs.

the energy of all hydrogen bridges. As a consequence, the difference of energy between the hard and the soft bridges remains practically constant when two sulfur atoms replace oxygen. In Figures 3-5 there are the atomic charge variations of the A-T system as a function of the hydrogen position in the hydrogen bridges. In particular, in Figures 3 and 4 we have shown the charge variations of the atoms directly involved in the S f O substitution and in Figure 5 that of the oxygen (or

Figure 5. Charges of the oxygen or sulfur atom of the thymine not involved in the hydrogen bridges for A-T or thiobase pairs as a function of the hydrogen position in the bridge (in parentheses).

sulfur) atom of the thymine, indirectly involved in the hydrogen bridge. The atomic charge variations are the most significant modifications, induced by the S f O substitution, for understanding the different inter- and intra-base-pair interactions. From the analyses of Figures 3 and 4, first of all we note the typical atomic charge variation due to movement of the hydrogen atom from a base to the other (see Figure 7a in ref 27). The meaning of this behavior is that in these hydrogen bridges there is not a proton transfer separated from the electron one. In fact, the hydrogen atomic charge changes through the transfer from the donor to the acceptor, but never one has a proton passage and an electron that remains in the original fragment. As a consequence, the process of hydrogen transfer in both the A-T and the corresponding thiobase pairs systems is a case of proton-coupled electron transfer,33 similar to other biological processes.34 In particular, the charges of both the hydrogen atoms in the NsH-N and in the N-HsO bridges are only a little changed, comparing the initial to the final situation where these atoms move from a base to the other. In both cases, the heavy atoms supporting the bridge have complementary charge variation with the acceptor that becomes less negative and the donor more negative. When the oxygen atom is substituted from the sulfur, the behavior is practically unchanged in the NsH-N bridge, indirectly involved in the substitution, and the change is larger in the N-HsO(S) bridge, with a formation of a less polar atomic region. Different is the

Thiobase Pairs Hydrogen Bridges

Figure 6. (a) Charges of the atoms involved in the N-HsO(S) hydrogen bridge of G-C or modified base pairs, when only the oxygen atom involved in this bridge has been substituted with sulfur atom. (b) Charges of the atoms involved in the N-HsO(S) hydrogen bridge of G-C base pairs when also the oxygen atom not involved in this bridge has been substituted with sulfur atom.

behavior of the charge of the oxygen atom of the thymine, not involved in the hydrogen bridges. This atomic charge changes considerably and in an opposite way as a function of the hydrogen position in one or in the other bridge. This behavior is increased when this oxygen atom is substituted with the sulfur (Figure 5). In Figures 6-8, we have shown the atomic charge variations of the G-C base pair and of the substituted ones as a function of the hydrogen position in the bridges. In particular, in Figure 6 we have shown the charge variations of the three atoms of the N-HsO bridge, in Figure 7 that of the OsH-N, and in Figure 8 of the N-HsN ones. From the analysis of Figure 6a, we note that the hydrogen charge remains practically unchanged as a consequence of the oxygen substitution and that of the nitrogen atom becomes less negative. The behavior of the atomic charge of the sulfur is similar to that of the oxygen atom as a function of the hydrogen position in this bridge, but the amount of change is twice for the sulfur compared to the oxygen. In particular, this sulfur charge starts more negative than the oxygen, when the hydrogen atom is on the nitrogen atom, and it becomes largely less negative when this hydrogen arrives on the O or S atom. In Figure 6b, we note that the addition of the other oxygen substitution (on the guanine) does not change the atomic charge situation. In Figure 7a,b, we have shown the same atomic charges of Figure 6, but for the OsH-N hydrogen bridge. The modifica-

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Figure 7. (a) Charges of the atoms involved in the O(S)sH-N hydrogen bridge of G-C or modified base pairs, when only the oxygen atom involved in this bridge has been substituted with sulfur atom. (b) Charges of the atoms involved in the O(S)sH-N hydrogen bridge of G-C or modified base pairs, when also the oxygen atom not involved in this bridge has been substituted with sulfur atom.

tions induced from the oxygen substitution are similar. The only difference is in the relative charge of oxygen and sulfur atoms: practically identical when the hydrogen of this bridge is on the nitrogen atom and more different when this hydrogen atom arrives on O or S atom. In Figure 8, we have shown the atomic charge variations of the N-HsN hydrogen bridge. This bridge is different from the others since in every case it is only indirectly involved in the S f O substitution. In this bridge, the only charge modification, as a function of its hydrogen atom position, is that of the nitrogen atom of the base where the S f O substitution has been applied, and this atom becomes less negative. In any case, the difference between the oxygen or sulfur atomic charge is practically constant as a function of the position of the hydrogen atom in the bridge (Figure 8a,b). When the S f O substitution has been applied at both bases, both nitrogen atoms have this behavior and the hydrogen charge has a little positive variation (Figure 8c). Finally, the thiobases are also characterized by increased vertical and total molecular polarizabilities and increased intermolecular electron correlation stabilization is expected. The electronegativity of oxygen is larger than that of sulfur, which results in reduced polarity of the CdS bond, and when the sulfur

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Villani TABLE 3: Energy and Dipole Moment of Base Pairs and Thiobase Pairs A-T A-thioT A-(thio)iT A-(thio)2T G-C thioG-C G-thioC thioG-thioC

Figure 8. (a) Charges of the atoms involved in the NsH-N hydrogen bridge of G-C or modified base pairs, when only the oxygen atom involved in N-HsO bridge has been substituted with sulfur atom. (b) Charges of the atoms involved in the NsH-N hydrogen bridge of G-C or modified base pairs, when only the oxygen atom involved in OsH-N bridge has been substituted with sulfur atom. (c) Charges of the atoms involved in the NsH-N hydrogen bridge of G-C or modified base pairs, when both the oxygen atoms involved in N-HsO and OsH-N bridges have been substituted with sulfur atoms.

atom participates in the hydrogen bond, the H-bond length increases by about 0.5 Å and the pairs are weaker than the parent structures. The systems under the focus of the present work, as we said, are widely implicated for a variety of biological activities. Now,

energy (au)

dipole moment (D)

-921.5500 -1244.5152 -1244.5165 -1567.4785 -937.6029 -1260.5707 -1260.5668 -1583.5328

1.44 2.29 2.67 1.95 6.03 6.77 4.52 5.84

we would like to analyze if our calculations can give support to the common interpretation of those and/or can suggest new ideas. In the literature, the principal experimental data on these systems are NMR studies. There is not NMR data on the A-T thiobase pairs, at our knowledge. All NMR data on the G-C thiobase pairs suggest that the O f S substitution leads to only moderate changes in the stability of the DNA duplex, and these data suggest also that the presence of thioG leads to partial opening of the G-C Watson-Crick thiobase pair into the major groove, probably as a consequence of the larger size of the sulfur atom.35-37 Particularly, NMR exchange measurements by Somerville et al.38 indicated an anomalous fast proton exchange rate for the G-C thiobase pairs, corresponding to a ∼80-fold decrease of the base pair lifetime. In addition, the duplex stabilities of the oligonucleotides were observed as melting temperatures (Tms). An increase in Tm shows an increase in the duplex stability and vice versa. A change in Tm of the thiobase pairs is therefore an index of the substituent effect. In the case of sulfur f oxygen substitution in the G-C system, the melting temperature of this modified G-C duplex (Tm ) 39.4 °C) is ∼6 °C lower than that of the G-C duplex (Tm ) 45.0 °C).37-39 Our results on the stability of these thiobase pairs are shown in Table 3. As we can note, there is a very small difference between the case where the sulfur atom substitute an oxygen atom directly involved in an hydrogen bond with respect to the case where this atom is only indirectly involved. For example, the two cases of A-thioT base pairs have a difference of energy of ∼0.8 kcal/mol. This means that there is not a difference in the stability of the hydrogen bridge with oxygen or sulfur atom. Otherwise, in the PES of Figures 1 and 2, one can note that the energy change is significant if we consider also the nonequilibrium structures generate from the variation of the hydrogen atom position in the bridge. In the case of A-T systems, the two hydrogen bridges have different behavior when the oxygen atom is substituted from the sulfur one: the N-HsO is destabilized and the NsH-N stabilized, as a function of hydrogen position in the bridge. Otherwise, all the hydrogen bridges of the G-C systems are destabilized from the sulfur substitution of the oxygen. This means that the modified A-T base pair is broadly unchanged, but the G-C is destabilized. These different PES can give different biological properties. In particular, it is different the amount of the rare tautomers, the imino-enol forms, generate from the movement of hydrogen atoms form a base to the other. The relevance of these tautomers on biological processes has been discussed in the literature for a long time.40-42 The role of these tautomeric forms as mutation points of the DNA chains has been contested by our studies,27,28,30-32 in the case of base pairs with oxygen atoms. The NMR data and our calculations suggest that the substitution of O with S in guanine compromised base pair interactions in the G-C thiobase pairs compared to those in the G-C one. Moreover, from the analyses of the G-C and the substituted

Thiobase Pairs Hydrogen Bridges G-G structures,38 some structural perturbations in this thiopurine have been found, but only localized to the modified base pair. Those could not account for the significant differences observed in the thermal stability, the large chemical shift resonance positions difference between the G-C hydrogen atom in the thiobase pairs compared with that of G-C, and the biochemical and biological effects of thioG-modified DNA on enzyme activities, protein recognition, and cellular response. However, it must be considered that base pairs DNA are highly dynamic and, therefore, are continually in motion and local disruption and transient opening of individual base pairs occur naturally. As base pairs fluctuate between opened and closed states, the rate at which an individual imino proton exchanges with solvent, for example, is governed also by the lifetime of that base pair or the amount of time spent in the closed, base paired state. The effects of thioG in the base pair dynamics are much more pronounced than those on structure alone, and the large upfield shift of the thioG imino proton resonance suggests that the hydrogen bond formed by this atom is significantly weakened and/or that there may be significant differences in dynamics between the two systems: thioG-C and G-C. It is well-known in the literature, and supported here, that the thiobases have larger dipole moments than the standard bases, though the direction of the dipole moment is not changed. This means that the electrostatic dipole-dipole interaction in stacked and H-bonded complexes of thiobases will be enhanced. Moreover, due to the larger atomic radius of the sulfur atom, the exchange repulsion is larger as well, and this may cause steric problems in some configurations allowed for oxobases. Moreover, the increase of dipole moment in thioG-C base pair (and the decrease in the G-thioC one) can be relevant in the solvation of these systems. In summary, due to its biomolecular compatibility, thioG is metabolized and ultimately incorporated into DNA by DNA polymerase during replication. Other DNA processing enzymes (i.e., RNase H, Topo II, and DNA ligase), however, are not immune to the physical-chemical effects of thioG. These enzymes are exquisitely sensitive to the presence of thioG in DNA strands, and in the literature this is attributed to the striking and localized effects of thioG on base pair stability and dynamics. Thus, the structural and dynamic results for thioG-modified duplex DNA offer unique insights into its dichotomous mechanism of action, being structurally silent on the one hand and producing lethal effects on base pair stability and dynamics on the other.17 3. Conclusion In this paper we have analyzed the thio-substituted A-T and G-C base pairs. Three important conclusions can be schematized for the structures, the energies, and the atomic charges of these systems. 1. In both A-T and G-C base pairs, the substitution of the oxygen atom with the sulfur generates a HsS hydrogen bond ∼0.5 Å larger than the OsH one. In the thioG-thioC base pair, the N-H-N hydrogen bridge, indirectly involved in the substitution of the oxygen atoms, is lengthened by ∼0.55 Å. The A-(thio)2T base pair is a largely nonplanar system (∼40° between the A base and the (thio)2T one). 2. In the A-T system, the S f O substitution reduces the energy of the indirectly involved NsH-N bridge and increases that of directly involved N-HsO one. The addition of the other S f O substitution gives an ulterior reduction of the energy of the NsH-N bridge but is irrelevant for the N-HsO one. In the G-C system, the S f O substitution on the guanine base increases the energy of both the directly and indirectly involved

J. Phys. Chem. B, Vol. 113, No. 7, 2009 2133 hydrogen bridges. The addition of the other S f O substitution gives a reduction of the energy of all the hydrogen bridges. 3. When the oxygen atom is substituted by the sulfur in the A-T base pair, the charge situation is practically unchanged in the NsH-N bridge, indirectly involved in the substitution, and the change is larger for the N-HsO(S) bridge, with a formation of a less polar atomic region. When the oxygen atom is substituted by the sulfur in the G-C base pair, the charge of the hydrogen atom remains practically unchanged in the bridge directly involved in the substitution and that of the nitrogen atom becomes less negative. The behavior of the atomic charge of the sulfur is similar to that of the oxygen atom, but the amount of change (when the hydrogen moves from a base to the other) is twice for the sulfur compared to the oxygen. The addition of the other oxygen substitution does not largely changes the atomic charge situation. In the case of the N-HsN hydrogen bridge, as a function of the hydrogen position in the bridge, the only atomic charge modification is that of the nitrogen atom of the base where the S f O substitution has been applied and this atom becomes less negative, but this change is practically constant. When the S f O substitution has been applied at both bases, both nitrogen atoms have this behavior. Three are the key effects in order to understand the thio substitution and, probably, interrelated: the dramatically changes of the structural features, the drastic change of their functional ones expressed in terms of intermolecular interactions, and the energetic modification. The first two effects are well-known in the literature. As we have shown in the present work, there is a noticeable structural effect in the hydrogen-bonding patterns of the thiobase pairs. This is related to the fact that the sulfur atom involved in the formation of the hydrogen bond is longer compared to that of the oxygen one (the classical van der Waals radii of the sulfur atom, 1.85 Å, exceeds that of the oxygen one, 1.40 Å), and this may cause a marked strain in the doublehelix structure of DNA. The other implications of the thio substitution is due to the well-documented lower electronegativity of sulfur compared to oxygen atom. This likely underlies the suggestion that the modified biochemical activity of thiobases arises from their altered molecular interactions. The energetic modification induced by the sulfur substitution of the oxygen atom, instead, is denied in the literature since all energetic calculations are done in the equilibrium configuration. One of the most important result of this paper is to revalue this effect by the computation of the potential energy curves. In the comparison of literature and our dates, we would like to underline that the structural and energy changes have been underestimated in the first, since all considerations have been done on the equilibrium structures and not followed as a function of the movement of hydrogen atom in the bridges. Contrarily, the dynamical situation supposed in the literature can be supported from our analyses and estimated here. As a consequence, we believe that the biological and chemistry differences between base pairs and thiobase pairs can be explained by both structural and energy modifications and dynamical ones. References and Notes (1) Krynetskaia, N. F.; Cai, X. J.; Nitiss, J. L.; Krynetski, E. Y.; Relling, M. V. FASEB J. 2000, 14, 2339. (2) Krynetskaia, N. F.; Feng, J. Y.; Krynetski, E. Y.; Garcia, J. V.; Panetta, J. C.; Anderson, K. S.; Evans, W. E. FASEB J. 2001, 15, 1902. (3) Massey, A.; Xu, Y. Z.; Karran, P. DNA Repair 2002, 1, 275. (4) Presta, M.; Belleri, M.; Vacca, A.; Ribatti, D. Leucemia 2002, 16, 1490. (5) Herrlinger, K. R.; Kreisel, W.; Schwab, M.; Schoelmerich, J.; Fleig, W. E.; Ruhl, A.; Reinshagen, M.; Deibert, P.; Fellermann, K.; Greinwald, R.; Stange, E. F. Aliment. Pharmacol. Ther. 2003, 17, 503.

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