Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 29, Issue Number 1, (2011) Adenine Press (2011) Abstract Intramolecular CH O Hydrogen Bonds in the AI and BI DNA-like Conformers of Canonical Nucleosides and their Watson-Crick Pairs. Quantum Chemical and AIM Analysis http://www.jbsdonline.com The aim of this work is to cast some light on the H-bonds in double-stranded DNA in its AI and BI forms. For this purpose, we have performed the MP2 and DFT quantum chemical calculations of the canonical nucleoside conformers, relative to the AI and BI DNA forms, and their Watson-Crick pairs, which were regarded as the simplest models of the doublestranded DNA. Based on the atoms-in-molecules analysis (AIM), five types of the CH O hydrogen bonds, involving bases and sugar, were detected numerically from 1 to 3 per a conformer: C2 H O5, C1 H O2, C6H O5, C8H O5, and C6H O4. The energy values of H-bonds occupy the range of 2.3-5.6 kcal/mol, surely exceeding the kt value (0.62 kcal/mol). The nucleoside CH O hydrogen bonds appeared to survive turns of bases against the sugar, sometimes in rather large ranges of the angle χ values, pertinent to certain conformations, which points out to the source of the DNA lability, necessary for the conformational adaptation in processes of its functioning. The calculation of the interactions in the da T nucleoside pair gives evidence, that additionally to the N6H O4 and N1 N3H canonical H-bonds, between the bases adenine and thymine the third one (C2H O2) is formed, which, though being rather weak (about 1 kcal/mol), satisfies the AIM criteria of H-bonding and may be classified as a true H-bond. The total energy of all the CH O nontraditional intramolecular H-bonds in DNA nucleoside pairs appeared to be commensurable with the energy of H-bonds between the bases in Watson-Crick pairs, which implies their possible important role in the DNA shaping. Yevgen P. Yurenko 1 Roman O. Zhurakivsky 1 Svitlana P. Samijlenko 2 Dmytro M. Hovorun 2,3 * 1 Laboratory of computational structural biology, Department of molecular and quantum biophysics, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 150 Zabolotnoho Street, Kyiv 03143, Ukraine 2 Department of molecular and quantum biophysics, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 150 Zabolotnoho Street, Kyiv 03143, Ukraine 3 Department of molecular biology, Biotechnology and biophysics, Institute of High Technologies, Kyiv National Taras Shevchenko University, 2 Hlushkova Ave., Kyiv 03127, Ukraine Key words: The AI and BI DNA forms; Watson-Crick 2 -deoxyribonucleosides pairs as the simplest DNA models; The CH O hydrogen bond energy; Quantum chemical; AIM analysis. Introduction Since Watson and Crick s determination of the DNA spatial structure (1), hydrogen bonds (H-bonds) stabilizing its structure, became a subject of numerous investigations (see, e.g., works (2-4) and refs. therein). For a discussion of different hydrogen bonds such as intramolecular H-bonds in expanded synthetic nucleic acid bases see Palamarchuk et al. (5) and Sharma et al. (6). Speaking of H-bonds in DNA, one implies first of all canonical ones between the pairs of complementary nucleotide bases Gua Cyt and Ade Thy. According to the Watson-Crick initial model (1), chains of a right-handed double helix are connected by two H-bonds between complementary nucleotide bases in each of two pairs. Later Pauling and Corey (7), based on analysis of electronic structure of the Gua Cyt base pair and geometric considerations, postulated the third H-bond N2H O2 on the side of the minor *E-mail: dhovorun@imbg.org.ua 51

52 Yurenko et al. groove (see also an essay (8)). This H-bond was detected experimentally in 1963, when O Brien managed to get by X-ray diffraction the structure of the crystal complex between 9-ethylguanine and 1-methylcytosine (9). At early 1990s a discussion was launched in literature, this time on the third H-bond in the Ade Thy DNA base pair (C2H O2). Noteworthy, this idea was born as early as in 1966, when Crick (10) accentuated, that the absence of such a contact in the minor groove of DNA might be a reason for its structural-dynamical instability. Based on X-ray data for polynucleotides, Hunter and co-workers (11) supposed the idea of formation of a weak C2H O2 hydrogen bond in Ade Thy and Ade Ura base pairs. Grounding on geometric characteristics, they anticipated its energy to be less than 1 kcal/mol, but perceived importance of this H-bond formation for alleviating the destabilization of the Ade Ura pair with unsatisfied hydrogen-bond acceptor (oxygen atom of carbonyl group). Shortly after, applying ab initio quantum chemical modeling (at the HF/6-31G (d,p) and MP2/6-31G (d,p) levels of theory) to experimentally observed crystal geometry of the adenylyl-3-5 -uridine dinucleotide, Starikov and Steiner (12) estimated a ~6% contribution of the C2H O2 interaction to the total binding energy in the Watson-Crick Ade Ura base pair of RNA and assumed that this contact may be considered cum grano salis as a weak hydrogen bond. Somewhat later, additional quantum chemical evidence for the C2H O2 hydrogen bond existence appeared, such as a critical point and a gradient bond path on the electron density topological map in the DNA base pair Ade Thy (13). However, the nature of the CH O contact in the Watson-Crick DNA and RNA base pairs Ade Thy and Ade Ura, respectively, is still being discussed. For example, some authors (14) consider the C2H O2 interaction to be rather a van der Waals one than a weak H-bond. In the work of Hobza et al. (15) the existence of the third H-bond in Ade Ura pair was definitely ruled out. On the contrary, in the study of Grunenberg (16) the C2H O2 interaction in Ade Thy pair was proved to be a real H-bond with a compliance constant equal to 23.131 Å/mdyn. Besides H-bonds between the bases in Watson-Crick base pairs, DNA is most likely netted with the lace of so-called non-canonical H-bonds CH O between the bases and the sugar phosphate backbone and backbone-backbone H-bonds, as well as cross-strand interactions between non-complementary Watson-Crick pairs, some of which were studied experimentally (17-19) and theoretically (20-22). It should be noted that base-stacking interactions, stabilizing the DNA double helix as well, were extensively studied both experimentally and theoretically (see, e.g., works 23, 24 and refs. therein). In a seminal paper by Frank-Kamenetskii and coworkers (23) the DNA stacking parameters were obtained experimentally for wide ranges of temperatures and salt concentrations. Energetic features of stacking interactions between the Watson-Crick pairs were determined by quantum chemical calculations (e.g., see (25) and refs therein). A curious fact that the n2π* vertical interactions, similar to stacking (between the O4 deoxyribose atom and the aromatic base) are possible in the dcpg dinucleotide adopting Z form, was theoretically obtained (26). DNA is known to be a polymorphic molecule (2, 4). Its main conformational families AI, BI, BII, ZI and ZII were extensively investigated (e.g., see (2, 4, 27) and refs therein) and their biological importance was demonstrated (e.g., see work (28) and refs therein). Recently AII form, called crankshaft DNA, was discovered (29) and currently is being intensively studied (30). Reasonably, such a conformational variety of DNA can be ensured both by the CH O non-canonical H-bonds and the canonical Watson-Crick H-bonds between the bases in nucleotide/nucleotide pairs. Quite recently the following criteria have been considered (31) as evidence of a hydrogen bond (X H Y): charge transfer between a donor X and an acceptor Y; polarization of the X H bond; the preferable range of the X H Y angle should be 110 2180 ; lengthening of the X H bond and concomitant red shift and

increase in intensity of its infrared stretching frequency band; the X H bond proton deshielding in a NMR spectrum; the Gibbs energy of a hydrogen bond surpassing the kt value. The last criterion is important for identification of weak hydrogen bonds, as in the present study. The greater the number of criteria satisfied, the more reliable is the characterization of a X H Y interaction as a hydrogen bond, but they may not be used as rigorous indicators of hydrogen bonding (31). Some of these criteria may not be satisfied for hydrogen bonds in networks. Not all the above criteria hold true for well established weak hydrogen bonds with shortened X H bonds, and with concurrent blue shifts and reduced intensity in infrared spectra (32). 53 CH O Hydrogen Bonds in DNA Structure Based on calculation of complete conformational spaces of nucleosides, we have previously elucidated elementary physico-chemical mechanisms of biological action of 2 -deoxy-6-azacytidine (33) and 2 -deoxyuridine (34) and showed, that each of full conformational families of all canonical nucleosides contains only four DNA-like conformers relative to the AI, AII, BI and BII forms of a right doublehelical DNA (35-39). The chief aim of the present work was to study conformational durability of all the CH O intramolecular H-bonds in the nucleoside conformers, pertaining to the AI and BI forms of DNA, and estimate their contributions to the stability of the Watson- Crick nucleoside pairs, which can be regarded as the simplest periodic structural motifs of the DNA double helix (40-42). To achieve this purpose, we have performed systematic quantum chemical calculations of geometric and energetic features of the nucleosides and their pairs and also evaluated energies of all the H-bonds, using the electron density topological analysis (43). In such a way, in this paper we made an attempt to study intrinsic conformational properties of the DNA nucleosides and their canonical pairs, which are determined by the CH O intramolecular H-bonds. Materials and Methods Chemical structure, atom numbering and designation of the conventional torsion angles (according to (2)) in canonical 2 -deoxyribonucleosides are presented in Figure 1. Figure 1: Chemical structure, atom numbering and nomenclature of dihedral angles of canonical 2 -deoxyribonucleosides as in ref. 2.

54 Yurenko et al. The nucleoside geometry was calculated at two levels of theory DFT (B3LYP hybrid exchange-correlation functional (44, 45)) and MP2 (46), using the standard Gaussian basis set 6-31G(d, p). The nucleoside conformers with the values of conformational parameters χ anti, P C3 endo, β t, γ g +, ε t and the conformers with the parameters χ anti, P C2 endo, β t, γ g +, ε t were regarded as the AI DNA-like and the BI DNA-like ones (40), correspondingly. Next, the H-bond stability in the AI and BI conformers was investigated as a function of the angle χ. For this purpose, the potential energy profile of all the AI and BI conformers of canonical 2 -deoxyribonucleosides was scanned via changing angle χ by the 1º step in the range, corresponding to the anti conformation for a given nucleoside. The B3LYP/6-31G(d,p) geometry optimization was applied to every fixed value of χ. For geometry optimization convergence and integration grid specification, the following Gaussian 03 keywords (47) were applied: Opt = (Tight, GDIIS) and Grid = UltraFine. The ΔG Gibbs free energy values for all structures were obtained at the MP2/6-311++G(d,p)// B3LYP/6-31G(d,p) level of theory in the following way: MP2 was used for electronic energy, while B3LYP for thermal correction and entropy contribution. All the quantum chemical calculations were performed using program package Gaussian 03 for Win32 platform (47). Wave functions were obtained at the theory level used for geometry calculations. Topology of electronic density was analyzed using program package AIMAll (48). Identification of H-bonds was performed according to the Koch and Popelier criteria (49). A bond critical point (BCP, the so-called (3, -1) point) and a bond path between a hydrogen donor and acceptor, as well as a positive value of Laplacian at BCP, were considered as necessary conditions for an H-bond formation (43). All the H-bonds were also tested for compliance with classic geometric criteria: the sum of van der Waals radii (50) of the hydrogen and acceptor atoms should be larger than a distance between them. Energies of H-bonds were evaluated by the Espinosa-Molins-Lecomte (EML) method (51), based on the electronic density distribution at critical point of a hydrogen bond: E HB = 0.5V(r), V(r) being the value of a local potential energy at critical point. The adequacy of this formula in evaluation of the H-bond energy was shown recently (52, 53). Energies of intermolecular H-bonds and intramolecular CH O hydrogen bonds were calculated for Watson-Crick nucleoside pairs da T and dg dc in the AI and BI DNA-like conformations. The geometries of Watson-Crick nucleoside pairs and wave functions were obtained at the B3LYP/6-31G(d,p) level of theory. The harmonic vibrational frequency calculations to determine local minima on the energy landscape were performed at the same level of theory. It was shown earlier that the B3LYP/6-31- G(d,p) calculations reproduce quite reliably low-temperature matrix IR spectra of 2 -deoxyuridine (34) and thymidine (35). To check up the theory level influence on the H-bond energies, we optimized geometry and calculated wave functions of the Ade Thy and Gua Cyt Watson-Crick base pairs using several approximations (B3LYP/6-31G(d,p), B3LYP/6-31++G(d,p), B3LYP/6-311++G(d,p), B3LYP/cc-pVDZ, MP2/6-31G(d,p), MP2/6-31++G(d,p) and MP2/cc-pVDZ). To detect the charge transfer in the CH O hydrogen bonds, stabilizing the nucleosides, we have resorted to the Natural Bond Orbital (NBO) analysis, which interprets the electronic wave function in terms of a set of occupied Lewis and a set of unoccupied non-lewis localized orbitals (54, 55). The second order Fock matrix analysis was carried out to evaluate interaction between donor (i) and acceptor ( j) bonds. The result of such an interaction is a migration of the electron density from the idealized Lewis structure into an empty non-lewis orbital. For each donor (i) and acceptor ( j) bonds, the stabilization energy E (2) is estimated as E (2) = ΔE ij = q i [1] F (i, j) 2 e j 2 e i

where q i is the donor orbital occupancy, ε j and ε i are diagonal elements and F(i, j) is the off diagonal element of NBO Fock matrix. Results and Discussion A comparison of geometric features of the DNA-like nucleoside conformers, calculated at the B3LYP/6-31G(d,p) and MP2/6-31G(d,p) levels of theory (Table I), shows that conformational parameters in both cases are rather close, which confirms an acceptability of the applied DFT method instead of much more expensive method MP2. The greatest differences between χ values, obtained at both theory levels, are observed in the cases of the AI-like conformers of da and dg (15.3º and 12.5º respectively). It should be also noted, that B3LYP method underestimates the sugar puckering amplitude on the average by 3.5º in comparison with MP2 one. Calculated values of the Gibbs relative energy (as reference points were chosen energetically the most favourable conformers from complete families of each nucleosides (34-39)) give evidence (Table I), that the BI-like conformers for all the DNA-like nucleosides, except da, are more stable, than the AI-like ones. It should be noted, that authors of the works (56, 57) established unusual conformational behavior of 2 -deoxycytidine (dc), which consists in the fact that, in contrast to other canonical 2 -deoxyribonucleosides, electronic energy of its BI-like conformer exceeds electronic energy of the AI-like conformer. It has been suggested (40) that dc plays an important role in the equilibrium between the A and B forms of DNA. However, in our case, such irregularity (i.e., ratio of the BI/AI conformer stability) is eliminated, when operating with G values (Table I). The fact of a larger dispersion of the χ angle values and corresponding larger changes in the Gibbs free energy for the BI conformers, as compared to the AI ones, trails the way for understanding, at microstructural level, of well-known experimental data (58, 59), showing that the BI form of DNA is more heterogeneous, than the AI one. 55 CH O Hydrogen Bonds in DNA Structure As it was shown by topological analysis of an electron density (43), the investigated conformers are stabilized by intramolecular H-bonds of various types ( Figure 2, Table II). H-bonds C8H O5 or C6H O5 are formed in all the conformers of purine and pyrimidine nucleosides, accordingly. In the nucleosides there are also H-bonds susceptible to both the sugar conformation and to the base type. So, the C6H O4 hydrogen bond is specific for the AI-like conformers of pyrimidine nucleosides. The C2 H2 O5 (which may be qualified as intrasugar and is also present in the model sugar residue 1, 2 -dideoxyribose (60)) and C1 H O2 hydrogen bonds are intrinsic exclusively to BI-like conformers, the latter one being Table I Structural and energetic characteristics of AI and BI form DNA-like conformers of canonical 2 -deoxyribonucleosides at the B3LYP/6-31G(d,p) and MP2/6-31G(d,p) levels of theory. Nucleoside Conformation P ν max χ γ β δ ε DFT MP2 DFT MP2 DFT MP2 DFT MP2 DFT MP2 DFT MP2 DFT MP2 ΔG da AI 9.6 6.5 34.0 38.8 2153.3 2168.6 51.3 52.9 172.2 178.7 85.1 82.1 2167.3 2168.7 4.02 BI 172.1 169.1 33.4 35.9 2125.5 2128.6 50.6 50.4 174.6 175.3 145.0 148.0 173.9 173.0 4.25 T AI 13.7 12.3 34.2 38.5 2158.8 2162.1 52.9 52.3 175.6 176.0 84.0 80.8 2168.8 2170.5 1.34 BI 164.6 163.3 34.4 37.0 2129.5 2128.8 51.3 50.1 176.2 175.9 142.7 146.0 174.1 172.9 0.65 dg AI 11.0 9.6 33.5 38.5 2149.2 2161.7 49.9 50.8 167.2 173.1 85.5 81.7 2164.5 2165.5 4.66 BI 172.5 169.6 33.4 35.9 2123.0 2126.1 49.6 49.3 171.8 172.8 145.2 148.2 175.5 174.2 4.49 dc AI 12.3 11.6 34.4 38.6 2163.0 2165.3 54.1 53.3 178.0 178.2 83.7 80.7 2172.6 2173.4 1.62 BI 163.7 164.1 34.3 36.5 2145.8 2152.3 53.9 53.2 178.7 179.3 142.0 145.9 169.8 168.2 1.32 Note: AI and BI DNA-like conformers corresponding to AI and BI forms of DNA. The nomenclature dihedral angles (in degrees) are the same as in Figure 1 and reference 2; ΔG-relative Gibbs energy at the MP2/6-311++G(d,p)//B3LYP/6-31G(d,p) level of theory (T = 298.15 K).

56 Yurenko et al. Figure 2: Intramolecular H-bonds CH O in nucleoside conformers, corresponding to DNA-like AI and BI forms. peculiar to the pyrimidine conformers only. However, according to the MP2 calculation (Table II), in the dc BI-like conformer the C1 H O2 hydrogen bond is not formed at the χ value 152.3, corresponding to the local minimum, but appears at χ = 148 ± 0.5 and exists in a rather wide range. All the H-bonds studied satisfy classical geometric criteria of H-bonding: distance between hydrogen and oxygen (acceptor) atoms proved to be less than the sum of their van der Waals radii (according to Bondi (50), the sum equals 2.72 Å); the CH O angle always appeared to be obtuse. At both levels of theory, DFT and Table II Geometric, energetic and electron density topological characteristics of intramolecular H-bonds shaping canonical 2 -deoxyribonucleosides calculated at the B3LYP/6-31G(d,p) and MP2/6-31G(d,p) levels. Nucleoside Conformer H-bond CH O d CO a d HO b CHO c ρ d Δρ e E HB f ΣE HB g DFT MP2 DFT MP2 DFT MP2 DFT MP2 DFT MP2 DFT MP2 DFT MP2 da AI C8H O5 3.312 3.208 2.305 2.314 154.2 139.4 0.014 0.014 0.039 0.043 3.19 3.38 3.19 3.38 BI C8H O5 3.455 3.423 2.518 2.499 144.5 143.2 0.009 0.010 0.029 0.032 1.96 2.20 4.07 5.13 C2 H2 O5 3.076 2.993 2.533 2.423 109.5 111.2 0.010 0.012 0.037 0.046 2.11 2.93 T AI C6H O5 3.315 3.237 2.286 2.224 158.0 155.5 0.014 0.016 0.041 0.047 3.34 4.09 7.64 9.08 C6H O4 2.742 2.718 2.319 2.287 101.1 101.6 0.017 0.019 0.074 0.079 4.30 4.99 BI C6H O5 3.454 3.409 2.414 2.372 160.0 160.1 0.011 0.013 0.033 0.037 2.53 3.00 9.03 10.58 C2 H2 O5 3.118 3.026 2.590 2.467 108.7 110.6 0.009 0.011 0.034 0.043 1.87 2.67 C1 H O2 2.782 2.788 2.256 2.274 107.2 106.6 0.019 0.019 0.077 0.080 4.63 4.91 dg AI C8H O5 3.339 3.252 2.319 2.283 156.6 148.9 0.014 0.015 0.038 0.044 3.10 3.59 3.10 3.59 BI C8H O5 3.464 3.428 2.528 2.501 144.4 143.6 0.009 0.010 0.028 0.032 1.91 2.19 4.04 5.17 C2 H2 O5 3.069 2.986 2.528 2.415 109.4 111.2 0.010 0.012 0.038 0.047 2.13 2.98 dc AI C6H O5 3.313 3.245 2.314 2.259 152.4 150.8 0.014 0.015 0.039 0.045 3.14 3.78 7.58 8.89 C6H O4 2.719 2.701 2.307 2.279 100.3 100.9 0.018 0.019 0.077 0.081 4.44 5.11 BI C6H O5 3.498 3.451 2.532 2.539 147.9 141.6 0.009 0.009 0.029 0.031 1.89 2.00 8.52 4.61 C2 H2 O5 3.139 3.038 2.612 2.475 108.7 110.8 0.008 0.011 0.033 0.042 1.77 2.61 C1 H O2 2.706 2.695* 2.260 2.277* 102.0 100.4* 0.020 0.020* 0.081 0.089* 4.86 5.37* a Distance between C (donor) and O (acceptor) atoms (in Å). b Distance between H and O atoms (in Å). c H-bond angle (in degrees). d Electron density at the bond critical point (atomic units). e Laplacian of the electron density at the bond critical point (atomic units). f H-bond energy calculated by Espinosa-Molins-Lecomte formula (in kcal/mol). g Sum of energies of conformer H-bonds, kcal/mol. *Data for H-bond appearing at χ 5 148 ± 0.5.

MP2, electronic characteristics electron density ρ and Laplacian of electron density Δρ (Table II) appeared to be within the limits, established in the work (49). Energies of CH O hydrogen bonds, calculated by the EML method (51) are higher on average by 16% at MP2 level of theory in comparison with DFT. Noteworthy, the C6H O5 hydrogen bond in dc more significantly contributes to the energy of its AI-like conformer (Table II), as compared to the BI-like one, which is in agreement with the results reported in (57). The energies of C6H O4 and C1 H O2 hydrogen bonds unexpectedly appeared to be rather high. 57 CH O Hydrogen Bonds in DNA Structure Previously (36, 61) some of the H-bonds in canonical 2 -deoxyribonucleosides conformers were tested by other five criteria, which deal with changes of atomic properties of atoms O and H and require a reference conformation without H-bond under consideration (so-called two molecules criteria ). These criteria are: increase in the hydrogen atom positive charge q; decrease in the hydrogen atom polarization moment M; diminution of the hydrogen atom volume v; growth of the hydrogen atom energy E; mutual penetrability between hydrogen and acceptor atoms, defined as the sum of changes in atomic radii Δr H + Δr B. It turned out, that these criteria hold true for all H-bonds detected in the nucleosides (36). To make sure that two molecules criteria are valid in our case, we applied them to every H-bond in AI and BI form DNA-like conformers of canonical 2 -deoxyribonucleosides (Table III). Therefore we have chosen structurally close conformers without a given H-bond as reference molecules (34-39). The results of QM/AIM analysis (Table III) clearly prove that all weak CH O interactions reported in this work fulfill all these H-bonding criteria. Moreover, we should emphasize that we observed rather strong mutual penetration of the H and O atoms, which was considered to be a sufficient criterion for H-bonding (49). Some H-bonds are characterized by rather high values of ellipticity ε, which according to Matta and coworkers (22) pointed out to their dynamic nature, i.e., their energy values are modulated Table III The two-molecules criteria applied to H-bonds in AI and BI form DNA-like conformers of canonical 2 -deoxyribonucleosides. Nucleoside Conformer H-bond CH O q(h) a Δq M(H) b -ΔM ν(h) c 2Δν 2E(H) d ΔE r(h) e Δr(H) r(o) f Δr(O) da AI C8H O5 0.129 0.064 0.104 0.021 37.0 9.7 0.581 0.024 1.69 0.87 2.67 0.72 BI C8H O5 0.097 0.032 0.111 0.014 41.0 5.7 0.592 0.013 1.88 0.67 2.88 0.51 C2 H2 O5 0.031 0.021 0.128 0.009 43.4 6.4 0.621 0.012 2.06 0.45 2.75 0.65 T AI C6H O5 0.122 0.062 0.098 0.027 34.7 9.6 0.588 0.023 1.67 0.88 2.65 0.74 C6H O4 0.122 0.013 0.098 0.030 34.7 3.4 0.588 0.002 1.91 0.63 2.54 0.80 BI C6H O5 0.102 0.042 0.107 0.018 37.8 6.5 0.594 0.017 1.78 0.78 2.79 0.60 C2 H2 O5 0.020 0.007 0.128 0.008 44.0 5.2 0.624 0.011 2.12 0.39 2.80 0.60 C1 H O2 0.075 0.032 0.120 0.014 39.6 4.2 0.620 0.008 1.79 0.72 2.51 0.77 dg AI C8H O5 0.118 0.060 0.102 0.023 37.6 9.5 0.585 0.022 1.70 0.86 2.68 0.71 BI C8H O5 0.093 0.035 0.112 0.013 41.3 5.8 0.594 0.013 1.89 0.66 2.89 0.50 C2 H2 O5 0.026 0.018 0.123 0.010 43.2 6.1 0.624 0.009 2.05 0.46 2.74 0.66 dc AI C6H O5 0.123 0.041 0.100 0.020 35.1 6.6 0.588 0.015 1.70 0.84 2.67 0.72 C6H O4 0.123 0.014 0.100 0.027 35.1 3.6 0.588 0.003 1.91 0.63 2.53 0.81 BI C6H O5 0.103 0.021 0.111 0.009 38.7 3.0 0.593 0.010 1.89 0.66 2.89 0.50 C2 H2 O5 0.017 0.008 0.128 0.012 45.0 4.6 0.625 0.007 2.14 0.37 2.82 0.58 C1 H O2 0.076 0.016 0.126 0.003 39.9 2.6 0.618 0.005 1.82 0.69 2.50 0.78 a Atomic charge of hydrogen atom (H). b Dipolar polarization moment of H. c Volume of H. d Energy of H. e Radius of H. f Radius of the oxygen (O) atom. The Δ symbol denotes changes of the above-mentioned values upon H-bonding. The structurally close conformers were taken as reference molecules in each case. All units are atomic. The integration over atomic basins was performed in natural coordinates. Wave functions were calculated at the B3LYP/6-31G(d, p) level of theory.

58 Yurenko et al. by vibrations of the C-H O angle and the extent of these modulations becomes deeper, as ellipticity increases. To check up the conformational limits of the CH O hydrogen bonds existence, we performed potential energy surface scanning for all the AI and BI conformers with respect to χ angle in the ranges, peculiar to anti conformers (27, 41). Almost all CH O hydrogen bonds proved to survive in rather large ranges of χ, their energy values covering the 1.33-5.30 kcal/mol interval (Table IV, Figure 3). This finding allows us to suppose, that these H-bonds retain also in the DNA right-handed helices and together with the backbone-backbone CH O hydrogen bonds (22), as well as with cross-strand interactions (21) may assure variety of conformations, necessary in the processes of its functioning. Table V depicts the results of NBO analysis of H-bonds in nucleosides. For all H-bonds we observed hyperconjugative interactions LP(O)-σ * (CH) between oxygen lone pair and antibonding orbital σ * (CH). The strength of this interaction is characterized by the E (2) energy values (Table V). The greatest values of E (2) are observed in case of C8H/C6H O5 hydrogen bonds in purine and pyrimidine nucleosides: 1.50 2.74 kcal/mol and 1.42 2.94 kcal/mol, respectively. At the same time the C2 H2 O5 hydrogen bonds are characterized by the least E (2) values: from 0.60 to 0.68 kcal/mol. The small values of E (2) indicate that, most probably, the stabilizing charge transfer is counterbalanced by the Pauli repulsion, which may result in blue-shifted nature of these H-bonds (62). To determine contribution of the CH O hydrogen bonds to stabilization of the DNA nucleoside pairs, we calculated four Watson-Crick pairs of nucleosides da T and dg dc with the AI and BI DNA-like sugar conformations (Figure 4, Table VI). Total energy of the intramolecular CH O hydrogen bonds appeared to be commensurable with total energy of the H-bonds between the DNA base pairs (Tables VI, VII). For the da T and dg dc nucleoside pairs in the AI form the total energy of the CH O hydrogen bonds, estimated according to EML method (51), comes up respectively to 66% and 47% of the total energies of the H-bonds between bases; in the case of the da T and dg dc nucleoside pairs in the BI form corresponding values are 79% and 57%. In other words, the impact of CH O H-bonds is greater for the da T pairs as compared to the dg dc ones and for the BI sugar conformations in comparison with the AI ones. This finding may be regarded as an argument to the statement, that non-canonical H-bonds in DNA are significant in formation of its spatial structure and conceivably play an important role in processes of protein- Table IV Stability of H-bonds in AI and BI DNA-like conformers of canonical 2 -deoxyribonucleosides as to rotation of a nucleotide base around sugar moiety. dc T da dg H-bond Conformer χ range Energy range χ range Energy range χ range Energy range χ range Energy range C6H O5 BI 234-272 2.09-2.63 234-272 2.23-2.75 AI 179-221 2.28-3.47 179-221 2.31-3.50 C8H O5 BI 234-272 1.67-2.06 234-272 1.63-1.99 AI 179-221 1.99-3.21 179-221 1.99-3.13 C1 H O2 BI 234-272 5.05-5.30 234-272 4.66-4.77 AI 209-221 5.03-5.28 216-221 4.70-4.74 C6H O4 AI 179-201 4.41-4.92 179-205 4.27-4.91 C2 H2 O5 BI 234-272 1.86-2.23 234-272 1.89-2.25 234-272 2.08-2.38 234-272 2.11-2.43 Note: The χ angle ranges (in degrees), for which the corresponding H-bonds remain intact, i.e., do not break, are indicated. Calculations were performed at the B3LYP/6-31G(d,p) level of theory, energy ranges are in kcal/mol. The χ angle scanning was performed with step of 1º for nucleosides in the AI and BI DNA forms. Geometry optimization was performed without any variable constrained but angle χ, which was fixed at every step within the scanning range.

nucleic acid recognition. Particularly, under the DNA synthesis high fidelity DNA polymerases are very sensitive to conformation of sugar residues and base orientation against the sugar (63). Energy of the third H-bond C2H O2 between Ade and Thy in the da T nucleoside pair appeared to be about 1 kcal/mol (Tables VI and VII), which is acceptable for a weak hydrogen bond of the CH O type (64-67). It is also worth mentioning that C2H O2 hydrogen bond in (da T)/AI and (da T)/BI nucleoside pairs, despite blue-shift of stretching mode ν(c2h), 14 cm -1 and 16 cm 1 correspondingly, is characterized by a noticeable increase in out-of-plane-mode γ (C2H) frequency (which is normal for traditional hydrogen bonds) as against free deoxyribonucleosides, by 24 cm 1 and 26 cm 1, respectively. Moreover, the third H-bond in the DNA base pair Ade Thy and in da T nucleoside pair with both AI and BI conformations of sugar proved to satisfy five two-molecules Koch and Popelier criteria, which evidences that C2H O2 interaction undoubtedly corresponds to quantum chemical requirements for H-bonding (Table VIII). The calculation of isolated Ade Thy pair at different levels of theory (Table VII) attested, that the C2H O2 H-bond energy in the Ade Thy pair lies within the 0.73 1.17 kcal/mol range. 59 CH O Hydrogen Bonds in DNA Structure In the recent work (14) the C2H O2 interaction in the Ade Thy isolated base pair was considered as a blue-shifted van der Waals contact rather than a hydrogen bond. However, the authors of this paper clearly showed, that all three contacts (two strong N6H O4 and N3H N1 and one weak C2H O2) are results of different interplays between the same five factors: intermolecular and intramolecular hyperconjugations, structural reorganization, rehybridization and repolarization, accompanying the Ade Thy pair formation. In our opinion, the results of this work Figure 3: Dependence between energies of intramolecular H-bonds (in kcal/mol) and values of χ torsion angle (in degrees) in DNA-like conformers of canonical nucleosides. A (left) for AI DNA-like conformers. H-bonds are numbered as follows: 1 - C6H O5' (dc), 2 -C1'H O2 (dc), 3 - C6H O4' (dc), 4 - C6H O5' (T), 5 - C1'H O2 (T), 6 - C6H O4' (T), 7 - C8H O5' (da), 8 - C8H O5' (dg). B (right) for BI DNA-like conformers. H-bonds are numbered as follows: 1 - C6H O5' (dc), 2 - C1'H O2 (dc), 3 - C2'H2 O5' (dc), 4 - C6H O5' (T), 5 - C1'H O2 (T), 6 - C2'H2 O5' (T), 7 - C8H O5' (da), 8 - C2'H2 O5' (da), 9 - C8H O5' (dg), 10 - C2'H2 O5' (dg).

60 Yurenko et al. could not be rigorously considered as an argument against the C2H O2 hydrogen bond formation in the Ade Thy pair. The data reported in (16) also witness for the third H-bond in this base pair. What is more, cooperativity of three H-bonds in Ade Thy (da T) pair may be a reason of deviations from six criteria of an H-bond listed in the work (31). The third hydrogen bond N2H O2 between Gua and Cyt in the dg dc nucleoside pair appeared to be energetically congruent to the N1H N3 hydrogen bond Table V NBO characteristics of H-bonds in the AI and BI DNA-like conformers of canonical 2 -deoxyribonucleosides calculated at the DFT B3LYP/6-31G(d,p) level of theory. Nucleoside Conformer H-bond CH O E (2)a sp n (C) b %C c %H c n(σ CH ) d n(σ * CH ) e q f C q f H q f O da AI C8H O5 2.66 (1.44) 1.72 63.24 36.76 1.984 0.027 0.220 0.251 20.773 BI C8H O5 1.53 1.77 62.65 37.35 1.984 0.024 0.223 0.241 20.779 C2 H2 O5 0.64 3.13 63.38 36.62 1.976 0.013-0.527 0.266 T AI C6H O5 2.94 (1.49) 2.06 64.17 35.63 1.976 0.027 0.115 0.163 20.534 C6H O4 0.71 20.533 BI C6H O5 2.42 2.10 63.65 36.35 1.977 0.024 0.032 0.262 20.778 C2 H2 O5 0.68 3.14 63.17 36.83 1.976 0.013-0.530 0.262 C1 H O2 1.29 2.60 64.15 35.85 1.979 0.033 0.272 0.266 20.636 dg AI C8H O5 2.74 (1.28) 1.71 63.07 36.93 1.984 0.027 0.195 0.248 20.773 BI C8H O5 1.50 1.75 62.49 37.51 1.984 0.024 0.199 0.238 20.778 C2 H2 O5 0.64 3.11 63.49 36.51 1.978 0.013-0.527 0.269 dc AI C6H O5 2.39 (1.41) 2.03 64.10 35.90 1.979 0.026 0.069 0.270 20.773 C6H O4 0.72 20.591 BI C6H O5 1.42 2.07 63.58 36.42 1.980 0.022 0.071 0.262 20.775 C2 H2 O5 0.60 3.17 63.11 36.89 1.976 0.013-0.529 0.261 C1 H O2 0.87 2.59 64.09 35.91 1.978 0.034 0.274 0.264 20.639 a Energy of hyperconjugative interaction (kcal/mol) between acceptor oxygen s lone pair of electrons (LP) and antibonding orbital σ * CH. When two LPs are involved in charge transfer, the corresponding value for the second LP is indicated in parenthesis. b sp-hybridization number n. c Contributions of C and H natural hybrid orbitals (NHO) to the natural bond orbital σ CH. d Occupancy of the σ CH bonding orbital. e Occupancy of the σ * CH antibonding orbital. f NBO charges of carbon C (H-bond donor), hydrogen H and oxygen O (H-bond acceptor) atoms. (Table VI); the range of energetic difference between the two H-bonds is about 1-2 kcal/mol. It is interesting that the exceeding in energy of the N4H O6 H-bond over the N2H O2 one by more than 2 kcal/mol may elucidate the NMR data (68), showing the less retarded rotation of the Gua amino group in the Gua Cyt pair, as compared to the Cyt base. Figure 4: Hydrogen bonds in Watson-Crick nucleoside pairs, corresponding to the AI and BI forms of DNA.

Shapes of the nucleosides in Watson-Crick pairs were found to be slightly deformed as compared to their free states (Tables I and IX). The deviations in values of nomenclature dihedral angles do not exceed 3.7º, except the angles between exocyclic NH 2 amino group and the C6N, C4N and C2N bonds (in da, dc and dg, respectively), which undergo a perceptible decrease (from 21 to 28 ) upon formation of the Watson-Crick nucleoside pairs. At that, the total energy of intramolecular CH O H-bonds in nucleosides manifests insignificant changes upon the nucleoside pairing only up to 0.36 kcal/mol. 61 CH O Hydrogen Bonds in DNA Structure Concluding Remarks We have performed, for the first time, a systematic quantum chemical analysis of energy, geometry and electron density topology study of the CH O hydrogen bonds in AI and BI DNA-like conformers of canonical 2 -deoxyribonucleosides. Energetic evaluation of intramolecular H-bonds between a base and a sugar residue, as well as intrasugar ones, showed that their energies markedly exceed the kt value at room temperature (0.62 kcal/mol), which implies their possible role in stabilizing the structure of the DNA-like nucleoside conformers. Specifically, our calculations of the H-bond energies in Watson-Crick pairs of the AI and BI DNA like nucleosides the simplest structural models for double-stranded DNA (40) have demonstrated an unexpected commensurability of the total energy of the intramo- Table VI Energetic and electron density topological characteristics of Watson-Crick nucleoside pairs da T and dg dc, corresponding to AI and BI DNA forms, calculated at the B3LYP/6-31G(d,p) level of theory. Nucleoside pair/ conformation H-bond AH B d AB, Å d HB, Å AHB, degree ρ, a.u. Δρ, a.u. E HB, kcal/mol ΣE HB, kcal/mol (da T)/AI N6H O4 2.929 1.909 174.8 0.028 0.078 6.46 16.26 N3H N1 2.850 1.803 178.1 0.043 0.095 8.91 (16.30) C2H O2 3.620 2.795 132.5 0.005 0.018 0.89 C8H O5 (da) 3.329 2.313 155.8 0.014 0.039 3.14 10.70 C6H O5 (T) 3.318 2.302 155.4 0.014 0.040 3.23 (10.83) C6H O4 (T) 2.738 2.312 101.2 0.017 0.074 4.33 (da T)/BI N6H O4 2.939 1.919 174.6 0.028 0.075 6.31 16.23 N3H N1 2.849 1.801 178.9 0.043 0.095 8.95 (16.30) C2H O2 3.590 2.762 132.7 0.005 0.019 0.97 C8H O5 (da) 3.477 2.546 143.8 0.009 0.028 1.82 12.85 C2 H2 O5 (da) 3.080 2.540 109.4 0.010 0.037 2.07 (13.10) C6H O5 (T) 3.468 2.435 158.7 0.011 0.032 2.41 C2 H2 O5 (T) 3.122 2.595 108.7 0.009 0.034 1.85 C1 H O2 (T) 2.769 2.247 107.0 0.019 0.077 4.70 (dg dc)/ai N4H O6 2.798 1.762 179.2 0.039 0.112 8.88 22.59 N1H N3 2.935 1.902 177.8 0.034 0.080 6.98 (22.76) N2H O2 2.915 1.892 178.4 0.029 0.081 6.73 C8H O5 (dg) 3.364 2.346 156.3 0.013 0.037 2.93 10.63 C6H O5 (dc) 3.294 2.290 153.3 0.014 0.041 3.30 (10.68) C6H O4 (dc) 2.723 2.309 100.5 0.017 0.076 4.40 (dg dc)/bi N4H O6 2.792 1.754 179.1 0.040 0.113 9.05 22.75 N1H N3 2.938 1.905 177.8 0.034 0.080 6.93 (22.76) N2H O2 2.910 1.888 178.5 0.030 0.082 6.77 C8H O5 (dg) 3.506 2.576 143.7 0.008 0.027 1.69 12.92 C2 H2 O5 (dg) 3.074 2.530 109.6 0.010 0.038 2.12 (12.56) C6H O5 (dc) 3.447 2.431 155.5 0.011 0.032 2.42 C2 H2 O5 (dc) 3.133 2.608 108.6 0.008 0.033 1.80 C1 H O2 (dc) 2.732 2.228 105.6 0.020 0.078 4.89 Notation is the same as in Table II. Values in brackets refer to total energies of free base pairs Ade Thy and Gua Cyt and free complementary nucleosides (i.e. the DNA-like AI and BI conformers).

62 Yurenko et al. Table VII Energies (in kcal/mol) of H-bonds E HB in Watson-Crick Ade Thy and Gua Cyt base pairs calculated by Espinosa-Molins-Lecomte formula at DFT and MP2 levels of theory using different basis sets. The geometry optimization and wave function calculations were performed at the same levels of theory. B3LYP MP2 Base pair H-bond 6-31G (d,p) 6-31++G (d,p) 6-311++G (d,p) 6-31G (d,p) 6-31++ G(d,p) ccpvdz ccpvdz Ade Thy N6H O4 6.31 6.20 5.97 6.17 6.08 6.14 5.88 N3H N1 9.13 8.09 9.69 9.91 9.65 9.25 10.70 C2H O2 0.86 0.73 0.74 0.94 1.12 1.08 1.17 ΣE HB 16.30 15.02 16.40 17.02 16.85 16.47 17.75 Gua Cyt N1H N3 7.07 6.65 7.40 7.18 7.27 7.29 7.70 N4H O6 9.12 8.84 10.02 10.27 8.79 8.73 9.99 N2H O2 6.57 6.31 6.13 6.35 6.59 6.47 6.17 E HB 22.76 21.80 23.55 23.80 22.65 22.49 23.86 lecular CH O hydrogen bonds with the total energy of H-bonds between the bases in corresponding Watson-Crick nucleoside pairs. A conformational durability of the CH O hydrogen bonds, shaping the DNA nucleosides, was studied in this work for the first time: most of them proved to exist in rather large ranges of the χ angle, describing a mutual orientation of a base against a sugar. Hence, it is reasonable to suggest that the CH O hydrogen bonds may determine the DNA intrinsic architectural features and also be important in the processes of specific protein-nucleic acid interactions (69, 70), all the more similar the CH O hydrogen bonds are also essential for the protein structure (71). Such weak, but numerous H-bonds of the CH O/N type are likely a potential tool for a fine mutual adaptation of conformations of proteins and nucleic acids in the processes of their recognition. According to our data, the C2H O2 interaction between Ade and Thy in the AI and BI like da T nucleoside pairs, though being rather weak, appeared to be a real H-bond. Its energy was shown to be about 1 kcal/mol, which is an acceptable value for weak H-bonds of the CH O type. Finally, our results cast light on the well-known fact of greater conformational heterogeneity of the DNA BI form in comparison with the AI form. The plausible Table VIII The two-molecules electron density topological criteria for the C2H O2 third H-bond between Ade and Thy in their pair and in the AI and BI DNA-like nucleoside pairs. Nucleoside pair and its conformation, pair of DNA bases q(h) Δq M(H) 2ΔM ν(h) 2Δν 2E(H) ΔE r(h) r(o) Δr(H) Δr(O2) (da T)/AI 0.076 0.043 0.130 0.005 45.3 2.3 0.604 0.019 2.17 3.12 0.36 0.16 (da T)/BI 0.075 0.039 0.127 0.008 44.9 2.5 0.605 0.017 2.14 3.09 0.39 0.19 Ade Thy 0.075 0.039 0.125 0.008 44.9 2.6 0.605 0.018 2.15 3.10 0.39 0.20 Note: All units are atomic. Two-molecules criteria include changes in atomic charges Δq, dipolar polarization moment ΔM, volume Δν, energy ΔE and radii Δr(H) and Δr(O2). The integration over atomic basins was performed in natural coordinates at the B3LYP/6-31G(d,p) level of theory.

Table IX Structural and energetic characteristics of the AI and BI DNA-like conformers of canonical 2 -deoxyribonucleosides in Watson-Crick nucleoside pairs at the B3LYP/6-31G(d,p) level of theory. Nucleoside pair Nucleoside P ν max χ γ β δ ε ΔG 63 CH O Hydrogen Bonds in DNA Structure da T da/bi 171.9 33.4-126.8 50.7 174.2 144.8 173.2 0.00 T/BI 164.3 34.4-131.0 51.4 175.6 142.5 173.0 da/ai 10.0 34.0-152.7 51.1 171.0 85.1-167.1 0.66 T/AI 13.1 34.3-160.5 53.3 176.3 83.8-170.0 dg dc dg/bi 171.4 33.5-124.1 49.5 169.8 144.7 173.1 0.00 dc/bi 164.2 34.1-137.6 53.1 178.4 142.2 173.3 dg/ai 10.9 33.8-148.3 49.4 163.5 85.1-165.7 0.15 dc/ai 12.0 34.3-162.7 54.4 178.3 84.0-170.2 Notes are the same as for Table I. reason is the greater dispersion of a base orientation relative to a sugar residue and respectively greater scattering of the Gibbs energy for the BI form-like nucleosides, as well as more heterogeneous base stacking (20). Acknowledgments The authors thank Gaussian Corporation (USA) for granting molecular modelling package Gaussian 03. The work was partly supported by Ukrainian-French Program Dnipro (2009-2010). Y.P.Y. was supported by Ukrainian State Fund for Fundamental Research (grant no. GP/F27/0060). The authors also greatly appreciate useful discussion of the results with M. Ghomi, M. B. Hernandez (Université Paris 13) and I. Kolomiets (Institute of Molecular Biology and Genetics NASU). References 1. J. D. Watson and F. H. C. Crick. Nature 171, 737-738 (1953). 2. W. Saenger. Principle of Nucleic Acid Structure. Springer Verlag, New York (1984). 3. G. A. Jeffrey and W. Saenger. Hydrogen Bonding in Biological Structures. Springer- Verlag, Berlin, Heidelberg, New York (1991). 4. V. A. Bloomfield, D. M. Crothers, and I. Tinoco, Jr. Nucleic Acids: Structures, Properties and Functions. University Science Books, Sausalito, CA (2000). 5. P. Sharma, S. Sharma, A. Mitra, and H. Singh. J Biomol Struct Dyn 27, 65-81 (2009). 6. G. V. Palamarchuk, O. V. Shishkin, L. Gorb, and J. Leszczynski. J Biomol Struct Dyn 26, 653-661 (2009). 7. L. Pauling and R. B. Corey. Arch Biochem Biophys 65, 164-181 (1956). 8. S. Wain-Hobson. Nature 439, 539 (2006). 9. E. J. O Brien. J Mol Biol 7, 107 (1963). 10. F. H. C. Crick. J Mol Biol 19, 548-555 (1966). 11. G. A. Leonard, K. McAuley-Hecht, T. Brown, and W. N. Hunter. Acta Crystallogr Sect D: Biol Crystallogr 51, 136-139 (1995). 12. E. B. Starikov and T. Steiner. Acta Crystallogr Sect D: Biol Crystallogr 53, 345-347 (1997). 13. R. Parthasarathi, R. Amutha, V. Subramanian, B. U. Nair, and T. Ramasami. J Phys Chem A 108, 3817-3828 (2004). 14. P.-P. Zhou and W.-Y. Qiu. J Phys Chem A 113, 10306-10320 (2009). 15. P. Hobza, J. Sponer, E. Cubero, M. Orozco, and F. J. Luque. J Phys Chem B 104, 6286-6292 (2000). 16. J. Grunenberg. J Am Chem Soc 126, 16310-16311 (2004). 17. I. Berger, M. Egli, and A. Rich. Proc Natl Acad Sci USA 93, 12116-12121 (1996). 18. M. Egli and R. V. Gessner. Proc Natl Acad Sci USA 92, 180-185 (1995). 19. C. Yoon, G. G. Prive, D. S. Goodsell, and R. E. Dickerson. Proc Natl Acad Sci USA 85, 6332-6336 (1988). 20. A. Ghosh and M. Bansal. J Mol Biol 294, 1149-1158 (1999). 21. D. Bandyopadhyay and D. Bhattacharyya. Biopolymers 83, 313-325 (2006). 22. C. F. Matta, N. Castillo, and R. J. Boyd. J Phys Chem B 110, 563-578 (2006). 23. P. Yakovchuk, E. Protozanova, and M. D. Frank-Kamenetskii. Nucl Acids Res 34, 564-574 (2006).

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