yDNA versus xDNA Pyrimidine Nucleobases: Computational Evidence

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J. Phys. Chem. B 2008, 112, 12526–12536

yDNA versus xDNA Pyrimidine Nucleobases: Computational Evidence for Dependence of Duplex Stability on Spacer Location Linda A. Lait, Lesley R. Rutledge, Andrea L. Millen, and Stacey D. Wetmore* Department of Chemistry and Biochemistry, UniVersity of Lethbridge, 4401 UniVersity DriVe, Lethbridge, Alberta, T1K 3M4, Canada ReceiVed: June 23, 2008; ReVised Manuscript ReceiVed: August 5, 2008

The structural and binding properties of the natural and x- and y-pyrimidines were compared using computational methods. Our calculations show that although the x-pyrimidines favor different orientations about the glycosidic bond compared to the natural pyrimidines, which could have implications for the formation and resulting stability of xDNA duplexes and jeopardize the selectivity of expanded nucleobases, y-pyrimidines have rotational profiles more similar to the natural bases. Increasing the pyrimidine size using a benzene spacer leads to relatively minor changes in the hydrogen-bond strength of isolated Watson-Crick base pairs. However, differences in the anomeric carbon distances in pairs composed of x- or y-pyrimidines suggest yDNA may yield a more optimal expanded structure. By stacking two monomers via their centers of mass, we find that the expanded nucleobases stack much stronger than the natural bases. Additionally, although replacing xT by yT changes the stacking energy by less than 5 kJ mol-1, replacing xC by yC significantly strengthens complexes with the natural nucleobases (by up to 30%). Calculations on larger duplex models composed of four nucleobases reveal that x- and y-pyrimidines can increase duplex stability of natural helices by strengthening both the intra and interstrand stacking interactions. Furthermore, when the total stability (sum of all hydrogen-bonding and (intrastrand and interstrand) stacking interactions) of the larger models is considered, y-pyrimidines lead to more stable complexes than x-pyrimidines for all but three duplex sequences. Thus, through analysis of a variety of properties, our calculations suggest that the location of the benzene spacer affects the properties of expanded nucleobases and the stability of expanded duplexes, and therefore should be carefully considered when designing future expanded analogues. Introduction DNA,1

When Watson and Crick unlocked the structure of many new research fields quickly emerged that focused on applications of modified DNA components.2 For example, oligonucleotides with modified sugar-phosphate backbones have been extensively studied for use as antisense therapeutics,3-7 which target mRNA using complementary single-stranded oligomers. Modifications to the backbone, such as locked sugars8,9 or phosphorothioate linkages,10 have proven promising in these applications due to improved stability and cellular uptake, as well as decreased toxicity, when compared with natural oligonucleotides. However, an abundance of research has also considered modifications to the four natural nucleobases.2,11-15 Changes to the nucleobases are particularly intriguing since this is how DNA stores genetic information. Therefore, expansion of the genetic code has potential implications in, for example, information storage. In 1975, Leonard and co-workers introduced the world to “expanded” versions of adenine,16-19 which were used as bioprobes to explore the active sites of ATP-dependent enzymes.20-22 This was the first report of a modified nucleobase generated by the insertion of a (benzene) spacer. More recently, Kool and co-workers extended the applicability of Leonard’s design by synthesizing expanded analogues of all four natural bases,23-27 and subsequently incorporating these into helices.28-33 The so-called xDNA (“expanded” DNA) is composed of an expanded base in one strand hydrogen bonded to the comple* Corresponding author. E-mail: [email protected].

mentary natural base in the neighboring strand, which thereby generates a complete eight-base expanded system. The expanded bases have been shown to selectively pair with the appropriate natural counterpart and generate expanded helices with greater stability than natural duplexes.24-33 The original goal of the Kool group for developing expanded DNA was to design a new, fully functioning, genetic system that can be used for multiple tasks, which include designing the building blocks to developing enzymes that replicate the new genetic set.23 It was hoped that this work would provide insight into how biological information is stored and transferred in nature, and generate new biophysical or biomedical tools. Recent studies have shown that expanded nucleobases have many other biochemical applications, including fluorescent probes for biophysical analysis and applications in biotechnology.34 They may also have properties that are promising for the design of new molecular nanowires or other relevant devices.35,36 Due to the widespread applications of Kool’s xDNA helices, several other types of expanded DNA nucleobases have been synthesized. For example, other ring spacers have been considered including thiophene, 37,38 benzo[b]thiophene,39,40 and naphthalene,41-43 and a variety of heteroring expanded versions of guanine have also been studied.38 Furthermore, spacers that extend the nucleobases in other ways have been developed. For example, the two purine rings have been separated by single carbon-carbon bonds44-46 and single bonds have been used to attach new ring systems to the pyrimidines.47

10.1021/jp805547p CCC: $40.75  2008 American Chemical Society Published on Web 09/05/2008

yDNA versus xDNA Pyrimidine Nucleobases

J. Phys. Chem. B, Vol. 112, No. 39, 2008 12527

Figure 2. The (a) anti and (b) syn orientations of (natural) thymine.

Computational Details58-69

Figure 1. Structure and chemical numbering of the (a) natural DNA, (b) xDNA, and (c) yDNA pyrimidines as modeled in the present work.

The abundance of expanded base analogues recently synthesized raises questions about how different spacers affect the electronic structure and properties of the natural bases, where understanding the effects of the composition of the base spacer at the molecular level is necessary to aid future molecular design. However, before these questions can be answered, we must first understand the effects of the location of the spacer. For example, in addition to xDNA bases, the Kool group has synthesized y-pyrimidines,48,49 where the benzene ring is added along a different vector from the natural pyrimidines (Figure 1). When incorporated into helices, these unique bases generate so-called yDNA (“wide” DNA). It has been proposed that a change in the location of the benzene ring in expanded pyrimidines likely has little effect on the width of the base or its hydrogen-bonding interactions with the natural bases. 48,49 However, the spacer location will likely affect the shape of the major and minor grooves, and may also affect the stacking with neighboring bases.48,49 A greater understanding of the effects of the spacer location on the properties of expanded bases can be obtained from computational studies, which can provide an accurate comparison of the discrete interactions between expanded and natural nucleobases that stabilize expanded duplexes. Indeed, several computational studies have considered the properties of xDNA bases.50-56 However, to the best of our knowledge, only one computational study of yDNA nucleobases has appeared in the literature,57 which primarily focused on structure and HOMOLUMO gaps to understand the utility of these bases in nanotechnology. The present study complements previous computational and experimental work on yDNA by considering the energy barriers for rotation about the glycosidic bond, the (Watson-Crick) hydrogen-bond strengths, and the stacking interactions of y-pyrimidines. Through comparison to our previous study on natural DNA and xDNA nucleobases, 54 this work will reveal the effects of the location of the benzene spacer on properties that are essential for understanding the stability of expanded helices.

Since the methodologies required to accurately describe structure, hydrogen-bond strengths and stacking energies significantly differ, a variety of computational approaches and models were implemented in the present work. These techniques were previously used by our group to gain a greater understanding of the properties of xDNA nucleobases.54 To study the profile for rotation about the glycosidic bond, as well as the structure and strength of hydrogen-bonding interactions, the nucleobases, nucleosides and hydrogen-bonded pairs were optimized with B3LYP/6-31G(d,p) in the gas phase. Singlepoint calculations were subsequently performed at the B3LYP/ 6-311+G(2df,p) level to determine more accurate relative energies, which include zero-point vibrational energy (ZPVE) and basis set superposition error (BSSE) corrections (where applicable). To study the stacking interactions between two nucleobases, the monomers were optimized in Cs symmetry using MP2/631G(d), and the MP2 BSSE-corrected potential energy surfaces of stacked dimers were investigated using the 6-31G*(0.25) basis set, which replaces standard d-exponents (0.8) with a value of 0.25.58 This combination of methods has proven to accurately reproduce higher-level (CCSD(T)/CBS) stacking energies of natural nucleobase dimers59-63 and has been used to study the interactions between the natural nucleobases and a variety of other molecules.64-69 MP2/6-31G*(0.25) was also used to calculate the hydrogen-bonding and stacking interactions in larger DNA models composed of four nucleobases. We note that although both xT and xC were originally synthesized with methyl groups attached to the benzene spacer,27 our previous study considered models closer to the natural base analogues where xC did not contain a methyl group (Figure 1).54 In the present study, we directly model the structures of the synthesized y-pyrimidines, 48,49 where only yC contains a methyl group (Figure 1). Additional details of our methodologies will be provided in the appropriate Results and Discussion sections for clarity and to avoid redundancy. All calculations were performed using Gaussian 03.70 Results and Discussion (1) Rotation about the Glycosidic Bond. The relative orientation of nucleobases with respect to the deoxyribose moiety plays an important role in the structure and formation of DNA duplexes, as well as in a variety of biological processes. There are primarily two distinct orientations of the nucleobase about the (glycosidic) bond connecting the base and the sugar, which are referred to as anti and syn.71 In natural DNA, the pyrimidines adopt the anti orientation, which maximizes the distance between H1′ of the sugar moiety and H6 of the base and exposes the Watson-Crick hydrogen-bonding face to the neighboring strand (for example, see Figure 2 for the anti and syn orientations of thymine).

12528 J. Phys. Chem. B, Vol. 112, No. 39, 2008

Figure 3. Comparison of the relative energy as a function of rotation about the glycosidic bond for (a) thymine and (b) cytosine, where χ (deg) equals ∠(O1′C1′N1C2) for the natural pyrimidines (b), ∠(O1′C1′C4bC6) for the x-pyrimidines (9) and ∠(O1′C1′C3bC4b) for the y-pyrimidines (2). (χ values between approximately 90 and 270° represent anti conformations).

To ensure selective binding of expanded bases to the complementary natural base, and thereby generate an efficient alternative genetic alphabet, new nucleobases should preferentially adopt the anti orientation. To determine the effects of the benzene spacer, as well as its location on the preferred nucleobase orientation, we compare the rotational profiles about the glycosidic bond of the natural and expanded pyrimidine nucleotides. Specifically, a series of optimizations were performed with χ fixed in 10° increments between 0 and 360°, where χ equals ∠(O1′C1′N1C2) in the natural pyrimidines, ∠(O1′C1′C4bC6) in the x-pyrimidines and ∠(O1′C1′C3bC4b) in the y-pyrimidines. In these calculations, a simplified DNA sugar backbone was implemented, which replaces the C3′ and C5′ phosphates with hydroxyl groups. Additionally, the HO5′C5′C4′ dihedral angle was restricted to 180°, which prevents unrealistic interactions between the C5′-hydroxyl group and the nucleobase, and the sugar puckering was initially set to C2′-endo, which is the preferred puckering in B-DNA. Figure 3 compares the rotational profiles of the natural and x- and y-pyrimidines, where χ values in the approximate range of 90 to 270° represent the anti orientation. Our calculations reveal that the syn conformation of the natural pyrimidines is highly unfavorable due to repulsive interactions between O5′ and the O2 carbonyl (Figure 4a).54 However, the syn conformation of the x-pyrimidines is more favorable due to a relatively strong O5′ · · · H-N1 hydrogen bond (Figure 4b). This suggests that the selectivity of the expanded bases is jeopardized since the (anti) base orientation required for Watson-Crick binding

Lait et al.

Figure 4. Comparison of anti and syn minima for rotation about the glycosidic bond in (a) natural, (b) x- and (c) y-thymine, where χ (deg) equals ∠(O1′C1′N1C2) for the natural pyrimidines, ∠(O1′C1′C4bC6) for the x-pyrimidines and ∠(O1′C1′C3bC4b) for the y-pyrimidines.

is destabilized compared with other (syn) orientations that may yield base mismatches. Furthermore, two different anti minima are prevalent on the rotational profiles of the x-pyrimidines (Figure 3), where the lowest energy anti conformation (χ ) 310°, Figure 4b) is highly distorted due to close contacts between sugar and nucleobase hydrogens. This suggests that the resulting structure and stability of the expanded duplex may not be optimal despite experimental evidence that x-pyrimidines adopt Watson-Crick binding and xDNA is more stable than natural DNA helices.24,27 The glycosidic bond rotational profiles of the y-pyrimidines differ from those of the x-pyrimidines (Figure 3). Indeed, the rotational profiles of the y-pyrimidines more closely mimic those of the natural nucleobases where only one anti minimum is found. However, the rotational barriers between the anti and syn orientations of the yDNA pyrimidines are less than 10 kJ mol-1 (8.8 kJ mol-1 for yC and 8.9 kJ mol-1 for yT) due to the lack of strong hydrogen bonds or large repulsion between the base and sugar moiety in either orientation (Figure 4c). Although the y-pyrimidines exhibit a slight thermodynamic preference for the anti orientation, the low barrier to rotation likely indicates that there is a competition between the anti and syn orientations for the y-pyrimidines. Nevertheless, the barriers for rotation in the natural nucleosides are also fairly low (26.9 kJ mol-1 for C and 23.7 kJ mol-1 for T). Therefore, we suggest that expanded duplexes formed using yDNA pyrimidines may not suffer from the same potential instabilities as strands involving xDNA pyrimidines due to rotation about the glycosidic bond. This is the first indication that the location of the benzene spacer may affect the properties of expanded nucleobases and thereby affect the structure and/or stability of expanded duplexes. We acknowledge, however, that the effect of the charged phosphate group and the surrounding solvent may provide additional



TABLE 1: Hydrogen-Bond Strengths (∆EHBond, kJ mol-1) and Anomeric Carbon Distances (R(C · · · C), Å) for Dimers Involving Natural and Expanded Pyrimidinesa dR ) Hb

dR ) CH3c

dimer

∆EHBond

∆EHBond

R(C · · · C)

A:T A:xT A:yT xA:T xA:xT xA:yT G:C G:xC G:yC xG:C xG:xC xG:yC

-43.8 -43.5 -43.7 -46.2 -46.0 -46.0 -96.1 -99.0 -105.8 -89.7 -92.9 -98.9

-44.1 -43.7 -44.2 -46.6 -45.8 -46.4 -95.7 -99.2 -105.9 -89.6 -92.7 -99.1

10.6 12.7 13.1 13.1 15.1 15.6 10.8 12.8 13.3 13.2 15.2 15.8

a Relative energies calculated at the B3LYP/6-311+G(2df,p)// B3LYP/6-31G(d,p) level of theory, and include ZPVE and BSSE corrections. Data for pairs involving x-primidines was previously published in ref . b Deoxyribose was replaced with a hydrogen atom (dR ) H). c Deoxyribose was replaced with a methyl group (dR ) CH3).

important information regarding the geometric preference of expanded nucleobases in double helices and should be the focus of future studies. (2) Watson-Crick Hydrogen-Bond Strengths. Computational studies can provide information about both the structure and strength of hydrogen-bonding interactions. Indeed, calculations on doubly expanded DNA nucleobases have suggested that the Watson-Crick binding mode is not always the most stable since the canonical form is not the most stable tautomer for all expanded bases.43 Nevertheless, experimental base-pairing studies have shown that xDNA28-33 and yDNA48,49 pyrimidines bind to their Watson-Crick purine partner. Therefore, we focus our present efforts on comparing the Watson-Crick structures and hydrogen-bond strengths for pairs involving natural, x- and y-pyrimidines. The binding strengths of complexes between the natural, xor y-pyrimidines and the natural purines are provided in Table 1. We have considered two different models where the deoxyribose moiety is replaced with either a hydrogen atom or a methyl group (dR ) H or CH3, Figure 1). Consideration of the dR ) CH3 model is particularly important since this allows us to provide an accurate estimate of the distance between the anomeric carbons (R(C · · · C), Table 1), which directly relates to the width of DNA duplexes. To understand the geometry and binding strength in the context of an eight-base expanded DNA system,18-23 we also considered the complexes between the natural pyrimidines and the x-purines, where a benzene spacer is inserted between the pyrimidine and imidazole rings of the purines. All hydrogen-bonded pairs optimize to a near planar geometry. Furthermore, as postulated from experimental studies,48,49 the structures of hydrogen-bonded pairs involving the x- and y-pyrimidines are very similar (see, for example, the adeninethymine pairs in Figure 5). However, the anomeric carbon distances in x-purine-natural-pyrimidine pairs are 2.5 Å larger than those in the natural pairs (Table 1), while those in x-pyrimidine-natural-purine pairs are only 2 Å larger. This difference in the anomeric carbon distance could cause instabilities or lead to weaker binding of the x-pyrimidine-natural-purine pairs in xDNA duplexes. However, when the y-pyrimidines are paired with the natural bases, the anomeric distances mirror

Figure 5. Comparison of the structure of the (a) A:T, (b) A:xT and (c) A:yT hydrogen-bonded pairs.

those of the x-purine-natural-pyrimidine pairs. This suggests that y-pyrimidines and x-purines may be a better choice when designing an eight-base information storage system based on benzene-expanded nucleobases. Table 1 reveals that there is not a significant difference in the hydrogen-bond strength when the deoxyribose moiety is modeled as a hydrogen atom or a methyl group. More importantly, there is a very small change in the binding strength upon expansion of thymine in the A:T pair, and both xT and yT bind to adenine with a similar strength. However, expansion of cytosine in the G:C pair leads to larger changes in hydrogenbond strengths. Indeed, replacing cytosine with xC increases the G:C binding strength by 3 kJ mol-1, while replacing xC with yC increases the binding strength by an additional 7 kJ mol-1. Furthermore, as mentioned above, the slightly shorter anomeric carbon distances in pairs involving x-pyrimidines may lead to longer hydrogen-bond lengths, and therefore weaker interactions, when the bases are incorporated into duplexes. This once again suggests that pairs involving y-pyrimidines may lead to slightly more stable expanded duplexes than pairs involving x-pyrimidines. We also considered the hydrogen-bonded complexes between the x-purines and x- or y-pyrimidines (Table 1) due to potential future interest in designing doubly expanded DNA. First, we must consider the effect on the natural base pair binding strengths due to expansion of the purines. Expansion of adenine in the natural A:T pair to xA:T increases the hydrogen bond strength only slightly (by 3 kJ mol-1), while expansion of guanine in the G:C pair to xG:C leads to a 6 kJ mol-1 decrease in the binding strength. When doubly expanded DNA is considered by expanding both the purine and pyrimidine, the change in the G:C binding strength is slightly greater than for A:T. Furthermore, the hydrogen-bond strengths of xA:xT and xA:yT are equal, while the xG:yC pair is approximately 5 kJ mol-1 stronger than the xG:xC pair. Once again, this suggests that the y-pyrimidines may lead to slightly more stable expanded duplexes than x-pyrimidines. (3) Stacking Interactions between Nucleobase Monomers. The above results suggest that there are subtle changes in hydrogen-bond strength upon expansion of the natural nucleobase pairs. However, these changes are likely not large enough to account for the increased stability reported in experimental


Lait et al.

TABLE 2: Summary of the Largest (Most Negative) and Smallest (Least Negative) Stacking Interactions (kJ mol-1) and Corresponding Angle of Rotation (r, deg.) between the Natural Nucleobases and the Natural Pyrimidines (n ) DNA), x-Pyrimidines (n ) xDNA) or y-Pyrimidines (n ) yDNA) Stacked According to Their Centers of Massa,b n ) DNA largest nT · · · A nT · · · C nT · · · G nT · · · T nC · · · A nC · · · C nC · · · G nC · · · T nC · · · nC nC · · · nT nT · · · nT

n ) xDNA smallest

largest

n ) yDNA smallest

largest

smallest

∆E

R

∆E

R

∆E

R

∆E

R

∆E

R

∆E

R

-41.0 -36.9 -47.9 -30.0 -34.2 -36.1 -40.9 -36.9 -36.1 -36.9 -30.0

150 120 270 60 240 180 330 120 180 120 60

-22.9 -9.3 -22.2 21.6 -10.9 14.2 -3.1 -9.3 14.2 -9.3 21.6

60 0 30 0 0 0 210 0 0 0 0

-44.3 -41.6 -53.0 -44.8 -43.9 -39.6 -49.2 -45.8 -51.7 -52.7 -55.5

120 60 270 180 270 210 60 210 120 180 180

-30.8 -19.2 -30.0 -22.9 -24.3 -14.0 -17.7 -20.7 -1.0 -25.0 9.0

330 240 90 330 30 0 120 330 0 0 0

-44.6 -39.8 -47.8 -38.6 -50.8 -52.1 -62.9 -47.0 -72.5 -57.1 -52.2

150 150 300 180 240 210 0 210 150 210 180

-29.8 -23.5 -30.2 -23.8 -30.9 -14.3 -21.6 -16.2 25.5 -22.0 -9.7

330 330 90 240 330 330 120 330 0 0 0

Stacking interactions were calculated with MP2/6-31G* (0.25) and include BSSE corrections, where data for n ) DNA and xDNA were previously reported in ref.54 b Bases were separated vertically by 3.3 Å, and a 0° angle of rotation is defined as the orientation where the model glycosidic bonds (dR ) H, Figure 1) are aligned in parallel. a

melting temperature studies of xDNA24,27 and yDNA48,49 helices. Indeed, data from dangling end experiments suggests that enhanced stacking interactions lead to the observed increased stability.27,48 Previous computational studies of xDNA provide additional support that the benzene spacer significantly increases the stacking potential of the natural nucleobases in stacked dimers,54 as well as expanded helices.52,54 As a first step toward understanding and comparing the stacking abilities of expanded DNA pyrimidines, we consider the interactions between two isolated nucleobases (dR ) H, Figure 1). Specifically, we compare the interaction energies between two natural nucleobases, one x- or y-pyrimidine and one natural pyrimidine or purine, and two x- or two ypyrimidines. Since previous computational studies have revealed that the stacking interactions involving a natural nucleobase monomer are most dependent on the angle of rotation between monomers,59,64-67,72,73 we stacked the nucleobases according to their centers of mass with a vertical separation of 3.3 Å, which is the average separation in B-DNA and the favored calculated separation between expanded and natural nucleobases.74 Initial structures were generated by aligning the glycosidic bonds of the nucleobase monomers (i.e., the bond involving dR ) H in Figure 1), which defines an angle of rotation (R) equal to 0°. This alignment corresponds to nucleobases within the same DNA strand (i.e., intrastrand stacking) in the absence of the 36° helical twist. Although base orientations that correspond to interactions between opposing strands (i.e., interstrand stacking) can also be considered, which involve a 180° flip of one nucleobase with respect to the other. These orientations are not investigated since center-of-mass stacking does not realistically model interstrand interactions due to minimal overlap between opposing strands in DNA helices.75-78 We note that the effects of horizontally shifting one monomer in its molecular plane on the stacking energy can also be considered. Although the so-called parallel displaced structure is a local minimum for the benzene dimer, 75-78 horizontal displacement of one monomer with respect to the other has been shown to only slightly affect stacking energies involving the natural nucleobases.64-67 Therefore, these shifts are not considered in our potential energy surface scans. We note, however, that the effects of the horizontal shift will be discussed in the following section when a larger DNA model is implemented. From the initial monomer orientations described above, one nucleobase was rotated in 30° increments from 0 to 360°. As

noted previously for a variety of stacked complexes,64-67 the interaction energy varies significantly with this rotation, where the largest (most negative) and smallest (least negative) interaction energies are summarized in Table 2. In natural nucleobase pairs, the stacking energy varies with the angle of rotation by approximately 18 - 52 kJ mol-1. The variation is less when one natural pyrimidine is replaced by an x-pyrimidine (14 40 mol-1), and even less when one natural pyrimidine is replaced by a y-pyrimidine (14 - 26 kJ mol-1). The variation in the stacking energy with the relative orientation of the monomers is found to be larger when complexes between two x-pyrimidines (28 to 65 kJ mol-1) or y-pyrimidines (35 - 98 kJ mol-1) are considered. To understand the large variation in the stacking energy with the relative monomer orientations, we must understand the structures of complexes with the smallest and largest interactions. The smallest interactions for dimers of two natural or two expanded pyrimidines typically occur when the overlap of ring atoms is maximized (R ) 0°), which increases the repulsive contribution to stacking. However, these repulsive interactions are larger for some complexes. Indeed, the weakest interaction energy for nucleobase dimers containing methyl groups is positive (+21.6 kJ mol-1 for T · · · T, +9.0 kJ mol-1 for xT · · · xT and +25.0 kJ mol-1 for yC · · · yC) due to large repulsion between the out of plane methyl hydrogens. Due to these repulsive interactions, dimers between two nucleobases of the same size have the largest variation in the stacking energy upon rotation of one monomer. In contrast, when a natural nucleobase is paired with an expanded pyrimidine, the difference in base size implies that the center of mass stacking arrangement with R ) 0° leads to negligible atomic overlap. Thus, the variation in the stacking energy upon rotation of one monomer is not as large in these mixed dimers. In structures with the largest stacking interactions, the dipole moments of the monomers are typically antialigned (Figure 6), which reveals the importance of electrostatics to the overall stacking energy. Repulsion between ring nitrogens or exocyclic (carbonyl, amino) substituents also plays a role in determining the structure with the largest stacking energy and explains slight deviations from structures with perfectly antialigned dipole moments. This finding is in agreement with previous studies of the stacking interactions when natural nucleobases are bound with a variety of other aromatic molecules.64-67 Furthermore, for each pyrimidine, the strongest stacking interactions occur



Figure 6. Dipole moment vectors (magnitude (Debyes) included in parentheses) of the x- and y-pyrimidines, as well as the natural nucleobases, and the corresponding dimers with the largest stacking energies after adjusting the angle of rotation (R).

upon complexation with guanine due to the relatively large size, dipole moment, and therefore polarizability of this natural nucleobase. This result is also in agreement with studies of the stacking interactions between natural DNA nucleobases,58-60,62,63 where the guanine dimer has the largest interaction energy. The trend with respect to the other nucleobases is unclear due to the importance of both size and dipole moment for determining the interaction strength. Perhaps the most important information presented in Table 2 is the increase in the maximum stacking energy upon expansion of the pyrimidines by benzene. Specifically, our calculations show that the largest stacking interaction involving the natural pyrimidines range between -30 to -48 kJ mol-1. This range increases to fall between -40 and -53 kJ mol-1 when one natural pyrimidine in the pair is expanded to an x-pyrimidine, where the increase for any given dimer is up to 15 kJ mol-1. If natural pyrimidine is replaced by a y-pyrimidine, the range in the largest interaction energy increases to fall between -39 and -63 kJ mol-1, where the stacking energy of any given dimer increases by up to 22 kJ mol-1. Further expansion of the pyrimidine complexes by considering interactions between two expanded bases leads to even greater interaction energies. Specifically, stacking interactions in xpyrimidine dimers range between -52 and -56 kJ mol-1, which represents an up to 30 kJ mol-1 increase compared with the

corresponding natural pyrimidine complex, while interaction energies of y-pyrimidine dimers range between -52 and -73 kJ mol-1, which represents an up to 36 kJ mol-1 increase. Interestingly, the large increases in stacking with respect to the corresponding natural base pair for dimers between two expanded pyrimidines are greater than the sum of the effects of expanding each monomer. For example, replacing T or C in the C · · · T complex with the corresponding y-pyrimidine increases the stacking energy by 6 or 11 kJ mol-1, respectively. Therefore, the additive effect of expanding both pyrimidines in the C · · · T pair is 17 kJ mol-1. However, calculations on the yC · · · yT complex reveal that replacing both pyrimidines in the C · · · T dimer increases the interaction energy of the corresponding natural pair by 22 kJ mol-1. The greater than additive effects of base expansion occur due to an increase in the overlap of both the pyrimidine and benzene π-systems when both bases in the complex increase in size. The ranges in the interaction energies reported above reveal that there is a significant increase in stacking upon expanding a pyrimidine with a benzene spacer, which primarily arises due to increased size, and therefore polarizability, of the nucleobase and increased monomer overlap in the dimer. However, the ranges also imply that there is a difference in the stacking properties of x- and y-pyrimidines. In other words, the location of the benzene spacer affects the stacking of the expanded


Figure 7. MP2/6-31G(d) electrostatic potentials (kJ mol-1) for natural, x- and y- (a) thymine and (b) cytosine.

nucleobase. However, the magnitude of this effect and the spacer location that affords the maximum stacking energy are different for thymine and cytosine. Specifically, in dimers between one natural nucleobase and one expanded pyrimidine, yT generally stacks slightly weaker than xT by up to 5 kJ mol-1. However, yC generally stacks stronger than xC by up to 15 kJ mol-1, which represents an up to 30% increase in stacking upon replacing xC with yC. The dependence of stacking on the location of the benzene spacer is at least in part because of changes in the nucleobase dipole moments. Differences in the dipole moments arise due to changes in the benzene ring location, inclusion of methyl groups in some structures, and rearrangement of the ring nitrogen atoms. Specifically, the dipole moment of xT (5.324 D, Figure 6) is slightly larger than that of yT (5.039 D), while the dipole moment of xC (7.803 D) is significantly smaller than that of yC (8.821 D). These changes can be seen as differences in the electrostatic potentials (Figure 7). Additionally, the methyl group of yC and xT, which is absent in xC and yT, directly contributes to the overall stabilization of stacked complexes. For example, the yC dimer is stabilized by C-H · · · O (and/or C-H · · · N) interactions between the methyl group hydrogens and nucleobase carbonyls (and/or ring nitrogens, see Figure 8). xT dimers have similar interactions, but the structure with the largest stacking energy also has significant repulsion between ring atoms due to direct atomic overlap (Figure 8) and therefore the effects of C-H...O interactions are less pronounced. Indeed, the role of the methyl group in strengthening thymine stacking interactions over those of uracil has been previously reported.79 Additionally, methane has been shown to have significant interactions with a variety of biomolecules.80-82 Our results suggest that there is a significant increase in stacking upon expansion of a natural pyrimidine due to the increased size of the molecule. Furthermore, the magnitude of the increase is dependent on the location of the benzene spacer, which is primarily due to changes in dipole moment with a smaller role also played by weak interactions by nucleobase methyl groups. The largest differences occur for complexes with the corresponding natural nucleobase (i.e., nC · · · C and nT · · · T, Table 2), the corresponding expanded nucleobase (i.e., nC · · · C and nT · · · nT, Table 2) or guanine. This suggests that, depending on the oligonucleotide sequence, duplexes containing y-pyrimidines may be more stable than those containing x-pyrimidines.

Lait et al.

Figure 8. Relative monomer orientations in dimers involving x- or y-pyrimidines that lead to the largest stacking energies after adjusting the angle of rotation (R).

Indeed, although dangling-end experiments indicate that duplex melting temperatures are nearly equivalent for x- and ypyrimidines, melting temperatures of DNA strands with expanded pyrimidines paired opposite natural purines are up to 6 °C larger for yC48 than xC.27 The true implications of the center-of-mass stacking on duplex stability are unclear, where the 36° helical twist within DNA leads to stacking orientations that significantly differ from the center of mass stacked geometries considered in the present section. Specifically, previous studies have shown that horizontal displacement of one monomer with respect to the center of mass stacked geometries typically decreases the interaction energy.59,64-67,72,73 Therefore, the next section considers a more accurate model in attempts to gain a greater understanding of these interactions within expanded duplexes. (4) Interactions between Stacked Nucleobase Pairs. The current lack of experimental crystal structures for a variety of oligonucleotide sequences containing x- or y-pyrimidines means that a simplified computational model must be implemented in order to gain a greater understanding of the stacking interactions of these modified biomolecules within duplex environments. Indeed, no crystal structures are currently available for expanded helices and NMR solution structures are only available for select xDNA sequences.28,33 We adapt the methodology previously used to study the stacking interactions of B-DNA,79,83,84 where the model yields good agreement with calculations using fiber or crystal geometries. In our approach, models composed of four nucleobases (Figure 9) were built by first identifying the centers of B3LYP/6-31G(d,p) optimized hydrogen-bonded pairs as midpoints of the line connecting C8 of the natural purines and C6 of the natural pyrimidines, C3b of the x-pyrimidines or C2b of the y-pyrimidines. Next, two base pairs were stacked according to these centers and separated by 3.3 Å, which is the average separation in B-DNA. Finally, a helical twist of 36° was imposed in the right-hand sense with respect to the C8-C6, C8-C3b or C8-C2b axes. Using this model, the total MP2/ 6-31G*(0.25) binding energy in the four nucleobase sequence was calculated as the sum of six (dimer) interactions: two intrastrand stacking (X1 · · · Y1, X2 · · · Y2), two interstrand stacking (X1 · · · Y2, X2 · · · Y1) and two hydrogen bonding (X1 · · · X2 and Y1 · · · Y2), where each interaction energy includes a BSSE correction and the total errors arising due to this additivity assumption are anticipated to be small.83 We previously used the analogous four-nucleobase duplex model to study the stacking interactions in expanded duplexes


Figure 9. Nomenclature for individual bases and the hydrogen-bonding (a, b), intrastrand stacking (c, d), and interstrand stacking (e, f) interactions within stacked hydrogen-bonded pairs considered in the present work.

composed of (mixed) base pairs between x- and natural purines and pyrimidines.54 This model was justified since, although some differences between DNA and xDNA helices have been identified such as a greater number of base pairs per turn, a reduction in the helical twist, and a slightly bent xDNA helix,28,33 the overall structures are very similar.22 Specifically, xDNA helices are right-handed, composed of Watson-Crick hydrogen-bonded pairs, involve anti nucleobase orientations with respect to the sugar moieties and exhibit C2′-endo sugar puckering.28,33 Furthermore, calculations on model xDNA helices indicate that slight changes in the helical twist or vertical separation between base pairs leads to minor changes in interaction energies between bases,54 and a similar conclusion has been obtained for natural helices.79 The same arguments can be used to justify the use of our duplex model to study yDNA helices. We also note that even if crystal structures were available, the coordinates cannot be directly used in ab initio calculations since unrelaxed geometries can distort wave functions. 83,84 Furthermore, the use of force-field optimized structures for ab initio calculations has been criticized.83 Table 3 compares the individual interaction energies obtained using our simplified duplex model for all combinations of natural purines hydrogen bonded to x- or y-pyrimidines in the neighboring strand, as well as the corresponding natural DNA sequences. The (X1 · · · Y1 and X2 · · · Y2) intrastrand stacking interactions in natural helices fall between -4 and -40 kJ mol-1. Any given interaction is typically 15-30 kJ mol-1 weaker than the optimum center-of-mass stacking interaction, which represents an up to 60% decrease. In mixed duplexes, the intrastrand interactions between natural nucleobases remain close to those in natural helices, and decreases of up to 5 kJ mol-1 occur due to slight differences in nucleobase orientations. In expanded duplexes, the intrastrand stacking interactions between an expanded pyrimidine and natural purine range between -14 and -48 kJ mol-1 for xDNA helices and -14 and -59 kJ mol-1 for yDNA. Although these intrastrand interactions are 20-80% smaller than the optimal center-ofmass stacking arrangement, expansion of one base in the duplex increases intrastrand interactions by up to 14 kJ mol-1. Furthermore, interactions between two expanded pyrimidines are 10-24 kJ mol-1 stronger than interactions between the corresponding natural pyrimidines. In general, most intrastrand stacking interactions involving y-pyrimidines in mixed helices are within 5 kJ mol-1 of the corresponding interaction with x-pyrimidines. This is a different conclusion from that based on our center of mass stacking study, which suggested these interactions can significantly differ. However, there are two exceptions. First, yC · · · yC interactions are up to 13 kJ mol-1 less than xC · · · xC interactions. This

J. Phys. Chem. B, Vol. 112, No. 39, 2008 12533 occurs in part due to steric repulsions between the yC methyl groups and in part due to repulsions between negatively charged regions of yC, where the larger dipole moment of yC leads to a more highly charged Watson-Crick binding face (Figure 7). In our center-of-mass stacking study, we found that yC dimers are 20 kJ mol-1 stronger than xC dimers due to interactions involving the methyl group and the large dipole moment of yC. Second, yC · · · G interactions are up to 12 kJ mol-1 more stable than xC · · · G interactions. This can be at least in part explained by close contact (2.5 Å) between the yC methyl group and N9 in G, as well as the larger dipole moment of yC. These two examples reemphasize the important role methyl substituents play in altering the stacking interactions of expanded nucleobases in double helices as recently discussed in a comparison of thymine and uracil interactions.79 It is anticipated that (X1 · · · Y2 and X2 · · · Y1) interstrand interactions are very weak. Indeed, the calculated interstrand interactions in natural helices are generally very small. However, sometimes these interactions can be significantly repulsive. For example, C · · · C interactions are up to +12 kJ mol-1 since the highly charged regions of the nucleobases interact across the strand. It is more intriguing that some interactions are highly favorable and can provide up to 20 kJ mol-1 net stabilization to the four nucleobase sequence. These stabilizing interactions are typically due to overlap between two purines or interactions between oppositely charged regions of cytosine and guanine. In mixed duplexes, interstrand interactions can decrease, remain the same or increase relative to the corresponding interaction in natural helices. For example, upon expansion of the pyrimidines, the natural purines in neighboring strands are separated by greater distances and therefore the corresponding interstrand stacking interactions are weakened compared with natural base interactions. Indeed, upon expansion to the GCGC (X1Y1X2Y2) sequence to GyCGyC, the G · · · G interstrand interaction decreases from -16.5 kJ mol-1 to -5.5 kJ mol-1. Alternatively, the natural purine-expanded pyrimidine interstrand interactions are very similar to the corresponding interactions in natural helices due to similar overlap of Watson-Crick binding edges. Indeed, upon expansion of the ATAT (X1Y1X2Y2) sequence to AyTAyT, the T · · · A interstrand interactions change by less than 0.4 kJ mol-1. However, interstrand interactions between two expanded pyrimidines can add as much as 20 - 25 kJ mol-1 net stabilization to mixed helices, which is much larger than pyrimidine-pyrimidine interactions in natural helices. For example, upon expansion of the ATTA (X1Y1X2Y2) sequence, the T...T interstrand interactions increase by 12.6 kJ mol-1. This indicates that expansion of the pyrimidines can increase duplex stability by enhancing both intra and interstrand stacking interactions. Most xDNA and yDNA interstrand interactions are similar. However, the interactions between two expanded pyrimidines can vary by up to 16 kJ mol-1 and there is no clear trend in which expanded pyrimidines lead to stronger interactions. For example, in the X1X2Y1Y2 ) nCGGnC sequence, the xC · · · xC interaction is 16 kJ mol-1 stronger than the yC...yC interaction. This difference occurs since the G:yC hydrogen-bonded pair is wider than the G:xC pair (Table 1) and therefore there is less overlap between two y-cytosines. On the other hand, in the X1X2Y1Y2 ) AnTnTA sequence, the yT · · · yT interaction is 11 kJ mol-1 stronger than xT · · · xT due to differences in overlap arising because of differences in how the pyrimidines are twisted into the major and minor groves. From the above discussion, it is clear that inclusion of either x- or y-pyrimidines in DNA helices increases the stacking


Lait et al.

TABLE 3: Summary of the Stacking and Hydrogen-Bonding Interactions Calculated for Duplexes Composed of Two Hydrogen-Bonded Pairs between a Natural Purine and a Natural, x-Pyrimidine, or y-Pyrimidine DNAa,b intrastrand stacking X1

X2

Y1

interstrand stacking

hydrogen bonding

Y2

X1 · · · Y1

X2 · · · Y2

X1 · · · Y2

X2 · · · Y1

X1:X2

Y1:Y2

Σ(stacking)c

Σ(H bond)d

totale

A A A A T T T T G G G G C C C C

DNA-DNA T A T T T G T C A A A T A G A C C A C T C G C C G A G T G G G C

T A C G T A C G T A C G T A C G

-27.3 -22.9 -37.0 -16.4 -21.6 -14.9 -16.8 -20.1 -30.5 -20.1 -13.6 -25.6 -18.8 -16.0 -40.6 -3.9

-14.9 -22.8 -16.0 -20.1 -22.3 -27.3 -18.8 -30.5 -20.1 -16.4 -3.9 -25.6 -16.8 -37.0 -39.9 -13.6

-6.3 -6.9 1.5 -16.7 2.0 -6.3 -0.7 -2.1 -2.1 -16.7 -14.3 -16.5 -0.7 1.5 11.8 -14.3

-5.0 1.6 0.1 -4.2 -3.3 -5.0 -15.2 -0.3 -0.3 -4.2 -19.7 4.0 -15.2 0.1 5.6 -19.7

-56.8 -56.8 -56.8 -56.8 -56.8 -56.8 -56.8 -56.8 -114.8 -114.8 -114.8 -114.8 -114.8 -114.8 -114.8 -114.8

-56.8 -56.8 -114.8 -114.8 -56.8 -56.8 -114.8 -114.8 -56.8 -56.8 -114.8 -114.8 -56.8 -56.8 -114.8 -114.8

-53.5 -51.0 -51.4 -57.4 -45.2 -53.5 -51.5 -53.0 -53.0 -57.4 -51.5 -63.7 -51.5 -51.4 -63.1 -51.5

-113.6 -113.6 -171.6 -171.6 -113.6 -113.6 -171.6 -171.6 -171.6 -171.6 -229.6 -229.6 -171.6 -171.6 -229.6 -229.6

-167.2 -164.6 -223.0 -229.1 -158.8 -167.2 -223.3 -224.7 -224.7 -229.1 -281.1 -293.3 -223.3 -223.0 -292.8 -281.1

A A A A xT xT xT xT G G G G xC xC xC xC

xDNA-DNA xT A xT xT xT G xT xC A A A xT A G A xC xC A xC xT xC G xC xC G A G xT G G G xC

xT A xC G xT A xC G xT A xC G xT A xC G

-22.4 -28.3 -37.5 -20.1 -31.8 -34.4 -25.4 -43.4 -24.5 -27.0 -14.0 -36.9 -27.2 -38.1 -47.6 -26.8

-34.4 -28.3 -38.1 -27.0 -31.8 -22.4 -27.2 -24.5 -43.4 -20.1 -26.8 -36.8 -25.4 -37.5 -47.1 -14.0

-8.0 0.1 0.8 -12.8 -8.2 -8.0 -14.8 -3.1 -3.1 -12.8 -13.4 -3.8 -14.8 0.8 -2.3 -13.4

-4.8 -0.5 1.4 -7.0 -1.9 -4.8 -8.0 -1.5 -1.5 -7.0 -19.2 6.7 -8.0 1.4 12.8 -19.2

-56.1 -56.1 -56.1 -56.1 -56.1 -56.1 -56.1 -56.1 -115.8 -115.8 -115.8 -115.8 -115.8 -115.8 -115.8 -115.8

-56.1 -56.1 -115.8 -115.8 -56.1 -56.1 -115.8 -115.8 -56.1 -56.1 -115.8 -115.8 -56.1 -56.1 -115.8 -115.8

-69.6 -57.0 -73.4 -66.9 -73.7 -69.6 -75.4 -72.5 -72.5 -66.9 -73.4 -70.8 -75.4 -73.4 -84.2 -73.4

-112.2 -112.2 -171.9 -171.9 -112.2 -112.2 -171.9 -171.9 -171.9 -171.9 -231.6 -231.6 -171.9 -171.9 -231.6 -231.6

-181.8 -169.2 -245.2 -238.8 -185.8 -181.8 -247.4 -244.3 -244.3 -238.8 -305.0 -302.3 -247.4 -245.2 -315.7 -305.0

A A A A yT yT yT yT G G G G yC yC yC yC

yDNA-DNA yT A yT yT yT G yT yC A A A yT A G A yC yC A yC yT yC G yC yC G A G yT G G G yC

yT A yC G yT A yC G yT A yC G yT A yC G

-22.9 -23.7 -37.4 -16.4 -29.9 -29.3 -30.6 -38.9 -25.8 -28.0 -15.0 -34.1 -20.6 -29.8 -58.2 -13.7

-29.3 -23.7 -31.4 -27.4 -29.9 -22.9 -23.9 -25.2 -39.1 -16.4 -15.1 -34.4 -29.5 -36.4 -59.3 -14.1

-5.9 -0.3 2.7 -10.6 -0.1 -5.9 -7.3 -4.4 -3.9 -11.4 -16.3 -5.5 -8.0 2.8 13.4 -17.2

-4.8 -11.0 -0.5 -10.8 -0.6 -4.8 -9.1 -0.1 0.1 -10.8 -25.7 11.2 -9.7 -0.5 11.2 -25.8

-56.4 -56.4 -56.4 -56.4 -56.4 -56.4 -56.4 -56.4 -124.6 -124.6 -124.6 -124.6 -124.6 -124.6 -124.6 -124.6

-56.4 -56.4 -124.6 -124.6 -56.4 -56.4 -124.6 -124.6 -56.4 -56.4 -124.6 -124.6 -56.4 -56.4 -124.6 -124.6

-62.9 -58.7 -66.6 -65.2 -60.6 -62.8 -70.9 -68.6 -68.7 -66.6 -72.1 -62.8 -67.8 -63.9 -92.8 -70.8

-112.9 -112.9 -181.1 -181.1 -112.9 -112.9 -181.1 -181.1 -181.1 -181.1 -249.3 -249.3 -181.1 -181.1 -249.3 -249.3

-175.7 -171.6 -247.7 -246.2 -173.4 -175.7 -251.9 -249.6 -249.7 -247.7 -321.4 -312.1 -248.8 -245.0 -342.1 -320.1

a See Figure 9 for the DNA helix model and nomenclature of the interactions. Data for natural DNA duplexes and xDNA-DNA duplexes were previously reported in ref 54. b Interaction energies were calculated with MP2/6-31G*(0.25) and include BSSE corrections. c Sum of the intra and interstrand stacking interactions. d Sum of the hydrogen-bonding interactions. e Sum of all (stacking and hydrogen-bonding) interactions.

interactions of natural duplexes. However, any given interaction is sometimes larger in xDNA and sometimes larger in yDNA. Furthermore, whether x- or y-pyrimidines lead to stronger stacking is highly dependent on the strand sequence. Therefore, to gain a greater appreciation of how the differences discussed above combine to yield overall differences in helix stabilization, Table 2 summarizes the total stacking interaction energies (Σ(stacking)). The first important conclusion is that expanded helices always have a greater stacking contribution to the total

stabilization than natural helices. However, the amount of stabilization is significantly dependent on the base sequence, where the increase in total stacking ranges between 6 and 30 kJ mol-1 (or a 10-50% increase). The second important conclusion is that xDNA generally has a greater total contribution to stacking than yDNA by 0-8 kJ mol-1. Indeed, only two strand sequences (X1X2Y1Y2 ) nCGGnC and AnTnTA) have total stacking energies greater for the y-pyrimidines (by 8.6 and 1.2 kJ mol-1, respectively).

yDNA versus xDNA Pyrimidine Nucleobases In addition to stacking interactions, we must consider the hydrogen-bond strengths between neighboring strands in order to gain a total picture of duplex stability. The individual and total hydrogen-bonding interactions calculated at the MP2/631G*(0.25) level of theory are reported in Table 3, which are larger than those calculated with B3LYP (Table 1). More importantly, there is a large difference (8.8 kJ mol-1) in the hydrogen-bond strength of the xC:G and yC:G pairs. This results in an up to 18 kJ mol-1 larger contribution of hydrogen bonding to the total stabilization for helices containing y-pyrimidines compared with x-pyrimidines in a four nucleobase sequence. We note that although MP2/6-31G*(0.25) has been shown to recover stacking energies obtained with CCSD(T) at the complete basis set limit, a MP2 to CCSD(T) correction is recommended when comparing hydrogen-bonding and stacking interactions of the natural bases.85 Therefore, our reported differences in the magnitude of the hydrogen-bonding and stacking interactions may be slightly exaggerated. Nevertheless, the number and large size of the molecules considered currently excluded the accurate prediction of this correction in the present studies. Since the trends are generally accurate with MP2, we anticipate that our major conclusions will hold at higher-levels of theory. The implications of the greater stacking in xDNA and stronger hydrogen-bonding in yDNA can be understood by considering the total interaction energies (see “total” column in Table 3). Replacing the natural pyrimidines with x-pyrimidines generally leads to a net increase in the stabilization of the four nucleobase sequence of 10 - 25 kJ mol-1 (8-10%) while y-pyrimidines lead to a 15 - 40 kJ mol-1 (10-14%) stabilization. Indeed, replacement of x-pyrimidines with y-pyrimidines stabilizes the complex for all but three nucleobase sequences, which are composed of only expanded A:T pairs. However, there is a strong sequence dependence on the magnitude of the increase in stabilization provided by base expansion. For example, in the X1X2Y1Y2 ) nCGGnC combination, yC leads to 25 kJ mol-1 net stronger stabilization than xC, while in the X1X2Y1Y2 ) nTAAnT combination, xT leads to 12 kJ mol-1 net stronger stabilization than yT. Thus, our previous conclusion based on the center-of-mass stacking interactions that the y-pyrimidines may lead to more stable strands through enhanced stacking does not hold when larger models are considered. Instead, xDNA pyrimidines generally stack stronger in model helices. Nevertheless, when the total stabilization of the helix is considered, which includes all intrastrand and interstrand stacking and hydrogen-bonding interactions, y-pyrimidines lead to more stable four nucleobase sequences than x-pyrimidines for all but three base sequences. Conclusions The present study complements previous computational work on expanded nucleobases designed through incorporation of a benzene spacer by comparing the energy profile for rotation about the glycosidic bond, the (Watson-Crick) hydrogen-bond strengths, and the stacking interactions for natural, x- and y-pyrimidines. We find that the favored orientation of yDNA pyrimidines about the glycosidic bond more closely mimics that of the natural DNA pyrimidines compared with the xDNA counterparts. This suggests that the yDNA pyrimidines may not lead to as many mismatches or destabilize the duplex to the extent of xDNA pyrimidines. Calculations of the hydrogen-bonding and stacking interactions between one expanded and one natural nucleobase reveal important information about the relative binding ability of x-

J. Phys. Chem. B, Vol. 112, No. 39, 2008 12535 and y-pyrimidines. Specifically, although there is a large (10 kJ/mol) difference in the hydrogen-bond strength of G:yC versus G:xC pairs, there is not a significant difference in the binding strengths of various expanded A:T pairs. More important to the overall structure and stability of duplexes is the fact that the anomeric carbon distances of mixed pairs involving the ypyrimidines more closely mimic those of mixed pairs involving expanded x-purines than when x-pyrimidines are incorporated into the pair. This suggests that yDNA may yield a more optimal size-extended helices. Additionally, although the binding strengths of xT and yT are within 5 kJ mol-1, yC can bind up to 30% stronger than xC. These properties indicate that y-pyrimidines may lead to more stable duplexes than x-pyrimidines. Expansion of our computational model by considering two stacked hydrogen-bonded pairs reveals that both x- and ypyrimidines yield mixed expanded duplexes of greater strength than natural duplexes due to increases in both the intra and interstrand stacking interactions. Furthermore, the total duplex stability, which is calculated as the sum of all hydrogen-bonding, intrastrand stacking and interstrand stacking interactions, reveals that y-pyrimidines lead to a greater total net stabilization compared to x-pyrimidines for all but three combinations of nucleobase sequences. In summary, our results reveal that the location of the benzene spacer affects the preferred structure and binding properties of expanded pyrimidines. Therefore, in addition to spacer composition, spacer location must be carefully considered when designing building blocks for widened helices. Future studies should more closely consider how the location of the benzene spacer affects the dependence of strand stability on the nucleobase sequence. The effects of the environment, which is known to attenuate binding strengths, on the total calculated strand stabilities must also be considered. References and Notes (1) Watson, J. D.; Crick, F. H. C. Nature 1953, 1171, 737. (2) Cobb, A. J. A. Org. Biomol. Chem. 2007, 5, 3260–3275. (3) Uhlmann, E.; Peyman, A. Chem. ReV. 1990, 90, 543–584. (4) Demesmaeker, A.; Haner, R.; Martin, P.; Moser, H. E. Acc. Chem. Res. 1995, 28, 366–374. (5) Kvaerno, L.; Wengel, J. Chem. Commun. 2001, 1419–1424. (6) Leumann, C. J. Chimia 2005, 59, 776–779. (7) Sahu, N. K.; Shilakari, G.; Nayak, A.; Kohli, D. V. Curr. Pharm. Biotechnol. 2007, 8, 291–304. (8) Vester, B.; Wengel, J. Biochemistry 2004, 43, 13233–13241. (9) Kaur, H.; Babu, B. R.; Maiti, S. Chem. ReV. 2007, 107, 4672– 4697. (10) Cosstick, R.; Buckingham, J.; Brazier, J.; Fisher, J. Nucleos. Nucleot. Nucl. 2007, 26, 555–558. (11) Krueger, A. T.; Kool, E. T. Curr. Opin. Chem. Biol. 2007, 11, 588– 594. (12) Rozners, E. Lett. Org. Chem. 2005, 2, 496–500. (13) Hirao, I. Curr. Opin. Chem. Biol. 2006, 10, 622–627. (14) Wilson, J. N.; Kool, E. T. Org. Biomol. Chem. 2006, 4, 4265– 4274. (15) Henry, A. A.; Romesberg, F. E. Curr. Opin. Chem. Biol. 2003, 7, 727–733. (16) Leonard, N. J.; Morrice, A. G.; Sprecker, M. A. J. Org. Chem. 1975, 40, 356–363. (17) Czarnik, A. W.; Leonard, N. J. J. Am. Chem. Soc. 1982, 104, 2624– 2631. (18) Leonard, N. J.; Sprecker, M. A.; Morrice, A. G. J. Am. Chem. Soc. 1976, 98, 3987–3994. (19) Leonard, N. J.; Hiremath, S. P. Tetrahedron 1986, 42, 1917–1961. (20) Leonard, N. J.; Scopes, D. I. C.; Vanderlijn, P.; Barrio, J. R. Biochemistry 1978, 17, 3677–3685. (21) Leonard, N. J. Acc. Chem. Res. 1982, 15, 128–135. (22) Lessor, R. A.; Gibson, K. J.; Leonard, N. J. Biochemistry 1984, 23, 3868–3873. (23) Krueger, A. T.; Lu, H. G.; Lee, A. H. F.; Kool, E. T. Acc. Chem. Res. 2007, 40, 141–150. (24) Liu, H. B.; Gao, J. M.; Lynch, S. R.; Saito, Y. D.; Maynard, L.; Kool, E. T. Science 2003, 302, 868–871.

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JP805547P

yDNA versus xDNA Pyrimidine Nucleobases: Computational Evidence

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